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Role of Protein Kinase C and Its Adaptor Protein p62 in Voltage-Gated Potassium Channel Modulation in Pulmonary Arteries

【关键词】  Role

    Voltage-gated potassium (KV) channels play an essential role in regulating pulmonary artery function, and they underpin the phenomenon of hypoxic pulmonary vasoconstriction. Pulmonary hypertension is characterized by inappropriate vasoconstriction, vascular remodeling, and dysfunctional KV channels. In the current study, we aimed to elucidate the role of PKC and its adaptor protein p62 in the modulation of KV channels. We report that the thromboxane A2 analog 9,11-dideoxy-11,9-epoxymethano-prostaglandin F2 methyl acetate (U46619[GenBank]) inhibited KV currents in isolated mice pulmonary artery myocytes and the KV current carried by human cloned KV1.5 channels expressed in Ltk– cells. Using protein kinase C (PKC)–/– and p62–/– mice, we demonstrate that these two proteins are involved in the KV channel inhibition. PKC coimmunoprecipitated with KV1.5, and this interaction was markedly reduced in p62–/– mice. Pulmonary arteries from PKC–/– mice also showed a diminished [Ca2+]i and contractile response, whereas genetic inactivation of p62–/– resulted in an absent [Ca2+]i response, but it preserved contractile response to U46619.[GenBank] These data demonstrate that PKC and its adaptor protein p62 play a key role in the modulation of KV channel function in pulmonary arteries. These observations identify PKC and/or p62 as potential therapeutic targets for the treatment of pulmonary hypertension.

    Voltage-gated potassium (KV) channels play an essential role in regulating vascular smooth muscle function. They make a substantial contribution to whole-cell K+ conductance and resting membrane potential in pulmonary artery smooth muscle cells (PASMCs), and its inhibition causes membrane depolarization, activation of L-type Ca2+ channels (CaL), increases in [Ca2+]i, and vasoconstriction (Barnes and Liu, 1995; Archer et al., 1998; Yuan et al., 1998b). These channels are common targets of pulmonary vasoconstrictor stimuli such as hypoxia, thromboxane A2 (TXA2), 5-hydroxytryptamine, endothelin-1 or angiotensin-II (Archer et al., 1998; Shimoda et al., 2001; Cogolludo et al., 2003, 2006). In addition, decreased expression or function of KV channels in PASMCs has been involved in the pathogenesis of pulmonary arterial hypertension (PH) (Weir et al., 1996, Yuan et al., 1998a; Pozeg et al., 2003). From the variety of KV channels expressed in PASMC (Platoshyn et al., 2006), special interest has been paid to KV1.5, because decreased expression or activity and mutations of KV1.5 occur in human (Yuan et al., 1998a; Remillard et al., 2007) and experimental (Archer et al., 1998; Pozeg et al., 2003) idiopathic and hypoxic PH, and in vivo gene transfer of KV1.5 reduces PH (Pozeg et al., 2003).

    TXA2 is a prostanoid synthesized by cyclooxygenase with potent vasoconstrictor, mitogenic, and platelet aggregant properties via activation of thromboxane-endoperoxide (TP) receptors (Halushka et al., 1989). The vasoconstrictor effects of TXA2 are particularly pronounced in the pulmonary vascular bed, where it participates in the control of vessel tone under physiological and pathological situations, including PH. We have previously reported that in intact PAs and freshly isolated PASMCs, TXA2, via activation of TP receptors, inhibits KV channels, leading to membrane depolarization, activation of L-type Ca2+ channels, and vasoconstriction. Furthermore, using a protein kinase C (PKC) pseudosubstrate inhibitory peptide (PKC-PI), we provided evidence for the role of this kinase as a link between TP receptor activation and KV channel inhibition (Cogolludo et al., 2003, 2005). PKC (together with PKC/) belongs to the atypical PKC (aPKC) subclass. Both aPKCs play key roles in different signaling pathways regulating cell growth, survival, and differentiation (Moscat and Díaz-Meco, 2000). The aPKCs share with other members of their family a conserved catalytic domain, but they display a clearly distinct regulatory region because they have been shown to be independent of Ca2+, diacylglycerol, and phorbol esters, all of which are potent activators of other PKC isoforms. PKC is activated by phosphatidylinositols, arachidonic acid, and other lipids (Hirai and Chida, 2003) as well as by a variety of mediators, including insulin (Liu et al., 2006), thromboxane A2 (Shizukuda and Buttrick, 2002; Cogolludo et al., 2003, 2005), angiotensin II (Gayral et al., 2006; Godeny and Sayeski, 2006), or proinflammatory cytokines (Frey et al., 2006).

    The mechanism underlying the activation of aPKCs responsible for its diverse physiological functions remains unclear, but several groups have identified a number of aPKC-interacting proteins, including p62 (also called ZIP1 or sequestosome 1), Par-4, Par-6, and MEK5 (Moscat and Diaz-Meco, 2000). It is noteworthy that nerve growth factor and catecholamines have been reported to increase the expression of p62, enabling the formation of the PKC-p62-KV complex, which results in a hyperpolarizing shift in the KV current activation curve (Gong et al., 1999; Kim et al., 2004, 2005).

    The role of PKC on pulmonary vasoconstriction has been widely reported (Ward et al., 2004); however, many of these studies have been conducted with PKC modulators of dubious selectivity, thereby limiting their conclusions. Molecular biology and genetic approaches and the currently available isoform-selective PKC inhibitors have made possible the elucidation of the involvement of specific PKC isoforms in cellular processes (such as vascular contractility) (Salamanca and Khalil, 2005). However, recent evidence suggests that some considered isoform-specific PKC inhibitors, such as myristoylated PKC pseudosubstrate peptide, may exert other effects unrelated to inhibition of PKC; thus, they should be used with caution (Krotova et al., 2006).

    Therefore, in the present study, we aimed to further characterize the signaling pathway modulating KV currents in PAs. Using PKC–/– and p62–/– mice, we provide evidence for the interaction of PKC with KV channels, which further support the role of this interaction in TXA2-induced effects. In addition, we hypothesized that the PKC-KV-L-type Ca2+ channels pathway might involve other proteins such as p62. This possibility was tested by analyzing the modulation of KV channels in wild-type and p62 homozygous null mice.

    All experiments were carried out in accordance with the European Animals Act 1986 (Scientific Procedures), and they were approved by our institutional review board.

    Animals. Lungs from PKC–/– (mixed C57BL/6 and SV129J background), p62–/– (C57BL/6), and corresponding wild-type mice (6–8 weeks old; either sex) were generously supplied by Drs. J. Moscat and M. T. Diaz-Meco (both from the Genome Research Institute, University of Cincinnati, Cincinnati, OH). These mice were generated as described previously (Leitges et al., 2001; Duran et al., 2004). PAs from male Wistar rats (250–300 g) were also used in these experiments.

    Tissue Preparation and Cell Isolation. Second-order branches of the PA (internal diameter, 0.5 mm) isolated from mice were dissected into a nominally calcium-free physiological salt solution (PSS) of the following composition: 130 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 10 mM glucose, and 10 mM HEPES, pH 7.3, with NaOH. Endothelium denuded PAs were cut into small segments (2 x 2 mm), and cells were isolated in Ca2+-free PSS containing 1 mg/ml papain, 0.8 mg/ml dithiothreitol, and 0.7 mg/ml albumin. Cells were stored in Ca2+-free PSS (4°C) and used within 8 h of isolation.

    Electrophysiological Studies. Membrane currents were measured using the whole-cell configuration of the patch-clamp technique (Cogolludo et al., 2003) normalized for cell capacitance and expressed in picoamperes per picofarad. Membrane potential (Em) was measured under current-clamp configuration. KV currents were recorded under essentially Ca2+-free conditions using an external Ca2+-free PSS and a Ca2+-free pipette (internal) solution (see Solutions and Chemicals). Ltk– cells stably expressing hKV1.5 channels (Valenzuela et al., 1995) were superfused with PSS containing 1 mM CaCl2. Currents were evoked after the application of 200-ms depolarizing pulses from –60 mV to test potentials from –60 to +40 mV in 10-mV increments. All experiments were performed at room temperature (22–24°C).

    [Ca2+]i Recording. PA rings were incubated for 80 min at room temperature in Krebs' solution containing the fluorescent dye fura-2 acetoxymethyl ester (5 x 10–6 M) and 0.05% cremophor EL, and then they were mounted in a fluorimeter (model CAF 110; Jasco, Tokyo, Japan). PA rings were alternatively illuminated (128 Hz) with two excitation wavelengths (340 and 380 nm), and the emitted fluorescence was filtered at 505 nm (Pérez-Vizcaíno et al., 1999). The ratio of emitted fluorescence (F340/F380) obtained at the two excitation wavelengths was used as an indicator of [Ca2+]i. Arteries were stimulated with 30 and 300 nM U46619[GenBank], added in a cumulative manner. In preliminary experiments in wild-type mice, these concentrations produced 60 and 80% of the maximal response, respectively. The [Ca2+]i signal in each vessel was calibrated according to the Grynkiewicz equation by sequential addition of 15 µM ionomycin and 10 mM EGTA at the end of the experiment.

    Coimmunoprecipitation and Western Blot Analysis. Mice lungs were rapidly frozen in liquid nitrogen. In some experiments, rat PA were placed in warm Krebs' solution and then in the absence or presence of 1 µM U46619[GenBank] for 30 s and then rapidly frozen. Frozen tissues were homogenized in a glass potter in 200 µl of a buffer of the following composition: 10 mM HEPES, pH 8, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 6 µM aprotinin, 9 µM leupeptin, 11 µM N-p-tosyl-L-lysine chloromethyl ketone, 5 mM NaF, 10 mM Na2MoO4, 1 mM NaVO4, 0.5 mM phenylmethanesulfonyl fluoride, and 10 nM okadaic acid. Homogenates were centrifuged at 13,000g for 5 min at 4°C, and the supernatant fraction was collected. For immunoprecipitation, 60 µg of protein was incubated for 2 h with anti-PKC or anti-KV1.5 antibody at 4°C, followed by the addition of protein A/G beads and further incubation overnight. These immune complexes or 20 µg of the homogenates from mice lungs or rat PA were separated by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane for Western blotting as described previously (Cogolludo et al., 2003). Membranes were probed for KV1.5-, PKC-, and p62-like immunoreactivity.

    Solutions and Chemicals. For the single cell electrophysiological studies, the composition of the Ca2+-free bath solution (external Ca2+-free PSS) was as follows: 130 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 10 mM glucose, and 10 mM HEPES, buffered to pH 7.3 with NaOH. The Ca2+-free pipette (internal) solution contained 110 mM KCl, 1.2 mM MgCl2, 5 mM Na2ATP, 10 mM HEPES, and 10 mM EGTA, pH adjusted to 7.3 with KOH. The Krebs' solution used for tissue bath experiments included 118 mM NaCl, 4.75 mM KCl, 25 mM NaHCO3, 1.2 mM MgSO4, 2.0 mM CaCl2, 1.2 mM KH2PO4, and 11 mM glucose. This solution was gassed with a 95% O2, 5% CO2 mixture at 37°C. U46619[GenBank] was obtained from Sigma Chemical Co. (Tres Cantos, Spain). [1S-1,2,5]-[5-Methyl-2-(1-methylethyl) cyclohexyl] diphenyl phosphine oxide (DPO-1) was from Tocris Cookson Inc. (Bristol, UK), secondary horseradish peroxidase-conjugated antibodies and fura-2 acetoxymethyl ester were from Calbiochem (Barcelona, Spain), rabbit anti-KV1.5 was from Alomone Labs (Jerusalem, Israel), goat anti-PKC was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and guinea pig anti-p62 was from Progen (Heidelberg, Germany).

    Statistical Analysis. Data are expressed as means ± S.E.M.; n indicates the number of arteries or cells tested. All experiments were conducted in arteries or cells from at least four different animals. Statistical analysis was performed using Student's t test for paired or unpaired observations. Differences were considered statistically significant when p was less than 0.05.

    Role of PKC in KV Current Inhibition Induced by TXA2. A family of KV currents [IK(V)] were obtained in mice PASMCs when eliciting depolarizing steps from –60 to +40 mV (Fig. 1, A and B) from a holding potential of –60 mV. The magnitude of the currents, the threshold voltage for activation, and the current-voltage relationship (Fig. 1, C and D) was similar in PASMCs from wild-type and PKC–/– mice (e.g., current density at +40 mV was 9.1 ± 1.9 and 8.7 ± 0.8 pA/pF, respectively). Current inactivation was also similar in both strains (i.e., at 200 ms, the current decayed by 11.5 ± 3 and 12.1 ± 3.8%, respectively). Currents were recorded before (control) and after addition of the TXA2 analog U46619.[GenBank] U46619 (100 nM) caused a significant inhibition of KV currents in the whole range of channel activation in PASMCs from wild-type mice (Fig. 1A). The degree of current inactivation at +40 mV was increased by U46619[GenBank] (i.e., at 200 ms, the current decayed by 25.6 ± 4.8%; p < 0.05). In addition, U46619[GenBank] induced membrane depolarization in wild-type PASMCs (Fig. 1E). However, U46619[GenBank] had no effect on either KV currents or membrane potentials in PASMC from PKC–/– mice (Fig. 1, B, D, and F).

    Fig. 1. TP receptor activation leads to KV current inhibition and depolarization in PASMCs from PKC+/+ (A, C, and E) but not from PKC–/– (B, D, and F) mice. A and B, current traces for 200-ms depolarization pulses from –60 to +40 mV (in 10-mV increments) from a holding potential of –60 mV before (control) and after application of the TXA2 analog U46619 (100 nM). C and D, current-voltage relationship measured at the end of the 200-ms pulse (means ± S.E.M. of five cells). E and F, effects of 100 nM U46619 on membrane potential recorded under current clamp conditions. *, p < 0.05 and **, p < 0.01, respectively, versus control (paired Student's t test).

    Role of PKC in [Ca2+]i Increase and Contraction Induced by TXA2. Changes in [Ca2+]i and contraction induced by U46619[GenBank] were simultaneously analyzed in fura-2-loaded PAs from wild-type and from PKC–/– mice. Basal levels of [Ca2+]i in PKC–/– (203 ± 40 nM; n = 6) were not significantly different from those in wild-type mice (160 ± 40 nM; n = 6). Stimulation of endothelium-denuded PA rings with 30 and 300 nM U46619[GenBank] induced a sustained elevation in [Ca2+]i and a contractile response in PAs from wild-type and PKC–/– animals (Fig. 2, A and B). However, the increase in [Ca2+]i (Fig. 2C) and the contractile response (Fig. 2D) was significantly reduced in PKC–/– mice compared with wild-type mice.

    Fig. 2. PA from PKC–/– mice show reduced [Ca2+]i and contractile responses induced by TP receptor activation. A and B, simultaneous recordings of [Ca2+]i (top trace) and force (bottom trace) in PAs from PKC+/+ and PKC–/–, respectively, stimulated by 30 and 300 nM U46619. The averaged values (means ± S.E.M. of five to seven PAs) of U46619-induced increase in [Ca2+]i and force are shown in C and D, respectively. *, p < 0.05 versus PKC+/+ (unpaired Student's t test).

    Role of p62 in KV Current Inhibition, [Ca2+]i Increase, and Contraction. To analyze the functional role of p62 and the PKC-p62-KV1.5 interaction, we analyzed the effects of 100 nM U46619[GenBank] on KV currents in p62–/– and the corresponding wild-type mice. The magnitude of the currents, the threshold voltage for activation, the current-voltage relationship, and the current inactivation (Fig. 3, C and D) were similar in PASMCs from wild-type and p62–/– mice (e.g., current density at +40 mV was 10.9 ± 1.3 and 11.6 ± 1.7 pA/pF, respectively; and at 200 ms, current decayed by 12.7 ± 2.8 and 14.3 ± 2.9%, respectively). As expected, U46619[GenBank] caused a significant inhibition in the whole range of channel activation and depolarized the membrane in PASMCs from wild-type mice (Fig. 3, A, C, and E). Current inactivation at +40 mV was also increased by U46619[GenBank] (i.e., at 200 ms, the current decayed by 21.7 ± 2.9%; p < 0.05). However, the TXA2 analog had no effect on KV currents in PASMCs from p62–/– mice (Fig. 3, B, D, and E).

    Fig. 3. TP receptor activation leads to KV current inhibition and depolarization in PASMCs from wild-type (A, C, and E) but not from p62–/– (B, D, and F) mice. A and B, current traces for 200-ms depolarization pulses from –60 to +40 mV (in 10-mV increments) from a holding potential of –60 mV before (control) and after application of the TXA2 analog U46619 (100 nM). C and D, current-voltage relationship measured at the end of the 200-ms pulse (means ± S.E.M. of five to six cells). E and F, effects of U46619 on membrane potential recorded under current-clamp conditions.*, p < 0.05 versus control (paired Student's t test).

    Basal levels of [Ca2+]i in p62–/– (184 ± 35 nM; n = 5) were not significantly different from those in wild-type mice (170 ± 45 nM; n = 6). We found that genetic inactivation of p62 abolished the increase in [Ca2+]i induced by U46619[GenBank] (Fig. 4, B and C). However, the contractile response induced by the two concentrations of U46619[GenBank] tested was remarkably similar in p62–/– and wild-type mice (Fig. 4D).

    Fig. 4. PAs from p62–/– mice show no [Ca2+]i responses but preserved contractions induced by TP receptor activation. A and B, simultaneous recordings of [Ca2+]i (top trace) and force (bottom trace) in PA from p62+/+ and p62–/–, respectively, stimulated by 30 and 300 nM U46619. The averaged values (means ± S.E.M. of five PAs) of U46619-induced increase in [Ca2+]i and force are shown in C and D, respectively. *, p < 0.05 versus p62+/+ (unpaired Student's t test).

    Role of KV1.5 Channels in TXA2-Induced Effects. KV1.5 channels have been reported to be major contributors of KV currents in PASMCs in several animal species. Figure 5A shows hKV1.5 current traces recorded in Ltk– cells stably expressing hKV1.5 channels. U46619[GenBank] (100 nM) significantly inhibited hKV1.5 currents. This inhibitory effect was only observed at the end of the depolarizing pulse; e.g., currents were almost unaffected at the peak (4.6 ± 2.4% decrease; not significant), but they were reduced by 17.8 ± 4.2% after 200 ms (n = 4; p < 0.05). In rat PASMCs, U46619[GenBank] also inhibited KV currents (Fig. 5B) as described previously (Cogolludo et al., 2003). The KV1.5 channel blocker DPO-1 (Lagrutta et al., 2006) inhibited KV currents in rat PASMCs. In the presence of DPO-1, U46619[GenBank] produced no further inhibitory effects (Fig. 5B). Therefore, we analyzed a possible interaction between PKC, KV1.5 channels and p62. Rat pulmonary arteries were incubated for 30 s in the absence (control) or presence of U46619.[GenBank] Homogenates were immunoprecipitated with anti-PKC or anti-KV1.5 antibodies, and the content of KV1.5, PKC, or p62 in the immunoprecipitates was analyzed via Western blot. Figure 5C shows that in immunoprecipitates of KV1.5 both PKC and p62 were present. The KV1.5-PKC and the KV1.5-p62 association were 135 ± 13% (n = 8; p = 0.06, not significant) and 163 ± 31% (n = 7; p < 0.05), respectively, in U46619[GenBank]-treated versus untreated arteries. The KV1.5-PKC interaction was also observed in immunoprecipitates of PKC immunoblotted with the anti-KV1.5 antibody (data not shown).

    Fig. 5. Role of KV1.5 channels: TP receptor activation inhibits KV1.5 currents and increases coimmunoprecipitation of KV1.5 with PKC. A, current traces recorded in Ltk– cells stably transfected with human KV1.5 before (control) and after 100 nM U46619 and current-voltage relationship (means ± S.E.M. of four cells) measured at the end of the 200-ms pulse. Depolarizing steps from –60 to +60 mV were applied from a holding potential of –60 mV. *, p < 0.05 versus control (paired Student's t test). B, current traces recorded in rat PASMCs cells before (control) and after the 100 nM U46619 (top) or before (control), after DPO-1 (300 nM), and after DPO-1 plus U46619 (bottom). Current-voltage relationships are shown at the right (means ± S.E.M. of three to four cells). *, p < 0.05 versus control (paired Student's t test). C, rat pulmonary arteries were incubated for 30 s in the absence (control) or presence of 100 nM U46619, frozen, and homogenated. Homogenates were immunoprecipitated with anti-KV1.5 antibodies and immunoblotted with anti-PKC or anti-p62. Results are representative of samples from seven to eight mice. Each pair of bands (control and U46619) is obtained from the same animal.

    Interaction of PKC with KV Channels: Role of p62. To determine the potential role of the PKC scaffold protein p62, the PKC-KV1.5 interaction was analyzed by coimmunoprecipitation in lungs from wild-type and p62–/– mice. Genetic inactivation of p62 in mice did not modify the expression levels of either PKC or KV1.5 channels in PASMCs (Fig. 6A). In immunoprecipitates of PKC from wild-type mice immunoblotted with the anti-KV1.5 antibody, a band of approx. 80 kDa was observed, which presumably reflects the mature (glycosylated) form of the channel expressed in the membrane (Li et al., 2000). However, p62-deficient mice showed a weak PKC-KV1.5 coimmunoprecipitation (Fig. 6B).

    Fig. 6. Similar expression of PKC and KV1.5 but reduced interaction between PKC and KV 1.5 in p62–/– versus p62+/+. A, representative Western blots of lung homogenates using anti-KV1.5 and anti-PKC antibodies. B, lung homogenates were immunoprecipitated with anti-PKC antibodies and immunoblotted with anti-KV1.5; membranes were reblotted with the anti-PKC antibody as a loading control. The graph shows the densitometric analysis of the KV1.5 protein relative to PKC and expressed as a percentage of values in wild-type mice. **, p < 0.01 versus wild type.

    By using nonselective PKC inhibitors and the PKC-selective inhibitor PKC-PI, we suggested that PKC was involved in the KV channel inhibition and the contractile response induced by TXA2 in rat pulmonary artery myocytes (Cogolludo et al., 2003, 2005). Herein, we confirmed the role of PKC in native KV currents by using PASMCs from PKC–/– mice. Consistent with the essential role of KV1.5 channels in the pulmonary vasculature, we show that the KV1.5 inhibitor DPO-1 inhibited KV currents in native rat PASMCs by approx. 50% and that the TXA2 analog U46619[GenBank] had no further inhibitory effects. In addition, cloned human KV1.5 channels expressed in Ltk– cells were also inhibited by U46619.[GenBank] Moreover, our results demonstrate the interaction between PKC and KV1.5 in both rat PAs and mouse lungs, which was minimal in p62–/– mice. Deletion of p62 abolished KV channel inhibition and Ca2+ responses induced by TXA2, further supporting the role of p62 as a key mediator between PKC and KV1.5. However, our study also showed that the contractile response induced by U46619[GenBank] in PA was similar in wild-type and p62–/– mice.

    In both rat and newborn porcine PASMCs, U46619[GenBank] inhibited KV currents, depolarized cell membrane, increased [Ca2+]i through CaL channels, and induced a contractile response (Cogolludo et al., 2003, 2005). U46619[GenBank] had no direct effect on CaL channels in voltage-clamped cells, indicating that increased Ca2+ entry through CaL channels is secondary to membrane depolarization. Herein, we demonstrated that, in mice, U46619[GenBank] also inhibits KV currents in PASMCs and induces a [Ca2+]i response and vasoconstriction in isolated PA. The degree of KV channel inhibition in mice PASMCs (25% at 100 nM U46619[GenBank]) was similar to that observed in porcine and in rat PA, and it was accompanied by a significant membrane depolarization. In rat and porcine PAs, all these effects were inhibited by calphostin C and PKC-PI (Cogolludo et al., 2003, 2005). These experiments suggested a role for PKC as a link between TP receptors and KV channels, which was confirmed in the present study using PKC–/– mice. The magnitude and current-voltage relationship of KV currents were similar in the wild-type and knock-out animals, suggesting no changes in the channel proteins underlying KV currents. Thus, genetic inactivation or pharmacological inhibition of PKC abolished the effects of U46619[GenBank] on KV currents or membrane potential in PASMCs. In contrast, both approaches only partially inhibited (50–70%) the Ca2+ signal induced by U46619[GenBank] in rat and mice PAs, indicating that, in addition to the PKC-KV-CaL pathway, mechanisms increasing [Ca2+]i (e.g., Ca2+ release from intracellular stores) are also activated in response to U46619[GenBank] (Snetkov et al., 2006).

    The present experiments also indicate that in mice, PKC contributes to the vasoconstriction induced by TP receptor activation. These results are in agreement with those obtained in rats and newborn piglets using PKC-PI (Cogolludo et al., 2003, 2005). However, in 2-week-old piglets (Cogolludo et al., 2005), PKC-PI and the Ca2+ channel blocker nifedipine almost fully inhibited U46619[GenBank]-induced increases in [Ca2+]i, but they had no effect on U46619[GenBank]-induced contractile responses; i.e., there was a contractile response in the absence of changes in [Ca2+]i. Therefore, in these animals, the up-regulation of Ca2+-independent mechanisms for contraction (Somlyo and Somlyo, 2000) makes PKC and the [Ca2+]i signal redundant.

    KV currents recorded in native PASMCs reflect the contribution of multiple KV channel proteins [e.g., in human PAs, 22 transcripts of KV subunits: KV1.1 to KV1.7, KV1.10, KV2.1, KV3.1, KV3.3, KV3.4, KV4.1, KV4.2, KV5.1, KV6.1 to -6.3, KV9.1, KV9.3, KV10.1, and KV11.1, and three of KV subunits KV1 to -3 have been identified by reverse transcription-polymerase chain reaction]. However, KV1.5 subunits are thought to be major contributors of the native KV currents in PAs from different species, and their activity is regulated by vasoactive factors such as 5-hydroxytryptamine (Cogolludo et al., 2006) and hypoxia (Platoshyn et al., 2006). Therefore, we analyzed the effects of U46619[GenBank] on the KV current carried by human cloned KV1.5 channels expressed in mouse fibroblast (Ltk–) cells. This cell line expresses endogenously the KV2.1 subunit, which assembles with the transfected hKv1.5 protein (Uebele et al., 1996). U46619[GenBank] induced a weak but significant inhibitory effect on this current, suggesting that KV1.5 channels are involved in the effects of TP receptor activation in native PASMCs. The small inhibition in this cell type probably reflects a lower efficacy of the signaling pathway compared with rat or mouse PASMCs. Furthermore, after pharmacological inhibition of KV1.5 channels with DPO-1, U46619[GenBank] had no further inhibitory effects on KV currents in rat PASMCs.

    In the present article, we show that PKC coimmunoprecipitates with KV1.5 channels. In a previous study (Cogolludo et al., 2003), we reported that U46619[GenBank] induced the translocation of PKC from the cytosolic to the membrane fraction. Therefore, TP receptor-induced KV channel inhibition is associated with the translocation of PKC to the plasma membrane where it interacts with KV1.5 channels. This PKC-KV1.5 interaction is not necessarily a direct protein-protein interaction; it seems more likely that it is mediated by adaptor proteins. In this regard, it has been described that PKC can interact with the  subunit KV2 of the KV channel via the p62 adaptor protein (Gong et al., 1999). In immunoprecipitation experiments, we found that p62 was present in the KV1.5-PKC complex. Even when the complex was constitutive, the association of p62 with KV1.5 increased significantly by U46619.[GenBank] Furthermore, the PKC-KV1.5 coimmunoprecipitation was strongly reduced in p62–/– mouse lung, indicating that p62 physically associates PKC into the KV channel complex.

    KV subunits function as molecular chaperones, and they can directly regulate channel inactivation, voltage dependence, and current amplitude (Martens et al., 1999). p62 overexpression stimulates PKC-dependent phosphorylation of KV2 (Gong et al., 1999), and it induces a hyperpolarizing shift of KV current activation in pheochromocytoma cells (Kim et al., 2004). Thus, we analyzed the effect of genetic inactivation of p62 on KV currents and its modulation by TP receptor activation. KV currents in PASMCs from p62–/– were similar to wild type. As expected, U46619[GenBank] had no effect on KV currents in p62–/– PASMCs, indicating that the p62-dependent PKC-KV1.5 interaction is required for the inhibitory effect of TP receptor activation on KV current.

    Thus, genetic inactivation of p62 had a similar effect to genetic or pharmacological inactivation of PKC regarding KV current modulation. We were surprised to find that p62 gene deletion fully inhibited the Ca2+ response induced by U46619[GenBank] in isolated PAs compared with a 50 to 70% inhibition by PKC inactivation. More intriguingly, the contractile response to U46619[GenBank] was not affected in PA from p62–/– mice. This contractile response in the absence of changes in [Ca2+]i must then be attributed to Ca2+-independent mechanisms (i.e., Ca2+ sensitization; Somlyo and Somlyo, 2000). This response to U46619[GenBank] in p62–/– mice PA is similar to that observed in 2-week-old piglet PAs after inhibition of PKC (i.e., contraction without [Ca2+]i signal) (Cogolludo et al., 2005). In these animals, there is an up-regulation of Rho kinase (Bailly et al., 2004), a key enzyme in Ca2+-sensitizing mechanisms. In addition, Rho kinase inhibitors were more effective inhibiting U46619[GenBank] contractions in these piglets than in newborn piglets or adult rats (Cogolludo et al., 2005). Thus, we speculate that the chronic down-regulation of the PKC-p62-KV-CaL-dependent pathway, either at the level of KV channel activity (as occurs in older piglets) or p62 (p62–/– mice), but not PKC (PKC–/– mice), is compensated by up-regulation of Ca2+ sensitization mechanisms.

    In conclusion, PKC modulates KV channel function, and it is involved in pulmonary vasoconstriction induced by TP receptor activation. The interaction between PKC and KV1.5 and the inhibitory effect of U46619[GenBank] in cloned human KV1.5 channels suggest that these specific channel subtypes are functional targets for PKC. The adaptor protein p62 is required for the PKC-KV1.5 interaction and hence for the inhibition of KV currents after TP receptor activation.

    ABBREVIATIONS: KV, voltage-gated K+; PASMC, pulmonary artery smooth muscle cell; CaL, voltage-dependent L-type Ca2+ channel; TXA2, thromboxane A2; PH, pulmonary hypertension; TP, thromboxane-endoperoxide; PKC, protein kinase C; PA, pulmonary artery; IP, inhibitory peptide; aPKC, atypical PKC; PSS, physiological salt solution; h, human; U46619[GenBank], 9,11-dideoxy-11,9-epoxymethano-prostaglandin F2; DPO-1, [1S-1,2,5]-[5-methyl-2-(1-methylethyl) cyclohexyl] diphenyl phosphine oxide.

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作者单位:Department of Pharmacology, School of Medicine, Universidad Complutense de Madrid, Madrid, Spain

日期:2009年8月25日 - 来自[2007年第69卷第11期]栏目
循环ads

Functional Characterization of the Promoter of Human Carbonyl Reductase 1 (CBR1). Role of XRE Elements in Mediating the Induction of CBR1 by Ligands of the Ar

【关键词】  Functional

    Human carbonyl reductase 1 (CBR1) metabolizes a variety of substrates, including the anticancer doxorubicin and the antipsychotic haloperidol. The transcriptional regulation of CBR1 has been largely unexplored. Therefore, we first investigated the promoter activities of progressive gene-reporter constructs encompassing up to 2.4 kilobases upstream of the translation start site of CBR1. Next, we investigated whether CBR1 mRNA levels were altered in cells incubated with prototypical receptor activators (e.g., dexamethasone and rifampicin). CBR1 mRNA levels were significantly induced (5-fold) by the ligand of the aryl hydrocarbon receptor (AHR) -naphthoflavone. DNA sequence analysis revealed two xenobiotic response elements (–122XRE and –5783XRE) with potential regulatory functions. CBR1 promoter constructs lacking the –122XRE showed diminished (9-fold) promoter activity in AHR-proficient cells incubated with -naphthoflavone. Fusion of –5783XRE to the –2485CBR1 reporter construct enhanced its promoter activity after incubations with -naphthoflavone by 5-fold. Furthermore, we tested whether the potent AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced Cbr1 expression in Ahr+/– and Ahr–/– mice. TCDD induced hepatic Cbr1 mRNA (TCDD, 2-fold) and Cbr1 protein levels (TCDD, 2-fold) in Ahr+/– mice compared with vehicle-injected controls. In contrast, no significant Cbr1 mRNA and Cbr1 protein induction was detected in livers from Ahr–/– mice treated with TCDD. These studies provide the first insights on the functional characteristics of the human CBR1 gene promoter. Our data indicate that the AHR pathway contributes to the transcriptional regulation of CBR1.

    Human carbonyl reductase 1 (CBR1) catalyzes the NADPH-dependent reduction of a variety of xenobiotic compounds including smoke derived carcinogens and many relevant pharmacological agents. For example, CBR1 catalyzes the two-electron reduction of the C-13 carbonyl group of the anticancer anthracyclines doxorubicin and daunorubicin to generate their corresponding alcohol metabolites (doxorubicinol and daunorubicinol) (Forrest and Gonzalez, 2000). Anthracycline C-13 alcohol metabolites are cardiotoxic, have diminished tumor cell killing activities, circulate in plasma at various levels, and contribute to the unpredictable pharmacology of anthracycline drugs (Frost et al., 2002; Minotti et al., 2004). Significant interindividual variability in carbonyl reductase activity (CBR) has been documented in liver, erythrocytes, and in breast and lung tumors (Iwata et al., 1993; Wong et al., 1993; Rady-Pentek et al., 1997; Lopez de Cerain et al., 1999). We observed wide ranges of CBR activities in liver cytosols from black (range, 4.1–21.5 nmol/min · mg) and white donors (range, <0.1–28.0 nmol/min · mg) (Covarrubias et al., 2006). However, the molecular basis of such disparities and its potential impact on CBR-mediated drug metabolism remain to be elucidated. We hypothesize that interindividual differences in CBR activity may in part reflect variable rates of CBR1 gene transcription. CBR1 spans approximately 3.2 kb on chromosome 21 (21q22.13), contains three exons, and encodes for a monomeric 277 amino acid protein with a molecular weight of 30,375 (Wermuth et al., 1988). It is noteworthy that despite the major role of CBR1 in the biotransformation of xenobiotics, there is a paucity of reports focused on the functional characterization of the human CBR1 gene promoter. Therefore, the first aim of our study was to investigate the potential promoter activities of progressive DNA deletion constructs encompassing up to 2485 base pairs (bp) of genomic sequence 5' upstream the translation start site of CBR1 by using gene-reporter assays.

    Our second aim was to test whether CBR1 mRNA levels were induced in cell cultures incubated with prototypical activators of the nuclear glucocorticoid receptor, the constitutive androstane receptor, the pregnane X receptor, and the aryl hydrocarbon receptor (AHR), respectively. We detected significant induction of CBR1 mRNA expression in HepG2 and MCF-7 cells treated with the AHR ligand -naphthoflavone. AHR is a ligand-activated basic helix-loop-helix transcription factor that participates in the regulation of several key mammalian genes involved in the metabolism of xenobiotics (e.g., CYP1A1 and CYP1B1). After ligand binding, AHR translocates from the cytoplasm into the nucleus to form a complex with aryl hydrocarbon receptor nuclear translocator. The resulting ligand/AHR/aryl hydrocarbon receptor nuclear translocator complex interacts with specific DNA sequences termed xenobiotic responsive elements (XREs) to induce the transcription of target genes (Nebert et al., 2000; Nioi and Hayes, 2004). The consensus XRE sequence (5'-T/GNGCGTG-3') contains the substitution intolerant XRE core motif (5'-GCGTG-3') (Lusska et al., 1993). It is interesting that we identified two perfect XRE motifs located at 122 bp and 5783 bp upstream the translation start site of CBR1 (–122XRE, and –5783XRE). Therefore, our third aim was to investigate the functional impact of the proximal (–122XRE), and distal (–5783XRE) XRE motifs by performing gene reporter assays with engineered CBR1 promoter constructs.

    The development of Ahr-deficient mice (Ahr–/–) has contributed to the identification of a battery of genes regulated through the AHR pathway (Fernandez-Salguero et al., 1995; Zaher et al., 1998; Sugihara et al., 2001; Jiang et al., 2004). AHR mediates the induction of several key xenobiotic-metabolizing enzymes such as CYP1A1, CYP1B1, glutathione transferase, and NAD(P)H:quinone oxidoreductase (NQO1) (Nebert et al., 2000; Shimada et al., 2002). Thus, we extended our observations by testing whether the potent AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced Cbr1 mRNA and Cbr1 protein levels in livers from Ahr-proficient (Ahr+/–), and Ahr-deficient (Ahr–/–) mice. Together, our findings provide insights on the regulation of CBR1 and lay the foundation for future studies aimed toward the elucidation of the molecular bases that govern variable CBR activity in humans.

    Cell Culture and Reagents. HepG2 (human hepatocarcinoma, HB-8065), and MCF-7 (human breast adenocarcinoma, HTB-22) cell lines were obtained from the American Type Culture Collection (Manassas, VA). Minimum essential medium, fetal bovine serum, and other cell culture reagents were purchased from Invitrogen (Carlsbad, CA). Cells were routinely cultured in 75-cm2 vented flasks using -minimum essential medium supplemented with 10% fetal bovine serum. Cultures were grown in an incubator at 37°C, 5% CO2, and 95% relative humidity. Cultures were maintained at low passage numbers (n <12) and were free of mycoplasma contamination.

    Dexamethasone, clotrimazole, 1,4-bis-[2-(3,5-dichloropyridyloxy)]-benzene (TCPOBOP), and rifampicin were purchased from Sigma-Aldrich (St. Louis, MO). -Naphthoflavone was purchased from Indofine (Hillsborough, NJ).

    Cloning of CBR1 Promoter Constructs. Approximately 5 kilobases of DNA sequence upstream from the translation start codon (ATG) of CBR1 were amplified from human DNA sample HD17030 (Coriell Institute for Medical Research, Camden, NJ) by using the Expand Long Template PCR system (Roche, Indianapolis, IN). PCR primers were 5'-CCCCTGACTGCCCTTTCTTA-3' (forward) and 5'-TCACCAGCGCTACATGGAT-3' (reverse). A derivative fragment of 2485 bp was cloned into a pGL3 basic luciferase vector (Promega, Fitchburg, WI) by using the following primers: 5'-GCTCTTACGCGTGCTAGCCCGAGCTCTGAATTATCCTGAGTGG-3' (forward), and 5'-CCGCGCGCCCCGTTCAGCCGAATTCATCTGCGATCTAAG-3 (reverse). Eight 5' progressive deletion constructs were made by PCR using primers listed below. The resulting products were cloned into pGL3 basic firefly luciferase reporter vectors. The identity of each construct and the absence of cloning artifacts were verified by direct sequencing with the dye-terminator method in a 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA).

    The –122XRE substitution intolerant core (5'-GCGTG-3') was deleted from the –413CBR1 promoter construct using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) with the following primers: 5'-CCTGCGCGCTCAGCGGCCGGTAACCCACGGGTGCGCGCCC-3', and 5'-GGGCGCGCACCCGTGGGTTACCGGCCGCTGAGCGCGCAGG-3'. Deletion of –122XRE was confirmed by direct sequencing analysis.

    A 12-bp sequence containing the distal –5783XRE element (5'-TTGCGTGCCTTG-3', bases –5790 to –5779) was added to the 5' end of the –2485CBR1 construct by using QuikChange with the following primers: 5'-CGCGTGCTAGCCTTGCGTGCCTTGGAGCTCTGAATTATCC-3', and 5'-GGATAATTCAGAGCTCCAAGGCACGCAAGGGCTAGCACGCG-3'. The addition of –5783XRE was verified by direct sequencing.

    List of primers. The forward primers included –1847CBR1:5'-CCTAAATCTGTACTGCCCAATACGCGTACAGTGACCACTAACACATGC-3'; –1561CBR1:5'-GAGGGAGTCACTCTGTTGACGCGTCCCAGGCTGGAGTGCAG-3'; –1101CBR1:5'-CCAGACCCCTCACCTGCAACGCGTGCCTGATGCCTGTTGAC-3'; –746CBR1: 5'-GGTACATCCTAGAGTGTACGCGTTATTGTCCGTGTAAAATAGGG-3'; –600C- BR1: 5'-CTGGCTAAGTCAGTAGCACGCGTTTTGTTTTCATATACTTAGGGG-3'; –413CBR1: 5'-CACAACTAGGAATGAACGCGTTTGAACAGCTGGGAG-3'; –205CBR1:5'-GCTCCGCACCCCGGACGCGTGGTTCCGGTGG-3'; and –101CBR1:5'-GGGCGTGTAACCCACGCGTGCGCGCCCACG-3'; The reverse primer was RevCBR1:5'-CCGCGCGCCCCGTTCAGCCGAATTCATCTGCGATCTAAG-3'.

    Transient Transfections and Luciferase Activity Assays. Cells were plated 24 to 48 h before transfections in 12-well plates. Reporter gene constructs (firefly luciferase) and the SV40-driven Renilla reniformis luciferase pRL-SV40 plasmid (Promega) were cotransfected into 60 to 70% confluent cell cultures by using FuGENE 6 (Roche). Twenty-four hours after cotransfection, cultures were washed once with phosphate-buffered saline solution, and the cells were lysed with passive lysis buffer (250 µl/well) (Promega). Cell lysates were incubated at room temperature (15 min), mixed with a vortex blender (10 s), and centrifuged at 4°C (1500 rpm for 30 s). Luciferase reporter gene activities were determined with the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. Light intensity was measured in a Synergy HT luminometer equipped with proprietary software for data analysis (BioTek, Winooski, VT). Light intensity values from cell cultures transfected with the promoterless (pGL3) vector were used to correct for background. Corrected firefly luciferase activities were normalized to R. reniformis luciferase activities and expressed as fold increases with respect to the values obtained with pGL3-basic empty vector. In all cases, three to five independent experiments were performed in duplicate to evaluate reproducibility. Unpaired Student's t tests (two groups) and analysis of variance (ANOVA, three or more groups) were used to compare experimental means. In all cases, differences were considered to be significant at p < 0.05. Computations were performed with Microsoft Excel 2000 version 9.0 (Microsoft, Redmond, WA) and SigmaPlot version 8.02 (SPSS Inc., Chicago, IL).

    Quantification of CBR1 mRNA in Cell Cultures by Real-Time RT-PCR. Cell cultures (70–80% confluence) were treated for 24 h with dexamethasone (10 µM), rifampicin (10 µM), -naphthoflavone (10–50 µM), clotrimazole (20 µM), TCPOBOP (0.250 µM), or vehicle (DMSO). Total RNA was extracted with RNeasy Mini kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. RNA was eluted with molecular biology-grade water, and stored at –80°C until use. RNA concentrations were measured by spectrophotometric analysis at 260 nm in a Shimadzu UV-1601 PC spectrophotometer (Shimadzu, Kyoto, Japan). Total RNA (100 ng) was reverse-transcribed and amplified by using one-step QuantiTect SYBR Green RT-PCR kits (QIAGEN). RT-PCR reaction mixtures were incubated on a MX4000 engine thermal cycler equipped with proprietary software for data analysis (Stratagene). The comparative quantitation method was used to determine relative CBR1 mRNA levels in drug-treated samples. Vehicle-treated samples were used as references, and individual -actin mRNA levels were used as normalizers (Blanquicett et al., 2002; Bustin, 2002). CBR1 primers were: 5'-CTGATCCCACACCCTTTCAT-3' (forward), and 5'-TTAAGGGCTCTGACGCTCAT-3' (reverse); -actin primers, 5'-ACGGCTCCGGCATGTGCAAG-3' (forward), and 5'-TGACGATGCCGTGCTCGATG-3' (reverse). Cycling parameters for the amplifications in parallel of CBR1 and -actin mRNAs were: 50°C for 30 min (reverse transcription), 95°C for 10 min (Taq polymerase activation); 40 cycles of 95°C for 15 s (denaturation), 51°C for 30 s (annealing), 72°C for 30 s (extension), and 78°C for 30 s (fluorescence collection). Standard curves for CBR1 and -actin mRNA (10-fold dynamic range) were run in parallel to ensure accurate mRNA quantifications. In all cases, the regression coefficients (r) of the standard curves were r  0.9. Amplification efficiencies for CBR1 and -actin mRNAs were similar and ranged between 125 and 175%. In all cases, experimental samples and standards for calibration curves were analyzed in quadruplicate.

    Animals and Treatments. Ahr+/– and Ahr–/– mice were procured from the laboratory of Dr. Christopher Bradfield (University of Wisconsin). The Institutional Animal Care and Use Committee approved the experimental protocol. Animals were housed in a temperature- and humidity-controlled room under a light cycle with free access to food and water. Mice (aged 81 ± 14 days) were treated with intraperitoneal injections of TCDD (50 µg/kg; AccuStandard Inc., New Haven, CT), or corn oil vehicle (200 µl), respectively. Animals were sacrificed by CO2 inhalation. Livers were removed, snap-frozen in liquid nitrogen, and stored at –80°C until use.

    Quantification of Hepatic Cbr1 mRNA by Real-Time RT-PCR. Liver RNA was extracted with RNeasy Mini kits (QIAGEN). RNA samples (100 ng) were subjected to one-step quantitative real-time RT-PCR using QuantiTect SYBR green RT-PCR kit (QIAGEN). Mouse Cbr1 primers were: 5'-ATCACTCGTGACCTGTGTCG-3' (forward), and 5'-GGTGTCGTCATTGACCTTGA-3'(reverse); -actin primers: 5'-GACCCAGATCATGTTTGAGACCTTC-3' (forward), and 5'-GGAGTCCATCACAATGCCAGTG-3' (reverse). Amplification conditions for murine Cbr1 and -actin mRNAs were: 50°C for 30 min (reverse transcription), 95°C for 10 min (Taq polymerase activation); 40 cycles of 95°C for 15 s (denaturation), 52°C for 30 s (annealing), 72°C for 30 s (extension), and 78°C for 30 s (fluorescence collection). Standard curves (10-fold dynamic range) for Cbr1 and -actin mRNA were run in parallel. Relative Cbr1 mRNA levels were calculated by using the comparative quantitation method as described above. Samples were analyzed in quadruplicate.

    Detection of Hepatic Cbr1 by Immunoblotting. Fragments of frozen mouse liver were homogenized in three volumes of ice-cold lysis buffer (Promega). The homogenates were centrifuged at 13,000g for 20 min at 4°C. The resulting supernatants (100 µg) were separated on 4 to 20% precast polyacrylamide gels (Pierce, Rockford, IL) and transferred onto Hybond ECL nitrocellulose membranes (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). Membranes were first incubated with a monoclonal anti-human CBR1 antibody (1:1000 dilution) that cross-react with murine Cbr1 (Abnova Corporation, Taipei City, Taiwan) and with a secondary anti-mouse IgG conjugated with horseradish peroxidase (1:1000 dilution; GE Healthcare). The membranes were also probed with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:10,000 dilution; Chemicon International, Temecula, CA) to correct for differences in protein loading. Immunoreactive bands were visualized with the ECL Plus Western blotting detection system (GE Healthcare) and quantified by using a ChemiDoc XRS gel documentation system equipped with Quantity One software (Bio-Rad Laboratories, Hercules, CA).

    CBR Activity. Maximal CBR activity was measured in cellular lysates, and in mice liver cytosols by using the specific NQO1 inhibitor dicoumarol in the presence of the substrate menadione and the NADPH cofactor (Wermuth et al., 1986; Bello et al., 2004; Covarrubias et al., 2006). Typical incubation mixtures (1 ml) contained sodium phosphate buffer (0.1 M), pH 7.4, 200 µM NADPH (Sigma-Aldrich), 200 µM menadione (Sigma-Aldrich), and 5 µM dicoumarol. Mixtures were equilibrated for 2 min at 37°C after the addition of cytosols (200 µg). The rates of NADPH oxidation were recorded for 4 min at 37°C in a Cary Varian Bio 300 UV-visible spectrophotometer (Palo Alto, CA). Enzymatic velocities were automatically calculated by linear regression of the abs/time points (2400 readings) and expressed as micromoles per minutes per milligram. Protein concentrations were determined with the Bradford assay (Bio-Rad).

    Cloning and Functional Analysis of CBR1 Promoter Constructs. First, we cloned a 2485-bp DNA fragment from the 5'-flanking region of CBR1 to perform functional characterization studies. Sequencing of the insert revealed 100% identity with a segment of nucleotide sequence from locus AP001724 (Homo sapiens genomic DNA, chromosome 21q, section 68/105; Entrez Nucleotide Database, available at http://www.ncbi.nlm.nih.gov/sites/entrez?db=Nucleotide). According to the Data Base of Transcriptional Start Sites (DBTSS), CBR1 has a predominant transcription start site (TSS, 123/146 cDNA clones) located 92 bp upstream of the ATG codon (Fig. 1). Analysis of the core promoter sequence of CBR1 revealed the presence of a typical initiator element containing the TSS [Inr, Py-Py(C)-A+1-N-T/A-Py-Py]. The CBR1 core promoter region has no downstream core promoter element (A/G+28-G-A/T-C/T-G/A/C), no TATA box, and no CAAT box. The core promoter is embedded in a CpG island of approximately 0.65 kb that encompasses –273 bp of 5'-flanking sequence, and extends 369 bp downstream of the ATG codon. There are two contiguous GC boxes located at –165 and –152 bp, respectively. In addition, there is a proximal SP1 motif at –53 bp and a relatively more distal SP1 motif embedded in the –152 bp GC box. Together, these findings indicate that the core promoter of CBR1 has the configuration of a typical CpG island promoter (Butler and Kadonaga, 2002).

    Fig. 1. Annotated sequence from the 5'-flanking region of human CBR1. The transcription start site (–92 bp, DBTSS) is indicated with a solid arrow, and the Inr element is underlined. The different fragments corresponding to the series of deletion promoter constructs are indicated with dotted arrows. The proximal XRE motif (–122XRE) is indicated in a gray box, and putative transcription factor binding sites are indicated in clear boxes. AP1, activator protein 1; HINF A, histone nuclear factor A; SF1, steroidogenic factor 1; NF-B, nuclear factor  B; Oct 1, octamerbinding transcription factor; CAC, CACCC binding protein; IK-2, Ikaros 2 protein; GATA, GATA or GATAA sequence; HNF 3, hepatic nuclear factor 3; YY1, Yin Yang 1.

    Computer-assisted searches for additional cis-acting elements using the TESS and TRANSFAC databases pinpointed potential consensus motifs for a number of transcription factors including hepatic nuclear factor-3, Ikaros 2 protein, and octamer-binding transcription factor (Fig. 1). We identified one proximal sequence motif for a xenobiotic response element (–122XRE; Fig. 1). Further analysis of up to 6 kb upstream of the ATG codon revealed a distal XRE motif containing the substitution-intolerant core sequence 5'-GCGTG-3' at position –5783 (–5783XRE).

    Next, we generated a series of progressive 5'-deletion constructs and performed gene reporter assays in HepG2 and MCF-7 cells (Fig. 2, A and B). Results from both cell lines suggested the presence of a negative regulatory element in the –2485/–1847 region, because deletion of the 653-bp segment resulted in significant increases in luciferase activities (HepG2, Student's t test, p < 0.05; MCF-7, Student's t test, p < 0.05). In both cell lines, further 5' truncation of up to 746 bp resulted in no significant changes in the promoter activities of constructs –1847CBR1, –1561CBR1, and –1101CBR1, respectively (HepG2, ANOVA, p = 0.75; MCF-7, ANOVA, p = 0.83). In HepG2 cells, the –413CBR1 construct exerted the highest promoter activity from the series suggesting that the –600/–413 region may harbor an element whose regulatory role depends on the cellular context. Further deletion of 208 bp (–205CBR1) decreased the promoter activity in HepG2 (2.7-fold) and MCF-7 cells (2.4-fold). Data from both cell lines showed that the –205/–101 region contains cis-acting elements that are crucial to sustain gene transcription because deletion of 104 bp resulted in substantial decreases in the promoter activities by 22-fold (HepG2, Student's t test, p < 0.01) and 41-fold (MCF-7, Student's t test, p < 0.001), respectively. The –205/–101 segment contains two GC boxes and the proximal –122XRE. Thus, it is likely that the removal of these elements resulted in a construct (–101CBR1) with diminished promoter activity (Figs. 1 and 2). In both cell lines, the –101CBR1 showed minimal although significant increases in transcriptional activity compared with the pGL3-Basic vector (HepG2 = 3-fold, Student's t test, p < 0.05; MCF-7 = 8-fold, Student's t test, p < 0.05). It is possible that the Inr element (–93 bp) and the proximal SP1 site (–53 bp) dictate the minimal promoter activity of the –101CBR1 construct.

    Fig. 2. Functional analysis of human CBR1 promoter constructs in HepG2 cells (A) and MCF-7 cells (B). Panels show schematic representations of each CBR1 promoter construct (left) and its corresponding luciferase activity from gene reporter experiments (right). Luciferase activities were measured as described under Materials and Methods. Light intensity values from transfections with the promoterless vector were used to correct for background. Corrected luciferase activity values were normalized to R. reniformis luciferase activity and expressed as fold increases with respect to the values obtained with pGL3-basic empty vector. Each value represents the mean ± S.D. of four independent experiments performed in duplicate.

    Induction of CBR1 mRNA and CBR Activity by a Ligand of the Aryl Hydrocarbon Receptor. To pinpoint pathways potentially involved in the transcriptional regulation of CBR1, we analyzed the effect of different receptor activators on CBR1 mRNA levels. Cultures of HepG2 cells were incubated with different receptor activators at concentrations known to affect the regulation of other drug-metabolizing enzymes (Schuetz et al., 1993; Zhang et al., 2003; Hempel et al., 2004). CBR1 and -actin (normalizer) mRNA levels were determined simultaneously by quantitative real-time RT-PCR (see Materials and Methods). We detected no changes in CBR1 mRNA levels after incubations with the glucocorticoid receptor agonist dexamethasone. Likewise, incubations with activators of constitutive androstane receptor (clotrimazole and TCPOBOP) and pregnane X receptor (rifampicin) did not significantly affect CBR1 mRNA levels. In contrast, incubations with the prototypical AHR ligand -naphthoflavone (50 µM, 24 h) induced CBR1 mRNA levels by 5.5-fold (Student's t test, p < 0.005) compared with controls (Fig. 3). In MCF-7 cells, -naphthoflavone exerted moderate cytotoxicity (20–30%) at the 50 µM concentration, whereas incubations with 10 µM resulted in negligible cytotoxicity ( 5%) and induced CBR1 mRNA by 2.5-fold (Student's t test, p < 0.05; Fig. 3). The increase in CBR1 mRNA levels in MCF-7 cells treated with -naphthoflavone was paralleled by a 3-fold increase in maximal cytosolic CBR activity (CBRcontrols (DMSO):50 ± 13 pmol/min · mg versus CBR-naphthoflavone: 140 ± 2 pmol/min · mg).

    Fig. 3. Induction of CBR1 mRNA in HepG2 cells (A) and MCF-7 cells (B) by prototypical receptor activators. HepG2 cells were incubated with vehicle (DMSO, 0.01%), dexamethasone (10 µM), -naphthoflavone (50 µM), clotrimazole (20 µM), TCPOBOP (0.250 µM), and rifampicin (10 µM) for 24 h. MCF-7 cells were incubated with vehicle (DMSO, 0.01%) and -naphthoflavone (10 µM) for 24 h. The expression of CBR1 mRNA was analyzed by quantitative real-time RT-PCR using specific primers as described under Materials and Methods. Each value represents the mean ± S.D. from three independent experiments analyzed in quadruplicate. Asterisks indicate significant differences from the CBR1 mRNA levels from vehicle treated cells (*, p < 0.005; **, p < 0.05).

    Transcriptional Activation of CBR1 Promoter Constructs by -Naphthoflavone. Next, we tested whether -naphthoflavone induced the gene reporter activities of different CBR1 promoter constructs encompassing up to 1561 bp of the 5' flanking region. In all cases, incubations with -naphthoflavone (10 µM, 48 h) or vehicle (DMSO) were performed 24 h after the cotransfections with reporter constructs (see Materials and Methods). On average, -naphthoflavone induced the luciferase activities of the constructs by 11-fold (–1561CBR1, p < 0.05), 5-fold (–600CBR1, p < 0.05), and 15-fold (–413CBR1, p < 0.001) in MCF-7 cells compared with vehicle-treated controls (Fig. 4).

    Fig. 4. Effect of the AHR ligand -naphthoflavone on the gene reporter activities of CBR1 promoter constructs. Cultures of MCF-7 cells were cotransfected with CBR1 reporter constructs (–1561CBR1, –600CBR1, and –413CBR1) and the normalizer plasmid pRL-SV40. Twenty-four hours after cotransfections, cells were treated with -naphthoflavone (10 µM) or vehicle (DMSO, 0.01%) for 48 h. Luciferase activities were measured as described under Materials and Methods. For each construct, normalized luciferase activities were expressed as fold increases with respect to the values from control incubations, which were set arbitrarily at 1. Data represent the mean ± S.D. from three independent experiments performed in duplicate. Asterisks indicate significant difference from vehicle-treated cells (*, p < 0.05; **, p < 0.05; ***, p < 0.001).

    Fig. 5. Effect of -naphthoflavone on the gene reporter activities of the promoter constructs –413CBR1 and –413CBR1-–122 XRE. Cotransfections included the normalizer construct (pRL-SV40) and either the intact –413CBR1 construct or the engineered –413CBR1-–122XRE construct (without –122XRE motif). Twenty-four hours after cotransfections, cells were treated with -naphthoflavone (10 µM) or vehicle (DMSO, 0.01%) for 48 h. Luciferase activities were measured as described under Materials and Methods. For each construct, normalized luciferase activities were expressed as fold increases with respect to the values from control incubations (DMSO), which were set arbitrarily at 1. Data represent the mean ± S.D. from three independent experiments performed in duplicate. The asterisk indicates significant difference from the luciferase activity exerted by the –413CBR1 construct in the presence of -naphthoflavone (*, p < 0.001).

    Functional XRE motifs in the promoters of drug-metabolizing enzymes are necessary to activate gene transcription in response to AHR ligands (Nioi and Hayes, 2004). Thus, we first we tested whether the –122XRE motif was necessary to induce luciferase reporter gene expression in the presence of the ligand -naphthoflavone. The removal of –122XRE decreased the -naphthoflavone response by 9-fold in MCF-7 cells (Student's t test, p < 0.001; Fig. 5).

    In another set of experiments, we evaluated the ability of the distal –5783XRE to augment reporter gene activity in response to -naphthoflavone. To achieve this end, a 12-bp sequence (bases, –5790 to –5779) containing the –5783XRE was fused into the –2485CBR1 reporter construct. Treatment with -naphthoflavone increased the reporter activity of –2485CBR1 by 4-fold compared with incubations with the vehicle DMSO (Student's t test, p < 0.05). Fusion of the distal –5783XRE to –2485CBR1 further enhanced the -naphthoflavone response by 5-fold (Student's t test, p < 0.0001; Fig. 6).

    Fig. 6. Effect of -naphthoflavone on the gene reporter activities of –2485CBR1 and –2485CBR1 + –5783XRE. Both constructs are schematized at the top of the graph. Cotransfections included the normalizer construct (pRL-SV40) and either the intact –2485CBR1 construct or the engineered –2485CBR1 + –5783XRE construct. Twenty-four hours after cotransfections, cells were treated with -naphthoflavone (10 µM) or vehicle (DMSO, 0.01%) for 48 h. Luciferase activities were measured as described under Materials and Methods. For each construct, normalized luciferase activities were expressed as fold increases with respect to the values from control incubations, which were set arbitrarily at 1. Data represent the mean ± S.D. from three independent experiments performed in duplicate. Asterisks indicate significant difference from vehicle treated cells (*, p < 0.05; **, p < 0.0001).

    Induction of Hepatic Cbr1 by TCDD Treatment in Ahr+/– and Ahr–/– Mice. We extended our observations by evaluating whether the administration of the potent AHR ligand TCDD affected the expression of Cbr1 in livers from Ahr+/– and Ahr–/– mice. First, TCDD (50 µg/kg) was administered by a single intraperitoneal injection, and the expressions of Cbr1 mRNA and protein were analyzed from livers collected 72 h after treatments. In heterozygous Ahr+/– animals, TCDD treatment resulted in a 2-fold induction of Cbr1 mRNA levels compared with vehicle-treated heterozygous controls. In contrast, TCDD treatment failed to induce the expression of hepatic Cbr1 mRNA in homozygous null (Ahr–/–) mice (Fig. 7, A and B). It is noteworthy that the induction of hepatic Cbr1 mRNA in heterozygous Ahr+/– animals treated with TCDD was paralleled by a 2-fold increase in Cbr1 protein levels as determined by semiquantitative immunoblotting (Fig. 7, C–F). In line, hepatic Cbr activity increased by 40% in TCDD-treated mice with one active Ahr allele (Ahr+/–), whereas the levels of Cbr activity remained essentially unchanged in the livers of TCDD-treated Ahr–/– mice (data not shown). Moreover, Cbr activity was induced by 5-fold in livers of Ahr+/– mice treated with three consecutive doses of TCDD (50 µg/kg/day for 3 days) compared with vehicle-treated animals (p < 0.05; Fig. 8A). Identical TCDD treatments failed to induce hepatic Cbr activity in Ahr–/– mice (p = 0.32; Fig. 8B).

    Fig. 7. Effect of TCDD on Cbr1 mRNA and Cbr1 protein expression in livers from Ahr+/– and Ahr–/– mice. Hepatic Cbr1 mRNA levels in Ahr+/– (A) and Ahr–/– (B) mice treated with vehicle (n = 2) or TCDD (n = 2), respectively. The expression of Cbr1 mRNA was analyzed by using specific primers as described under Materials and Methods. Bars represent the mean ± S.D. from two quantifications performed in quadruplicate for each animal. Immunodetection of hepatic Cbr1 in Ahr+/– (C) and Ahr–/– mice (E). Hepatic Cbr1 and GAPDH were detected with specific antibodies as described under Materials and Methods. Immunoreactive bands were visualized in a ChemiDoc XRS gel documentation system. The intensities from the immunoreactive GAPDH bands were used to correct for differences in protein loading during densitometric analyses. Densitometric analyses of Cbr1 in livers from Ahr+/– (D) and Ahr–/– mice (F). Each bar represent the level of Cbr1 expressed as fold induction with respect to the average intensity value obtained from vehicle-treated animals.

    Fig. 8. Effect of TCDD on hepatic Cbr activity in heterozygous Ahr+/– (A) and homozygous Ahr–/– (B) mice. Each bar represents the average from two measurements performed in duplicate. Insets, hepatic Cbr activity expressed as fold induction with respect to the average activity value obtained from vehicle-treated animals.

    The first aim of our study was to perform the functional characterization of the promoter of human CBR1. Our sequence annotation showed that the core promoter of CBR1 has the features of a prototypical CpG promoter including two GC boxes, proximal SP1 sites, and the absence of TATA and downstream core promoter element elements (Fig. 1). In humans, approximately half of the promoter regions are located in CpG islands, and gene transcription may occur at different start sites (Butler and Kadonaga, 2002). In line, CBR1 has a predominant start site at –92 bp, and 16% of the clones reported in DBTSS showed alternative start sites (e.g., –101 and –125 bp). Our results from gene reporter experiments in HepG2 and MCF-7 cells demonstrated the presence of regulatory regions that seem to be relevant to promote transcription under basal conditions in both cell types. For example, deletion of the segment that contains the two GC boxes and the proximal –122XRE (–205/–101) significantly reduced the reporter gene activity of the –205CBR1 construct compared with the –413CBR1 construct in HepG2 (22-fold) and MCF-7 (41-fold) cells. It has been demonstrated that SP1 binding sites together with an Inr motif can activate transcription in CpG promoters (Smale and Baltimore, 1989; Butler and Kadonaga, 2002). Consequently, the CBR1 –101/+1 region harboring both SP1 and Inr consensus displayed minimal although significant promoter activities in both cell lines. Functional mutagenesis analysis within the context of the minimal CBR1 promoter will provide further evidence on the role of the Inr and SP1 elements.

    The second aim of this study was to evaluate the ability of prototypical receptor activators to induce the expression of CBR1 mRNA. In agreement with the seminal observation by Forrest et al. (1990), the AHR ligand -naphthoflavone was the only compound that significantly induced CBR1 mRNA levels in HepG2 and MCF-7 cells. Furthermore, our data with engineered reporter constructs suggest that –122XRE, and –5697XRE may act as bona fide functional elements to activate AHR-mediated gene transcription in the presence of AHR ligands.

    The overall identity between the human CBR1 proximal promoter region (600 bp) and the mouse Cbr1 putative promoter region is 33% (global alignment analysis). Similar overall identity values (36%) were obtained when comparisons were extended up to 2500 bp. Further analysis by using the sequence comparison tool from DBTSS pinpointed 3 DNA fragments (size range, 38–42 bp) with relatively high sequence identity values (average, 72%). In addition, we identified a proximal XRE and a GC box element on the murine sequence that correspond with similar motifs on the human CBR1 promoter (Fig. 9). Sun et al. (2004) analyzed the positional conservation of XRE core motifs between several murine and human genes and found that only 39% of the human-mouse orthologs contain positionally conserved XREs. Thus, the positional conservation of the substitution-intolerant XRE core in both murine and human CBR1 sequences is interesting and supports the notion that the transcription of CBR1 in both species is controlled by similar key regulatory factors (e.g., AHR). Furthermore, our experiments with heterozygous Ahr+/– and homozygous Ahr–/– mice clearly showed that Ahr plays a pivotal role in mediating Cbr1 induction in vivo. It is noteworthy that the presence of one active Ahr allele was essential to induce Cbr1 mRNA, Cbr1 protein, and Cbr activity in Ahr+/– mice treated with the AHR ligand TCDD. In contrast, TCDD treatment failed to induce Cbr1 expression in homozygous null animals (Ahr–/–).

    Fig. 9. DNA sequence alignment of the human and mice CBR1 5'-flanking regions. Sequences were aligned by using the global positioning alignment algorithm. XRE core motifs and GC boxes are highlighted in gray.

    The reduction of carbonyl moieties catalyzed by CBR1 is an important step in the metabolism of a wide variety of clinically relevant drugs such as the anticancer daunorubicin, the antipsychotic haloperidol, and the antidiabetic acetohexamide (Ohara et al., 1995; Forrest and Gonzalez, 2000; Rosemond and Walsh, 2004). CBR1 also catalyzes the reduction of toxins such as the potent tobacco carcinogen 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK). In humans, NNK is detoxified through two-electron reductions catalyzed mainly by cytosolic CBR1 and microsomal 11-hydroxysteroid dehydrogenase type I. The resulting alcohol metabolite 4-methylnitrosamino-1-(3-pyridyl)-1-butanol can be further subjected to glucuronidation to form 4-methylnitrosamino-1-(3-pyridyl)-1-butanol-glucuronide, which is excreted in urine (Maser et al., 2000). Variable CBR1 mRNA expression has been described in human lung, and a recent study on 59 patients with non–small-cell lung carcinoma reported higher postoperative survival rates among patients having tumors containing "high" CBR1 mRNA expression compared with those with tumors presenting "low" CBR1 mRNA expression (5-year survival CBR1-high, 68.3%, versus 5-year survival CBR1-low, 36.5%; p = 0.03) (Finckh et al., 2001; Takenaka et al., 2005). The polycyclic aromatic hydrocarbon benzo-(a)pyrene (BP) is one of the best-characterized carcinogens in cigarette smoke and is also a powerful AHR ligand (Denison and Nagy, 2003). Moreover, BP induces Cbr1 expression significantly in Ahr-proficient mice but fails to induce Cbr1 in Ahr-deficient animals (S. S. Lakhman, E. G. Schuetz, and J. G. Blanco, unpublished observations). Thus, it is reasonable to hypothesize that BP may modulate CBR1 expression in the lungs of smokers via the AHR pathway, which in turn has an impact on the CBR1-mediated detoxification of other smoke carcinogens relevant to the pathogenesis of lung cancer such as NNK. In conclusion, our results describe the first functional characterization of the promoter of human CBR1 and indicate that AHR is a key mediator in dictating variable CBR activity.

    Acknowledgements

    We gratefully acknowledge the excellent assistance of Dr. Lubin Lan.

    ABBREVIATIONS: CBR1, human carbonyl reductase 1; AHR, aryl hydrocarbon receptor; CBR, carbonyl reductase activity; XRE, xenobiotic response element; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCPOBOP, 1,4-bis-[2-(3,5-dichloropyridyloxy)]-benzene; kb, kilobase; bp, base pair; PCR, polymerase chain reaction; ANOVA, analysis of variance; RT-PCR, reverse transcription-polymerase chain reaction; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DBTSS, Data Base of Transcriptional Start Sites; SP1, specificity protein 1; NNK, 4-methylnitrosamino-1-(3-pyridyl)-1-butanone; BP, benzo(a)pyrene.

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作者单位:Department of Pharmaceutical Sciences, the State University of New York at Buffalo, Buffalo, New York (S.S.L., X.C., V.G.-C., J.G.B.); Department of Pharmaceutical Sciences, St. Jude Children Research Hospital, Memphis, Tennessee (E.G.S.)

日期:2009年8月25日 - 来自[2007年第69卷第9期]栏目

Involvement of MRP4 (ABCC4) in the Luminal Efflux of Ceftizoxime and Cefazolin in the Kidney

【关键词】  Involvement

    The purpose of the present study was to investigate the role of multidrug resistance-associated protein 4 (MRP4) in the tubular secretion of cephalosporin antibiotics. Most of the injectable cephalosporins have an inhibitory effect on the ATP-dependent uptake of [3H]dehydroepiandrosterone sulfate by membrane vesicles expressing hMRP4, whereas cephaloridine, cefsulodin, and cefepime do not. Aminocephalosporins have a weak inhibitory effect. Significant ATP-dependent transport of ceftizoxime (Km, 18 µM), cefazolin (Km, 80 µM), cefotaxime, and cefmetazole has been observed only in the membrane vesicles expressing hMRP4. Ceftizoxime and cefazolin were given by a constant intravenous infusion to wild-type and Mrp4–/– mice. The steady-state plasma concentrations of ceftizoxime and cefazolin were unchanged in Mrp4–/– mice. The urinary recovery of ceftizoxime was significantly reduced in Mrp4–/– mice, whereas it was unchanged for cefazolin. The kidney-to-plasma concentration ratio of ceftizoxime and cefazolin was increased 2.0- and 2.7-fold in Mrp4–/– mice, respectively; thus, the renal clearance with regard to the kidney concentration was reduced in Mrp4–/– mice, to 7.5 and 34% of the corresponding control values, respectively. These results suggest that Mrp4 is involved in the tubular secretion of ceftizoxime and cefazolin, in concert with basolateral uptake transporters.

    Cephalosporins are one of the most important groups of antibiotics, and inhibit synthesis of the bacterial peptidoglycan cell wall. They have a cephem nucleus with various side-chains at the 3 and 7 positions of the -lactam and dihydrothiazine rings, respectively. They are currently classified into four generations based on 1) the general features of their antimicrobial activities and 2) their stability to hydrolysis by -lactamase. Most cephalosporins are excreted into the urine in unchanged form; only a few cephalosporins, such as cefoperazone, cefpiramide, and cefodizime are excreted predominantly into the bile (Petri, 2006). In the kidney, active secretion by the proximal tubules, together with glomerular filtration, has been suggested to be the renal clearance mechanism of cephalosporins. In particular, inhibition of renal elimination of cephalosporins by probenecid, a well known inhibitor of organic anion transporters, suggests that multispecific organic anion transporters play a major role in their renal elimination (Brown, 1993; Lepsy et al., 2003). Indeed, in vitro transport studies using cDNA-transfected cells or cRNA-injected oocytes have shown that cephalosporins are substrates of renal basolateral multispecific organic anion transporters (OAT1 and OAT3) (Jariyawat et al., 1999; Jung et al., 2002; Uwai et al., 2002; Ueo et al., 2005), and probenecid is a potent inhibitor of these transporters (Tahara et al., 2005). Because cephalosporins are efficiently transported by hOAT3, Ueo et al. (2005) speculated that hOAT3 plays a major role in the renal uptake of cephalosporins.

    Because of the hydrophilic nature of cephalosporins, transporters will be involved in the luminal efflux process for efficient directional transport into the urine from the blood. Facilitated diffusion has been suggested as a mechanism for the luminal efflux of cephalosporins. Saturable uptake of cefixime by brush-border membrane (BBM) vesicles exhibits membrane-voltage dependence and is inhibited by probenecid and p-aminohippurate (Tamai et al., 1988). Because benzylpenicillin, cephalothin, and cephradine exhibit trans-stimulation of the efflux of cefixime from the BBM vesicles, the transporter also accepts these compounds as substrates. Renal clearance of aminocephalosporins exhibit nonlinearity that increases as the plasma concentrations increased (Garcia-Carbonell et al., 1993; Granero et al., 1994). This can be explained by saturation of reabsorption in the kidney. The uptake of aminocephalosporins by BBM is pH-dependent, and dipeptide transporters (PEPT1 and PEPT2) have been considered to be responsible for the reabsorption of aminocephalosporins in the kidney (Boll et al., 1996; Terada et al., 1997).

    During preliminary studies, we found that some cephalosporins are substrates of an ATP binding cassette transporter, multidrug resistance-associated protein 4 (MRP4/ABCC4). MRP4 is characterized by its broad substrate specificity, and cumulative studies have demonstrated that cAMP, cGMP, p-aminohippurate, urate, dehydroepiandrosterone sulfate, methotrexate, prostaglandins, estradiol-17-D-glucuronide, and adefovir are MRP4 substrates (Schuetz et al., 1999; van Aubel et al., 2002, 2005; Reid et al., 2003; Zelcer et al., 2003). In addition, glutathione modulates the substrate recognition of MRP4, and MRP4 accepts taurocholate as a substrate only in the presence of reduced glutathione or its S-methyl derivative, which lacks reducing activity (Rius et al., 2003). MRP4 is abundantly expressed in the kidney (Maher et al., 2005; Nishimura and Naito, 2005), where it is localized on the BBM of the proximal tubules (van Aubel et al., 2002; Leggas et al., 2004); thus, it has been considered to play an important role in the tubular secretion of drugs. We have found Mrp4 to be involved in the tubular secretion of diuretics (hydrochlorothiazide and furosemide) and antiviral drugs (adefovir and tenofovir) (Hasegawa et al., 2007; Imaoka et al., 2007).

    The purpose of the present study was to elucidate the role of MRP4 in the tubular secretion of cephalosporins in the kidney. The chemical structure of the cephalosporins used in this study are shown in Fig. 1. In vitro transport study using membrane vesicles expressing hMRP4 was carried out to show that these cephalosporins are MRP4 substrate. In addition, to obtain direct evidence, in vivo pharmacokinetic parameters related to the renal elimination of the two cephalosporins, ceftizoxime and cefazolin, were compared in wild-type and Mrp4–/– mice.

    Fig. 1. Structures of ceftizoxime, cefazolin, cefmetazole, and cefotaxime

    Materials. Ceftizoxime was a gift from Kowa Co., Ltd. (Tokyo, Japan). Other unlabeled cephalosporins (cefazolin, cefotaxime, and cefmetazole) were purchased from Sigma-Aldrich (St. Louis, MO). [14C]Inulin (8 mCi/mmol) and [3H]dehydroepiandrosterone sulfate (DHEAS) (74.0 Ci/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). All other chemicals and reagents were of analytical grade and readily available from commercial sources.

    Animals. Female Mrp4–/– and wild-type BL6-129 mice (12–16 weeks old) were used in the present study. Mrp4 knockout mice were established previously (Assem et al., 2004; Leggas et al., 2004). Mrp4–/– mice are fertile and exhibit no physiological abnormalities. The hepatic expression of Sult2a1 is down-regulated (Assem et al., 2004), whereas the mRNA expression of Oat1, Oat3, Mrp2, and Bcrp is unchanged in the kidney of Mrp4–/– mice (Imaoka et al., 2007). All animals were maintained under standard conditions with a dark-light cycle and were treated humanely. Food and water were available ad libitum. The studies reported in this manuscript were carried out in accordance with the guidelines provided by the Institutional Animal Care Committee (Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan).

    Cell Culture. HEK293 cells were grown in low-glucose Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C with 5% CO2 at 95% humidity.

    Construction and Infection of Recombinant Adenovirus and Membrane-Vesicle Preparation. Membrane vesicles were prepared from HEK293 cells infected with recombinant adenovirus harboring hMRP4 and GFP gene according to the published method (Hasegawa et al., 2007; Imaoka et al., 2007). For preparation of the isolated membrane vesicles, HEK293 cells cultured in a 15-cm dish were infected by recombinant adenovirus (multiplicity of infection of 5). Forty-eight hours after the infection, the membrane vesicles were isolated from 1 to 2x108 cells. In brief, cells were diluted 40-fold with hypotonic buffer (1 mM Tris-HCl and 0.1 mM EDTA, pH 7.4, at 37°C) containing protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 1 µg/ml pepstatin, and 5 µg/ml aprotinin). After gentle stirring for 1 h on ice, the cell lysates were centrifuged at 100,000g for 30 min at 4°C. The resulting pellets were suspended in 10 ml of isotonic TS buffer (10 mM Tris-HCl and 250 mM sucrose, pH 7.4 at 4°C) and homogenized using a Dounce B homogenizer (glass/glass, tight pestle, 30 strokes). The crude membrane fraction was layered on top of a 38% (w/v) sucrose solution in 5 mM Tris-HEPES, pH 7.4, at 4°C, and centrifuged in a rotor centrifuge (SW41; Beckman Coulter, Fullerton, CA) at 280,000g for 60 min at 4°C. The turbid layer at the interface was collected, diluted to 23 ml with TS buffer, and centrifuged at 100,000g for 30 min at 4°C. The resulting pellet was suspended in 400 µl of TS buffer. Vesicles were formed by passing the suspension 30 times through a 27-gauge needle using a syringe. The membrane vesicles were finally frozen in liquid nitrogen and stored at –80°C until use. Protein concentrations were determined by the Lowry method (Lowry et al., 1951), and bovine serum albumin was used as a standard.

    Transport Studies with Membrane Vesicles. The transport studies were performed using a rapid filtration technique. In brief, 15 µl of transport medium (10 mM Tris-HCl, 250 mM sucrose, and 10 mM MgCl2, pH 7.4) containing radiolabeled compound (0.03 µCi), with or without unlabeled substrate, was preincubated at 37°C for 3 min and then rapidly and gently mixed with 5 µl of membrane vesicle suspension (5 µg of protein). The reaction mixture contained 5 mM ATP or AMP along with the ATP-regenerating system (10 mM creatine phosphate and 100 µg/µl creatine phosphokinase). The transport reaction was terminated at designated times by addition of 1 ml of ice-cold buffer containing 10 mM Tris-HCl, 250 mM sucrose, and 0.1 M NaCl, pH 7.4. The stopped reaction mixture was passed through a 0.45-µm hemagglutinin filter (GVWP; Millipore Corporation, Billerica, MA), and then washed twice with 5 ml of stop solution. The radioactivity retained on the filter was determined in a liquid scintillation counter (LS6000SE; Beckman Coulter) after the addition of scintillation cocktail (Clear-sol I; Nacalai Tesque, Tokyo, Japan). For the uptake of cephalosporins, 20 µg of protein was used for each point. The stopped reaction mixture was passed through a 0.45-µm filter (JH-filter; Millipore). Unlike GVWP, JH-filter is not dissolved in organic solvents for extraction of the cephalosporins. Substrates retained on the filter were recovered in 1 ml of methanol containing internal standard by sonication for 15 min. After centrifugation, the supernatants were evaporated using a centrifugal concentrator (CC-105; TOMY, Tokyo, Japan), and dissolved in 45 µl of 0.05% HCOOH. Then, 40-µl aliquots were used for liquid chromatography/mass spectrometry quantification as described below.

    In Vivo Infusion Study in Mice. Female BL6-129 and Mrp4–/– mice weighing approximately 20 to 30 g were used. Under pentobarbital anesthesia (30 mg/kg), the femoral vein was cannulated with a polyethylene catheter (PE-50) for the injection. Ceftizoxim (20.8 nmol/min/kg) and cefazolin (12.5 nmol/min/kg) were infused via the jugular vein. Blood samples were collected from the jugular vein at 30, 60, 90, and 120 min after administration and centrifuged. Urine was collected in preweighed test tubes at 30-min intervals throughout the experiment. At the end of the experiment, kidneys were excised. To determine the GFR, [3H]inulin (0.4 mg; 0.9 mCi/min/kg) was infused via the jugular vein. Blood and urine specimens were collected in the same way as for ceftizoxime and cefazolin.

    Liquid Chromatography/Mass Spectrometry analysis. The quantification of ceftizoxime, cefazolin, cefmetazole, and cefotaxime was performed by high-performance liquid chromatography (Alliance 2690; Waters, Milford, MA) connected to a mass spectrometer (ZQ; Micromass, Manchester, UK). In this method, 10 µl of plasma, 10 µl of 10-fold-diluted urine, and 10 µl of 30% (w/v) kidney homogenate were deproteinized with 90 µl of acetonitrile containing an internal standard (cephalexin), mixed, and centrifuged. Then, 100-µl samples of the supernatants were concentrated. The concentrated samples were dissolved in 45 µl of 0.05% HCOOH, and 40-µl aliquots were injected into the liquid chromatography/mass spectrometry. High-performance liquid chromatography analysis was performed on a CAPCELL PAK C18 column (MGII, 3 µm, 2.0 mm ID, 50 mm; Shiseido, Tokyo, Japan) at 40°C. Elution was performed with a 95% to 40% linear gradient (for ceftizoxime and cefazolin) or 95% to 5% linear gradient (for cefmetazole) of 0.05% HCOOH over 5 min at 0.4 ml/min. The eluate was introduced into the MS via an electrospray interface. Detection was performed by selected ionization monitoring in positive ion mode (m/z 384, m/z 455, m/z 472, m/z 456, and m/z 348 for ceftizoxime, cefazolin, cefmetazole, cefotaxime, and cephalexin, respectively).

    Pharamacokinetic Analysis. Total plasma clearance (CLtotal), renal clearance normalized by the circulating plasma concentration (CLrenal,p), and renal clearance normalized by the kidney concentration (CLrenal,k) were calculated from the equations CLtotal = I/Css,p, CLrenal,p = Vurine/Css,p, and CLrenal,k = (Vurine – Css,p x fb x GFR)/Css,k, where I, Css,p, Vurine, and Css,k represent the infusion rate (nanomoles per minute per kilogram), plasma concentration at steady state (micromolar), urinary excretion rate at steady state (nanomoles per minute per kilogram), and kidney concentration at steady-state (micromolar), respectively. Css,p was determined using the mean value of the plasma concentrations from 30 to 120 min. Vurine was determined as the mean value of the urinary excretion rate from 30 to 60 min, 60 to 90 min, and 90 to 120 min. GFR was determined in a separate experiment and calculated from the mean value of the urinary excretion rate of [3H]inulin from 30 min to 120 min divided by the mean value of the plasma concentration of [3H]inulin from 30 to 120 min.

    Statistical Analysis. Statistical differences were determined using one-way analysis of variance followed by Fisher's least significant difference method.

    Effects of Various Cephalosporins on hMRP4-Mediated Transport. The uptake of [3H]DHEAS, a typical hMRP4 substrate, in the presence of ATP by membrane vesicles expressing hMRP4 was significantly greater than that in the presence of AMP (204 ± 17 and 4.0 ± 0.7 µl/mg of protein for 2 min), and that obtained in the control vesicles (8.3 ± 1.7 and 4.9 ± 3.5 µl/mg of protein for 2 min). The effect of various cephalosporins on the ATP-dependent uptake of [3H]DHEAS by MRP4-expressing membrane vesicles was examined (Fig. 2). The cephalosporins tested were first- to fourth-generation cephalosporins. Most of the tested cephalosporins had an inhibitory effect on the MRP4-mediated transport of [3H]DHEAS, whereas cephaloridine (a first-generation cephalosporin), cefsulodin (a third-generation cephalosporin), and cefepime (a fourth-generation cephalosporin) had no effect. The inhibitory effect of aminocephalosporins (cefaclor, cephalexin, and cefadroxil) was weak.

    Fig. 2. Effect of various cephalosporins on the ATP-dependent uptake of [3H]DHEAS by hMRP4 expressing membrane vesicles. MRP4-expressing membrane vesicles were incubated with medium containing [3H]DHEAS (0.1 µM) for 2 min in the presence or absence of various cephalosporins at designated concentrations. Each point represents the mean ± S.E. (n = 3). *, p < 0.05; **, p < 0.01, significantly different from control hMRP4-expressing membrane vesicles in the absence of cephalosporins by analysis of variance followed by Dunnett's test.

    ATP-Dependent Transport of Ceftizoxime, Cefazolin, Cefmetazole, and Cefotaxime via Human MRP4. The uptake of cephalosporins by membrane vesicles was determined in the presence of ATP or AMP. Addition of ATP markedly stimulated the uptake of ceftizoxime, cefazolin, cefmetazole, and cefotaxime only in the membrane vesicles expressing hMRP4. AMP lacked this effect, and the uptake by membrane vesicles expressing hMRP4 in the presence of AMP was similar to the that observed in control vesicles determined in the presence of ATP and AMP (Fig. 3). The ATP-dependent uptake of ceftizoxime and cefazolin by membrane vesicles expressing hMRP4 was saturable (Fig. 4). Nonlinear regression analyses showed that the Km values of ceftizoxime and cefazolin, respectively, for hMRP4 were 18.3 ± 2.2 and 80.9 ± 10.9 µM, respectively, and the Vmax values were 0.529 ± 0.026 and 3.24 ± 0.25 nM/mg of protein, respectively.

    Fig. 3. Time-profiles of the uptake of ceftizoxime, cefazolin, cefmetazole, or cefotaxime by human MRP4-expressing vesicles. Membrane vesicles (20 µg) prepared from human embryonic kidney 293 cells infected with hMRP4 (, ) or GFP (, ) adenovirus were incubated at 37°C in uptake medium containing ceftizoxime (10 µM) (A), cefazolin (10 µM) (B), cefmetazole (5 µM) (C), or cefotaxime (5 µM) (D). , , uptake from medium containing 5 mM ATP; , , uptake from medium containing 5 mM AMP. Each point represents the mean ± S.E. (n = 3).

    Fig. 4. Saturation of the ATP-dependent uptake of ceftizoxime and cefazolin by human MRP4-expressing vesicles. ATP-dependent uptake of ceftizoxime (A) or cefazolin (B) by human MRP4 ()-expressing membrane vesicles was determined for a 2-min incubation at a substrate concentration ranging from 5 to 1000 µM. The results are also given as the Eadie-Hofstee plots. Each point represents the mean ± S.E. (n = 3). The data were fitted to the Michaelis-Menten equation by nonlinear regression analysis, and the solid line represents the fitted curve.

    Renal Excretion of Ceftizoxime and Cefazolin in Wild-Type and Mrp4–/– Mice. The renal clearance of [3H]inulin in wild-type and Mrp4–/– mice were 8.59 ± 0.80 and 11.9 ± 1.2 ml/min/kg, respectively, and these were used as the GFR. Ceftizoxime and cefazolin were given by intravenous infusion. In the case of ceftizoxime, the plasma concentration after intravenous infusion was similar in wild-type and Mrp4–/– mice (Fig. 4A). The urinary excretion of ceftizoxime was significantly reduced, and the kidney concentration was significantly increased in Mrp4–/– mice (Fig. 5A). The pharmacokinetic parameters are summarized in Table 1. Taking the unbound fraction of ceftizoxime in mouse plasma (0.7) (Tawara et al., 1992) and the GFR into consideration, the renal clearance of ceftizoxime was greater than its glomerular filtration rate (6.0 ml/min/kg). There was no significant difference in the total body and renal clearances between the two strains (Table 1). The kidney-to-plasma concentration ratio (Kp,kidney) was 2.0-fold higher in Mrp4–/– mice than in wild-type mice (Table 1). The tubular secretion clearance with respect to the kidney concentration (CLrenal,k) was significantly reduced in Mrp4–/– mice.

    Fig. 5. Time-profiles of the plasma concentration, urinary excretion, and kidney concentration of ceftizoxime and cefazolin in Mrp4–/– and wild-type mice. The plasma concentration, urinary excretion, and kidney concentration of ceftizoxime (A) and cefazolin (B) in Mrp4–/– () and wild-type mice () were examined. Ceftizoxime and cefazolin were infused at 20.8 and 12.5 nmol/min/kg, respectively. Each symbol represents the mean ± S.E. (n = 4–10). *, p < 0.05; **, p < 0.01, significantly different from wild-type mice.

    TABLE 1 Pharmacokinetic parameters of ceftizoxime and cefazolin during constant infusion into wild-type and Mrp4-/- mice

    Data are taken from Figure 5 and represent the mean ± S.E. GFR was determined by the renal clearance of inulin, which was 8.59 ± 0.80 and 11.9 ± 1.2 ml/min/kg in wild-type and Mrp4-/- mice, respectively. The unbound fractions of ceftizoxime and cefazolin have been reported to be 0.7 and 0.2, respectively (Tawara et al., 1992).

    The plasma concentration after intravenous infusion of cefazolin was slightly higher in Mrp4–/– mice than in wild-type mice, although there was no significant difference in the urinary excretion rate between Mrp4–/– mice and wild-type mice (Fig. 5B). The kidney concentration of cefazolin was significantly greater in Mrp4–/– mice than in wild-type mice (Fig. 5B). Considering the low unbound fraction of cefazolin in mouse plasma (0.2) (Tawara et al., 1992), the major urinary excretion route of cefazolin is tubular secretion in mice. Similar to ceftizoxime, there was no significant difference in the total body and renal clearances between Mrp4–/– mice and wild-type mice (Table 1). The kidney-to-plasma concentration ratio (Kp,kidney) was 2.9-fold greater in Mrp4–/– mice than in wild-type mice (Table 1). The tubular secretion clearance with respect to the kidney concentration (CLrenal,k) was significantly reduced in Mrp4–/– mice (Table 1).

    For the first time, in the present study, we have identified cephalosporins (ceftizoxime, cefazolin, cefmetazole, and cefotaxime) as MRP4 substrates. Furthermore, in vivo pharmacokinetic studies using Mrp4–/– mice have shown that Mrp4 is involved in the luminal efflux of ceftizoxime and cefazolin in the kidney.

    In the transport studies using membrane vesicles, we found that most of the cephalosporins are inhibitors of MRP4 (Fig. 2). Injectable cephalosporins were more potent inhibitors of hMRP4, except for cefepime, cefsulodin, and cephaloridine, than aminocephalosporins (Fig. 2). The generation of the cephalosporins is probably unrelated to their inhibitory potency. Ceftizoxime, cefazolin, cefmetazole, and cefotaxime could be measured by our LC/MS system with sufficient sensitivity, enabling direct measurement of the accumulation by membrane vesicles. ATP-dependent uptake of these cephalosporins by hMRP4 was observed with similar transport activities only in hMRP4-expressing membrane vesicles (Fig. 3). Thus these four cephalosporins, at least, are substrates of hMRP4.

    Among the four cephalosporins, involvement of Mrp4 in the urinary excretion was tested for ceftizoxime and cefazolin. Urinary excretion is the predominant elimination pathway of ceftizoxime and cefazolin in wild-type mice, and this is mediated by both tubular secretion as well as glomerular filtration (Table 1). This is consistent with a previous report in which probenecid and p-aminohippurate inhibited the renal elimination of ceftizoxime and reduced its kidney-to-plasma concentration ratio (Terakawa et al., 1981). The total body clearances of ceftizoxime and cefazolin were almost unchanged, and the renal clearance (CLrenal,p) of ceftizoxime and cefazolin was slightly, but not significantly, reduced in Mrp4–/– mice (Table 1). However, the kidney concentrations were significantly increased in Mrp4–/– mice; consequently, the tubular secretion clearances of ceftizoxime and cefazolin with respect to the kidney concentration (CLrenal,k), representing the intrinsic efflux activity across the BBM, were significantly reduced in Mrp4–/– mice. These results suggest that Mrp4 plays a significant role in the luminal efflux of these cephalosporins in the kidney. It should be noted that the effect of impairment of Mrp4 on the kidney concentrations may exhibit a gender difference because the present study was carried out using female mice, which exhibit a 3-fold higher mRNA expression of Mrp4 in the kidney than male mice (Maher et al., 2005).

    As far as cefazolin is concerned, the tubular secretion clearly remained in Mrp4–/– mice (Table 1). Indeed, the tubular secretion clearance of cefazolin with respect to the kidney concentration (CLrenal,k) exhibited moderate reduction (Table 1). Therefore, it is probable that the luminal efflux of cefazolin is also mediated by other transporters. Tamai et al. (1988) found that a membrane voltage-driven transporter is involved in the transport of cefixime in the BBM vesicles from the kidney. It is possible that this transporter is involved in the renal elimination of cefazolin. As membrane voltage-driven transporters for organic anions, OATv1 and RST have been identified in the BBM of the kidney (Jutabha et al., 2003; Imaoka et al., 2004). In addition, MRP2 and BCRP have been also identified on the BBM of the renal tubule cells (Schaub et al., 1999; Jonker et al., 2002; Mizuno et al., 2004). These transporters are candidates involved in the tubular secretion of cephalosporins in conjunction with Mrp4. The minimal change in the renal clearance (CLrenal,p) of cefazolin in Mrp4–/– mice can be explained by speculating that the efflux across the BBM is greater than that across the basolateral membrane, resulting in the uptake being the rate-limiting process of the net secretion. Under these conditions, the net elimination is hardly affected by the reduction in the luminal efflux clearance.

    The recovery of ceftizoxime in the urine was significantly reduced in Mrp4–/–, although the plasma concentrations of ceftizoxime were unchanged (Fig. 5). This suggests that impairment of Mrp4 produced a nonrenal elimination pathway for ceftizoxime, which apparently compensated for the reduced renal elimination. The nonrenal elimination pathway remains to be elucidated. Considering that some cephalosporins undergo biliary excretion (Wright and Line, 1980), hepatic elimination would account for the nonrenal elimination pathway of ceftizoxime in Mrp4–/– mice. Indeed, Mrp4 has been identified in the sinusoidal membrane in the liver (Rius et al., 2003), and reduction of sinusoidal efflux could increase the hepatic elimination. It is also possible that this is caused by adaptive regulation of detoxification systems in Mrp4–/–. Unlike ceftizoxime, the urinary recovery of cefazolin was unchanged in Mrp4–/– (Fig. 5), indicating that the nonrenal elimination pathway makes only a limited contribution for cefazolin. Further pharmacokinetic studies are necessary to elucidate the mechanisms underlying such a difference in the pharmacokinetics.

    The present study shows that Mrp4 plays an important role in the tubular secretion of ceftizoxime and cefazolin in addition to previously reported Mrp4 substrates, diuretics, and acyclic antivirus drugs (Hasegawa et al., 2007; Imaoka et al., 2007). MRP4 is also expressed in the BBM of the human kidney (van Aubel et al., 2002). It may also make a significant contribution to the luminal efflux of these drugs in the human kidney. Caution must be paid when extrapolating the result of animal studies to humans considering the possibility of species difference in the protein expression, substrate specificity/transport activity of not only MRP4 but also other luminal transporters. It is necessary to obtain clinical data to support the functional role of MRP4 in human kidney. A population pharmacokinetic analysis has suggested a bimodal distribution of the renal clearance of ceftizoxime with regard to the plasma concentration (Facca et al., 1998), although the underlying mechanism remains to be elucidated. Cumulative studies suggest that the allele frequencies non-synonymous with functional change and nonsense mutations of OAT1 and OAT3 are too low to account for the bimodal distribution (Fujita et al., 2005; Erdman et al., 2006). Whether this is associated with interindividual variations in MRP4 activity as a result of genetic polymorphisms and/or other factors is a topic for future investigation.

    In conclusion, some cephalosporins are MRP4 substrates, and Mrp4 makes a significant contribution to the luminal efflux of ceftizoxime and cefazolin in the kidney. This is the first report identifying the transporter responsible for the luminal efflux of cephalosporins. In the past, exchanger and/or facilitative transporters have been considered to play major roles in the urinary excretion of drugs, but an ATP binding cassette transporter, MRP4 is involved in the tubular secretion of some diuretics, acyclic nucleoside analogs, and cephalosporins. This finding will contribute to a better understanding of the luminal efflux mechanisms of drugs.

    ABBREVIATIONS: OAT, organic anion transporter; BBM, brush-border membrane; MRP, multidrug resistance-associated protein; DHEAS, dehydroepiandrosterone sulfate; GFP, green fluorescent protein; TS, Tris-sucrose; GFR, glomerular filtration rate; CL, clearance.

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作者单位:Department of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Tokyo, Japan (L.C., H.K., K.T., Y.S.); and Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee (M.A., J.S.)

日期:2009年8月25日 - 来自[2007年第69卷第6期]栏目
循环ads

Increased Enzyme Activity and β-Adrenergic–Mediated Vasodilation in Subjects Expressing a Single-Nucleotide Variant of Human Adenylyl Cyclase

【摘要】  Objective— cAMP is a critical regulator of metabolic and cardiovascular function. However, the role of genetic variability in the regulation of cAMP-mediated effects is unclear. Therefore, we assessed the effect of the expression of a recently identified missense genetic variant of adenylyl cyclase isoform 6 (ADCY6 S674).

Methods and Results— In rat vascular smooth muscle cells, gene transfer of ADCY6 S674 increased adenylyl cyclase activity and arborization to a greater extent than gene transfer of ADCY6 A674. Similarly, in adherent mononuclear leukocyte cells isolated from ADCY6 S674-expressing human subjects, both adenylyl cyclase activity and adenylyl cyclase–mediated cell retraction were significantly increased. Additionally, in dorsal hand vein LVDT studies, subjects expressing the hyper-functional ADCY6 S674 variant had significantly greater vascular sensitivity to the β-adrenergic agonist isoproterenol as assessed by both a greater potency and greater maximal effect than subjects expressing the ADCY6 A674 enzyme.

Conclusion— These data indicate that the expression of a novel, relatively common variant of ADCY6 parallels an increase in adenylyl cyclase activity and adenylyl cyclase–mediated function in humans.

We examined the phenotypic characteristics of an adenylyl cyclase 6 (ADCY6 S674) variant in human subjects and isolated human mononuclear leukocytes and rat smooth muscle cells. Our data demonstrate that expression of this ADCY6 S674 variant is associated with enhanced adenylyl cyclase activity and enhanced cAMP-mediated regulation of contractile responses.

【关键词】  smooth muscle adenylyl cyclase vasodilation


Introduction


Genetic variants have been described for a range of G protein–coupled receptors (as well as for G proteins) linked to adenylyl cyclase. 1,2 Further, expression of these variants leads to alterations in receptor-mediated activation of adenylyl cyclase as well as alterations in more "downstream" effector pathways. The identification of missense genetic variants of adenylyl cyclase had previously been limited to those in ADCY9, an isoform with much more restricted tissue distribution. 3 However, our recent studies have revealed a relatively prevalent missense single nucleotide variant of ADCY6. 4 Using an insect expression system we found that high expression of this ADCY6 variant was associated with decreased adenylyl cyclase enzymatic function. However, the impact of expression of this ADCY6 S674 variant on adenylyl cyclase activity in mammalian cell models at lower (more physiological) expression levels or the functional impact of endogenous expression of this variant in humans is unknown.


Variability in cAMP synthesis by adenylyl cyclase has previously been thought to be determined (predominantly) by the specific isoforms of adenylyl cyclase expressed in any individual cell. Nine membrane-bound isoforms of adenylyl cyclase arising from different genes have been cloned—grouped into 3 major subfamilies comprising: Group 1: ADCY1, ADCY3, ADCY8; Group 2: ADCY2, ADCY4, ADCY7; and Group 3: ADCY5, ADCY6. 5 Also, ADCY9 has been characterized as a distinct (and atypical) isoform 6 with restricted expression. Also, a soluble adenylyl cyclase has been characterized that is the predominant form in mammalian sperm. 7


Each isoform has a specific pattern of tissue distribution and a specific pattern of regulation. 5,6,8–10 Further, differential effects of specific isoforms on both PKA-mediated effects and growth regulation have been recently reported. 11 In this regard ADCY6 has been shown to be more tightly coupled to PKA-regulated functions, including cAMP-mediated cytoskeletal reorganization.


Changes in the regulation or expression of adenylyl cyclase are of physiological and pathological importance. In a model of acquired diabetes, the defect in vascular relaxation responses have been linked to impaired adenylyl cyclase function and reduced protein expression of ADCY5/ADCY6. 12 In mice with cardiomyopathy, cardiac expression of ADCY5 or ADCY6 improves cardiac function 13,14 and survival. 15 In transgenic mice, inhibition of adenylyl cyclase activity via activation of G i 2 results in insulin resistance. 16 However, the physiological or pathobiological significance of regulation of adenylyl cyclase function via "genetic" variability (ie, the expression of missense genetic variant enzymes) is unknown.


Given the critical role of ADCY6 in regulation of vascular smooth muscle contractility, 11 the expression of a genetic variant of ADCY6 that significantly alters ADCY6 function could lead to altered vascular adenylyl cyclase-mediated effects. The identification of dysfunctional genetic variants of adenylyl cyclase is presently limited to those in ADCY6 and ADCY9, of which the latter has a much more restricted distribution than the former. 3 Recently a single nucleotide polymorphism (SNP) in intron 17 of gene encoding the very widely expressed ADCY6 isoform, 17 was identified in a Japanese population. 18 However, the impact of this variant on adenylyl cyclase function is currently unknown.


In our initial studies, we discovered a relatively common ( 6% genotypic frequency in Whites) missense SNP in ADCY6 (g. T), with trivial name A674S. 4 This genetic variant was initially identified based on our screen of the ADCY6 sequence in normal subjects corresponding to residues 562 to 778, which include the C1b and IC4 regions of the molecule. This region was of particular interest because this includes domains critical in the regulation of catalytic activity. 5,8,9 Additionally, our interest in this region was based on previous findings that serine to alanine alterations in this domain are associated with significant alterations in the regulation of ADCY6 function. 19


Consequently, the present studies were undertaken to examine the impact of the expression of ADCY6 S674 variant both on vascular reactivity and on cellular adenylyl cyclase activity/function. Our data demonstrate that the expression of the ADCY6 S674 variant is associated with enhanced adenylyl cyclase activity and enhanced cAMP-mediated regulation of contractile responses.


Methods


Study Subjects


Recruitment of study subjects was based on mass advertising/emailing efforts within the Robarts Research Institute and the University of Western Ontario as part of an ongoing study of the phenotypic impact of the expression of the ADCY6 S674 variant. The age range of study subjects was 20 to 45 years. Subjects were normotensive- as determined by having a screening blood pressure measurement of less than 140/90 at the time of recruitment (based on the average of 5 readings, BPTru, Vancouver Canada) and healthy. Explicit exclusion criteria included: a history of cardiovascular events, average alcohol intake over 2 U per day, pregnancy, and use of antihypertensive/blood pressure altering drugs or anticoagulants.


Those subjects expressing the ADCY6 S674 variant allele (all were heterozygous) were subsequently invited to: (1) donate an additional blood sample for the in vitro assessment of adenylyl cyclase activity/function in adherent mononuclear leukocyte cultures or (2) participate in dorsal hand vein linear variable differential transformer studies assessing vascular reactivity. An otherwise similar population of subjects was recruited from among those expressing the ADCY6 A674 gene ( Table 1 ). Recorded blood pressure was based on the average of the last 5 measurements taken. A 10 mL blood sample was taken to confirm the ADCY6 genotype. Informed consent was obtained for all analyses, with approval from the University of Western Ontario Research Ethics Review Board.


Table 1. Age and Sex Distribution of ADCY6 A674 and ADCY6 S674 Subjects


Genomic DNA was extracted from whole blood as previously described. 20 Genotyping of ADCY6 A674S variants was performed using exon-specific DNA amplification followed by purification using shrimp alkaline phosphatase (Roche) and exonuclease I (ExoI; New England Biolabs) and DNA sequencing as recently described. 4,20


Construction of Adenovirus Expressing S674 and A674 Adenylyl Cyclase 6


cDNAs encoding flag-tagged ADCY6 A674 and ADCY6 S674 or GFP were used to generate adenoviral constructs (AdMax) as per manufacturer?s instructions (Microbix Biosystems Inc) as previously described. 21


Vascular Smooth Muscle Cell Primary Cultures


Rat aortic vascular smooth muscle cell (VSMC) primary cultures were isolated by a modification of the methods of Touyz et al. 22 Briefly, freshly isolated thoracic aortae from Wistar rats (Harlan, Indianapolis, Indiana) were digested using collagenase and elastase incubations as previously described. 11,23 Following digestion/isolation, vascular smooth muscle cells were resuspended in Dulbecco?s modified Eagle?s medium (DMEM) supplemented with 10% FBS, gentamicin and fungizone. Vascular smooth muscle cells were used between passages 4 to 12 for all experiments. The rats were cared for in accordance with the Canadian Council on Animal Care guidelines.


Gene Transfer in Vascular Smooth Muscle Cells by Adenovirus


Vascular smooth muscle cells were infected with adenoviral constructs (either adeno-GFP, adeno-ADCY6 A674, or adeno-ADCY6 S674) for 16 hours at 37°C after which infection media was replaced with fresh DMEM culture media. Cells were used for experimentation 48 hours postinfection. Under these conditions, infection efficiency was greater than 95%, as assessed in GFP-infected cells.


Using Adherent Mononuclear Leukocyte Cultures to Assess Alterations in Adenylyl Cyclase-Mediated Responses With Variant ADCY6 Expression


Adherent mononuclear leukocyte fractions with fibrocyte characteristics can be separated from peripheral blood samples. 24 Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from human blood by centrifugation over Histopaque-1077 (Sigma-Aldrich) following the manufacturer?s protocol. PBMCs were washed twice with sterile 0.9% NaCl, resuspended in RPMI 1640 supplemented with 20% fetal bovine serum and gentamycin and seeded onto culture plates. This population of cells, isolated from adherent cell fractions, appears in a "spindle form" within 5 days after seeding onto culture plates and persist for several weeks ( Figure 1 A). Preliminary studies demonstrated that after one week of culture these spindle cells are "positive" for phalloidin, fibronectin, and CD68 but "negative" for -smooth muscle actin—consistent with a "fibrocyte-type" cell of mononuclear leukocyte lineage ( Figure 1B through 1 D).


Figure 1. A, Characteristic spindle shape of adherent mononuclear leukocytes. Brightfield photomicrographic illustration of characteristic fibrocyte (spindle shape) of adherent mononuclear leukocytes after 1 week in primary culture. B, Assessment of monocytic lineage of adherent mononuclear leukocytes. Immunostaining with anti-CD68 depicting monocytic lineage. Nuclear staining (in blue) was assessed with Hoechst dye. C, Assessment of Actin content in adherent mononuclear leukocytes. Immunostaining with anti-phalloidin to detect the presence of actin. Nuclear staining was assessed with Hoechst dye. D, Assessment of fibronectin in adherent mononuclear leukocytes. Fibronectin content was detected with anti-fibronectin immunostaining. Nuclear staining was assessed with Hoechst dye.


Adenylyl cyclase activity in permeabilized adherent mononuclear leukocytes and in vascular smooth muscle cells was assessed using our previously described methods that we have used in a range of cell types including mononuclear leukocytes and vascular smooth muscle cells. 25 Briefly, digitonin-permeabilized cells were resuspended in a solution of Hanks? Balanced Salt Solution with 33 mmol/L HEPES, 0.5 mmol/L EDTA and 1 mmol/L magnesium sulfate (pH 7.4 at 4°C) were added in an aliquot of 40 µL to give a final incubation volume of 100 µL with 1 µCi [ 32 P] ATP, 0.3 mmol/L ATP, 2 mmol/L MgSO 4, 0.1 mmol/L cAMP, 5 mmol/L phosphoenol pyruvate, 40 µg/mL pyruvate kinase and 20 µg/mL myokinase. Incubations with GTP (1 µmol/L), isoproterenol (µmol/L) or forskolin (100 µmol/L) were carried out at 37°C for 10 minutes and terminated by addition of 1 mL of a solution containing 100 µg ATP, 50 µg cAMP, and approximately 15 000 cpm [ 3 H] cAMP. Cells were pelleted by centrifugation at 300 g for 5 minutes. cAMP was isolated from the supernatant by sequential Dowex and alumina chromatography and was corrected for recovery using [ 3 H] cAMP as the internal standard. Adenylyl cyclase activity was linear with time and cell number over the ranges used.


Adenylyl cyclase-mediated arborization response in rat vascular smooth muscle cells was assessed by video-microscopy, using our recently published techniques. 11,23 Dishes were mounted in a temperature-controlled chamber (Bionomic controller, 20/20 Technology, Inc) on an inverted microscope (Zeiss, Axiovert S100). Arborization was induced by the addition of forskolin (10 µmol/L). Progression of arborization was evaluated using time-lapse video microscopy with a digital recording system. Images were obtained every minute and the extent of arborization was determined by the change in image intensity (Northern Eclipse 6.0, Empix Imaging). The change in image intensity was expressed as a percent of basal intensity ie, before the addition of drug. The change in image intensity was plotted against time and slopes were determined from linear regression analysis using Prism 4.0 (GraphPad Software).


Adenylyl cyclase-mediated cell retraction as a manifestation of arborization in adherent mononuclear leukocyte fractions was assessed by video-microscopy as described above for vascular smooth muscle cells. However, in adherent mononuclear leukocytes, the "arborization" effect was assessed by measurement of change in cell perimeter during 15 minutes of basal recording and after 15 minutes of forskolin (100 µmol/L) stimulation. This mode of analysis was chosen based on the much lower extent of optical density changes associated with comparable extents of retraction seen in the arborization response in cultured mononuclear leukocytes (versus those seen in vascular smooth muscle cells). Cell perimeter was obtained by using the trace tool within the analysis software (Northern Eclipse 6.0). Typically 6 to 8 cells in the field of view (1 or 2 cells in each quadrant chosen randomly and prospectively) were analyzed under basal and forskolin-treated conditions. Change in cell perimeter was expressed a percentage of the initial cell perimeter.


Linear Variable Differential Transformer Studies Assessment of Vascular Sensitivity to Isoproterenol by Dorsal Hand Vein Linear Variable Differential Transformer Technique


Studies using the linear variable differential transformer (LVDT) technique in dorsal hand veins were performed according to our previously described methods. 26–29 Baseline venous distension was assessed after compression of the ipsilateral arm with a sphygmomanometer cuff inflated to 50 mm Hg. The extent of this distension at baseline was defined as 100%. Phenylephrine-mediated venoconstriction was assessed by infusion of increasing doses from 16 to 20 000 ng/min (in normal saline at an infusion rate of 0.1 mL/min). The maximal extent of phenylephrine-mediated constriction and the potency of phenylephrine (as defined by the dose that produced half-maximal effect [ED 50 ]) were determined by computerized nonlinear curve fitting (Sigmoid Plot, Subroutine, Prism 4.0, GraphPad Software). To assess the extent of isoproterenol-mediated attenuation of phenylephrine-mediated venoconstriction, veins were preconstricted with phenylephrine at a dose that achieved approximately 80% of the maximum phenylephrine-induced effect; the dose was individualized for each subject in each study. In the assessment of vasodilator responses, the extent of venous distension achieved with this dose of phenylephrine was defined as 0% venodilation. Isoproterenol was then concurrently infused at a dose of 0.32 to 200 ng/min in normal saline at an infusion rate of 0.1 mL/min. Maximum isoproterenol-mediated venodilation and ED 50 for isoproterenol were determined by analysis of the data by curve fitting techniques, as previously described. Maximal nitroglycerin-mediated vasorelaxation was determined at a dose of 100 ng/min.


Data Analysis


The nominal probability value for significance was <0.05. For 2-group comparisons, the statistical significance of differences was determined by student t test for unpaired data with Welch?s correction when necessary. For column statistics, the significance of differences from control was determined by 1-sample t tests. P <0.05 on a 2-sided test was taken as a minimum level of significance (Prism 4.0, GraphPad Software).


When conventionally expressed, measures of potency (eg, ED 50 ) are not normally distributed. 30 However, after log transformation these data are normally distributed. Therefore, these parameters are expressed as their geometric means.


Results


Effect of ADCY6 S674 Expression on Adherent Mononuclear Leukocyte Adenylyl Cyclase Activity


To determine whether the ADCY6 S674 variant was associated with any functional alterations in enzymatic activity, we examined adenylyl cyclase activity in adherent cell cultures derived from circulating mononuclear leukocytes isolated from whole blood samples. Basal (unstimulated) adenylyl cyclase activity did not differ significantly between subjects with ADCY6 A674 or the ADCY6 S674 variant (ADCY6 A674: 25±5 versus ADCY6 S674: 37±7 pmol/min/mg of protein, n=12 for both, P =0.2). However, both GTP- and forskolin-stimulated adenylyl cyclase activities were significantly increased in adherent mononuclear leukocytes obtained from ADCY6 S674 variant subjects as compared with ADCY6 A674 subjects ( Figure 2 A). Isoproterenol-stimulated adenylyl cyclase activity was also significantly increased in ADCY6 S674 adherent mononuclear leukocytes as compared with cells from subjects expressing WT ADCY6 ( Figure 2 A). However, isoproterenol-stimulated adenylyl cyclase activity actually represents isoproterenol+GTP-stimulated adenylyl cyclase activity. Therefore, we examined the proportional increase in isoproterenol-stimulated adenylyl cyclase activation. The proportional increase of isoproterenol-stimulated over GTP-stimulated adenylyl cyclase activity was not significantly different between ADCY6 S674- and ADCY6 A674-expressing adherent mononuclear leukocytes (141±15% versus 136±11% of GTP-ACA, n=12, both for the ADCY6 S674 variant and ADCY6 A674 respectively, P =0.75). Therefore, the elevation in forskolin-stimulated activity suggests an increase in the intrinsic activity of the enzyme, whereas the similar proportional increase in isoproterenol-stimulated activity suggests that the efficiency of GPCR-G protein coupling was not altered by the expression of the ADCY6 S674 variant.


Figure 2. A, Assessment of adenylyl cyclase activity in adherent MNL. GTP-, isoproterenol (ISO)-, and forskolin (FSK)-stimulated adenylyl cyclase activities were significantly higher in MNLs obtained from ADCY6 S674 subjects (n=12) as compared with ADCY6 A674 subjects (n=12). B, Assessment of cell retraction in adherent MNLs. FSK-induced retraction was significantly increased in MNLs obtained from ADCY6 S674 subjects (n=12) as compared with ADCY6 A674 subjects (n=12). Data represent the mean±SEM from 12 independent experiments. * P <0.05 vs MNLs from ADCY6 A674 subjects.


Effect of ADCY6 S674 Expression on Adenylyl Cyclase–Mediated Adherent Mononuclear Leukocyte Retraction


To determine whether the increase in forskolin-stimulated adenylyl cyclase activity in adherent mononuclear leukocytes from subjects with the ADCY6 S674 variant resulted in increased functional adenylyl cyclase-mediated responses, we performed adherent mononuclear leukocyte retraction assays. Forskolin treatment mediated both time- and concentration-dependent increases in cellular retraction (data not shown). Similar to the results obtained for the adenylyl cyclase activity assay, forskolin-mediated retraction was significantly increased in ADCY6 S674 variant-expressing adherent mononuclear leukocytes as compared with the ADCY6 A674 expressing cells ( Figure 2 B).


Assessment of the Impact of Expression of the ADCY6 Variant on Vascular Reactivity: LVDT Studies


In ADCY6 A674- expressing subjects, phenylephrine mediated a dose-dependent reduction in vascular distension with an ED 50 of 553 ng/min to a nadir of 36±13% of baseline distension. With infusion of a dose of phenylephrine sufficient to mediate approximately 80% of its maximal effect, concurrent infusion of isoproterenol caused dose-dependent vasodilation with an ED 50 of 8 ng/min reaching a maximum of 97±6% of initial baseline ( Table 2 ). Subsequent infusion of nitroglycerin at a dose of 100 ng/min resulted in a further increase in distension to 109±9% of baseline ( Table 2 ). These findings are similar to those we have previously reported in normal populations using this protocol. 26,29


Table 2. Venous Responsiveness in ADCY6 A674 and ADCY6 S674 Subjects


In subjects expressing ADCY6 S674, vascular sensitivity to isoproterenol was increased on average by approximately 10-fold (ie, the ED 50 isoproterenol was decreased by more than 90%, Table 2 ). Further, there was a significant increase in maximal isoproterenol-mediated vasodilation ( Table 2 ). In contrast, neither the extent of baseline distension nor maximal nitroglycerin-mediated relaxation differed between groups ( Table 2 ). Additionally, indices of vascular vasoconstrictor responses to phenylephrine (ED 50 phenylephrine, maximal phenylephrine-mediated vasoconstriction) did not differ between groups ( Table 2 ).


Assessment of Adenylyl Cyclase–Mediated Responses in Variant and ADCY6 A674–Expressing Vascular Smooth Muscle Cells


To determine whether the difference in adenylyl cyclase activity observed in adherent mononuclear leukocytes obtained from subjects expressing the ADCY6 S674 variant was a property of the adherent mononuclear leukocytes or of the intrinsic adenylyl cyclase activity, we examined the effect of ADCY6 S674 expression on adenylyl cyclase activity in another cell type, rat vascular smooth muscle cells, which express a highly homologous (93% compared with human) endogenous ADCY6. 31 With comparable expression of ADCY6 S674 and ADCY6 A674 in vascular smooth muscle cells ( Figure 3 A), gene transfer of the ADCY6 S674 variant resulted in a significant increase in forskolin-stimulated adenylyl cyclase activity as compared with ADCY6 A674-expressing smooth muscle cells ( Figure 3 B).


Figure 3. A, Assessment of ADCY6 expression in rat vascular smooth muscle cells. Comparable expression of ADCY6 A674 and ADCY6 S674 as assessed by Western blotting in rat vascular smooth muscle cells after gene transfer. B, Assessment of adenylyl cyclase activity in rat smooth muscle cells. ADCY6 S674-expressing smooth muscle cells demonstrate increased forskolin-stimulated adenylyl cyclase activity as compared with wild-type ADCY6 A674-expressing cells. Data represents the mean±SEM from 12 independent experiments. ** P <0.05 vs ADCY6 A674-expressing cells. * P <0.05 vs control cells. C, Assessment of arborization in rat smooth muscle cells. ADCY6 S674-expressing cells demonstrate increased forskolin-induced arborization as compared with ADCY6 A674-expressing cells. Data represent the mean±SEM from 6 independent experiments. ** P <0.05 vs ADCY6 A674-expressing cells. * P <0.05 vs control cells.


To determine whether increased ADCY6 S674-mediated enzymatic activity in vascular smooth muscle cells paralleled an increase in adenylyl cyclase-mediated function we examined forskolin-stimulated arborization in vascular smooth muscle cells. With gene transfer of ADCY6 S674 variant, forskolin-mediated arborization responses were significantly increased as compared with responses in ADCY6 A674-infected smooth muscle cells ( Figure 3 C).


Discussion


Although our most recent studies had identified a relatively common genetic variant of ADCY6, namely ADCY6 S674, the significance of its expression was unknown, either in mammalian systems or in humans. The present studies demonstrate that the expression of the ADCY6 S674 variant is associated with an increase in both adenylyl cyclase and β-adrenergic–mediated vascular reactivity.


The mechanism of the increase in adenylyl cyclase function in circulating adherent mononuclear leukocytes derived from whole blood samples taken from subjects expressing the ADCY6 S674 variant would appear to be best explained by an enhancement of ADCY6 function. This conclusion is supported by our findings that gene transfer of the ADCY6 S674 variant into rat vascular smooth muscle cells increased adenylyl cyclase effects, as assessed both enzymatically and functionally, to a significantly greater extent than expression of the WT ADCY6. It is important to note that in our prior evaluation of this variant we reported that, in the baculovirus/Sf9 insect cell system, the expression of ADCY6 S674 demonstrated reduced activity as compared with activity in cells expressing ADCY6 A674. 4 The reason for this discrepancy with our current findings is speculative. However, for membrane proteins, the "functional readouts" from insect cell systems may not predict their impact in mammalian systems. 32 This has been related to differences in functional responses of nonglycosylated protein forms as seen in insect models or to differences in intracellular scaffolding cytoskeletal structure that are critical for the functional effect of a number of membrane-associated proteins. 32 However, regardless of the explanation for the differences in effect of ADCY6 S674 in insect versus mammalian cells, our current studies indicate that increase adenylyl cyclase activity associated with expression of the ADCY6 S674 variant in a more relevant mammalian cell system (and at a much lower extent of overexpression) parallels the increase in "global" adenylyl cyclase activity and in adenylyl cyclase-mediated function in ADCY6 S674 adherent mononuclear leukocytes.


In the hypothetical case that ALL adenylyl cyclase isoforms would contribute comparably to regulation of contractile function in vascular smooth muscle cells, a 2-fold increase in adenylyl cyclase activity between variants of a single isoform (ie, as seen for the genetic variant of ADCY6 S674 versus the more common ADCY6 A674 when expressed in rat vascular smooth muscle cells) would NOT be expected ultimately to impact on "global" adenylyl cyclase-mediated function. However, ADCY6 has been identified as a predominant isoform expressed in a range of tissues important in cardiovascular regulation. 33 Further, our recent studies identified that among the AC isoforms, ADCY6 was selectively coupled to regulation of vascular smooth muscle contractile responses, 11 suggesting that the regulation of ADCY6 function would have an impact on contractile regulation far exceeding the proportional contribution of ADCY6 to total adenylyl cyclase expression. Our current findings support the hypothesis that a significant alteration of ADCY6 function leads to increased adenylyl cyclase–mediated contractile effects, both at a single–cell level as well as in vivo in humans.


The cardiovascular significance of the expression of this hyper-functional ADCY6 is supported by our LVDT studies demonstrating enhanced β-adrenergic–mediated vasodilatory responses in subjects expressing the S674 variant of ADCY6. Notably, the heritability of vasodilatory responses has been associated with genetic variants of both the beta 2 -adrenoceptor (Ile164 and Gln27 variants 34–37 ), as well as the G protein beta subunit GNB3 825T. 38–40 However, genetic variability in vasodilatory responses related to expression of an AC isoform genetic variant has not previously been reported.


The expression of missense genetic variants of proteins regulating adenylyl cyclase function have been linked to both hypertension and obesity. The expression of β-adrenoceptor variants and variants of GRK4 (an enzyme that regulates G protein–coupled receptors linked to AC activation) have been associated with variation in blood pressure and development of hypertension. 41 Further, hyper-functional G protein variants have been associated with variation in the development of obesity as well as hypertension (reviewed in 42 ). Expression of this ADCY6 variant and the consequent enhancement of ADCY6-mediated effects would be predicted to be associated with a "hyperdynamic" cardiovascular phenotype, demonstrating increased pulse pressure, systolic blood pressure, or increased pulse rate. Further, expression of this adenylyl cyclase variant would be predicted to be associated with a "leaner" phenotype, marked by decreased abdominal obesity.


In summary, our data indicate that the expression of a novel, relatively common variant of ADCY6 parallels an increase in adenylyl cyclase activity and adenylyl cyclase mediated function in humans. Whether there might be an altered frequency of the expression of the A674 allele in patients with metabolic syndrome, diabetes, and hypertension, ie, whether this genetic variant might prove to be a predictive marker for cardiovascular disease, is the focus of ongoing studies.


Acknowledgments


We gratefully acknowledge the important contributions made by Nancy Schmidt in assisting in the performance of the LVDT studies.


Sources of Funding


These studies were supported by grants-in-aid to R.D.F. from the Heart and Stroke Foundation of Ontario. R.G. is supported by a New Investigator Award from the Heart and Stroke Foundation of Canada.


Disclosures


None.

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作者单位:Departments of Medicine (S.V.U., A.H.J., J.G.P., R.A.H., R.D.F.) and of Physiology & Pharmacology (R.G., R.D.F.), University of Western Ontario, and Cell Biology (Q.D., R.D.F.) and Vascular Biology (R.G., J.G.P., R.A.H.) Research Groups, Robarts Research Institute, London, Ontario, Canada.

日期:2008年12月28日 - 来自[2007年第27卷第12期]栏目

Activated Protein C Decreases Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand by an EPCR- Independent Mechanism Involving Egr-1/Erk-1/2 Activati

【摘要】  Background— APC is an antithrombotic and antiinflammatory serine protease that plays an important role in vascular function. We report that APC can suppress the proapoptotic mediator TRAIL in human umbilical vein endothelial cells, and we have investigated the signaling mechanism.

Methods and Results— APC inhibited endothelial TRAIL expression and secretion and its induction by cell activation. To explore the mechanism, we examined factors associated with TRAIL regulation and demonstrated that APC increased the level of EGR-1, a transcriptional factor known to suppress the TRAIL promoter. APC also induced a significant increase in phosphorylation of ERK-1/2, required to activate EGR-1 expression. Activation of ERK-1/2 was dependent on the protease activated receptor-1 (PAR-1), but independent of the endothelial protein C receptor (EPCR). Using siRNA, we found that the effect of APC on the EGR-1/ERK signaling required for TRAIL inhibition was dependent on the S1P1 receptor and S1P1 kinase.

Conclusions— Our data suggest that APC may provide cytoprotective activity by activating the ERK pathway, which upregulates EGR-1 thereby suppressing the expression of TRAIL. Moreover, we provide evidence that APC can induce a cell signaling response through a PAR-1/S1P1-dependent but EPCR-independent mechanism.

We report that APC can suppress the proapoptotic mediator TRAIL by activating the ERK pathway to upregulate EGR-1, a negative regulator of TRAIL expression. The effect of APC was PAR-1– and S1P1-dependent, but independent of the endothelial protein C receptor, suggesting a mechanism to suppress injury in cells not expressing this receptor.

【关键词】  endothelial protein C receptor apoptosis protease activated receptor APC TRAIL


Introduction


The TNF family of cytokines mediates apoptosis and a variety of immune/inflammatory responses. The TNF-related apoptosis-inducing ligand (TRAIL) is constitutively expressed in most normal tissues and cell types 1 and is able to induce apoptosis on binding to either TRAIL-R1 or TRAIL-R2 receptors. 2,3 This binding interaction induces oligomerization of intracellular death domains, resulting in cleavage of pro-caspase 8 into its active form, which can activate caspase 3, the key mediator of apoptosis. 4 Early studies suggested that TRAIL induced apoptosis in malignant cells, but had minimal toxicity on normal cells because normal cells expressed high levels of decoy receptors. 5 However, subsequent studies have demonstrated that endothelial cells are susceptible to TRAIL-induced apoptosis. 6,7 Both soluble and membrane-associated TRAIL activate TRAIL-R1 on the cell surface 8 and human endothelial cells display little difference in response to membrane-bound versus soluble ligand. 9 Moreover, several studies have demonstrated both autocrine and paracrine activation of the TRAIL receptors from membrane-associated and soluble TRAIL. 10–13 In addition, recent studies in vivo have suggested a potential proinflammatory and proapoptotic role of TRAIL in vascular injury and atherothrombosis (reviewed in 14 ).


Apoptotic signals induced by TRAIL have been shown to be negatively regulated by the MAPK/ERK pathway, 15,16 and cells become susceptible to apoptosis when ERK is dephosphorylated. 17,18 Recent studies have also suggested that the early transcription factor, early growth response factor –1 (EGR-1), acts as a negative regulator of TRAIL. 19 Of interest, EGR-1 function has been shown to be dependent on ERK1/2 phosphorylation. 20


APC is a serine protease with a well characterized anticoagulant activity. Recent studies have demonstrated that APC has antiinflammatory and cytoprotective activities, which are not attributed to the anticoagulant activity of APC. 21–23 APC can initiate cell-signaling pathways in cells by activating protease-activated receptor 1 (PAR-1). 24 PAR-1 is a member of the 7 transmembrane domain G protein–coupled receptor family, and its activation requires cleavage at a specific site within its extracellular amino terminus. 25 This cleavage produces a new amino terminus, which then acts as its own tethered ligand, but optimal cleavage occurs when APC is juxtaposed to PAR-1. It is suggested that APC must bind to endothelial protein C receptor (EPCR) for it to be juxtaposed to PAR-1. Indeed, many studies have demonstrated antiinflammatory and antiapoptotic activities of APC that are dependent on PAR-1 and EPCR; however, previous studies have not elucidated alternative pathways that are activated by APC. 24,26,27


The present study was undertaken to determine whether APC could modulate the activity of endothelial TRAIL, an important mediator of vascular injury. We show that APC suppresses the expression of TRAIL mRNA by an EPCR-independent mechanism. We demonstrate that APC increases the phosphorylation and activation of ERK and increases the transcriptional activity of EGR-1, which in turn suppresses TRAIL mRNA expression. We also demonstrate that the activation of EGR-1 by APC is dependent both on PAR-1 and sphingosine 1-phosphate receptor (S1P1)-dependent signaling. These results describe a novel pathway by which APC may protect endothelial cells from mediators such as TRAIL and describe an APC-induced cellular response that is EPCR-independent.


Methods


TNF- –Induced TRAIL Expression


Confluent HUVECs were incubated with the EPCR blocking antibody JRK1494 (2.5 µg/mL) for 7 hours in serum-free media, and then concentrations of 8 to 160 nmol/L APC were added overnight. EPCR blocking experiments using RCR-252 were performed at 25 µg/mL for 2 hours. The concentration range was chosen to bracket the known 30 nmol/L Kd for APC interaction with EPCR, although these concentrations are above the therapeutic blood concentration achieved in APC-treated severe sepsis patients. The following morning, 1 ng/mL of TNF- was added to the cells for 1, 6, or 12 hours, then cells were lysed with M-PER and protein concentrations were determined by BCA assay according to the manufacturer?s protocol. The quantity of membrane-associated TRAIL was determined by ELISA, and the standard curve was generated using recombinant human TRAIL. Briefly, 96-well plates were coated with anti-TRAIL antibody overnight. The following day, wells were blocked for 1 hour with 4% bovine albumin in PBS. Samples or recombinant human TRAIL were added to the plate for 1 hour, followed by biotinylated anti-TRAIL Ab for 1 hour. Next, 100 µL of HRP streptavidin was added for 30 minutes. Wells were extensively washed with PBS-Tween between each of these steps. The ELISA was developed by adding TMB substrate solution for 20 minutes and stopped by adding H 2 SO 4. Plates were read at A 450 –A 595.


The effect of blocking EPCR with an siRNA on APC inhibition of TRAIL was performed in confluent HUVECs treated with a nonspecific control or EPCR-specific siRNA for 48 hours before treatment with 80 nmol/L APC. The level of EPCR expression in control and EPCR siRNA-treated cell was determined using a Western blot with S6 ribosomal protein (S6 RP) as a loading control.


Statistics


All experiments were performed at least in triplicate. Statistics were performed using JMP software (SAS Institute). Error bars indicate SEM. For the analysis of TRAIL response genes, comparison were displayed as a heat map generated using hierarchical clustering in JMP 5.1 (Ward method).


Please see supplemental materials, available online at http://atvb.ahajournals.org for additional details on Materials and Methods.


Results


TNF- –Induced TRAIL mRNA Expression Is Decreased by APC


As shown in Figure 1 A, the expression of endothelial TRAIL mRNA was suppressed by APC. Moreover, the induction of TRAIL mRNA by TNF- was blocked by APC treatment. To determine whether the effect of APC was concentration-dependent, cells were stimulated with various concentrations of APC (0 to 160 nmol/L), and the concentration of TRAIL was measured by ELISA. As Figure 1 B demonstrates, APC inhibited the amount of TRAIL secreted from HUVECs in a concentration-dependent manner, with a half-maximal effect at approximately 30 nmol/L. As was observed with the mRNA levels, APC also suppressed the induction of TRAIL secretion induced by TNF- ( Figure 1 C).


Figure 1. Influence of APC on TRAIL expression. A, mRNA levels of TRAIL determined in HUVECs treated with APC or APC plus TNF-. B, Concentration response for effect of APC on TRAIL secretion. C, Effect of APC on TNF- induction of TRAIL. D, Time course for effect of APC on TRAIL.


A time-course study was performed to determine how long APC was effective at reducing TRAIL. As shown in Figure 1 D, TRAIL was unchanged during the first hour after TNF- or APC treatment. At 6 hours, TRAIL was increased by 4.8-fold in TNF- –treated cells; however, TRAIL was reduced by 35% by APC treatment. At 12 hours, TRAIL was further increased by 1.3-fold in TNF- –stimulated cells, but was unchanged in cells that were costimulated with APC. These results suggest that a single exposure of cells to APC reduces TNF- –mediated TRAIL protein levels for at least 12 hours. Overall, our results suggest that APC can modulate TRAIL expression alone or after its induction with inflammatory mediators such as TNF-.


APC Decreases TRAIL Secretion Through an EPCR-Independent Mechanism


Several studies have shown that the antiapoptotic activity of APC can be mediated by the EPCR-dependent activation of PAR-1. To determine whether APC reduced TRAIL secretion via a similar mechanism, cells were treated with APC with or without TNF- administration in the presence of an antibody that prevents APC interaction with EPCR (JRK1494). As shown in Figure 2 A, the ability of APC to inhibit the expression of TRAIL was not affected by blocking the APC-EPCR interaction with anti-EPCR antibody JRK-1494 or with RCR-252 (data not shown). Moreover, blocking the APC-EPCR interaction had no effect on the ability of APC to reduce the TNF-induced secretion of TRAIL ( Figure 2 B). As a control to show that APC interaction with EPCR had in fact been blocked, in parallel experiments we were able to block the reported EPCR-dependent suppression of ICAM 23,25 with the anti-EPCR antibody (supplemental Figure I), and to suppress the known EPCR-dependent effects of APC on staurosporine-induced apoptosis 21,44 (supplemental Figure II) and on thrombin-induced changes in permeability determined by the BSA-Evans blue dye method described by Feistritzer and Riewald 28 (supplemental Figure III). While these data with blocking antibodies strongly suggested EPCR-independence of APC on TRAIL, we further confirmed this by treating cells with an siRNA, which ablated EPCR expression in the cell ( Figure 2 C, inset) but had no effect on the ability of APC to suppress the expression of TRAIL.


Figure 2. Effect of blocking EPCR on APC-mediated TRAIL suppression. Effect of EPCR blocking antibody JRK1494 on APC alone (A) or with TNF- (B). C, Effect of EPCR siRNA on the ability of APC to inhibit TRAIL expression. Error bars indicate SEM, n=3 to 5, * P <0.05 vs Control.


APC Increases the Expression and Functional Activity of EGR-1


Recent studies have suggested that the early transcription factor, EGR-1, acts as a negative regulator of TRAIL. 19 To further explore the mechanism for the inhibition of TRAIL by APC, we examined the relationship between the expression of EGR-1 and TRAIL across untreated and TNF-treated cells. As shown in Figure 3 A, the level of TRAIL expression was negatively associated with the expression of EGR-1. Moreover, we examined the level of EGR-1 and TRAIL at 6 hours after TNF treatment and observed a 53% decrease in EGR-1 expression and corresponding 61% increase in TRAIL expression (data not shown). These data are consistent with the previous report showing that TRAIL is negatively regulated by EGR-1. 19


Figure 3. APC Increases EGR-1 transcriptional activity and ERK-1/2 phosphorylation. A, Relationship of EGR-1 and TRAIL in cells treated with TNF. B, Serum starved HUVECs (30 minutes) were stimulated with 30 nmol/L APC (5 hours), and EGR-1 binding was determined by EMSA (lanes 1 to 2, untreated; 3 to 4, APC-treated, 5, cold probe competed).


To determine whether APC might suppress TRAIL via EGR-1, we analyzed the level of EGR-1 DNA binding activity by gel shift assay in untreated cells and those treated with APC. We observed significantly higher levels of EGR-1 in cells treated with APC ( Figure 3 B). As also shown, the binding was specific as cold binding site probe could completely inhibit the gel shift.


APC Increases pERK1/2 Independent of EPCR


EGR-1 binding activity has been shown to be dependent on ERK1/2 phosphorylation, 20 so we sought to determine whether APC might be affecting EGR-1 by modulating the level of ERK1/2. As shown in Figure 4 A, APC induced a time-dependent increase in the phosphorylation of ERK1/2. There was no change in the level of total ERK1/2 protein in these experiments as determined by Western blot analysis using an antibody to total ERK1/2 (data not shown). If the effect of APC on EGR-1 suppression of TRAIL was dependent on ERK1/2 phosphorylation, we would expect the effect to be EPCR independent as was shown in Figure 2. Therefore, we examined the effect of APC on ERK1/2 phosphorylation in the presence of the anti-EPCR antibody. As shown in Figure 4 B, the increase in pERK induced by APC was not suppressed by blocking the EPCR interaction. However, the effect of APC was completely suppressed by the blocking antibody to PAR-1. These data suggest that APC can induce EGR-1/ERK-1/2 cell signaling in a PAR-1–dependent but EPCR-independent manner.


Figure 4. Effect of APC on ERK-1/2 phosphorylation. A, HUVECs were stimulated with 30 nmol/L APC for various times, and the level of pERK1/2 was determined. B, Confluent HUVECs were incubated with the PAR-1 (ATAP2) or EPCR (RCR-252) blocking antibodies for 2 hours, then stimulated for 15 minutes with APC.


Effect of S1P1 Signaling on APC-Dependent EGR-1 Activation


Recent studies have demonstrated that the S1P1 receptor can effect cell signaling by APC. 28,29 As the effect of APC on TRAIL and the ERK1/2-EGR pathway appears to be PAR-1–dependent but EPCR-independent, we sought to determine the role of S1P1, especially as recent studies have shown that S1P1 signaling occurs with activation of the ERK1/2 pathway. 30–32 As shown in Figure 5 A, a siRNA blocking S1P1 (Edg1), but not a scrambled siRNA, significantly decreased the EGR1-DNA interactions induced by APC. We also examined the effect of SPHK1, the kinase required for generating S1P and activating the S1P1 receptor via PAR-1. As shown, the siRNA against SPHK1 also significantly suppressed the transcriptional induction of EGR-1 by APC. Our proposed model for the EPCR-independent activation of signaling by APC to suppress TRAIL expression, based on the model described by Camerer and Coughlin, 33 is shown in Figure 5 B.


Figure 5. Role of S1P1 signaling on EGR-1 activation. A, HUVECs were pretreated with specific siRNA to block S1P1 and SPHK1, serum starved for 30 minutes, followed by 30 nmol/L APC before determination of EGR-1 binding. B, Proposed pathway for the EPCR-independent signaling of APC to inhibit TRAIL expression.


TRAIL-Response Genes and APC Inhibition


We sought to assess the cellular consequence of APC-mediated TRAIL suppression by examining genes known to be suppressed or induced by TRAIL. For this analysis we examined cyclin-dependent kinase 4 (CDK4), previously shown to be suppressed by TRAIL, 34 and several genes (SOD2, IFI-15K/G1P2, NK4, HLA-A, HLA-C, TFPI-2) shown to be induced by TRAIL. Using both treated and untreated HUVECs, we first examined the relationship between the expression levels of TRAIL and the expression of these genes. Consistent with their known regulation by TRAIL, we observed a highly significant positive correlation between TRAIL and the set of TRAIL-induced genes, and a highly negative correlation between TRAIL and CDK4 ( Figure 6 A). We treated cells with APC to determine whether the APC-mediated reduction in TRAIL resulted in a suppression of TRAIL-induced genes. Figure 6 B shows the level of TRAIL in this experiment. As shown in Figure 6 C, the expression of SOD2, IFI-15K/G1P2, NK4, HLA-A, HLA-C, TFPI-2 were induced as TRAIL levels increased and suppressed by APC coincident with its ability to inhibit TRAIL secretion. Moreover, the TRAIL-suppressed gene CDK4 was increased by APC treatment, coincident with the suppression of TRAIL secretion. These data strongly suggest that the reduction in TRAIL expression by APC alters cellular pathways in HUVECs known to be mediated by TRAIL.


Figure 6. Effect of APC-mediated TRAIL reduction on TRAIL-responsive genes. A, Comparison of relative TRAIL expression levels versus genes modulated by TRAIL from untreated and TNF-treated cells (n=30). Relative level of TRAIL produced (B) and comparison of mRNA expression of genes known to be induced or suppressed by TRAIL (C).


Discussion


Apoptosis plays a major role in pathologic conditions where increased cell death has been associated with organ damage or failure. Indeed, this is true with severe sepsis where immunologic defense mechanisms initiate a cascade of inflammatory events leading to multi-organ failure (reviewed in 22 ). As indicated above, numerous studies have suggested that TRAIL induces apoptosis in cancer cells, 5 but recent studies have demonstrated that normal vascular endothelial cells are susceptible to TRAIL-mediated apoptosis. 6,7 Currently, the only therapy approved for treatment of patients with severe sepsis at high risk of death is recombinant human APC, drotrecogin alfa (activated). 35 Severely septic patients receiving recombinant human APC have better Sequential Organ Failure Assessment scores for cardiovascular and respiratory organ systems than severely septic patients treated with placebo. Although the role of TRAIL in human sepsis has not been defined, the data from the current study suggest that APC can inhibit TRAIL secretion activated by the proinflammatory cytokine TNF-, which is upregulated during sepsis. These observations provide insight into a potential mechanism by which APC might decrease apoptosis and improve organ function in severe sepsis patients. Moreover, the ability of APC to suppress TRAIL-regulated genes associated with proinflammatory and immunomodulatory activity ( Figure 6 ), ie, beyond the apoptosis pathway, suggests additional potential protective mechanisms.


The initial suggestion that APC could block apoptosis and promote cell survival came from a transcriptional profiling experiment, 23 and these results were confirmed in cell-based assays and animal models. 23,25,36 These studies suggested that APC mediates the suppression of apoptosis in a PAR-1/EPCR dependent mechanism by decreasing proapoptotic protein such as Bax, increasing antiapoptotic protein Bcl-2, and inhibiting the activation of caspase 3 and 8. In the current study, we show that APC can decrease the mRNA levels and secretion of the potent apoptosis factor TRAIL, via activation of the EGR-1/ERK-1/2 pathway. In contrast to previous reports, the mechanism of this effect appears to be PAR-1 dependent, but EPCR-independent. To the best of our knowledge, this is the first study to demonstrate that APC may activate cellular pathways in a PAR-1–dependent but EPCR-independent mechanism. Our data open the possibility of another cofactor involved in APC–PAR-1 interaction. A logical candidate would be thrombomodulin (TM); however, we were unable to demonstrate that APC-TM interactions are involved with the cell signaling we have described (data not shown).


Recent studies have suggested that APC signaling via PAR-1 can be transactivated by the S1P-S1P1 receptor interaction. 28,29 PAR-1 is coupled to several G proteins including G -β. The β subunit ultimately leads to the activation of the MAPK and ERK pathways, which can activate several transcription factors that transcribe genes containing a serum response element (SRE). 37 The transcription factor EGR-1 has 5 SREs, and studies have demonstrated that ERK can increase functional EGR-1. 38 Recent studies have also shown that S1P1 signaling activates the ERK1/2 pathway. 30–32 In the current study, we demonstrate that APC induces ERK-1/2 phosphorylation and increases the level of EGR-1 mRNA and functionally active protein. However, unlike previous studies, 28,29 the effect of APC signaling though PAR-1 and S1P1 signaling was independent of EPCR. EPCR has been shown to be expressed primarily on large vessels, but not in the normal microvasculature, and several studies have demonstrated that EPCR is differentially regulated during tissue injury. 39–41 Thus, protective signaling mechanisms independent of EPCR may be important under conditions of little or no EPCR expression.


APC plays an important role in the modulation of vascular function not only through inhibition of thrombin generation, but also by receptor-mediated effects via PAR-1 activation, which results in activation of cytoprotective and antiinflammatory pathways. The requirement of EPCR for APC to signal via PAR-1 has been documented, but its relevance and physiological role have been questioned based on kinetics and tissue distribution. 42 Our data suggest that the activation of protective signaling pathways, such as the ability to activate EGR-1/ERK-1/2 signaling to suppress TRAIL, can occur independent of EPCR. This suggests that conclusions on the role of signaling in the efficacy of APC may not be solely dependent on the tissue expression of this receptor. Recent studies have shown that the components required for the activation of PC and for APC signaling are colocalized to lipid rafts, 43 indicating the complex nature of the microenvironment on the membrane surface involved in the generation of APC and in PAR-1 signaling. Clearly, further studies will be needed to dissect the relative importance and balance of the EPCR-independent versus EPCR-dependent signaling in the in vivo function of APC, and to determine the importance of EPCR-independent signaling during endogenous APC generation versus exogenous APC exposure during therapy. Overall, the results described in this report provide a new mechanistic understanding for the antiapoptotic and antiinflammatory functions of APC and define a novel signaling pathway that is not dependent on EPCR.


Acknowledgments


We thank Michael Flagella for assistance with RNA preparation.


Disclosures


The authors are employees of Eli Lilly and Co, who produces human activated protein C for treatment of severe sepsis.

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作者单位:Division of Biotechnology Discovery Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Ind.

日期:2008年12月28日 - 来自[2007年第27卷第12期]栏目
循环ads

Aortic Msx2-Wnt Calcification Cascade Is Regulated by TNF- –Dependent Signals in Diabetic Ldlr –/– Mice

【摘要】  Objective— Aortic calcification is prevalent in type II diabetes (T2DM), enhancing morbidity and tracking metabolic syndrome parameters. Ldlr –/– mice fed high-fat "Westernized" diets (HFD) accumulate aortic calcium primarily in the tunica media, mediated via osteogenic morphogens and transcriptional programs that induce aortic alkaline phosphatase (ALP). Because elevated TNF- is characteristic of obesity with T2DM, we examined contributions of this inflammatory cytokine.

Methods and Results— HFD promoted obesity, hyperglycemia, and hyperlipidemia, and upregulated serum TNF- in Ldlr –/– mice. Serum haptoglobin (inflammatory marker) was increased along with aortic expression of BMP2, Msx2, Wnt3a, and Wnt7a. Dosing with the TNF- neutralizing antibody infliximab did not reduce obesity, hypercholesterolemia, or hyperglycemia; however, haptoglobin, aortic BMP2, Msx2, Wnt3a, and Wnt7a and aortic calcium accumulation were downregulated by infliximab. Mice with vascular TNF- augmented by a transgene ( SM22-TNF Tg ) driven from the SM22 promoter upregulated aortic Msx2, Wnt3a, and Wnt7a. Furthermore, SM22-TNF Tg;TOPGAL mice exhibited greater aortic β-galactosidase reporter staining versus TOPGAL sibs, indicating enhanced mural Wnt signaling. In aortic myofibroblast cultures, TNF- upregulated Msx2, Wnt3a, Wnt7a, and ALP. ALP induction was inhibited by Dkk1, an antagonist of paracrine Wnt actions.

Conclusions— TNF- promote aortic Msx2-Wnt programs that contribute to aortic calcium accumulation in T2DM.

Type II diabetes (T2DM) promotes medial artery calcification, a significant risk factor for lower extremity amputation. Using a murine disease model—the Ldlr –/– mouse fed high fat diabetogenic diets—we identified that arterial TNF-alpha signaling activates osteogenic Msx2-Wnt gene expression programs that direct medial calcification during disease initiation.

【关键词】  aortic calcification Wnt TNF metabolic syndrome diabetes


Introduction


Type II diabetes (T2DM) increasingly afflicts our dysmetabolic population, with concomitant increases in vascular disease burden. 1 Public health consequences of diabetic macrovascular disease are difficult to overestimate. Stroke, myocardial infarction, congestive heart failure, and lower- extremity amputation exact serious morbidity and economic cost. 1 Expenses associated with amputation are equivalent to the combined yearly costs of managing congestive heart failure and fatal and nonfatal myocardial infarction. 1 Lehto identified aortofemoral medial artery calcification as the most significant predictor of lower-extremity amputation. 2 A better understanding of mechanisms that control aortic calcium deposition will lead to novel strategies for improving macrovascular Windkessel function and thus disease burden.


Arterial calcification is a highly regulated form of tissue mineralization that proceeds via mechanisms resembling membranous and endochondral bone formation. 3 Osteo/chondrogenic transcription factors such as Sox9, Runx2, and Msx2 are upregulated in the mineralizing vasculature, activating expression of osteogenic enzymes and matrix proteins necessary for calcium deposition. 4 Both osteogenic and chondrogenic programs are elaborated in calcifying arterial specimens, including those from diabetic patients. 4 Rajamannan first demonstrated expression of Wnt3 and osteogenic Wnt signaling in calcifying human aortic valves. 5 The Multiethnic Study of Atherosclerosis established stepwise relationships between aortic valve calcium load and metabolic syndrome parameters. 6 Msx2-Wnt signaling participates in the aortic valve and medial calcification characteristic of T2DM in Ldlr –/– mice fed high-fat diets (HFD). 7,8 Thus, the obesity, hyperinsulinemia, hyperglycemia, dyslipidemia, and aortic calcification induced in the Ldlr –/– mouse fed HFD recapitulates key biological features of human disease. 5,6 Mechanisms that activate the aortic Msx2-Wnt cascade in this model have yet to be elucidated.


An emerging view of T2DM encompasses obesity-dependent low-grade systemic inflammation as a component of insulin resistance and vascular disease. 9,10 Adipose is an endocrine tissue that elaborates multiple adipocytokines, including TNF-. 10 Paracrine interactions between tissue adipocytes and interstitial macrophages control production of fat-derived humoral signals. 11 Chief among the inflammatory mediators is TNF-, a prototypic cytokine of activated macrophages 11 that induces insulin resistance. 9,10 TNF- activity of the fibro-fatty arterial adventitial layer is upregulated with diabetes. 12 Demer first identified that TNF- may participate in vascular calcification, upregulating ALP (alkaline phosphate) activity as a necessary component of CVC (calcifying vascular cell) mineralization in vitro. 13 However, contributions of TNF- to aortic calcification have not been studied in vivo.


In this work, we evaluated whether TNF- regulates the osteogenic signals recently identified as participating in aortic calcification in diabetic Ldlr –/– mice. 7,5 We identify that procalcific aortic Msx2-Wnt signaling cascades are entrained to TNF- signals activated by HFD-induced obesity and T2DM.


Materials and Methods


Materials, Cells, and Fluorescence RT-PCR


Recombinant TNF- and Dkk1 were obtained from R&D Systems. ELISA kits for haptoglobin (Life Diagnostics Inc), and TNF- and leptin (RND Systems) were commercially obtained. Biochemical assays for cholesterol and glucose were performed using commercial assays as per the manufacturer (Thermo). The TNF- neutralizing antibody infliximab 14 was purchased from Centocor. Other reagents were purchased from Sigma or Fisher. Primary mouse aortic adventitial myofibroblasts were generated as described 7 (detailed in the supplemental materials, available online at http://atvb.ahajournals. org). Fluorescence RT-PCR was used to quantify the relative mRNA accumulation of aortic osteogenic signaling molecules as previously detailed 7 (detailed in the online supplement).


Generation of SM22-TNFalphaTg and SM22-TNFalphaTg;TOPGAL Transgenic Mice


SM22-TNFalpha transgenic mice were generated as before 7 at the Washington University Mouse Genetics Core. Detailed methods are presented in the accompanying online supplement. TOPGAL mice were purchased from Jackson Laboratory, Bar Harbor, Maine.


Murine Studies and Cardiovascular Histochemistry


Male Ldlr –/– mice (Jackson Labs #002207; C57BL/6J background) were fed Picolab Rodent Diet 20 (#5053) until challenged at 2 months of age with HFD (Harlan Teklad diet TD88137; 42% fat calories, 0.15% cholesterol) for 5 weeks. All studies had 5 to 15 animals per treatment arm as indicated. Animals were dosed twice weekly with 10 µg/g subcutaneous infliximab in vehicle, 15 or with vehicle (500 mg sucrose, 0.5 mg polysorbate 80, 2.2 mg monobasic sodium phosphate monohydrate, and 6.1 mg dibasic sodium phosphate dihydrate in 10 mL of sterile water as per the package insert). At the end of the treatment period, mice were fasted overnight, then euthanized following protocols approved by the institutional Animal Studies Committee. Aortic segments from the arch to the renal arteries were dissected and individually analyzed for either (1) gene expression by RT-PCR or (2) calcium content after formic acid extraction. These published methods 7 are detailed in the accompanying online supplement.


Statistics


For histological staining results, chi-square analysis was performed with Yate correction 16 to compare SM22-TNFalphaTg;TOPGAL versus TOPGAL littermates. For all other data, analyses were performed using Student t test, with data presented as the mean±SEM.


Results


High-Fat Diets Induce TNF-, Low-Grade Systemic Inflammation, and Medial Artery Calcification in Ldlr –/– Mice


The male Ldlr –/– mice fed HFD typical of Western societies develop hyperglycemia, hyperinsulinemia, dyslipidemia, and obesity. 17 As observed in humans with metabolic syndrome or T2DM, 6 aortic calcification is enhanced in male Ldlr –/– with HFD—with the concomitant induction of osteogenic gene regulatory programs in aortic tissues. 8 Alizarin red staining for calcium deposition reveals that, after 1 month of HFD, patchy calcium deposition is observed in the aortic tunica media ( Figure 1A and 1 B). After 6 months of HFD, both medial and atherosclerotic calcium deposition are visible in proximal and distal thoracic aortic segments, with greater staining observed in the tunica media ( Figure 1C and 1 D). As observed in the coronary arteries ( Figure 1 E), regions of aortic media lacking overlying atheroma stained with Alizarin red with an intensity approximating that of mineralizing neonatal mouse bone ( Figure 1 F).


Figure 1. Diabetogenic diets promote calcification in male Ldlr –/– mice. Mice were fed HFD for 1 (A and B) or 6 (C through F) months. Alizarin staining of aorta histologically localized calcium deposition. 7 Initial patchy calcification (A and B) progressively becomes circumferential (C and D). Medial calcification predominates (black arrows, E and F), but some atherosclerotic calcification occurs (white arrows, D, F). A skeletal control for calcium staining is included in panel F.


Prior studies showed that TNF- plays a critical role in CVC mineralization in vitro. 13 Because TNF- signaling is implicated in pathogenesis of T2DM, 9 we evaluated serum TNF- levels in Ldlr –/– mice. Under basal conditions, fasting TNF- levels were undetectable; however, serum TNF- rose to 8.4±3.6 µg/L in animals fed HFD ( P =0.03). Moreover, HFD feeding significantly induced serum haptoglobin levels—a marker of inflammatory cytokine signaling—from 0.46±0.03 µmol/L to 1.27±0.16 ( P =0.025). To identify whether endogenous TNF- contributed to diet-induced inflammatory responses in Ldlr –/– mice, we tested the effects of infliximab (INX)—a clinically useful TNF- –neutralizing antibody—implementing a validated dosing regimen. 14 Circulating levels of haptoglobin were significantly reduced by infliximab, to 0.93±0.12 µmol/L ( P =0.046); parallel reductions in another serum inflammation marker, hemopexin, were also observed (not shown). Infliximab did not reduce the weight gain, hyperglycemia, hypercholesterolemia, or hyperleptinemia induced by HFD ( Table ). Thus, TNF- is upregulated in Ldlr –/– mice fed HFD. Infliximab administration reduced inflammation—reflected in the serum inflammatory biomarker haptoglobin—with little effect on weight gain, hyperglycemia, hypercholesterolemia, and hyperleptinemia induced by HFD consumption.


Table. Metabolic Parameters After 5 Weeks of Dietary Challenge and Infliximab Treatment *


Aortic Osteogenic BMP2-Msx2-Wnt Programs Induced by HFD Are Inhibited by Infliximab, With Concomitant Reduction in Aortic Calcium Content


In response to HFD, Ldlr –/– mice accrue arterial calcium accumulation as revealed by Alizarin red, most notable in the tunica media of the aorta and the coronary arteries ( Figure 1 ). We previously demonstrated that HFD upregulated both aortic BMP2 and Msx2 in Ldlr –/– mice. 8 Of note, paracrine Wnt signals activated by aortic Msx2 expression contribute to medial calcium accrual, 7 and a related paracrine Wnt signaling cascade controls BMP2-dependent ossification in cultured osteoblasts. 18 Therefore, we wished to assess whether antagonizing TNF- signaling with infliximab would ameliorate procalcific aortic BMP2, Msx2, and Wnt signaling in the Ldlr –/– mouse. As shown in Figure 2 A, HFD upregulated aortic mRNA accumulation for BMP2, Msx2, Wnt3 a, and Wnt7a as described. 7,8 Significantly, aortic expression of these HFD-induced signals was downregulated by infliximab ( Figure 2 A). This was paralleled by infliximab-induced downregulation of the cytokine OPN ( osteopontin ), 19 but not Runx2 or Dkk1 ( Figure 2 B). We next assessed effects of infliximab on aortic calcium accumulation after 5 weeks of HFD, an early disease stage when medial calcification predominates ( Figure 1 A), by quantifying acid-extractable calcium. Ldlr –/– animals were placed on HFD as before, and treated with either vehicle (n=10) or 10 µg/gm infliximab (n=5) twice weekly for 5 weeks. Aortic matrix calcium accumulation was reduced by 30% in animals treated with infliximab ( Figure 2 C, from 1.44 to 0.99 µg calcium/gm aortic tissue; P <0.05). Thus, infliximab, a specific TNF- neutralizing antibody, reduces aortic proosteogenic signaling and early calcium accumulation associated with HFD feeding in male Ldlr –/– mice.


Figure 2. Infliximab downregulates osteogenic BMP2-Msx2-Wnt programs in Ldlr –/– mice fed HFD. A, HFD upregulated aortic BMP2, Msx2, Wnt3a, and Wnt7a. 7,8 Infliximab treatment inhibited induction. B, Infliximab exerted little effect on Runx2 and Dkk1. C, Infliximab reduced aortic calcium in Ldlr –/– mice fed HFD for 5 weeks. At this early stage, only patchy medial calcification occurs ( Figure 1A and 1 B).


TNF- Directly Induces Msx2-Wn t Signaling in Aortic Myofibroblasts In Vitro


Endothelial production of BMP2 is enhanced by TNF-, 20 and BMP2 promotes osteogenic Msx2 8 and Wnt 18 signals. We wished to assess whether TNF- might exert direct actions on aortic Msx2-Wnt signaling. 7 We focused on primary aortic adventitial myofibroblasts, a mural progenitor cell population that expresses Msx2 and generates vascular cells that calcify in vivo and in vitro. 7,8,21 As shown in Figure 3 A, TNF- dose-dependently increased Msx2 mRNA accumulation in aortic adventitial myofibroblasts. Induction was an immediate-early response, because cycloheximide treatment did not prevent Msx2 upregulation (not shown). Subsequent time course studies revealed that Msx2 induction preceded that of BMP2 in myofibroblasts ( Figure 3 B). Moreover, addition of the BMP2 inhibitor, noggin, 18 did not alter TNF- induction of Msx2 (not shown).


Figure 3. TNF- directly activates Msx2-Wnt signaling with subsequent ALP induction. Aortic myofibroblasts 7 were treated with TNF- and analyzed for Msx2 expression. A, TNF- treatment (3 hours) increased Msx2. B, TNF- (10 ng/mL, 2 hours) upregulated Msx2 before BMP2. C, TNF- selectively upregulated Wnt3a and Wnt7a. D, Induction of ALP 7,8 is inhibited by the Wnt antagonist Dkk1. 18


We next evaluated effects of TNF- on expression of Wnt1, Wnt3a, and Wnt7a —canonical Wnt ligands previously identified as components of the aortic Msx2-Wnt signaling cascade. 7 TNF- treatment significantly upregulated expression of Wnt3a and Wnt7a, but had no effect on Wnt1 ( Figure 3 C). TNF- induction of these 2 canonical Wnt ligands was functionally important; recombinant Dkk1—an antagonist of Wnt signaling via LRP5 and LRP6 18 —inhibited TNF- upregulation of bone ALP ( Figure 3 D), a key genomic target of osteogenic Wnt signaling. 7,18 Thus, TNF- can stimulate Msx2-Wnt signaling in aortic adventitial myofibroblasts.


The SM22-TNFalpha Transgene Upregulates Aortic Msx2 Expression and Canonical Wnt Signaling In Vivo


To confirm that TNF- upregulates aortic Msx2-Wnt signaling in vivo, we generated SM22-TNFalpha transgenic (Tg) mice as a model for study. The SM22 promoter has proved useful for directing vascular smooth muscle cell (VSMC)-specific gene expression in vivo. 22 Therefore, we assembled an SM22 promoter— TNF - cDNA expression construct ( Figure 4 A), and generated and characterized SM22-TNFalphaTg mice. SM22-TNFalphaTg mice exhibit 2.4-fold elevated aortic TNF - message by RT-PCR as compared with nontransgenic littermates ( P =0.0003). Moreover, as observed in vitro, the SM22-TNFalpha transgene significantly upregulated expression of aortic BMP2, Msx2, Wnt3a, and Wnt7a gene expression in vivo ( Figure 4 B).


Figure 4. The SM22-TNFalpha transgene augments aortic Msx2-Wnt signaling. A, SM22-TNFalpha expression vector. Transgenic (Tg) mice exhibit 2.4-fold greater aortic TNF - vs nontransgenic littermates ( P =0.0003). B, SM22-TNFalpha Tg mice upregulate aortic BMP2, Msx2, Wnt3a, Wnt7a, and OPN. C and D, β-galactosidase/LacZ histochemistry revealed medial signal in SM22-TNFalphaTg;TOPGAL mice (4/5), not in TOPGAL siblings ( P =0.05, Yate Chi-squared test).


We wished to assess whether the TNF - transgene was capable of augmenting aortic Wnt signaling in vivo; this was important, because TNF- exerts time-dependent biphasic rapid inductive/delayed suppressive effects on myofibroblast Dkk1 expression (unpublished observations). The TOPGAL (TCF/LEF optimal promoter β-galactosidase/LacZ reporter) mouse has been used to monitor activation of canonical Wnt signaling in vivo. 7,23 Therefore, we generated SM22-TNFalphaTg;TOPGAL mice (mixed B6:CD1 background), and compared aortic LacZ staining to that observed with TOPGAL littermates lacking SM22-TNFalpha transgene. Four of 5 SM22-TNFalphaTg:TOPGAL mice exhibited prominent LacZ staining of mural cells ( Figure 4 C, right panels). By contrast, no LacZ staining was observed in 5 of 5 TOPGAL siblings lacking the SM22-TNFalpha transgene ( Figure 4 C, left panels; P =0.05, Yate corrected chi-squared test). Identical results were observed in coronary arteries ( Figure 4 D). Thus, TNF- promotes aortic Msx2, Wnt3a, and Wnt7a expression, and augments aortic canonical Wnt signaling in vivo.


Discussion


The epidemic of diabetes and obesity assailing Westernized societies threatens to interact with the prevalent, age-related incidence of aortic disease to increase macrovascular disease burden. 3 Primary prevention strategies are critically important; however, as many as one-third of patients with T2DM may be unaware of their disorder—and vascular disease processes that threaten life, limb, and autonomy progress from the earliest phases of the dysmetabolic state. 1,2 A better understanding of aortofemoral disease in T2DM is necessary to develop new strategies to address this burgeoning clinical need.


The Ldlr –/– mouse has emerged as one useful model for studying macrovascular injury in response to T2DM. 17 When fed HFD, these mice become obese, with concomitant hyperglycemia and dyslipidemia. 17 Unlike the Apoe –/– mouse—a model of atherosclerosis in the absence of hyperglycemia and obesity 17 Ldlr –/– mice fed the HFD elaborate key features of metabolic syndrome. 17 The relationships between metabolic syndrome parameters and aortic valve calcification in humans were recently established. 6 Thus, the Ldlr –/– murine model faithfully recapitulates key aortic pathobiology, including aortic calcification, entrained to the metabolic syndrome–T2DM continuum of disease severity.


Using this model, we describe a novel TNFalpha– Msx2-Wnt signaling axis that contributes to the pathobiology of diabetic macrovascular disease. TNF- is the prototypic inflammatory cytokine that exerts global influences on metabolism and innate immunity. 9,10 The TNF- neutralizing antibody infliximab downregulated aortic Msx2-Wnt programs without improving fasting serum glucose or cholesterol. Thus, based on the prior studies of Demer 24 and our results, we anticipate that programs elicited by TNF- divert multipotent CVCs—macrovascular mural pericytes 25 —to the osteogenic lineage via activation of the Msx2-Wnt pathway. Of note, in the dyslipidemic Apoe –/– mouse, migratory mesenchymal progenitors are recruited from the adventitia to populate mural VSMC populations. 26 This may include the pericytes, 25 myofibroblasts, 8 and CVCs 24 that program arterial calcification, because surgical resection of the adventitia reduces arterial calcification. 21 Our in vitro studies have emphasized responses in adventitial myofibroblast preparations. Like CVCs generated from tunica media, 24 aortic adventitial myofibroblast preparations are heterogeneous for calcifying cells. 8 As surface markers emerge that permit isolation of VSMCs at each stage of differentiation, it will be important to determine whether TNF- regulation of arterial Msx2-Wnt cascades change with phenotypic maturation.


Proosteogenic signals are counter-balanced by critically important negative regulators of arterial mineral deposition. Chief among these are OPN, inorganic pyrophosphate, fetuin, osteoprotegerin, and matrix Gla protein (MGP). 27,28 Regulatory cross-talk between proosteogenic signals and these mineralization inhibition mechanisms has recently been described. 3 BMP2 upregulates ALP, the ectoenzyme that degrades tissue pyrophosphate—a critical inhibitor of biomineralization and secretagogue for OPN. 19 For full elaboration of the vascular tissue mineralization process, these important defense mechanisms must become compromised with disease progression.


Other metabolic insults—such as the hyperphosphatemia, hypercalcemia, and oxidative stress of uremia 29 —clearly contribute to vascular calcium load via phosphate transporter activation of Cbfa1/Runx2. 30 Runx2 is an osteogenic and chondrogenic transcription factor exquisitely regulated by posttranslation modifications, subnuclear localization, and modulatory protein–protein interactions. 30 With atherosclerotic calcification, endochondral mineralization programs are recruited, primarily dependent on Runx2 and Sox9. 4 Oxysterols generated by lipoprotein oxidation enhance Runx2-dependent mineralization in culture. 31,32 In the male Ldlr –/– mouse fed HFD, Alizarin red staining for calcium accumulation reveals that medial calcification predominates as obesity and T2DM ensue. However, with prolonged dietary challenge of Ldlr –/– mice, atherosclerotic calcification clearly becomes super-imposed in the aorta ( Figure 1 ). Whether the TNF- –dependent Msx2-Wnt pathways and Runx2-regulated programs differentially contribute with disease progression has yet to be explored in this model.


Of note, biomineralization can be nucleated by phospholipid-rich matrix vesicles 33 and apoptotic bodies, 33 or by devitalized elastin. 34 Thus, disease pathobiology and histological characteristics will differ depending on the mechanisms used to initiate vascular calcium deposition. In early lesions of the Ldlr –/– mouse, arterial calcium deposition visualized by Alizarin red staining is overlapping yet distinct from that seen with von Kossa stain for phosphate deposition (unpublished observations); similar observations were noted almost 40 years ago by Puchtler et al in studies of early human atherosclerotic lesions. 35 Moreover, changes in bone resorption—coupled with common, clinically relevant agents such as warfarin that cripple Gla-dependent inhibitors of calcification—can enhance elastin-based arterial calcification by perturbing serum nucleator/nucleation inhibitor homeostasis. 36,37 Indeed, T2DM is frequently accompanied by chronic kidney disease that will increase bone turnover. 29 Thus, a comprehensive understanding of arterial calcification will require integration of the diverse mechanisms that control vascular mineral deposition—and recognition that mechanistic contributions likely change with clinical setting and disease stage. 3


We demonstrate that aortic mineralization programs characteristic of membranous bone formation 38 are entrained to signals elicited by TNF-. TNF- promotes aortic Msx2 expression and enhances procalcific arterial Msx2-Wnt cascades in vitro and in vivo. Treatment of aortic myofibroblasts with Dkk1, an antagonist of paracrine Wnt signals, inhibits TNF- induction of ALP, an early osteogenic gene downstream of Msx2-Wnt activation. Of note, the antiinflammatory compound salicylate also downregulates aortic Msx2 in vivo (not shown). Thus, strategies that attenuate proinflammatory TNF- signals may help ameliorate aortic calcium accumulation.


There are limitations to our study. The magnitude of aortic Msx2 and Wnt message downregulation by infliximab was consistently greater than the observed inhibition of aortic calcium accumulation (30% reduction) at this early disease stage. Regulatory mechanisms in addition to those emphasized in this study must also contribute. These include elastin metabolism, 34,39 fetuin- and calcium phosphate-dependent regulation of matrix vesicle metabolism, 33 osteoprotegerin-inhibited cytokines, 3,36 and other emerging inhibitors of calcification. 25 Terkeltaub 40 recently extended the seminal observations of Giachelli 19 that highlight the importance of vascular OPN; pyrophosphate generating systems not only inhibit mineralization directly, but also stimulate secretion of OPN 40 —a potent inhibitor of vascular calcium deposition and mediator of calcium egress. 19 Of note, infliximab therapy downregulates aortic OPN, consistent with its antiinflammatory properties but potentially compromising beneficial actions of OPN that limit vascular calcium deposition. 19 Moreover, unlike effects on Msx2-Wnt signals, infliximab did not reverse HFD-induced aortic Runx2 expression. With prolonged dietary challenge, endochondral mineralization mechanisms driven by Runx2 may continue unabated, even when Msx2-Wnt signals are mitigated. Finally, key redox enzymes impact the type and extent of oxidative stress—and thus generation of vascular oxysterols from LDL. 31,32 Oxysterols that drive Runx2 activation and endochondral mineralization 31,32 likely participate in aortic calcium load with prolonged dyslipidemia. Future studies will evaluate whether TNF- differentially impacts early versus late calcification mechanisms as disease progresses. Nonetheless, the identification that aortic calcification of T2DM occurs in part via TNF- driven Msx2-Wnt signaling 5 provides insights useful for developing novel multimodality strategies 5,36 to ameliorate diabetic vascular disease. 2,6


Acknowledgments


Sources of Funding


This work was supported by NIH grants HL81138 and HL69229 (to D.A.T.), the Barnes-Jewish Hospital Foundation (to D.A.T.), and St. Louis University (to Z.A.A.).


Disclosures


Dr Towler serves as a consultant for Program Project P01HL030568 and for the Center for Scientific Review Skeletal Biology Development and Disease Study Section.

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作者单位:Department of Medicine (Z.A.-A.), Division of Nephrology, St. Louis VA Medical Center, St. Louis University, and the Department of Medicine (J.-S.S., C.-F.L., E.H., J.C., A.B., S.-L.,C., D.A.T.), Center for Cardiovascular Research, Division of Bone & Mineral Diseases, Washington University, St.

日期:2008年12月28日 - 来自[2007年第27卷第12期]栏目

IL-6 Deficiency Protects Against Angiotensin II–Induced Endothelial Dysfunction and Hypertrophy

【摘要】  Objective— The goal of this study was to test the hypothesis that IL-6 mediates the increases in superoxide, vascular hypertrophy, and endothelial dysfunction in response to angiotensin II (Ang II).

Methods and Results— Responses of carotid arteries from control and IL-6–deficient mice were examined after acute (22-hour) incubation with Ang II (10 nmol/L) or chronic infusion of Ang II (1.4 mg/kg/d for 14 days). The hypertrophic response and endothelial dysfunction produced by Ang II infusion was markedly less in carotid arteries from IL-6–deficient mice than that in control mice. IL-6 deficiency also protected against endothelial dysfunction in response to acute (local) Ang II treatment (eg, 100 µmol/L acetylcholine produced 100±4 and 98±4% relaxation in vehicle-treated and 51±4 and 99±4% relaxation in Ang II–treated, control, and IL-6–deficient vessels, respectively). Endothelial dysfunction could be reproduced in vessels from IL-6–deficient mice with combined Ang II plus IL-6 (0.1 nmol/L) treatment. Increases in vascular superoxide and IL-6, as well as reductions in endothelial nitric oxide synthase mRNA expression, produced by Ang II were absent in IL-6–deficient mice.

Conclusions— These data demonstrate that IL-6 is essential for Ang II–induced increases in superoxide, endothelial dysfunction, and vascular hypertrophy.

The role of IL-6 in endothelial dysfunction and oxidative stress produced by angiotensin II was investigated. IL-6 deficiency was associated with reductions in angiotensin II–induced endothelial dysfunction, vascular hypertrophy, and superoxide. Thus, IL-6 produced locally, within the vessel wall, contributes substantially to the vascular dysfunction produced by angiotensin II.

【关键词】  geneticallyaltered mice inflammation oxidative stress endotheliumdependent responses


Introduction


IL-6 is an inflammatory cytokine that appears to play a key role in the activation/maintenance of the inflammatory response in chronic disease. 1 For example, plasma IL-6 levels are increased in cardiovascular diseases, including atherosclerosis, diabetes, heart failure, hypertension, as well as obesity. 2–6 Although plasma IL-6 levels have been shown to correlate positively with cardiovascular disease progression, 5,7 little is known regarding the effects of IL-6 within the vascular wall, particularly in response to angiotensin II (Ang II). As vascular inflammation has emerged as an important component of cardiovascular disease, 1 it is critical to elucidate the role of IL-6 in Ang II–induced hypertension and endothelial dysfunction.


Ang II, the main effector of the renin–angiotensin system, increases arterial pressure, produces oxidative stress, and impairs endothelium-dependent relaxation. 8–11 In addition to effects on vascular function, Ang II can activate components of the inflammatory cascade, including the production of IL-6, eg, Ang II increases expression of IL-6 in vascular cells as well as macrophages. 6,12–14 Although Ang II is generally regarded as having negative effects on vascular function, insight into the role IL-6 plays in this response is limited. Although Ang II increases IL-6 in vascular cells in culture, 12–14 the role of IL-6 in Ang II–induced endothelial dysfunction has not been previously examined.


The goal of this study was to examine the hypothesis that IL-6 plays an important role in mediating oxidative stress, vascular hypertrophy, and vascular dysfunction in response to Ang II. To test this hypothesis, we examined both acute and chronic effects of Ang II in genetically-altered IL-6–deficient mice using 2 experimental models. In one model, we examined local effects of Ang II and IL-6 on superoxide levels and vascular responses. In the second model, we examined the role of IL-6 in mediating increases in vascular hypertrophy as well as endothelial dysfunction produced by Ang II infusion.


Materials and Methods


Experimental Animals


Male C57Bl/6 (control; n=28), IL-6–deficient (n=39), and NAD(P)H oxidase (Nox)2-deficient (n=12) mice were obtained from the Jackson Laboratory (Bar Harbor, Me). 15,16 All experimental protocols and procedures conform to the NIH Guide for the Care and Use of Laboratory Animals and were approved by the institutional animal care and use committee of the University of Iowa.


Chronic Ang II Infusion, Blood Pressure Analysis, and Vascular Function Protocols


Mice were infused for 14 days with vehicle (saline) or a pressor dose of Ang II (1.4 mg/kg/d). 17 Briefly, mice were anesthetized with ketamine and xylazine (87.5 and 12.5 mg/kg, respectively, ip) and an osmotic minipump (Alzet Model 1002, DURECT Corporation) containing either vehicle or Ang II was implanted. Systolic blood pressure was measured in conscious mice using tail-cuff plethysmography (BP-2000 Visitech Systems). Briefly, mice were trained for 5 days, after which blood pressure was measured 1 day before (Day 0) and on Day 14 of vehicle or Ang II infusion.


After the last day of blood pressure measurement mice were euthanized with pentobarbital (100 mg/kg, ip) followed by removal of both common carotid arteries (for vascular studies as well as for measurement of medial cross-sectional area [CSA; see supplemental materials, available online at http://atvb.ahajournals.org, for methods]) and aorta (for real-time RT-PCR experiments). Arteries were placed in buffer, loose connective tissue was removed, and vessels were cut into rings for studies of vascular function. Relaxation of carotid arteries in response to acetylcholine and nitroprusside was measured after submaximal precontraction (50% to 60%) using the thromboxane analog, U46619 (9,11-dideoxy-11a,9a-epoxy-methanoprostoglandin-F 2 ) as described previously. 8,9,18,19 To confirm that the effect of Ang II on endothelial function is mediated by superoxide, some responses were examined in the absence and presence of Tempol (a superoxide scavenger; 1 mmol/L). We have shown previously that this concentration of Tempol is very efficacious in improving endothelium-dependent responses. 8


Ang II Incubation Protocol


Methods used for overnight incubation of arteries with Ang II have been described previously. 8 Briefly, carotid arteries and thoracic aorta were incubated in DMEM with either vehicle (ddH 2 O) or Ang II (10 nmol/L) for 22 hours at 37°C. Vascular responses were then examined (as described above) and superoxide was measured (in aorta, see below). To determine whether the effect of Ang II on superoxide levels and endothelial function in mouse carotid artery is mediated by NAD(P)H oxidase (a key potential source of superoxide in blood vessels), effects of Ang II were also examined in vessels from mice deficient in Nox2, a major component of the vascular NAD(P)H oxidase.


In reconstitution experiments, vessels from IL-6- and Nox2-deficient mice were coincubated with either vehicle or Ang II, with or without recombinant mouse IL-6 (0.1 nmol/L). To determine whether 0.1 nmol/L IL-6 is capable of producing endothelial dysfunction in the absence of Ang II, separate experiments examined responses of arteries from control mice incubated with IL-6 alone. This concentration of IL-6 was chosen based on preliminary experiments as well as reports in the literature. 5,14 A concentration of 0.1 nmol/L of IL-6 is within the range observed in plasma of hypertensive individuals. 5


Measurement of IL-6 Levels


Levels of IL-6 in conditioned DMEM (from overnight incubation experiments of carotid artery and aorta) were determined using a mouse IL-6 EIA kit (ALPCO Diagnostics) according to the manufacturer?s instructions.


Measurement of Vascular Superoxide


Relative superoxide levels were measured in aorta from control, Nox2-, and IL-6–deficient mice incubated overnight with either vehicle or Ang II using lucigenin (5 µmol/L)-enhanced chemiluminescence as described. 8,9,19


Real-Time RT-PCR


Expression of Nox2, Nox4, endothelial nitric oxide synthase (eNOS) and β-actin was assessed using RT-PCR using total aortic RNA from control and IL-6–deficient mice infused with vehicle or angiotensin II (see online supplement for details).


Drugs and Reagents


Acetylcholine, Ang II (human, acetate salt), lucigenin, nitroprusside, and Tempol were obtained from Sigma and all were dissolved in saline. U46619 was obtained from Cayman Chemical and dissolved in 100% ethanol with subsequent dilution being made with saline. Recombinant IL-6 (mouse; R&D Systems) was dissolved in sterile PBS containing 0.1% BSA.


Statistical Analysis


All data are expressed as means±SE. Relaxation to acetylcholine and nitroprusside is expressed as a percent relaxation to U46619 -induced contraction. Comparisons of relaxation and contraction as well as vessel cross-sectional area were made using ANOVA for repeated measures followed by Student-Newman-Keuls post-hoc test. Comparisons of IL-6 and superoxide levels, medial hypertrophy, as well as vascular expression of Nox2, Nox4, and eNOS, were made using unpaired t test, paired t test, or 1-way ANOVA followed by Dunnett post-hoc test. Statistical significance was accepted at P <0.05.


Results


Effect of Chronic Ang II Infusion on Blood Pressure


Baseline blood pressure was similar ( P 0.05) in control and IL-6–deficient mice, and infusion of vehicle had no significant effect ( P 0.05) in either group of mice ( Figure 1 A). In contrast, infusion of Ang II produced a marked increase in blood pressure that was similar ( P 0.05) in control and IL-6–deficient mice ( Figure 1 A).


Figure 1. A, Ang II infusion increased systolic blood pressure to a similar extent in control and IL-6–deficient mice. B, Vascular responses to acetylcholine in vehicle- and Ang II–infused control and IL-6–deficient mice. Means±SE; n=4 to 6; * P <0.05 vs respective vehicle.


Effect of Chronic Ang II Infusion on Vascular Responses and CSA


Acetylcholine produced relaxation in carotid artery that was similar in vehicle-infused control and IL-6–deficient mice ( Figure 1 B), suggesting that IL-6 deficiency does not alter endothelium-dependent responses under baseline conditions. In contrast to mice treated with vehicle, relaxation of arteries to acetylcholine was markedly impaired in Ang II-infused control mice and was much less in IL-6–deficient mice as compared with that in controls ( Figure 1 B).


Ang II produced a marked increase in medial CSA ( x 10L 3 µm 2 ) in carotid arteries from control mice (vehicle=4.1±0.2 and Ang II=21.1±6.6 x 10 3 µm 2 [means±SE; P <0.05]), consistent with previous studies. 20–22 This change was absent in carotid arteries from IL-6–deficient mice infused with Ang II (vehicle=3.1±0.1 and Ang II=5.3±1.1 [means±SE; P 0.05] and supplemental Figure I). Thus, the hypertrophic response to Ang II appears to be dependent on IL-6 expression. Taken together, the present findings provide strong evidence that IL-6 plays a major role in vivo in Ang II–induced vascular hypertrophy and endothelial dysfunction.


Effect of Acute (Local) Ang II on Vascular Responses


Acetylcholine produced relaxation that was similar ( P 0.05) in carotid arteries from control and IL-6–deficient mice incubated overnight with vehicle ( Figure 2 A). In contrast, 10 nmol/L Ang II produced marked impairment of acetylcholine-induced relaxation in arteries from control mice. Similar to results obtained from chronic (systemic) treatment with Ang II, relaxation of carotid arteries to acetylcholine in IL-6–deficient mice was not affected by incubation with Ang II ( Figure 2 A). Relaxation to nitroprusside was similar in carotid arteries from control and IL-6–deficient mice and was not affected by incubation with Ang II (supplemental Figure II). Consistent with these functional findings, IL-6 levels (in incubation media) were increased in vessels from control mice treated with Ang II, however this increase was absent in IL-6 deficient mice (supplemental Table I). Taken together, these data support the concept that IL-6 mediates endothelial dysfunction in response to Ang II. These findings are also important as they suggest that effects of IL-6 within the vessel wall (ie, local IL-6) are essential for Ang II–induced impairment of endothelium-dependent responses.


Figure 2. A, Vascular responses to acetylcholine in carotid arteries from control (n=7) and IL-6–deficient (n=9) mice treated (overnight) with vehicle or Ang II (10 nmol/L). Means±SE, * P <0.05 vs vehicle. B, Effect of IL-6 (0.1 nmol/L) on vascular responses in control (n=6) and IL-6–deficient (n=5) mice. * P <0.05 vs Ang II alone.


Effect of Treatment With IL-6 on Vascular Responses in IL-6–Deficient Mice


In reconstitution experiments, acetylcholine produced normal relaxation in carotid arteries from control mice incubated with 0.1 nmol/L IL-6 in the absence of Ang II ( Figure 2 B). Similarly, IL-6 had no effect ( P 0.05) on responses to nitroprusside in this group of mice (data not shown). These findings suggest that at this concentration, IL-6 alone is not sufficient to impair endothelial function. In contrast to the above, IL-6 produced impairment of endothelial function in carotid arteries from IL-6–deficient mice coincubated with Ang II ( Figure 2 B). IL-6 plus Ang II had no effect ( P 0.05) on responses to nitroprusside in this group of IL-6–deficient mice (data not shown). These findings further implicate IL-6 as an essential factor in mediating endothelial dysfunction produced by Ang II.


Endothelial Dysfunction Produced By Ang II Is Dependent on NAD(P)H Oxidase


Consistent with many previous findings, 8,21,23–26 the effect of Ang II on endothelial function in the present study was mediated by superoxide, as responses to acetylcholine in Ang II–infused control mice were normalized by Tempol ( Figure 1 B). Similarly, endothelial dysfunction produced by acute Ang II incubation involved NAD(P)H oxidase as Ang II had no effect on responses to acetylcholine or nitroprusside in carotid arteries from Nox2-deficient mice ( Figure 3 ) consistent with previous studies in aorta and the cerebral circulation. 23,27 Moreover, incubation of carotid arteries from Nox2-deficient mice with Ang II plus IL-6 had no effect on vascular responses to either acetylcholine or nitroprusside ( Figure 3 ), suggesting that the combined effect of Ang II and IL-6 on endothelial function in IL-6–deficient mice is dependent on Nox2 expression.


Figure 3. Vascular response to acetylcholine and nitroprusside in carotid arteries from Nox2-deficient mice (n=12) treated (overnight) with vehicle, Ang II (10 nmol/L), or Ang II plus IL-6. Means±SE; P 0.05.


Effect of IL-6- and Nox2-Deficiency on Ang II–Induced Increases in Superoxide


Basal superoxide levels tended to be lower in aorta from IL-6–deficient mice incubated with vehicle, but these values were not significantly different from that in vehicle-treated controls ( Figure 4 ). Importantly, Ang II treatment increased superoxide in aorta from control mice but not in IL-6–deficient or Nox2-deficient mice ( Figure 4 ). These findings are supportive of our functional data and clearly implicate a role for IL-6 and NAD(P)H oxidase in response to Ang II–induced increases in vascular superoxide.


Figure 4. Superoxide levels in aorta treated with vehicle or Ang II (10 nmol/L) from control (n=10), Nox2- (n=6), and IL-6–deficient (n=10) mice. Ang II produced a marked increase in superoxide levels in vessels from control mice, but not in Nox2- or IL-6–deficient mice. Means±SE, * P <0.05 vs vehicle.


Effect of Ang II on Expression Nox and eNOS


Aortic expression of Nox2, Nox4 (supplemental Figure III), and eNOS were similar in control and IL-6–deficient mice infused with vehicle ( Figure 5 and supplemental Table II), suggesting that deficiency of IL-6 per se does not alter expression of NAD(P)H oxidase or eNOS. In contrast, Ang II infusion produced a marked increase in Nox2 expression in both control and IL-6–deficient mice, suggesting that IL-6 is not necessary for Ang II to increase expression of Nox2. Ang II decreased eNOS expression in control, but not IL-6–deficient mice, suggesting that at least one protective mechanism of IL-6 deficiency involves maintenance of normal eNOS expression during Ang II–dependent hypertension.


Figure 5. Aortic Nox2 and eNOS expression in control and IL-6–deficient mice infused with vehicle or Ang II. Data were normalized to β-actin and are presented as relative expression to that in vehicle-infused control mice. Values are means±SE. n=3 in each group.


Discussion


The present study has several major findings. First, IL-6 deficiency protects against vascular hypertrophy and endothelial dysfunction produced by systemic administration of Ang II. At least one protective effect of IL-6 deficiency appears to be related to maintenance of normal eNOS expression in response to Ang II. Second, in vitro studies revealed that protective effects of IL-6 deficiency can occur within the vessel wall. Both the in vivo and in vitro effects of Ang II appear selective, as responses to nitroprusside were unaffected by Ang II or genotype. In addition, the effect of Ang II in the mouse carotid was mediated by NAD(P)H oxidase–derived superoxide as endothelial dysfunction could be inhibited with Tempol and was absent in Nox2-deficient mice. Third, treatment of vessels from IL-6–deficient mice with Ang II and IL-6 reproduced endothelial dysfunction observed in Ang II–treated control mice. Fourth, Ang II–induced increases in vascular superoxide were absent in IL-6– and Nox2-deficient mice. In addition, Ang II–induced increases in IL-6 from carotid artery and aorta were absent in IL-6–deficient mice, supporting the concept that IL-6 and NAD(P)H oxidase–derived superoxide contribute to Ang II–induced vascular dysfunction. Collectively, these findings provide very strong evidence that IL-6 is a major mediator of Ang II–induced vascular hypertrophy and endothelial dysfunction.


Ang II–Induced Hypertension and Endothelial Dysfunction


In the present study, infusion of Ang II produced marked hypertension and endothelial dysfunction in carotid arteries from control mice. The increase in blood pressure produced by this dose of Ang II is consistent with many previous studies. 17,20,21,22 In addition, Tempol was very effective in restoring the impaired endothelial responses in control mice implicating a role for superoxide in Ang II–induced endothelial dysfunction. This finding is consistent with studies where scavenging of superoxide or overexpression of CuZn-superoxide dismutase (CuZnSOD) was very effective in limiting Ang II–induced increases in superoxide and endothelial dysfunction. 8,10,24,25


Although studies involving systemic infusion of Ang II have been very important in elucidating the effects of Ang II on blood vessels and arterial pressure, these studies have limitations in that administration of Ang II has multiple potential effects in vivo. Unless additional controls are used, 8,24,25 it is difficult to distinguish between the effects of hypertension per se and nonvascular effects versus the direct effect of Ang II within the vessel wall in such studies. To directly examine the effects of local Ang II (independent of changes in blood pressure), we also performed studies using isolated vessels incubated with Ang II. Consistent with previous results, a relatively low concentration of Ang II produced endothelial dysfunction in carotid artery in control mice. 8


IL-6 Deficiency Largely Prevents Ang II–Induced Endothelial Dysfunction


Previous studies have shown that IL-6 deficiency does not alter blood pressure under normal conditions. 28,29 Consistent with this finding, we found that blood pressure was similar in control and IL-6–deficient mice. Ang II infusion produced similar levels of hypertension (as measured using tail-cuff) in control and IL-6–deficient mice in the present study. This conclusion is consistent with previous data where Ang II–dependent hypertension (as measured using telemetry) is not affected by IL-6 deficiency. 28 Taken together, IL-6 does not appear to be a major contributor to the pressor response in a commonly used model of hypertension (ie, infusion of Ang II in mice). In contrast, while our manuscript was in revision, a study was published which found that IL-6 deficiency reduced, the pressor response to 7 day Ang II infusion, however the effect was primarily over the first few days. 30 In addition, a role for IL-6 has been implicated in the pressor response in mice treated with high salt in combination with a very high dose of Ang II (5 mg/kg/d) as well as in mice exposed to psychosocial stress. 28,29


Exogenous IL-6 has been found to impair endothelium-dependent responses to acetylcholine via reductions in NO-cyclic GMP signaling. 31 Consistent with this, we found that the impairment of endothelial function in response to Ang II in our study was associated with increases in IL-6 from carotid artery and aorta. Moreover, Ang II reduced eNOS expression in control, but not IL-6–deficient, mice. This finding is consistent with a study which reported that IL-6 decreases eNOS expression in human aortic endothelial cells. 32 To our knowledge, the present study provides the first examination of endothelial function in IL-6–deficient mice. We found that IL-6 deficiency did not produce alterations of endothelial function, suggesting that IL-6 is not necessary for maintenance of normal vascular function. This result is not surprising as we would be predict that IL-6 levels would be relatively low in normal vessels. In addition, we also found that Ang II–induced hypertrophy was absent in IL-6–deficient mice. Taken together, these findings suggest an important role for IL-6 in limiting endothelial dysfunction and vascular hypertrophy in response to Ang II.


A major finding of this study is that IL-6 deficiency protects against endothelial dysfunction produced by Ang II both in vivo and in vitro. Moreover, the effect of IL-6 appears to occur independent of increases in blood pressure and to occur locally within the vessel wall. The magnitude of the protection produced by IL-6 deficiency was very large in both models and suggests that IL-6 within the vascular wall mediates vascular dysfunction produced by Ang II. Although IL-6 deficiency improved endothelial function, we did not detect any major effect on the pressor response to Ang II. Several possibilities could explain these findings. First, we examined endothelial dysfunction in carotid artery, which may or may not be representative of resistance blood vessels. Second, blood pressure is regulated at multiple levels, including central and renal mechanisms, in addition to vascular mechanisms. 33,34,35 Thus, although IL-6 deficiency may limit endothelial dysfunction, it is possible that other mechanisms are the key determinants of blood pressure in this model. Third, endothelial dysfunction may be a consequence, as opposed to a cause of hypertension. 36 Thus, it may be possible to improve endothelial function independent of reductions in blood pressure. Finally, there are many examples where endothelial dysfunction is present during disease but in the absence of hypertension. 37–39


Because Ang II increases oxidative stress, we considered the possibility that IL-6 was the mediator of increased vascular superoxide in response to Ang II. We found that a scavenger of superoxide restored endothelial responses to normal in arteries treated with Ang II. These findings are consistent with many previous studies and suggest that the effect of Ang II on endothelial function is attributable to superoxide-mediated inactivation of NO. 8,24–27,40 In this study, Ang II increased vascular superoxide levels in control mice and this increase was absent in mice deficient in IL-6 or Nox2 and suggest that NAD(P)H oxidase is a major source of superoxide and mediator of endothelial dysfunction in our model. Thus, IL-6 may be a critical link in NAD(P)H-derived superoxide-mediated impairment in NO-mediated vascular signaling. Whether activation of NAD(P)H oxidase by Ang II occurs upstream or downstream of IL-6 expression is not yet known. However, our data in Nox2-deficient mice suggest that expression of Nox2 is necessary for Ang II plus IL-6 to produce endothelial dysfunction.


In conclusion, the present findings clearly demonstrate an essential role of IL-6, most likely occurring within the vascular wall, in mediating effects of Ang II on vascular function. IL-6 expression may be an important link between Ang II–induced increases in NAD(P)H oxidase activity, thereby limiting the bioavailability of NO for normal vascular responses. The present findings have important implication in terms of our understanding of IL-6 in mediating the effects of Ang II, particularly in disease states associated with increased Ang II–mediated signaling.


Acknowledgments


The authors thank Mary L. Modrick, Darrin W. Kinzenbaw, and the Central Microscopy Core for excellent technical assistance.


Sources of Funding


This work was supported by National Institutes of Health grants NS-24621, HL-38901, HL-62984, and by a Bugher Award (0575092N), a National Scientist Development Grant (0230327N), and a Heartland Affiliate Beginning Grant-in-Aid (0565486Z) from the American Heart Association.


Disclosures


None.

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作者单位:Departments of Internal Medicine and Pharmacology, Cardiovascular Center, The University of Iowa Carver College of Medicine, Iowa City.

日期:2008年12月28日 - 来自[2007年第27卷第12期]栏目
循环ads

Novel Mechanism and Role of Angiotensin II–Induced Vascular Endothelial Injury in Hypertensive Diastolic Heart Failure

【摘要】  Objective— The mechanism and role of angiotensin II–induced vascular endothelial injury is unclear. We examined the molecular mechanism of angiotensin (AII)-induced vascular endothelial injury and its significance for hypertensive diastolic heart failure.

Methods and Results— We compared the effect of valsartan and amlodipine on Dahl salt-sensitive hypertensive rats (DS rats). Valsartan improved vascular endothelial dysfunction of DS rats more than amlodipine, by inhibiting endothelial apoptosis and eNOS uncoupling more. Moreover, valsartan inhibited vascular apoptosis signal-regulating kinase 1 (ASK1) more than amlodipine. Thus, AT1 receptor contributed to vascular endothelial apoptosis, eNOS uncoupling, and ASK1 activation of DS rats. Using ASK1 –/– mice, we examined the causative role of ASK1 in endothelial apoptosis and eNOS uncoupling. AII infusion in wild-type mice markedly caused vascular endothelial apoptosis and eNOS uncoupling accompanied by vascular endothelial dysfunction, whereas these effects of AII were absent in ASK1 –/– mice. Therefore, ASK1 participated in AII-induced vascular endothelial apoptosis and eNOS uncoupling. Using tetrahydrobiopterin, we found that eNOS uncoupling was involved in vascular endothelial dysfunction in DS rats with established diastolic heart failure.

Conclusion— AII-induced vascular endothelial apoptosis and eNOS uncoupling were mediated by ASK1 and contributed to vascular injury in diastolic heart failure of salt-sensitive hypertension.

We examined the mechanism and significance of angiotensin II (AII)-induced vascular endothelial injury. AII-induced vascular endothelial apoptosis and eNOS uncoupling were mediated by apoptosis signal-regulating kinase 1 and contributed to the exacerbation of vascular injury of salt-sensitive hypertensive rats with diastolic heart failure.

【关键词】  angiotensin endothelium heart failure nitric oxide signal transduction


Introduction


Salt-sensitive hypertensive patients are more prone to cardiovascular diseases than their salt-insensitive counterparts. 1,2 Therefore, it is a clinically important issue to determine the mechanism and the therapeutic strategy of cardiovascular diseases in salt-sensitive hypertension. Vascular endothelial function plays a key role in the pathophysiology 3,4 and the prognosis 5–7 of cardiovascular diseases, including atherosclerosis, ischemic heart disease, and heart failure. However, the detailed molecular mechanism and the pathological significance of vascular endothelial dysfunction in salt-sensitive hypertension are unknown.


Apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, has been identified as a proapoptotic signaling molecule. 8–11 ASK1 is activated in response to a variety of stress stimuli, such as reactive oxygen species (ROS), angiotensin II (AII), or cytokines, etc. Accumulating in vitro evidence indicates that ASK1 participates in not only apoptosis but also various cellular responses, including cell differentiation and growth, or gene expression. Previously, we have shown that ASK1 is responsible for cardiac hypertrophy and fibrosis, 12 vascular intimal hyperplasia, 13 and ischemia-induced angiogenesis. 14 Furthermore, other investigators have also reported that ASK1 is implicated in cardiac myocyte death and remodeling induced by ischemia. 15,16 However, the role of ASK1 in vascular endothelial injury is unclear.


In the present study, by using Dahl salt-sensitive hypertensive rats, the useful model of not only salt-sensitive hypertension but also diastolic heart failure, 17,18 and ASK1-deficient mice, we have obtained the first evidence that AII-induced vascular endothelial apoptosis and eNOS uncoupling are mediated by the activation of ASK1 and play a key role in exacerbation of vascular injury in salt-sensitive hypertensive rats at the stage of diastolic heart failure.


Materials and Methods


Animals


All procedures were in accordance with institutional guidelines for animal research. Dahl salt-sensitive hypertensive rats (DS rats) (Japan SLC Inc, Shizuoka, Japan) were used in the present study.


Male ASK1 –/– mice 19 and wild-type mice (C57BL/6J) were used in the present study.


Comparative Effect of Valsartan and Amlodipine on Vascular Injury and Survival Rate of DS Rats Fed High-Salt Diet


To elucidate the direct role of angiotensin II (AII) in vascular diseases of salt-sensitive hypertension, we compared the effect of valsartan (Novartis) and amlodipine (Pfizer) on DS rats. Twelve-week-old DS rats, which had fed a high-salt diet from 7 weeks of age, were given vehicle (0.5% carboxymethyl cellulose ), valsartan (10 mg/kg/d), or amlodipine (1 mg/kg/d), by gastric gavage once a day for 4 weeks (until 16 weeks of age).


In separate experiments, 12-week-old DS rats, fed a high-salt diet from 7 weeks of age, were orally given vehicle, valsartan (10 mg/kg/d), or amlodipine (1 mg/kg/d) in the same manner as the above experiment, and survival rate was examined until 24 weeks of age.


Effect of Angiotensin II Infusion on Wild-type and ASK1 –/– Mice


To examine the role of ASK1 in vascular endothelial injury by angiotensin II (AII), we compared the effects of chronic AII infusion on wild-type and ASK1 –/– mice. AII (600 ng/kg/min) was subcutaneously infused to mice via osmotic minipump (ALZA CO) for 4 weeks.


Role of eNOS Uncoupling in End-Stage Heart Failure of DS Rats


Using 20-week-old DS rats with overt heart failure, we examined the role of eNOS uncoupling in end-stage heart failure of DS rats. Tetrahydrobiopterin (BH4; sapropterin hydrochloride, Daiichi Suntory Pharma Co Ltd, Tokyo; 10 mg/kg/d), apocynin (0.3 mmol/kg/d), or hydralazine (20 mg/kg/d) was orally given to 20-week-old DS rats with overt heart failure, for 4 weeks (until 24 weeks of age). Furthermore, as a control, we also examined the effect of tetrahydroneopterin (H 4 N; Schircks Laboratories) (10 mg/kg/d), which has similar antioxidant properties to BH4 but is not directly linked to eNOS coupling and activity, in DS rats.


The authors had full access to the data and take responsibility for its integrity. All authors have read and agree to the manuscript as written.


Results


Effects of Valsartan and Amlodipine on Vascular Endothelial Function and Remodeling, and Survival of DS Rats


Valsartan and amlodipine slightly and comparably reduced blood pressure of DS rats, throughout 4 weeks of drug treatment (supplemental Figure IA, available online at http://atvb.ahajournals.org). Vascular endothelium-dependent relaxation to acetylcholine of 16-week-old salt-loaded DS rats was remarkably impaired, compared with control DS rats ( P <0.01) (supplemental Figure IB), whereas vascular endothelium-independent relaxation by sodium nitroprusside did not differ between the 2 groups of DS rats (data not shown). Despite comparable blood pressure lowering between valsartan and amlodipine, valsartan more potently improved vascular endothelium-dependent relaxation than amlodipine ( P <0.05; supplemental Figure IB). Valsartan significantly suppressed coronary arterial thickening, compared with vehicle ( P <0.05), whereas amlodipine did not significantly suppress it (supplemental Figure IC).


As shown in supplemental Figure ID, valsartan treatment significantly prolonged survival rate of DS rats, compared with vehicle ( P <0.01), whereas amlodipine did not prolong it.


Effects of Valsartan and Amlodipine on Vascular Endothelial Apoptosis of DS Rats


As shown in Figure 1, vascular endothelial apoptosis and ASK1 phosphorylation were significantly enhanced in salt-loaded DS rats. Valsartan attenuated vascular endothelial apoptosis of salt-loaded DS rats more than amlodipine ( P <0.05), which was associated with more inhibition of ASK1 by valsartan than amlodipine ( P <0.05).


Figure 1. Effect of valsartan (Val) and amlodipine (Am) on vascular endothelial apoptosis (A) and ASK1 phosphorylation (B) of DS rats. Top panels in A and B indicate representative terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) and representative Western blot, respectively. Arrows in A indicate vascular endothelial apoptosis. Val, Am, and Veh indicate DS rats treated with valsartan, amlodipine, and vehicle, respectively, from 12 to 16 weeks of age. Low Na indicates DS rats fed a 0.3% NaCl diet, throughout the experiments. P-ASK1 indicates phospho-ASK1. Each bar represents mean±SEM (n=6 to 8).


Effects of Valsartan and Amlodipine on Vascular NADPH Oxidase and Superoxide of DS Rats


As shown in supplemental Figure II, 16-week-old salt-loaded DS rats had more NADPH oxidase activity ( P <0.01) and vascular superoxide ( P <0.01) detected with the fluorescent probe dihydroethidium, than the age-matched control DS rats. The results on preincubation of vascular sections with polyethylene glycol (PEG)-SOD or Tiron confirmed that the increased vascular dihydroethidium fluorescence in salt-loaded DS rats indeed represented superoxide itself (supplemental Figure III). Treatment with valsartan or amlodipine from 12 to 16 weeks of age significantly reduced all the above mentioned parameters of DS rats. However, vascular superoxide levels were reduced by valsartan more than amlodipine ( P <0.05; supplemental Figure IIB).


Effects of Valsartan and Amlodipine on Vascular eNOS of DS Rats


As shown in Figure 2, the ratio of dimer to monomer of eNOS, eNOS activity, and plasma NOx levels in 16-week-old salt-loaded DS rats were reduced to 27% ( P <0.01), 47% ( P <0.01), and 37% ( P <0.01), respectively, compared with control DS rats. As shown in supplemental Table I, vascular BH4 levels in salt-loaded DS rats were lower than those in control DS rats ( P <0.01), whereas vascular oxidized biopterin levels in salt-loaded DS rats were higher than those in control DS rats ( P <0.05). As shown in supplemental Figure IV, using lucigenin chemiluminescence, we found that pretreatment of vascular segments with L-NAME significantly decreased superoxide production in salt-loaded DS rats, but not in control salt-unloaded DS rats, indicating that vascular superoxide in salt-loaded DS rats was at least partially derived from eNOS uncoupling. Valsartan prevented the decrease in the ratio of dimer to monomer of eNOS ( P <0.01) and the decrease in eNOS activity ( P <0.05) in DS rats more than amlodipine ( Figure 2 ). Valsartan significantly prevented the reduction of plasma NOx in DS rats ( P <0.01), whereas amlodipine did not.


Figure 2. Effect of valsartan (Val) and amlodipine (Am) on vascular eNOS uncoupling (A), vascular eNOS activity (B), and plasma NOx (C) of DS rats. Top panel in A indicates representative Western blot in each group. Abbreviations used are the same as in Figure 1. Each bar represents mean±SEM (n=6 to 8).


Effects of Angiotensin II Infusion on Blood Pressure, Vascular ASK1, and Vascular Endothelial Function of ASK1 –/– Mice


As shown in supplemental Figure VA, blood pressure elevation in ASK1 –/– mice by AII infusion was comparable to that in wild-type mice, throughout 4 weeks of the infusion. As shown in supplemental Figure VB, AII infusion increased the phosphorylation of vascular ASK1 x 1.7-fold ( P <0.01). On the other hand, as expected, ASK1 band was not detected in ASK1 –/– mice with or without AII infusion. As shown in Figure 3, there was no significant difference in the dose-response curve of vascular endothelium-dependent relaxation to acetylcholine between wild-type and ASK1 –/– mice without AII infusion. However, AII infusion in wild-type mice significantly impaired vascular endothelium-dependent relaxation to acetylcholine ( P <0.01). On the other hand, AII infusion in ASK1 –/– mice did not at all impair vascular endothelium-dependent relaxation to acetylcholine. Vascular endothelium-independent relaxation by sodium nitroprusside did not differ between wild-type and ASK1 –/– mice, regardless of AII infusion (data not shown).


Figure 3. Effect of angiotensin II infusion on acetylcholine-induced vascular endothelium-dependent relaxation of wild and ASK1 –/– mice. Wild (–), wild-type mice without angiotensin II infusion; Wild (+), wild-type mice infused with angiotensin II; ASK1 –/– (–), ASK1 –/– mice without angiotensin II infusion; ASK1 –/– (+), ASK1 –/– mice infused with angiotensin II. Values are mean±SEM (n=8 in each group).


Effects of Angiotensin II Infusion on Vascular NADPH Oxidase Activity, p22phox, and Superoxide of ASK1 –/– Mice


As shown in supplemental Figure VIA and VIB, AII infusion significantly increased vascular NADPH oxidase activity and p22phox expression in either wild-type or ASK1 –/– mice, to a comparable degree. On the other hand, the increase in vascular superoxide by AII infusion was smaller in ASK1 –/– mice than in wild-type mice ( P <0.01; supplemental Figure VIC).


Effect of Angiotensin II Infusion on Vascular Endothelial Apoptosis and eNOS of ASK1 –/– Mice


As shown in Figure 4 A, AII infusion markedly increased vascular endothelial apoptosis in wild-type mice ( P <0.01), but not in ASK1 –/– mice. AII infusion in wild-type mice significantly reduced the ratio of dimer to monomer of eNOS ( P <0.01), whereas AII infusion in ASK1 –/– mice did not alter it ( Figure 4 B). Vascular eNOS activity was reduced by AII infusion in wild-type mice ( P <0.05), whereas did not change by AII infusion in ASK1 –/– mice ( Figure 4 C). Vascular phospho-eNOS and total eNOS levels were not altered by AII infusion in wild-type or ASK1 –/– mice (supplemental Figure VII).


Figure 4. Effect of angiotensin II infusion on vascular endothelial apoptosis (A), vascular eNOS uncoupling (B), and eNOS activity (C) of wild-type and ASK1 –/– mice, with or without angiotensin II infusion. The upper panels in A and B indicate representative TUNEL and Western blot in each group. Arrows in A indicate vascular endothelial apoptosis. Each bar represents mean±SEM (n=8 per group). P <0.05 vs Wild AII (–). Abbreviations are the same as in Figure 3.


Effect of Tetrahydrobiopterin, Apocynin, and Hydralazine on Diastolic Heart Failure of 20-Week-Old DS Rats


In the present study, by echocardiography, we confirmed that 20-week-old salt-loaded DS rats used displayed diastolic heart failure (data not shown), being consistent with previous reports by us 17,20 and others. 18,21 BH4, apocynin, and hydralazine reduced blood pressure of DS rats to a comparable degree, throughout 4 weeks of the treatment (supplemental Figure VIII). BH4 significantly improved diastolic dysfunction (supplemental Figure IX) and prolonged survival rate of DS rats ( P <0.01; Figure 5 ), whereas apocynin or hydralazine did not improve them, despite their comparable hypotensive effects to BH4. Treatment of DS rats with the same dose of H4N as BH4 did not lower blood pressure of DS rats, and did not improve diastolic dysfunction or survival rate of DS rats (data not shown).


Figure 5. Effect of tetrahydrobiopterin, apocynin, and hydralazine on survival rate of DS rats with overt heart failure. Twenty-week-old DS rats with overt heart failure were given vehicle (n=14), tetrahydrobiopterin (n=12), apocynin (n=14), or hydralazine (n=15) for 4 weeks (until 24 weeks of age), and survival rates of DS rats were compared among each group. Veh indicates vehicle treatment; BH4, tetrahydrobiopterin treatment; Apo, apocynin treatment; Hyd, hydralazine treatment.


Furthermore, after 4 weeks of each drug treatment, we examined vascular endothelial function and coronary arterial thickening of surviving 24-week-old DS rats and compared with those of 20-week-old DS rats without drug treatment. As shown in supplemental Figure XA and XB, 20-week-old salt-loaded DS rats exhibited remarkable impairment of vascular endothelial function ( P <0.01) and prominent coronary arterial thickening ( P <0.01), compared with control DS rats. BH4 significantly reversed vascular endothelial dysfunction of DS rats ( P <0.05), whereas apocynin did not reverse it and hydralazine did not prevent further exacerbation of endothelial dysfunction. Furthermore, BH4, but not apocynin or hydralazine, prevented further progression of coronary arterial thickening of DS rats (supplemental Figure XB).


As shown in Figure 6 A, BH4 significantly increased the ratio of dimer to monomer of vascular eNOS ( P <0.01), compared with 20-week-old DS rats, whereas apocynin or hydralazine treatment did not alter it. Compared with 20-week-old DS rats, vascular eNOS activity was also increased by BH4 ( P <0.05) but was not altered by apocynin or hydralazine ( Figure 6 B). BH4 significantly reduced vascular superoxide levels of DS rats ( P <0.05), but apocynin or hydralazine could not alter it ( Figure 6 C). However, vascular NADPH oxide activity was significantly decreased by apocynin ( P <0.01), but not altered by BH4 ( Figure 6 D).


Figure 6. Effect of tetrahydrobiopterin, apocynin, and hydralazine on vascular eNOS uncoupling (A), eNOS activity (B), superoxide (C), and NADPH oxidase activity (D) of DS rats with heart failure. The upper panels in A indicate representative Western blot, which was obtained from the same gel. The upper panels in C indicate fluorescence photomicrograph in each group. Each bar represents mean±SEM (n=5 to 7). Abbreviations used are the same as in Figure 5.


Discussion


The major purpose of our work was to examine the mechanism of AII-induced vascular endothelial injury and its role in salt-sensitive hypertensive rats with diastolic heart failure. The major findings were that AII-induced vascular endothelial dysfunction was attributed to ASK1-mediated endothelial apoptosis and eNOS uncoupling, and was involved in vascular injury of hypertensive diastolic heart failure.


We 17,20 and others 21,22 have reported that AII contributes to not only cardiac hypertrophy and remodeling but also the progression of diastolic heart failure in DS rats. However, the precise role of AII in vascular endothelial injury in DS rats is still unknown. In the present work, to determine the potential role of AII in vascular endothelial injury, we compared the effect of valsartan and amlodipine on vascular injury of DS rats ( Figures 1 and 2 and supplemental Figures I and II). Recent report by Julius et al 23 on subanalysis of the Valsartan Antihypertensive Long-Term Use Evaluation (VALUE) trial has indicated that valsartan is superior to amlodipine in terms of the prevention of hypertensive heart failure, although its mechanism remains to be clarified. Therefore, our present study, comparing between valsartan and amlodipine in DS rats, is of clinical relevance. In the present work, valsartan more prevented the death of DS rats attributable to heart failure than amlodipine, being associated with greater improvement of vascular endothelial function and coronary arterial remodeling by valsartan. Notably, valsartan suppressed vascular endothelial apoptosis to a greater extent than amlodipine, indicating that the improvement of vascular endothelial dysfunction by valsartan was at least in part mediated by the suppression of endothelial apoptosis. Moreover, valsartan more ameliorated vascular eNOS uncoupling than amlodipine, which was accompanied by more decrease in vascular superoxide and more restoration of eNOS activity by valsartan than by amlodipine (supplemental Figure II and Figure 1 ). These results show that AII participated in vascular endothelial dysfunction of DS rats, by causing not only endothelial apoptosis but also eNOS uncoupling.


In our current work, we found that vascular ASK1 is activated in DS rats in accordance with the occurrence of endothelial apoptosis and eNOS uncoupling and that AII specifically contributed to ASK1 activation in DS rats, as shown by the significant inhibition of ASK1 by valsartan but not amlodipine ( Figure 1 ). Previously, we have reported that ASK1 is involved in vascular neointimal formation induced by balloon injury or cuff injury. 13 Furthermore, we have also reported that ASK1 is implicated in AII-induced cardiac hypertrophy and fibrosis. 12 Thus, ASK1 seems to be an important signaling molecule responsible for cardiovascular diseases. However, the precise role of ASK1 in AII-induced vascular endothelial injury remains to be defined. Therefore, in the present work, by using mice lacking ASK1, we examined the potential role of ASK1 in vascular endothelial dysfunction, apoptosis, and eNOS uncoupling induced by AII ( Figures 3 and 4 and supplemental Figures V through VII). Of note are the observations that AII infusion significantly activated vascular ASK1 and significantly impaired vascular endothelial function, whereas AII infusion did not at all impair vascular endothelial function in mice lacking ASK1. These observations provided the first evidence that ASK1 plays a critical role in AII-induced vascular endothelial dysfunction. To determine the reason for the absence of vascular endothelial dysfunction in ASK1-deficient mice subjected to AII infusion, we measured vascular endothelial apoptosis, NADPH oxidase, ROS, eNOS uncoupling, and eNOS activity. AII infusion markedly caused vascular endothelial apoptosis in wild-type mice, whereas it did not cause apoptosis in ASK1-deficient mice. These observations provide the evidence that ASK1 plays a key role in AII-induced vascular endothelial apoptosis. Furthermore, being consistent with the previous report, 24 AII infusion significantly induced vascular eNOS uncoupling in wild-type mice, which was accompanied by the significant increase in vascular superoxide and the significant reduction of eNOS activity. On the other hand, vascular eNOS uncoupling and the reduction of eNOS activity did not apparently occur in ASK1-deficient mice infused with AII, and the increase in vascular superoxide by AII infusion was less in ASK1-deficient mice than wild-type mice. All these results, taken together with the findings that AII infusion increased vascular NADPH oxidase activity and p22phox in wild-type and ASK1-deficient mice to a comparable degree and did not affect phospho-eNOS and total eNOS in either strain of mice, provided the evidence that ASK1 is specifically implicated in AII-induced vascular endothelial dysfunction by causing endothelial apoptosis and eNOS uncoupling.


eNOS uncoupling 25 and the increase in NADPH oxidase activity 26 have been reported in patients with heart failure. Therefore, the investigation on the relative role of eNOS uncoupling and NADPH oxidase in vascular endothelial injury in heart failure is of great clinical relevance. To further elucidate the potential role of eNOS uncoupling in hypertensive heart failure, we initiated treatment with BH4, 27,28 the essential cofactor of eNOS, or apocynin, a specific NADPH oxidase inhibitor, in 20-week-old DS rats with overt heart failure ( Figures 5 and 6 and supplemental Figures VIII through X). Of note are the observations that the suppression of vascular eNOS uncoupling by BH4 treatment at advanced stage of heart failure significantly improved cardiac diastolic dysfunction and prolonged survival rate of DS rats. Furthermore, BH4 treatment in DS rats with overt heart failure significantly reversed vascular endothelial dysfunction, and these beneficial effects were associated with the significant reduction of vascular superoxide and the restoration of eNOS activity. On the other hand, the significant NAPDH oxidase inhibition by apocynin treatment or the vasodilation by hydralazine treatment did not significantly improve survival rate of DS rats, despite their similar blood pressure lowering effects to BH4. Differing from BH4 treatment, apocynin significantly inhibited NADPH oxidase activity, but did not improve vascular endothelial function, not prevent the progression of coronary remodeling, not diminish vascular superoxide, and not restore eNOS activity. These results provided the solid evidence that eNOS uncoupling, via the production of superoxide, is involved in the exacerbation of vascular endothelial injury in DS rats, and suggested that eNOS uncoupling may play some role in the pathophysiology of diastolic heart failure in DS rats.


NADPH oxidase is reported to be involved in angiotensin II–induced ROS generation, as reviewed. 4,29 However, differing from our present study, previous studies have not examined animals at the stage of advanced vascular remodeling or heart failure. Therefore, the difference in the source of ROS between our present finding and previous findings might be explained by the difference in the stage of progression of vascular remodeling or cardiac dysfunction. Another possible reason is that our present findings might be specific for salt-sensitive hypertension or heart failure. Thus the relative role of eNOS uncoupling and NADPH oxidase in the generation of ROS seems to depend on the stage of vascular remodeling or the type of cardiovascular diseases.


Study Limitation


BH4 treatment of DS rats with diastolic heart failure improved cardiac diastolic dysfunction and survival rate, suggesting that vascular endothelial dysfunction caused by eNOS uncoupling may participate in the exacerbation of diastolic heart failure. However, the present study did not allow us to elucidate the potential role of vascular endothelial dysfunction in the pathogenesis of diastolic heart failure, because the main purpose of our present work was to examine the molecular mechanism of vascular endothelial injury in hypertensive diastolic heart failure. Hence, further study is needed to elucidate the accurate role of vascular endothelial injury in the pathophysiology of diastolic heart failure. Moreover, it remains to be determined whether or not our present findings are specific for diastolic heart failure.


In conclusion, in our current work, we obtained the evidence that AII-induced endothelial apoptosis and eNOS uncoupling are mediated by ASK1 activation and play a key role in the exacerbation of vascular injury in diastolic heart failure of salt-sensitive hypertension. Thus, our present work provided novel molecular mechanism underlying AII-induced vascular injury. Furthermore, ASK1 appears to be potentially the useful target for treatment of hypertensive diastolic heart failure.


Acknowledgments


Sources of Funding


This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology.


Disclosures


None.

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作者单位:Department of Pharmacology and Molecular Therapeutics (E.Y., K.K., T.Y., Y.T., Y.-F.D., S.M., S.K.-M.), Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan; the Department of Pediatrics (H.S.), Osaka City University Graduate School of Medicine, Osaka, Japan; the Laboratory of Ce

日期:2008年12月28日 - 来自[2007年第27卷第12期]栏目
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