Nov. 11, 2011 (Chicago) -- People with gout should make sure their uric acid levels are under control -- even if they're not experiencing symptoms of the painful arthritic disorder.
"Many people are walking around with uncontrolled uric acid levels and we used to not worry about it -- if they're not having symptoms, who cares?" says Eric Matteson, MD, MPH, head of rheumatology at Mayo Clinic in Rochester, Minn.
But new studies show that high uric acid levels in the blood are associated with a nearly 20% increased risk of developing diabetes and a more than 40% increased risk of developing kidney disease.
Uric acid is a chemical substance that can build up in the blood to a higher than normal level and lead to gout.
For the new studies, researchers reviewed the records of about 2,000 men with gout in a Veterans Administration database. None had diabetes or kidney disease at the start of the study.
Eswar Krishnan, MD, assistant professor of rheumatology at Stanford University, presented the findings here at the American College of Rheumatology's annual meeting. Krishnan consults for Takeda Pharmaceuticals International, which makes a gout medication and funded the study.
Over a three-year period, 9% of men with gout who had uncontrolled uric acid levels developed diabetes, compared with 6% of those whose uric levels were under control.
After taking into account other risk factors for diabetes, this corresponded to a 19% higher risk of diabetes in those with uncontrolled uric acid levels.
A blood uric acid level greater than 7 is considered uncontrolled.
The risk for an individual person might not be much. But the National Institutes of Health estimates that 6 million U.S. adults have had gout at some point in their lives, many with uncontrolled uric acid levels. That translates to tens of thousands of people at risk of diabetes and kidney disease, Matteson says.
A second study, conducted by the same researchers using the same database, showed that over a three-year period men with gout who had uncontrolled uric acid levels had a 40% greater risk of kidney disease compared to men with controlled uric acid levels.
The studies do not prove that uncontrolled uric acid levels cause the health problems but show an association of elevated levels to these health problems.
"Gout is a vastly undertreated disease," Matteson says. "Now we're finding that elevated uric acid, by itself, even if you have no gout, is associated with higher rates of heart attack, metabolic syndrome, diabetes, even death due to cardiovascular disease."
Still, it would be advisable to get uric acid levels under control, through diet or medication, he says.
Most importantly, maintain a healthy weight, Matteson says. Obesity is a major risk factor for all these conditions.
These findings were presented at a medical conference. They should be considered preliminary as they have not yet undergone the "peer review" process, in which outside experts scrutinize the data prior to publication in a medical journal.
【摘要】 目的 观察迷迭香酸的铁离子螯合作用。方法 采用邻二氮菲分光光度法和Western blot检测方法，分别观察不同浓度的迷迭香酸的铁离子螯合作用及对MES23.5细胞铁转出蛋白ferroportin1(FP1)的调节作用。采用四甲基偶氮唑盐(MTT)检测方法，观察不同浓度的迷迭香酸对MES23.5细胞存活率的影响。结果10-4～10-2 mol/L的迷迭香酸具有铁离子螯合作用(F=7.851，q=3.88～6.14，P<0.05)，10-5 mol/L的迷迭香酸没有表现出铁离子螯合作用(P>0.05)。10-5、10-4 mol/L的迷迭香酸对细胞无毒性作用(P>0.05)，10-3 mol/L的迷迭香酸使细胞存活率下降(F=4.948，q=4.04，P<0.05)。10-4 mol/L迷迭香酸不能调节MES23.5细胞内FP1的表达(F=0.333，P>0.05)。结论 迷迭香酸有明显的铁离子螯合作用，在治疗铁代谢相关疾病方面有着潜在的应用前景。
【关键词】 迷迭香属;铁螯合剂;帕金森病
IRON CHELATION OF ROSMARINIC ACID DU TINGTING, SONG NING, XIE JUNXIA, et al (Department of Physiology, Shandong Provincial Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology, Qingdao University, Qingdao 266071, China); [ABSTRACT] Objective To investigate the ironchelation properties of rosmarinic acid. Methods Orthophenanthroline spectrophometry was used to observe the iron chelation properties with different concentrations of rosmarinic acid, and western blots used to evaluate the regulation of ferroportin 1 (FP1) by rosmarinic acid in MES23.5 cells. MTT assay was used to observe the changes of cell viability with different concentrations of rosmarinic acid incubation in MES23.5 cell. Results Orthophenanthroline experiment indicated that the concentrations of 10-4-10-2 mol/L rosmarinic acid showed significant iron chelation properties (F=7.851,q=3.88-6.14,P<0.05), and that of 10-5 mol/L did not (P>0.05). MTT assay showed that 10-5 and10-4 mol/L rosmarinic acid did not have any toxic effects on MES23.5 cells (P>0.05). The cell viability decreased when treated with 10-3 mol/L rosmarinic acid (F=4.948,q=4.04,P<0.05). 10-4 mol/L rosmarinic acid had no effects on FP1 expression in MES23.5 cells (F=0.333,P>0.05). Conclusion Rosmarinic acid has an evident chelation of ferri ion, which is of potential prospect in the treatment of ironmetabolismrelated conditions.
[KEY WORDS] rosmarinus; iron chelator; Parkinson disease
帕金森病(PD)是一种多发于中老年的中枢神经系统退行性疾病，以运动不能、肌僵直、静止性震颤及姿势反射障碍为特征性表现[1]。PD主要病理学改变是黑质(SN)致密带多巴胺(DA)能神经元丢失及伴发的纹状体轴突末梢DA的耗竭。PD的发病过程有许多致病因子参与，如炎症递质的产生、DA分解过程中氧自由基的大量释放、线粒体的损伤、DA的毒性代谢产物、细胞凋亡以及神经营养因子的缺失等[2]。近来的研究发现，脑内铁代谢失衡所引起的铁水平升高与多种神经退行性疾病有关，在PD病人脑部特定的区域有明显的铁沉积，脑铁异常增高是DA能神经元死亡的重要原因，铁的异常增高和铁诱发的氧化应激反应可能是PD发病机制中的关键因素[3]。因此，实验开发一种铁离子螯合剂和抗氧化剂日益成为研究热点，寻找天然、高效、低毒的抗炎、抗氧化、自由基清除剂及铁离子螯合剂成为了一种必然趋势。迷迭香酸(RA) 是一种水溶性的含多酚羟基的酸，因1958年首次从迷迭香植物中分离提取而得名[4]。RA具有抗氧化、清除自由基、抗炎、免疫调节、抗菌、抗病毒等多种生物活性且作用确切，可用于治疗自由基引起的多种疾患。研究已证实，RA对维生素CNADPH 诱发的脂质过氧化均有较强的抑制作用，作用比维生素E强几百倍至千余倍，RA还能抑制山梨糖醇引起的细胞活性氧物质(ROS)和NO及一氧化氮合酶的增加，表明RA是一种很强的抗氧化剂[5]。但目前尚无RA铁离子螯合作用的研究，本实验拟采用邻二氮菲分光光度法和Western blot检测方法，分别观察RA的铁离子螯合作用以及对MES23.5细胞铁转出蛋白ferroportin1(FP1)的调节作用，以期为RA用于与铁代谢相关疾病的防治提供可靠的实验依据。现将结果报告如下。
1 材料和方法
1.1 材料
DMEM/F12为Gibco公司产品，FP1抗体为ADI公司产品，其他的化学试剂均为Sigma公司产品，RA由青岛大学医学院生物系提供，MES23.5细胞由美国休斯敦贝勒医学院神经科乐卫东教授友情馈赠。
1.2 细胞培养
将MES23.5细胞培养于含有体积分数0.05的胎牛血清、体积分数0.02的Satio溶液、10 g/L的L谷氨酰胺、105 U/L青霉素和100 g/L链霉素的DMEM/F12培养液中，置于体积分数0.05的CO2培养箱中传代培养，每2～3 d传代1次。
1.3 邻二氮菲分光光度法检测铁离子螯合作用
取标准液(RA浓度分别为0、10-5、10-4、10-3、10-2 mol/L，NH4Fe(SO4)2·12H2O 0.1 g/L)2 mL，分别加入体积分数0.1的盐酸羟胺1 mL， 1.5 g/L的邻二氮菲2 mL，1 mol/L NaAc 5 mL，每加入一种试剂都应初步混匀。然后，用去离子水定容至50 mL，充分摇匀。最后，用自动酶标读数仪比色(波长510 nm)，测定吸光度(A)值，A值越小表示铁离子螯合能力越强。
1.4 MTT方法检测细胞存活率
用新鲜配制的DMEM/F12培养液漂洗细胞1次，吹打制成单细胞悬液后离心，用DMEM/F12培养液稀释成108/L的细胞悬液，每孔100 μL接种于铺有多聚L赖氨酸的96孔板中，每孔细胞数量约为104个，置于37 ℃、体积分数0.05的CO2培养箱中培养24 h后，加入不同浓度的RA(10-5、10-4、10-3 mol/L)，对照组以配制的无血清DMEM/F12培养液替换，置于37 ℃、体积分数0.05的CO2培养箱中孵育24 h。培养结束后每孔加入5 g/L的MTT 20 μL， 37 ℃培养箱中培养4 h，弃上清后每孔加入二甲基亚砜100 μL，采用酶标检测仪测定570 nm处A值，以此来反映细胞的存活率。每组实验重复3次，取平均值。
1.5 Western blot检测FP1的表达
用新鲜配制的DMEM/F12培养液漂洗细胞1次，吹打制成单细胞悬液后离心，用DMEM/F12培养液稀释成108/L的细胞悬液，每孔2 mL接种于铺有多聚L赖氨酸的6孔板中，置于37 ℃、体积分数0.05的CO2培养箱中培养24 h后，加入10-4 mol/L RA，对照组以配制的无血清DMEM/F12培养液替换，置于37 ℃、体积分数0.05的CO2培养箱中孵育24 h后，提取蛋白。然后，进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDSPAGE)实验，转膜，将膜浸于100 g/L TBST脱脂奶中，过夜，以封闭非特异性结合位点;用50 g/L TBST脱脂奶稀释一抗，FP1和βactin均为1∶4 000，孵育2 h，用TBST摇洗3次，每次10 min;用TBST稀释二抗，FP1和βactin均为1∶10 000，孵育1 h，用TBST摇洗3次，每次10 min。最后，显影定影拍照，并用天能分析软件对条带进行分析，将条带的强度和净A值的乘积作为蛋白的含量。以FP1/βactin表示FP1的表达水平。每组实验重复3次，取平均值。
1.6 统计学处理
实验结果以±s表示，采用PPMS 1.5[6]软件进行统计学处理，组间比较采用单因素方差分析。
2 结 果
2.1 RA的铁离子螯合作用
对照组和10-5、10-4、10-3、10-2 mol/L RA处理组A值分别为0.133±0.005、0.126±0.010、0.116±0.008、0.109±0.008、0.100±0.010。10-4～10-2 mol/L RA处理组表现出铁离子螯合作用，与对照组相比差异具有统计学意义(F=7.851，q=3.88～6.14，P<0.05);但是10-5 mol/L RA处理组与对照组相比，差异无统计学意义(P>0.05);10-4、10-3、10-2 mol/L RA处理组之间相比，差异无统计学意义(P>0.05)。
2.2 RA对MES23.5细胞存活率的影响
对照组和10-5、10-4、10-3 mol/L RA处理组MES23.5细胞存活率分别为1.00±0.00、1.00±0.21、0.87±0.21、0.56±0.13。10-3 mol/L RA处理组细胞存活率明显下降，与对照组相比差异有统计学意义(F=4.948，q=4.04，P<0.05)。10-5、10-4 mol/L RA处理组细胞存活率与对照组相比，差异无统计学意义(P>0.05)。
2.3 RA对MES23.5细胞FP1的影响
对照组MES23.5细胞FP1表达水平为1.32±0.64，10-4 mol/L RA处理组为0.97±0.60，两组相比较差异无统计学意义(F=0.333，P>0.05)。见图1。
3 讨 论
RA是天然的抗氧化剂，其医学价值已经得到公认，尤其是它的抗氧化和抗炎作用。RA是非常强的过氧亚硝酸盐(ONOO-)和其他自由基的清除剂，并且RA的其他生物活性在诸多方面均发挥作用，所以对其进行研究开发具有一定的意义。已有文献报道，RA可以通过与α突触核蛋白相互作用来抑制α突触核蛋白的聚集，也可以通过抗氧化作用抑制ROS的生成，从而对PD起到保护作用[7]。
研究表明，铁在PD的发病中是一个关键因素。临床尸检结果、动物模型等都提供了越来越多的线索。经颅超声研究观察到PD病人SN铁水平增高早于临床症状的出现;且目前已证实除了少突胶质细胞内铁含量增高使SN脑区铁增高外，在残存的DA能神经元内铁含量也明显高于对照组[8]。本实验室的前期工作证实SN内Fe3+含量升高可使纹状体内DA释放量和含量均降低,铁含量高可能参与PD的病理改变[9]。本实验运用邻二氮菲分光光度计法，通过检测铁反映RA的铁螯合能力，结果表明10-4、10-3、10-2 mol/L RA具有铁离子螯合作用，而10-5 mol/L RA 没有表现出此作用，证实了RA是一种铁离子螯合剂。
铁转运相关蛋白可以通过调节铁的转入、储存、转出等过程来影响细胞内的铁水平，因此，通过观察细胞内铁转运相关蛋白的变化可以反映细胞内铁水平的变化。那么，RA作为铁离子螯合剂对细胞内铁转出蛋白FP1的表达又有何影响呢?由于高浓度(10-3、10-2 mol/L) RA对细胞有毒性作用，因此我们选择10-4 mol/L RA处理MES23.5细胞，用以观察RA对FP1表达的调节作用。实验结果显示，10-4 mol/L RA处理组FP1的表达水平与对照组相比差异无显著性，说明10-4 mol/L RA不能调节细胞内FP1的表达。在FP1 mRNA 5′未翻译区存在有功能性的铁反应元件(IRE)，提示其受细胞内IRE铁调节蛋白(IRP)系统的调节。在细胞内低铁时，细胞内IRP活性增强，导致FP1 mRNA 5′未翻译区IRE和IRP的结合力增强，因而可以通过IRE/IRP理论下调FP1的表达。邻二氮菲实验结果虽然证实了RA具有铁离子螯合作用，但是它又对细胞内FP1的表达没有影响。这可能是由于：①正常情况下细胞内游离铁很少，大部分铁以铁蛋白和含铁血黄素的形式被储存，这些铁在正常情况下不能被激活，因此少量的游离铁与RA结合不足以激活IRP调节的FP1的下调;②低浓度的RA铁离子螯合作用较弱，其尚不足以激活IRE/IRP调节的FP1的表达。
目前，已经有足够数据证明了在铁代谢相关疾病中铁起着重要作用，过量的游离铁，特别是Fe2+可以通过Fenton反应形成OH·，而OH·通过损伤蛋白质、核酸和含有大量未饱和脂肪酸的细胞膜，最终导致细胞死亡。本实验证实了RA是一种铁离子螯合剂，对其药理活性的进一步研究,必将使这种天然的铁离子螯合剂在治疗铁代谢相关疾病方面具有潜在的开发和应用前景。
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May 9, 2011 -- Long-term, regular users of drugs known as proton pump inhibitors (PPIs) such as Nexium, Prevacid, Prilosec, and Protonix appear to have a heightened risk of fractures, a study shows.
The researchers found that this class of acid-suppressive drugs raises the chances of breaking a bone by nearly 30%.
The study is published in the Annals of Family Medicine.
Available by prescription as well as over the counter, PPIs work by reducing the secretion of gastric acid. They are commonly recommended for patients with gastroesophageal reflux disease (GERD), peptic ulcer disease, erosive esophagitis, and Barrett’s esophagus.
PPIs are the third largest-selling class of therapeutic drugs on the market, with sales totaling $13.6 billion in 2010, according to IMS Health.
For patients with potentially serious conditions, the benefits offered by PPIs often outweigh the risks associated with them, says James M. Gill, MD, MPH, president of Delaware Valley Research Outcomes in Newark, Del., and lead author of an editorial accompanying the study.
“For certain things, PPIs are clearly indicated,” he says. The problem is that “many doctors don’t follow guidelines” and prescribe PPIs “willy-nilly.”
“This study is not a game changer in terms of guidelines,” Gill continues, “but it should encourage physicians to pay closer attention and be more cautious with these medications when they prescribe them.”
The present study, by researchers at Seoul National University Hospital in South Korea, is an analysis of 11 previously published studies in which researchers examined the possible link between fracture risk and PPIs. Overall, the risk of fracture increases by 29% with the use of PPIs. Hip fracture risk rises by 31%, vertebral fractures by 54%.
The researchers also report that they were unable to find a significant association between fracture risk and histamine H2-receptor antagonists, another class of acid-suppressing drugs, marketed under brand names such as Axid, Pepcid, Tagamet, and Zantac.
The researchers explain that the increased risk of fracture likely occurs in part because PPIs interfere with the body’s ability to absorb calcium, leading to weaker bones that are more prone to break.
Fractures are not the only risk factors associated with PPIs. PPIs may raise the risk of GI infections, while taking them for more than a year may lead to low serum magnesium levels, which can cause muscle spasms, irregular heartbeat, and convulsions, according to the FDA, which issued a warning to that effect in March of this year.
“As with all medications, there are risks and benefits,” a representative for Prilosec maker Procter & Gamble write in an email. “Like other OTC PPIs, Prilosec OTC should only be used as directed for 14 days for the treatment of frequent heartburn.”
As Gill writes in his editorial, physicians should not hesitate to prescribe PPIs to treat potentially serious conditions. But for patients with uncomplicated GERD, for example, Gill holds that patients would be better off taking a PPI “on-demand,” meaning on a short-term basis to control symptoms and reduce the risk of side effects.
“The over-the-counter PPIs warn consumers not to use them for more than two weeks at a time,” he says. “That’s probably a good rule of thumb overall.”
Nov. 17, 2009 -- There is new evidence that folic acid, taken in large doses, may promote some cancers.
Heart patients in Norway who took folic acid and vitamin B12 supplements were found to have a slightly increased risk for cancer and death from all causes, compared to heart patients who did not take the supplements in a study published in TheJournal of the American Medical Association.
Unlike the U.S., Norway does not fortify flour and grain food products with folic acid, which is the synthetic form of the B vitamin folate.
Because of this, Norwegians tend to have much lower blood folate levels than Americans, making the population a good one for studying the impact of folic acid supplementation on cancer risk, study researcher Marta Ebbing, MD, of Norway's Haukeland University Hospital tells WebMD.
Ebbing and colleagues analyzed data from two studies that included almost 7,000 heart patients treated with B vitamin supplements or placebo for an average of three and one-half years between 1998 and 2005.
The original intent of the studies was to determine if taking vitamin B supplements improved cardiovascular outcomes, which it didn't do.
During treatment, blood folate levels among patients who took 0.8 milligrams a day of folic acid plus 0.4 milligrams a day of vitamin B12 increased more than sixfold.
The patients were followed for an average of three years after supplementation ended, during which time 341 patients who took folic acid and B12 (10%) and 288 patients who did not (8.4%) were diagnosed with cancer.
Folic acid and B12 supplementation was associated with a 21% increased risk for cancer, a 38% increased risk for dying from the disease, and an 18% increase in deaths from all causes.
This finding was mainly driven by an increase in lung cancer incidence among the folic acid and B12-treated patients.
Seventy-five (32%) of the 236 cancer-related deaths among the study participants were due to lung cancer, and the cancer incidence among the study group was 25% higher than in the population of Norway as a whole.
Roughly 70% of all the patients in the study were either current or former smokers, including more than 90% of those who developed lung cancer.
In a statement issued in response to the study, a spokesman for the supplement-industry trade association Council for Responsible Nutrition (CRN) noted that the lung cancer finding has not been seen in other studies.
"The real headline of this study should be that smoking increases the risk of lung cancer -- the study found that a total of 94% of the subjects who developed lung cancer were either current or former smokers," CRN Vice President for Scientific and Regulatory Affairs Andrew Shao, PhD, says in a news release.
【关键词】 Regulation
Lysophosphatidic acid (LPA), via interaction with its G-protein coupled receptors, is involved in various pathological conditions. Extracellular LPA is mainly produced by the enzyme autotaxin (ATX). Using fibroblast-like synoviocytes (FLS) isolated from synovial tissues of patients with rheumatoid arthritis (RA), we studied the expression profile of LPA receptors, LPA-induced cell migration, and interleukin (IL)-8 and IL-6 production. We report that FLS express LPA receptors LPA1-3. Moreover, exogenously applied LPA induces FLS migration and secretion of IL-8/IL-6, whereas the LPA3 agonist L-sn-1-O-oleoyl-2-methyl-glyceryl-3-phosphothionate (2S-OMPT) stimulates cytokine synthesis but not cell motility. The LPA-induced FLS motility and cytokine production are suppressed by LPA1/3 receptor antagonists diacylglycerol pyrophosphate and (S)-phosphoric acid mono-(2-octadec-9-enoylamino-3-[4-(pyridine-2-ylmethoxy)-phenyl]-propyl) ester (VPC32183). Signal transduction through p42/44 mitogen-activated protein kinase (MAPK), p38 MAPK, and Rho kinase is involved in LPA-mediated cytokine secretion, whereas LPA-induced cell motility requires p38 MAPK and Rho kinase but not p42/44 MAPK. Treatment of FLS with tumor necrosis factor- (TNF-) increases LPA3 mRNA expression and correlates with enhanced LPA- or OMPT-induced cytokine production. LPA-mediated superproduction of cytokines by TNF--primed FLS is abolished by LPA1/3 receptor antagonists. We also report the presence of ATX in synovial fluid of patients with RA. LPA1/3 receptor antagonists and ATX inhibitors reduce the synovial fluid-induced cell motility. Together the data suggest that LPA1 and LPA3 may contribute to the pathogenesis of RA through the modulation of FLS migration and cytokine production. The above results provide novel insights into the relevance of LPA receptors in FLS biology and as potential therapeutic targets for the treatment of RA.
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by the destruction of articular cartilage and adjacent bone tissues (Feldmann et al., 1996). The critical events in RA have been suggested to be largely orchestrated by a complex interplay of proinflammatory cytokines, chemokines, and matrix metalloproteinases in both the synovial tissue and the synovial fluid (Wong and Lord, 2004). Besides inflammatory mediators, other invasive-promoting factors such as metastasis-associated protein (Senolt et al., 2006) or proliferation-mediated gene (Jang et al., 2006) have also shown to be activated in RA synovium. It is interesting that the mRNA of lysophospholipase D (lyso-PLD), also known as autotaxin (ATX), is expressed in FLS from patients with RA (Kehlen et al., 2001). ATX was originally identified as an autocrine tumor cell motility-stimulating factor and an enzyme that generates most of the extracellular lysophosphatidic acid (LPA) (Umezu-Goto et al., 2002).
LPA is a naturally occurring bioactive lipid belonging to the family of phospholipid growth factors, present in micromolar concentrations in serum and biological fluids and in higher concentrations at sites of inflammation and tumor growth (Ishii et al., 2004). LPA mediates many of its effects through interaction with a family of seven-transmembrane G-protein-coupled receptors that are encoded by the endothelial differentiation genes Edgs (Ishii et al., 2004). Five LPA receptors (LPA1-5) have been characterized. Among them, LPA1-3 share sequence homology with one another, whereas LPA4-5 sequences are more divergent (Noguchi et al., 2003; Lee et al., 2006). By binding to its cognate receptors, LPA activates various signaling pathways. The cellular signaling events linking LPA to its pleomorphic activities are complex, because these receptors couple to different pertussis toxin-sensitive and -insensitive G-proteins. Depending on the cell type, LPA receptors are coupled via Gi/o, Gq, and G11/12 to multiple effector systems, such as mitogen-activated protein kinase (MAPK), adenylate cyclase, phospholipase C, and small GTPases, Rho, Rac, and Ras (Ishii et al., 2004). Through interaction with LPA1, for instance, LPA stimulates cell migration and proliferation (Yamada et al., 2004), whereas binding to LPA2, LPA induces the synthesis of proangiogenic factors such as vascular endothelial growth factor, IL-8 and IL-6 (Palmetshofer et al., 1999). This phospholipid growth factor has been implicated in various diseases and injury states, such as angiogenesis and autoimmunity (Ishii et al., 2004). Although LPA receptors are functionally expressed in a broad variety of cells, including cells found in the sublining of the synovial membrane (Takuwa et al., 2002), little is known regarding LPA receptor biological activities and expression profile in human FLS. In RA, FLS play an important role as main effector cells in joint destruction through the production of matrix metalloproteinases, which are matrix-degrading enzymes (Firestein, 2003). FLS also migrate, invade, and degrade the connective tissue of cartilage and tendon (Pap et al., 2000).
In summary, the observation that the functional responses of FLS to inflammatory stimuli resemble those induced by LPA in various cell types, that ATX mRNA is expressed by RA synoviocytes (Kehlen et al., 2001), and that RA synovial fluid contains significant amounts of the LPA precursor lysophosphatidylcholine (LPC) (Fuchs et al., 2005), led us to the investigation of the expression profile and the functional responses of LPA1-3 receptors in FLS. We report that LPA1, LPA2, and LPA3 receptor mRNA is expressed in FLS. We also provide evidence that exogenous application of LPA induces cell migration and IL-8/IL-6 secretion by FLS. It is interesting that blocking LPA receptors with LPA1/3 receptor antagonists inhibits both LPA-induced cell motility and IL-8/IL-6 production. Moreover, the LPA-stimulated cytokine secretion is regulated by p42/44 MAPK, p38 MAPK, and Rho kinase, whereas LPA-induced cell motility requires p38 MAPK and Rho kinase but not p42/44 MAPK. In addition, we show that under an inflammatory microenvironment created by TNF-, both the expression of LPA3 receptor mRNA and the LPA- or OMPT-dependent secretion of IL-8/IL-6 is significantly increased. Finally, we demonstrate the presence of ATX in synovial fluid of RA patients and the reduction of synovial fluid-induced cell motility by LPA1/3 receptor antagonists and ATX inhibitors, which is suggestive of LPA production and LPA subsequent biological effects in human RA synovium. We therefore conclude that LPA/LPA receptor signaling may play essential role in the pathogenesis of RA.
Reagents. 1-Oleoyl-sn-glycero-3-phosphate (LPA) and LPC were purchased from Sigma (St. Louis, MO). LPA1/3-specific receptor antagonists, diacylglycerol pyrophosphate (DGPP), and VPC32183 were obtained from Avanti Polar Lipid Inc. (Alabaster, AL). ATX inhibitors 18:1 carbacyclic phosphatidic acid (18:1 ccPA, C22H41O5P), XY-44 (C22H40O4PSNa), and JGW-8 (C20H39NaBrO6P) were synthesized at The University of Utah by J. Gajewiak, Y. Xu, and G. Jiang. All of the compounds above were dissolved in phosphate-buffered saline containing 0.1% fatty acid-free bovine serum albumin from Sigma, and the aliquots were stored at -20°C. The specific LPA2 agonist, dodecylphosphate, and LPA3 agonist, L-sn-1-O-oleoyl-2-methyl-glyceryl-3-phosphothionate (2S-OMPT), were obtained from Biomol (Plymouth Meeting, PA) and Echelon Biosciences Inc. (Salt Lake City, UT), respectively. Tumor necrosis factor (TNF-), interleukin-1β (IL-1β), and tumor growth factor-β were from PeproTech Inc. (Rocky Hill, NJ). Human IL-8 and IL-6 enzyme-linked immunosorbent assay (ELISA) kits were purchased from BioSource International Inc. (Camarillo, CA). SYBR Green JumpStart Ready Mix was obtained from Sigma. TRIzol reagent was from Invitrogen (Burlington, ON, Canada). Inhibitors of p42/44 MAPK PD98059, of p38 MAPK SB203580, of Rho kinase Y27632, and of c-Jun N-terminal kinase (JNK) SP600125 were purchased from Calbiochem (San Diego, CA). Antibodies to total and phosphorylated forms of p42/44 MAPK, of p38 MAPK, of activating transcription factor-2 (ATF-2), and of JNK were purchased from Cell Signaling Technology (Waltham, MA). Antibodies to LPA1, LPA2, and LPA3 were obtained from MBL (Woburn, MA) and Exalpha Biologicals Inc (Watertown, MA). Cell culture reagents were purchased from Wisent Inc. (St. Bruno, QC, Canada).
Cell Culture. Human primary FLS were obtained from patients with RA who had received the diagnosis according to the criteria developed by the American College of Rheumatology Diagnostic Subcommittee for Arthritis who were undergoing arthroplasty (Faour et al., 2003). Cells were maintained under standard conditions (37°C and 5% CO2) and grown in DMEM supplemented with 10% fetal bovine serum, penicillin (100 IU), and streptomycin (100 µM). Cells were used at passages 5 to 15.
Cell Treatment. Semiconfluent cells were starved with serum-free medium for 24 h before treatment because the serum may contain up to 10 µM LPA. At the moment of cell treatment, the culture medium was replaced with fresh serum-free medium containing various concentrations of the tested compounds, as indicated in detail below.
Semiquantitative Reverse Transcription-PCR and Real-Time PCR Analysis of IL-8 and LPA Receptors. Cells were plated at a concentration of 5 x 104 cells/ml in six-well plates. For analysis of IL-8 mRNA expression, starved FLS were incubated with LPA (1-100 µM) and lysed for RNA extraction after 0.5 to 4 h. Where indicated, cells were pretreated for 30 min with the LPA receptor antagonists DGPP (1-100 µM) or VPC32183 (1-100 µM) and incubated with LPA (50 µM) in the presence or absence of the two antagonists for 2 h before RNA extraction. The expression of LPA receptors was monitored in starved FLS incubated in the absence or the presence of the indicated concentrations of TNF- (20-100 ng/ml) for up to 4 h before RNA extraction.
Total cellular RNA was extracted using TRIzol reagent according to the instructions from the manufacturer. Total RNA (0.5-1 µg) was reverse-transcribed using random priming and Superscript II Reverse Transcriptase system (Invitrogen, Burlington, ON, Canada) following the manufacturer's guidelines. All oligonucleotides used as primers were designed to recognize sequences specific for each target cDNA. Primer sequences and PCR conditions are as follows: LPA1 (432-bp product): sense, 5'-AAT-CGA-GAG-GCA-CAT-TAC-GG-3', and antisense, 5'-TGT-GGA-CAG-CAC-ACG-TCT-AG-3'; LPA2 (352-bp product): sense, 5'-CAT-CAT-GCT-TCC-CGA-GAA-CG-3', and antisense, 5'-GGG-CTT-ACC-AAG-GAT-ACG-CAG-3'; LPA3 (310-bp product): sense, 5'-TCG-CGG-CAG-TGA-TCA-AAA-ACA-GA-3', and antisense, 5'-ATG-GCC-CAG-ACA-AGC-AAA-ATG-AGC-3'; LPA4, (139-bp product): sense, 5'-AAA-GAT-CAT-GTA-CCC-AAT-CAC-CTT-3', and antisense, 5'-CTT-AAA-CAG-GGA-CTC-CAT-TCT-GAT-3'; LPA5, (350-bp product): sense, 5'-AGG-AAG-AGC-AAC-CAA-GCA-CAG-3, and antisense, 5'-ACC-ACC-ATA-TGC-AAA-CGA-TGT-G-3'; and IL-8 (562-bp product): sense, 5'-TGG-GTG-CAG-AGG-GTT-GTG-3', and antisense, 5'-CAG-ACT-AGG-GTT-GCC-AGA-TTT-3'. To ensure linear cDNA amplification, different amplifying cycles were tried. The experiments revealed linear amplification within 35 cycles. A total of 35 PCR cycles were run at 95°C (denaturation, 30 s), 63°C for LPA1, 64°C for LPA2, 66°C for LPA3, 60°C for LPA4, 60°C for LPA5, and 61°C for IL-8 (annealing, 30 s) and 72°C (extension, 30 s). The amount of ribosomal protein RPLP0 mRNA was used as an internal PCR control. RPLP0 (248-bp product) primer sequences are as follows: sense, 5'-GTT-GTAGAT-GCT-GCC-ATT-G-3'; and antisense, 5'-CCA-TGT-GAA-GTCACT-GTG-C-3'. The PCR products were subjected to electrophoresis on a 0.8% agarose gel and visualized by ethidium bromide staining. Densitometry analysis was used for band quantification using the software Alphamage 2000. The results were expressed as a ratio of the band intensity relative to the corresponding RPLP0 band obtained by amplification of the same template cDNA. Semiquantitative real-time PCR was also conducted using the SYBR Green PCR Master Mix kit in accordance with the manufacturer's instructions to examine the mRNA expression of LPA1-3 receptors and to evaluate the regulation of LPA3 mRNA expression upon TNF- treatment. In real-time PCR experiments, we used the same primers as for RT-PCR to amplify LPA1-3. The thermal cycling conditions were as follows: 95°C (initial denaturation, 3 min) followed by 40 cycles of 95°C (denaturation, 15 s), 63°C for LPA1-2, 66°C for LPA3 (annealing, 20 s), and 72°C (extension, 20 s).
Fig. 1. Expression of LPA1, LPA2, and LPA3 mRNA in human FLS. A, agarose gel electrophoresis of semiquantitative RT-PCR analysis of LPA1, LPA2, and LPA3. As a negative control, RT-PCR was performed without oligonucleotide primers. RPLP0 was used as an internal control. Experiments were repeated three times with identical results. B, the level of LPA receptor mRNA in human FLS relative to that of RPLP0 mRNA. Data shown are means ± S.E. of three independent experiments. C and D, LPA1, LPA2, and LPA3 mRNA expression in FLS measured using real-time PCR. Data shown are representative of three separate experiments.
Wound-Healing Assay. Cells were plated at a concentration of 5 x 104 cells/ml in 12-well plates. After routine starvation for 24 h, a plastic pipette tip was drawn across the center of the well to produce a clean wound area. Free cells were removed, and the medium was replaced with serum-free medium before stimulation with LPA, selective LPA receptor agonists/antagonists, and synovial fluid alone or in combination with LPA receptor antagonists, ATX inhibitors, PD98059, SB203580, SP600125, or Y27632 at indicated concentrations. Immediately after scratch wounding (0 h) and after incubation for 48 h, the wound healing process was photographed with an inverted microscope (Nikon TE300; Nikon, Tokyo, Japan). The migrated cells into the wound area were examined and monitored with the MetaMorph software.
IL-8 and IL-6 ELISA Assay. Cells were plated at a concentration of 5 x 104 cells/ml in 24-well plates. After routine starvation, cells were stimulated with LPA or LPA receptor agonists (1-5 µM). Where indicated, cells were pretreated with DGPP (20 µM), VPC32183 (10 µM), or the inhibitors of p42/44 MAPK, p38 MAPK, JNK, and Rho kinase for 30 min before stimulation with LPA or LPA receptor agonists. To evaluate the effect of TNF- on LPA receptor-mediated cytokine secretion, cells were either treated with TNF- (80 ng/ml) in combination with LPA for 2, 8, and 24 h or pretreated with TNF- (80 ng/ml) and washed extensively before stimulation. Cell culture supernatants were collected and stored at -80°C until the ELISA assay was performed. IL-8 and IL-6 protein concentrations were measured according to the manufacturer's protocol. All samples were analyzed in duplicate. Optical densities were determined using a SoftMaxPro 40 plate reader at 450 nm. The results were compared with a standard curve that was generated using known concentrations (in picograms per milliliter) of IL-8 and IL-6. The results were expressed in picograms per milliliter (5 x 104 cells).
Fig. 2. Stimulation of human FLS motility by LPA. A clean wound area was made on a monolayer of FLS. After removing free cells, the wound was allowed to heal for 48 h in serum-free medium containing LPA (5 µM) with or without DGPP (10 µM) or VPC32183 (10 µM). The wound-healing process was photographed at 0 and 48 h (top). The data shown are representative of four separate experiments. Migrated cell numbers were expressed as percentage of nontreated cells (bottom). Statistical comparative analyses were done between cells treated with LPA and cells treated with LPA + DGPP or LPA + VPC32183. Data shown are means ± S.E. of four independent experiments. *, p < 0.05.
Preparation of Cell Lysates and Western Blotting. Cells were plated at a concentration of 5 x 105 cells/well in six-well plates and were starved for 24 h before stimulation. To determine the activation state of p42/44 MAPK, p38 MAPK, ATF-2, and JNK, cells were exposed to LPA (5 µM) for various times (1-15 min). Where indicated, cells were pretreated with PD98059 (25 µM), SB203580 (10 µM), and Y27632 (10 µM) for 30 min before stimulation. Cells were lysed in lysis buffer [20 mM Tris/HCl, pH 7.4, 1% (v/v) Triton X-100, 150 mM NaCl, 5 mM EDTA, 100 mM NaF, and 1 mM Na3VO4]. Protein concentration was determined with a BCA protein assay kit (Pierce Chemical Co., Rockford, IL) using bovine serum albumin as standard. Equal amount of proteins (50 µg) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to methanol soaked Immobilon polyvinylidene difluoride membranes (Millipore Corporation, Bedford, MA). Primary antibody incubation was performed overnight in 5% (w/v) milk at 4°C. The membranes were then washed three times and incubated with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature. Membranes were washed three times, and antibody-antigen complexes were revealed using enhanced chemiluminescence according to the manufacturer's instructions (PerkinElmer Life and Analytical Sciences, Waltham, MA).
Synovial Fluid. This research was conducted according to the principles of the Declaration of Helsinki. Eleven synovial fluid samples were studied, originating from patients (eight women and three men with a mean age of 51.2 ± 3.4 years) with definite or classic RA according to the American College of Rheumatology criteria. After informed consent was obtained, synovial fluid was collected on heparin, centrifuged to eliminate cells and debris, and frozen at -20°C. Synovial fluid (2.5 µl) was mixed with one volume of boiling Laemmli sample buffer immediately before electrophoresis. To examine the contribution of ATX to FLS motility, synovial fluids were dialyzed using a 100,000 molecular weight cut off membrane to remove free or serum albumin-bound lysophospholipids including sphingosine-1-phosphate before addition to cell culture medium (Fuchs et al., 2005; Kitano et al., 2006).
ATX DNA Constructs, Cell Transfection, and Immunoblotting. The ATX cDNA was prepared and transfected into CHO2A cells as described previously (Murata et al., 1994). Cell lysates were resolved on 8% SDS-PAGE and transferred to polyvinylidene difluoride membranes before Western blotting with affinity-purified anti-ATX (1/2000) antibodies (Murata et al., 1994).
Statistical Analysis. Unless otherwise stated, experiments were performed in triplicates. Results presented are expressed as mean values ± S.E. or as representative studies. Statistical significance of the difference between treated and untreated samples was determined by analysis of variance (t test). Calculations were made with the Prism software 4.0 (GraphPad Software Inc., San Diego, CA). P values less than 0.05 were considered statistically significant.
mRNA Expression of LPA1, LPA2, and LPA3 in Human FLS. Because the biological activity of LPA is mediated through its interaction with specific cell surface receptors, we first examined the presence of LPA receptor transcripts in primary human FLS. Using semiquantitative RT-PCR and real-time PCR, we detected mRNA for LPA1, LPA2, and LPA3 (Fig. 1) but not LPA4 or LPA5 (data not shown) in cultured human FLS from RA patients. The most abundantly expressed receptor at the mRNA level was LPA1 compared with LPA2 and LPA3.
Fig. 3. LPA-induced IL-8 and IL-6 production in human FLS. A, concentration-dependent response to LPA on IL-8 mRNA expression. Primary FLS were incubated with LPA for 2 h at various doses as indicated and lysed for RNA extraction and RT-PCR. B, kinetics of LPA-induced IL-8 mRNA expression. Cells were incubated with LPA (2.5 µM) for various time points as indicated before lysing for RNA extraction and RT-PCR. C and E, concentration-dependent response to LPA on IL-8 (C) and IL-6 (E) secretion. Cells were treated with LPA for 24 h at various concentrations as indicated before collecting supernatants for protein quantification. D and F, kinetics of LPA-induced IL-8 (D) and IL-6 (F) secretion. Cells were treated with LPA at the indicated concentrations. Cell culture supernatants were collected at indicated time points, and cytokines were quantified by ELISA. Experiments were repeated three times, and the results are displayed as representative gels (A and B) or as mean value ± S.E. (C-F). Statistical comparative analyses were done between samples incubated with diluents and cells treated with the indicated concentrations of LPA (C and E), and for indicated times(D and F). *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Induction of Human FLS Migration by LPA via Its Receptors. The migration of FLS into cartilage and bone is central to RA pannus development. We therefore investigated whether LPA could directly alter the migratory behavior of these cells using a wound healing assay. As shown in Fig. 2, exogenously added LPA was able to induce the migration of FLS. The migratory response mediated by LPA was strongly reduced by specific antagonists against LPA1/3, namely DGPP (58% decrease) and VPC32183 (64% decrease). The results suggest that LPA, via LPA1 and/or LPA3 receptors, stimulates FLS migration.
Stimulation of IL-8 and IL-6 Production by LPA in Human FLS. LPA is known to induce both IL-8 and IL-6 secretion in several other cell lines (Fang et al., 2004; Saatian et al., 2006). Because infiltration of inflammatory cells into the synovium is another important characteristic of RA pathogenesis, we next investigated whether LPA could be involved indirectly in this process by regulating the production of IL-8 and IL-6 of FLS. We chose to investigate IL-8 and IL-6 because they are potent neutrophil chemoattractants involved in RA disease progression (Koch, 2005). As shown in Fig. 3A, no significant IL-8 mRNA expression was detected in control starved FLS from patients with RA. Upon treatment with LPA, however, FLS expressed IL-8 mRNA in a dose-(Fig. 3A) and time- (Fig. 3B) dependent manner, with a maximal induction observed at 50 µM LPA and at 2 h after stimulation. The effect of LPA induction seems to be specific because IL-8 expression in FLS was not induced by a treatment with related lipids such as LPC (data not shown).
The LPA-induced IL-8 and IL-6 protein secretion was also monitored. In this series of experiments, we used lower concentrations of LPA to avoid the cytotoxic effect of LPA, observed at 10 µM on starved FLS after an incubation of 24 h. A significant release of IL-8 (Fig. 3C) protein was detected with 1 to 5 µM LPA. LPA-stimulated IL-8 (Fig. 3D) secretion continued to increase for up to 24 h, the last time point tested. LPA also stimulated IL-6 secretion in a dose- and time-dependent manner (Fig. 3, E and F). These results demonstrate that LPA is able to induce an up-regulation of IL-8 and IL-6 production in human FLS.
Fig. 4. Inhibition of LPA-induced IL-8 and IL-6 production by LPA1/3 specific antagonists in human FLS. A and B, effect of DGPP and VPC32183 on LPA-induced IL-8 mRNA expression. Human FLS were pretreated for 30 min with DGPP (A) and VPC32183 (B) at various concentrations, as indicated, before adding LPA (50 µM) for another 2 h. Total RNA was extracted for RT-PCR. C and D, effect of DGPP and VPC32183 on LPA-induced IL-8 secretion. Cells were incubated with or without 20 µM DGPP (C) or 10 µM VPC32183 (D) for 30 min before stimulation with LPA (2.5 µM) for 24 h. E and F, effect of DGPP and VPC32183 on LPA-induced IL-6 secretion. Cells were incubated with or without 20 µM DGPP (E) or 10 µM VPC32183 (F) for 30 min before stimulation with LPA (2.5 µM) for 24 h. Results are presented as a representative electrophoresis agarose gel (top, A and B), or as ratios (means ± S.E.) of IL-8 and RPLP0 (bottom, A and B) from three independent experiments. For ELISA assay, experiments were repeated three times, and the results are displayed as mean value ± S.E. C to F, statistical comparative analyses were done between cells treated with LPA and cells treated with LPA + DGPP (C and E) or LPA + VPC32183 (D and F). *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Because the effects of LPA are probably receptor-mediated processes, we next analyzed the impact of two specific antagonists of LPA1/3, DGPP, and VPC32183 on LPA-induced IL-8 and IL-6 production in FLS from patients with RA. We observed that DGPP (Fig. 4A) and VPC32183 (Fig. 4B) significantly inhibit, in a concentration-dependent manner, the LPA-mediated IL-8 mRNA expression. DGPP and VPC32183 almost completely abolished IL-8 mRNA expression (up to 85%), with optimal inhibition observed at a concentration of approximately 20 µM for both antagonists. Application of the two antagonists also significantly blocked LPA-induced cytokine secretion; DGPP (20 µM) decreased IL-8 by 70% (Fig. 4C) and IL-6 by 42% (Fig. 4E), whereas VPC32183 (10 µM) inhibited IL-8 by 74% (Fig. 4D) and IL-6 by 94% (Fig. 4F), respectively. Taken together, these results suggest that the up-regulation of IL-8 and IL-6 production by LPA in FLS involves LPA/LPA receptor (LPA1 and LPA3) signaling.
Fig. 5. Effect of selective LPA receptor agonists on cell motility and cytokine secretion in human FLS. A, effect of the LPA2 agonist dodecylphosphate and the LPA3 agonist OMPT on cell motility. After removing free cells, the wound was allowed to heal for 48 h in serum-free medium containing dodecylphosphate (5 µM) or OMPT (5 µM). LPA (5 µM) was used as a positive control. Migrated cell numbers were expressed as a percentage of nontreated cells. Data shown are means ± S.E. of four independent experiments. B and C, secretion of IL-8 and IL-6 secretion in response to OMPT. Cells were treated with OMPT for 24 h at indicated concentrations before collecting supernatants for IL-8 (B) and IL-6 (C) measurements. Experiments were repeated three times, and the results are expressed as mean value ± S.E. Statistical comparative analyses were done between nontreated and OMPT-treated cells. *, p < 0.05; ***, p < 0.001.
Fig. 6. Involvement of MAPK and Rho kinase pathway in LPA-induced FLS migration. After removing free cells, the wound was allowed to heal for 48 h in serum-free medium containing LPA (5 µM), with or without inhibitor of p42/44 MAPK PD98059 (A), p38 MAPK SB203580 (B), and Rho kinase Y27632 (C), at the indicated concentrations. Migrated cell numbers were expressed as a percentage of nontreated cells. Data shown are means ± S.E. of three independent experiments. Statistical comparative analyses were done between LPA and LPA + inhibitors; *, p < 0.05.
Role of LPA Receptors in LPA-Mediated Functional Responses. To more accurately distinguish the contribution of LPA1, LPA2, and LPA3 receptors to LPA-mediated responses, we used the selective LPA2 agonist dodecylphosphate, and the LPA3 agonist OMPT (Fig. 5) in our functional assays. The LPA2 agonist had no effect on either cell motility (Fig. 5A) or cytokine secretion (data not shown). In contrast, the LPA3 agonist stimulated cytokine secretion (Fig. 5, B and C) but not cell motility (Fig. 5A). Taken together, the data suggest that LPA1 and LPA3 may play a major role in LPA-induced FLS motility and cytokine secretion, respectively.
Involvement of the Downstream Signaling Pathways Coupled to LPA Functional Responses. Because MAPK pathways play important roles in various cellular activities, including the induction of IL-8 and IL-6 (Oz-Arslan et al., 2006), and Rho kinase regulates actin reorganization and thus the cell motility (Tawara and Shimokawa, 2007), inhibitors of the p42/44 MAPK PD98059, of p38 MAPK SB203580, and of Rho kinase Y27632 were used to address the involvement of these signaling pathways in LPA-induced cell motility, cytokine secretion, and their effect on the activation of p42/44 MAPK and p38 MAPK. As shown in Fig. 6A, the inhibitor of p42/44 MAPK PD98059 had no significant effect on either the spontaneous or LPA-induced cell migration (p = 0.85 for LPA versus LPA + PD98059 10 µM, p = 0.57 for LPA versus LPA + PD98059 25 µM), whereas the inhibitor of p38 MAPK SB203580 at 10 µM decreased both spontaneous and LPA-mediated cell migration by 37 and 67%, respectively (Fig. 6B). The most significant effect was observed with Rho kinase inhibitor Y27632. It blocked LPA-induced cell migration by 81% at 10 µM (Fig. 6C). The results suggest a role for p38 MAPK and Rho kinase in LPA-modulated cell motility.
Fig. 7. Involvement of p42/44 MAPK, p38 MAPK, and of Rho in LPA-induced cytokine secretion. Cells were treated with LPA (5 µM) for 24 h in the presence or absence of p42/44 MAPK inhibitor PD98059 (A and D), p38 MAPK inhibitor SB203580 (B and E), and Rho kinase inhibitor Y27632 (C and F) at the indicated concentrations. Secreted IL-8 (A-C) and IL-6 (D-F) in cell culture supernatants were quantified by ELISA. Data shown are means ± S.E. of three independent experiments. Statistical comparative analyses were done between LPA and LPA + inhibitors. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Regarding cytokine production, all three inhibitors inhibited LPA-induced cytokine release (Fig. 7). The inhibitor of p42/44 MAPK PD98059 (25 µM) decreased LPA-induced IL-8 secretion by 78% (Fig. 7A) and that of IL-6 by 83% (Fig. 7D). The inhibitor of p38 MAPK SB203580 (10 µM) reduced LPA-mediated IL-8 and IL-6 secretion by 66% (Fig. 7B) and 67% (Fig. 7E), respectively. The inhibitor of Rho kinase Y27632 almost totally blocked LPA-stimulated IL-8 (Fig. 7C) and IL-6 (Fig. 7F) secretion at 10 µM, the highest concentration tested. These data suggest a role for p42/44 MAPK, p38 MAPK, and Rho in the modulation of IL-8 and IL-6 secretion by LPA.
The specificity of the signaling inhibitors was determined by assessing the activation state of p42/44 MAPK, p38 MAPK, and ATF-2, a downstream target of p38. LPA enhanced the phosphorylation of p42/44 MAPK and p38 MAPK. Phosphorylation peaked at 5 min for p42/44 MAPK and at 15 min for p38 MAPK (data not shown). As expected, PD98059 attenuated basal and LPA-induced phosphorylation of p42/44 MAPK (Fig. 8A) but had no significant effect on LPA-induced phosphorylation of p38 MAPK and ATF-2 (Fig. 8B). LPA-induced phosphorylation of p38 MAPK and ATF-2 (Fig. 8B) but not that of p42/44 MAPK (Fig. 8A) were blocked by the inhibitor of p38 MAPK SB203580. In this regard, the inhibition by SB203580 of LPA-induced p38 MAPK phosphorylation suggests that activation of this signaling pathway in FLS involves p38 MAPK autophosphorylation. It is interesting that the Rho kinase inhibitor Y27632 had no effect on LPA-induced activation of p42/44 MAPK but significantly diminished that of p38 MAPK (Fig. 8C). These results indicate that LPA is a potent activator of p42/44 MAPK and p38 MAPK in FLS. The data also suggest that p38 MAPK may act, at least in part, downstream of Rho/Rho kinase to promote LPA-induced FLS motility and cytokine secretion.
Fig. 8. Effect of p42/44 MAPK, p38 MAPK, and Rho kinase inhibitors on LPA-induced activation of p42/44 MAPK and p38 MAPK. Cells were pretreated with 25 µM PD98059, 10 µM SB203580 (A and B), and 10 µM Y27632 (C) for 30 min before LPA (5 µM) challenge for 15 min. Cell lysates were subjected to SDS-PAGE and samples were probed with antibodies to p-p42/44 MAPK, p42/44 MAPK, p-p38 MAPK, p38 MAPK, p-ATF-2, and ATF-2. Data shown are representative of three separate experiments.
We also explored the involvement of the JNK signaling pathway in LPA-induced FLS functional responses. Phosphorylated JNK was not detected after stimulation with LPA (data not shown). Furthermore, the JNK inhibitor SP600125 did not inhibit LPA-mediated IL-8 secretion and cell motility (data not shown). The results suggest that JNK is not activated by LPA in human FLS.
Regulation of LPA1, LPA2, and LPA3 Receptor mRNA Expression by Proinflammatory Stimuli in Human FLS. Because TNF- is a key inflammatory molecule in the RA cytokine network (Taberner et al., 2005), we also analyzed the mRNA expression profile of LPA receptors in response to TNF-. We found that treatment with TNF-, at concentrations of 20 to 80 ng/ml, up-regulated the mRNA expression of LPA3 in a dose-dependent manner (Fig. 9A). The maximal effect observed was a 3.5 ± 0.3-fold increase in LPA3 mRNA expression, induced by 80 ng/ml TNF-. To further investigate the kinetics of TNF--induced LPA3 expression, FLS were exposed to 80 ng/ml TNF- for 0.5 to 4 h. Semiquantitative RT-PCR analysis showed that LPA3 mRNA, normalized to that of RPLP0, peaked after 2-h stimulation with TNF- and decreased thereafter (Fig. 9B). On the other hand, under the same conditions, LPA1 and LPA2 expression was not altered by TNF- (data not shown). It is noteworthy that no up-regulation of LPA3 mRNA was observed in FLS treated with other cytokines, such as IL-1β and tumor growth factor-1β (data not shown). Up-regulation of LPA3 expression by TNF- (Fig. 9, A and B) was further confirmed by quantitative real-time PCR (Fig. 9C). The results indicate that the expression of LPA receptors, at least that of LPA3, can be up-regulated by TNF-. Receptor expression in human FLS was lower than the threshold for detection by LPA1, LPA2, and LPA3 antibodies (data not shown).
Fig. 9. Regulation of LPA3 mRNA expression by TNF- in human FLS. A, concentration-dependent response to TNF- on LPA3 mRNA expression. Cells were treated with TNF- (2 h, at various concentrations as indicated) before lysing for RNA extraction and RT-PCR. B, kinetics of LPA3 mRNA expression in response to TNF-. Cells were incubated with TNF- (80 ng/ml) for the indicated time lengths before RNA extraction and RT-PCR. Results are presented as a representative electrophoresis agarose gel (top) and/or as ratios (means ± S.E.) of LPA3 and RPLP0 from three separate experiments (bottom). C, real-time PCR analysis of LPA3 mRNA expression. Cells were incubated with TNF- (80 ng/ml) for 2 h before RNA extraction and real-time PCR. Statistical comparative analyses were done between nontreated and TNF--treated cells (bottom of A) and between cells transiently treated with TNF- at various time points (bottom of B). *, p < 0.05.
Effect of TNF- on LPA-Induced Functional Responses in Human FLS. The next series of experiments was undertaken to examine the effect of a proinflammatory environment, established by a pretreatment of FLS with TNF-, on both of the functional experiments we performed previously (i.e., LPA- or OMPT-mediated cell migration and cytokine secretion). To monitor the effect of TNF-, starved FLS were pretreated with a range of TNF- concentrations (1-80 ng/ml) and, after washing with serum-free medium, LPA- or OMPT-induced cell motility and cytokine secretion were determined. TNF- alone did not show a significant effect either on spontaneous FLS migration or LPA-induced migration using our wound-healing assay (data not shown). As shown previously in Figs. 3 and 5, LPA or OMPT alone weakly stimulated cytokine production compared with TNF--primed FLS (Fig. 10). However, after priming with TNF- for 2, 8, and 24 h, LPA-induced IL-8 secretion was strongly enhanced. TNF- pretreatment (80 ng/ml, 24 h) increased up to 38 times the LPA-induced IL-8 release (Fig. 10A). Moreover, LPA-induced IL-8 production was strongly enhanced after priming with a concentration of TNF- (80 ng/ml) shown previously to up-regulate LPA3 mRNA expression by FLS (Figs. 5 and 10B). The production of IL-8 (Fig. 10C) and of IL-6 (Fig. 10D) induced by the selective LPA3 agonist OMPT was also superstimulated after a pretreatment of FLS with TNF-. The results emphasize the potential contribution of LPA and signaling through LPA receptors in the inflamed synovium. To determine the relevance of the LPA receptor(s) to LPA-mediated enhanced cytokine production after priming with TNF-, FLS were treated with the LPA receptor antagonists DGPP and VPC32183. DGPP and VPC32183 had no effect on TNF--mediated IL-8 or IL-6 secretion but almost completely inhibited the enhanced secretion of cytokine induced by LPA in TNF--primed cells (Fig. 11). The results indicate that TNF- modulates LPA (LPA3) receptor functional expression and responses in human FLS.
Fig. 10. Priming with TNF- on LPA- or OMPT-induced cytokine secretion. A, kinetics of TNF- pretreatment on LPA-induced IL-8 secretion. Cells were pretreated with TNF- (80 ng/ml) for different time lengths as indicated before stimulation with 2.5 µM LPA for 24 h. Cell culture supernatants were harvested for IL-8 measurement. B, effect of increased concentrations of TNF- on LPA-mediated IL-8 secretion. Cells were pretreated with TNF- (1, 10, and 80 ng/ml) for 8 h and subsequently washed extensively before stimulation with 2.5 µM LPA for 24 h. Cell culture supernatants were harvested for IL-8 measurements. C and D, effect of TNF- priming on OMPT-induced IL-8 and IL-6 secretion. Cells were pretreated with TNF- (80 ng/ml) for 8 h before adding OMPT at indicated concentrations. Cell culture supernatants were collected for IL-8 (C) and IL-6 (D) measurement after 24 h. The results are presented as means ± S.E. of three separate experiments. Statistical comparative analyses were done between LPA and LPA + TNF- (B) and between nontreated and OMPT-stimulated cells (C and D). *, p < 0.05; **, p < 0.01.
Fig. 11. Effect of LPA1/3 receptor antagonists on LPA-induced super production of cytokines after priming of FLS with TNF-. A and B, effect of TNF- on LPA-induced IL-8 secretion. Cells were stimulated with TNF- (80 ng/ml) for 8 h before washing and stimulation with LPA (2.5 µM) for another 24 h in the presence/absence of 20 µM DGPP (A) and 10 µM VPC32183 (B). C and D, effect of TNF- priming on LPA-induced IL-6 secretion. Cells were stimulated with TNF- (80 ng/ml) for 8 h before washing and stimulation with LPA (2.5 µM) for another 24 h in the presence/absence of 20 µM DGPP (C) and 10 µM VPC32183 (D). The results are presented as means ± S.E. of three separate experiments. Statistical comparative analyses were done between cells treated with TNF- + LPA and TNF- + LPA + LPA receptor antagonists. **, p < 0.01; ***, p < 0.001.
Contribution of ATX to Synovial Fluid-Mediated FLS Motility. ATX was originally identified as an autocrine tumor cell motility-stimulating factor and was shown to be a lyso-PLD, an enzyme that generates LPA from lysophospholipids such as LPC (Umezu-Goto et al., 2002). Because synovial fluid contains significant amounts of the LPA precursor LPC (Fuchs et al., 2005), we monitored the presence of ATX in synovial fluid from patients with RA. As shown in Fig. 12A, using the affinity-purified anti-ATX antibody, we detected the presence of the full-length ATX protein in synovial fluid from patients with RA. The addition of dialyzed synovial fluid to cell culture medium (2.5%, final) strongly stimulated the motility of FLS (Fig. 12, B and C). To link the role of ATX to LPA production and synovial fluid-mediated FLS motility, we examined with wound-healing assay the effect of ccPA 18:1, a pure inhibitor of ATX with no significant agonist or antagonist activity at LPA receptors (Baker et al., 2006; Xu et al., 2006; Jiang et al., 2007), and of JGW-8 (Baker et al., 2006; Jiang et al., 2007) and XY-44 (Xu et al., 2006), two compounds that exhibit both submicromolar inhibition of ATX and submicromolar antagonist activity for four LPA receptors. As shown in Fig. 12B, treatment of cells with 5 µM ccPA 18:1, JGW-8, or XY-44 diminished synovial fluid-induced FLS motility by 58, 54, and 48%, respectively. Synovial fluid-mediated FLS motility was also inhibited by LPA1/3 antagonists (Fig. 12C). DGPP (10 µM) and VPC32183 (10 µM) reduced synovial fluid-induced cell motility by 64 and 84%, respectively. Together, these results provide strong support to the hypothesis that ATX in synovial fluid produces LPA and stimulates human FLS through activation of LPA1/3.
Fig. 12. Role of ATX in synovial fluid-induced FLS motility. A, expression of ATX in synovial fluids from patients with RA. Synovial fluids (2.5 µl) from 11 patients with RA was electrophoresed on an 8% SDS-PAGE and probed with anti-ATX antibody. Positive and negative controls were CHO2A cells transfected with or without pcDNA6/V5 His-ATX construct. Results shown are representative of two experiments with similar results. B and C, effect of ATX inhibitors and LPA1/3 antagonists DGPP and VPC32183 on synovial fluid-induced cell motility. After removing free cells, the wound was allowed to heal for 48 h in serum-free medium containing 2.5% of synovial fluid for up to 48 h in the presence/absence of ATX inhibitors ccPA, JGW-8, and XY-44 (B) or the LPA1/3 antagonists DGPP and VPC32183 (C) at the indicated concentrations. LPA (5 µM) was used as a positive control. Migrated cell numbers were expressed as a percentage of nontreated cells. Data shown are means ± S.E. of four independent experiments. Statistical comparative analyses were done between cells treated with synovial fluid and synovial fluid + ATX inhibitors or synovial fluid + LPA1/3 receptor antagonists.
In the present study, we report several novel findings regarding LPA receptor expression, regulation, and function in FLS from patients with RA. We provide direct evidence for the mRNA expression of LPA1-3 receptors, LPA-induced cell migration, and secretion of IL-8 and IL-6 by FLS. The LPA-induced effects were shown to be driven by signaling through the LPA1/3 receptors and regulated by p42/44 MAPK, p38 MAPK, and Rho kinase. Moreover, both LPA3 receptor expression and LPA-induced cytokine secretion by FLS are modulated by the inflammatory cytokine TNF-. We also demonstrate that ATX is present in synovial fluid and that ATX inhibitors or LPA1/3 receptor antagonists can reduce the synovial fluid-induced FLS motility. To the best of our knowledge, this is the first report of the functional expression and regulation of LPA receptors in human FLS.
The main histological characteristic of RA is the hyperplasia of the synovial intimal lining cells. As a constituent of synovial pannus in RA, FLS have long been considered as key players in the aggressive invasion of cartilage and bone (Shiozawa et al., 1983). FLS are believed to migrate over the cartilage and erode into the subchondral bone, eventually resulting in the formation of erosions. However, the potential factors that direct FLS migration to form the pannus are not well known. Gilat et al. (1996) have reported that the expression of adhesion molecules can chemotactically guide cells with the appropriate receptors. Our data show that LPA induces a strong migration of FLS, suggestive that activated LPA receptors on FLS may act directly as a driving force in the pannus invasion of cartilage in RA.
Among others, IL-8 and IL-6 have been demonstrated to affect the regulation of the signaling steps leading to neutrophil recruitment and activation (Lin et al., 2004). Previous studies have shown that LPA could stimulate the production of IL-8 and IL-6 by ovarian cancer, breast cancer, and bronchial epithelial cells (Fang et al., 2004; So et al., 2004). In this regard, we show in the present study that serum-starved FLS do not express detectable IL-8 mRNA or secrete IL-8 in the resting state. In contrast, exogenously applied LPA strongly promotes IL-8 mRNA expression and IL-8/IL-6 secretion by FLS. Thus, LPA may contribute to the regulation and the maintenance of the inflammatory response in RA, in part through stimulation of IL-8 and IL-6 secretion by FLS. The two cytokines may subsequently increase the recruitment of neutrophils and thus promote inflammation and neovascularization on the synovium (Middleton et al., 2004).
The biological effects of LPA are mediated by one or more LPA receptors, depending on the cell type studied. Forced expression of LPA1-3 has been reported to increase IL-8/IL-6 production in ovarian cancer cells, with LPA2 being more efficient in stimulating IL-8 secretion (Fang et al., 2000). In contrast, in bronchial epithelial cells, LPA1 and LPA3 were shown to be the major receptors regulating IL-8/IL-6 production (Saatian et al., 2006). In the present study we used a pharmacological approach to identify the specific involvement of LPA receptors in the FLS responses. The selective antagonists against LPA1/3 DGPP and VPC32183 strongly abrogated the LPA-driven cell motility, whereas the specific LPA2 agonist, dodecylphosphate, and LPA3 agonist, OMPT, had no effect on this function. These findings indicate that LPA1 receptors are more efficient in inducing the LPA-driven motility of FLS. Regarding cytokine production, the selective LPA1/3 antagonists severely diminished LPA-dependent cytokine production. In addition, the LPA3-but not the LPA2-specific agonist robustly stimulated cytokine secretion. Although a role for LPA1 in LPA-induced cytokine production cannot be excluded, our pharmacological approach suggests that LPA3 drives IL-8 and IL-6 secretion in FLS.
Previous studies have implicated the p38 MAPK pathway in LPA1 receptor-mediated migration of glioma cells (Malchinkhuu et al., 2005) and Rho kinase in LPA-induced migration of airway smooth muscle cells (Hirakawa et al., 2007). Other studies also illustrated that LPA-induced IL-8 and IL-6 secretion is regulated by p38 MAPK (Saatian et al., 2006), p42/44 MAPK, and Rho kinase (Oz-Arslan et al., 2006; Saatian et al., 2006). Here we show that LPA-induced FLS motility depends on the activation of p38 MAPK and Rho but not p42/44 MAPK or JNK. We also demonstrated that signal transduction through p42/44 MAPK, p38 MAPK, and Rho kinase but not JNK is involved in LPA-dependent cytokine secretion. Our results suggest that coupling of LPA receptors to various heterotrimeric G-proteins and thereof distinct downstream signaling pathways contribute to LPA-mediated cytokine synthesis and motility of human FLS.
In an attempt to fully understand the effects of LPA on RA synovium in vivo, we have analyzed the expression pattern of LPA receptors and the LPA-mediated biological responses in FLS pretreated with TNF- to more closely parallel the critical events that take place in the RA synovium. Although a large number of genes regulated by TNF- and IL-1β in FLS have been identified in previous gene expression profiling studies (Taberner et al., 2005), regulation of LPA receptors by TNF- has not been reported. In the current study, we observed the selective up-regulation of LPA3 mRNA by TNF-, which was suggestive that LPA receptor(s) expression is regulated in a proinflammatory environment, such as in the inflamed synovium. TNF- did not have an impact on FLS migration, either by itself or that induced by LPA (data not shown), suggesting that LPA itself is sufficient to induce a full migratory response of these cells. It must be pointed out that in comparison with LPA, TNF- is a stronger stimulator of cytokine secretion. It is noteworthy that a preincubation of FLS with TNF-, at a concentration found to up-regulate LPA3 expression (80 ng/ml) for several hours before stimulation with LPA markedly enhanced LPA-induced IL-8 secretion (up to 38 times). Whereas TNF--induced cytokine secretion was not inhibited by the LPA1/3 receptor antagonists, we observed that the enhanced LPA-induced IL-8 and IL-6 secretion after cell priming with TNF- was totally inhibited by the LPA1/3 receptor antagonists DGPP and VPC32183. This raises the interesting possibility of a causal relationship between the enhanced expression of LPA3 receptor after a treatment with TNF- and the resultant increase in cytokine secretion. Nonetheless, the synergy of LPA and TNF- on IL-8 and IL-6 production may be intimately associated with the inflammation of the synovium in RA. The hypothesis that LPA could be a critical mediator of cytokine secretion in RA inflammatory synovium is currently under investigation in our laboratory. Several pathways can contribute to the production of LPA (Aoki et al., 2002). Recent studies suggest a major contribution of ATX in the production of extracellular LPA (Umezu-Goto et al., 2002; Hama et al., 2004). Moreover, LPA content is increased in the extracellular fluid of inflamed tissues (Croset et al., 2000) and upon challenge with inflammatory stimuli (Sasagawa et al., 1998). Another finding of our study is the presence of ATX protein in synovial fluid from patients with RA, of which we believe is the first report for this issue. Although the extracellular concentration of LPA in RA synovial fluid is not known, synovial fluid from patients with RA contains significant amounts of LPC, which is a substrate from which ATX produces LPA (Fuchs et al., 2005). It is also interesting to point out that the levels of sphingosine-1-phosphate, another metabolic product of ATX (Clair et al., 2003), in synovial fluid from patients with RA are much higher than those found in serum and plasma (Kitano et al., 2006). Therefore, it is tempting to speculate that elevated production of LPA by ATX in the joint microenvironment may contribute to the inflammation of the synovium. Our results show that RA synovial fluid strongly stimulated FLS migratory activity, and this effect was inhibited by ATX inhibitors and LPA1/3 antagonists. We thus suggest that LPA may be constantly generated from LPC by ATX in synovial fluid, building up the concentration of LPA that led to the subsequent responses of FLS to LPA through activation of LPA1/3.
In summary, our data demonstrate the functional expression of LPA receptors in RA FLS, implicating this lysophospholipid in synovial cell motility and chemokine secretion such as IL-8 and IL-6. Furthermore, our data suggest that up-regulation of LPA3 receptor expression and enhanced LPA-induced cytokine secretion by TNF--primed FLS would strengthen the inflammatory responses. In addition, we report the presence of ATX/lyso-PLD in synovial fluid from patients with RA. In this context, it can be suggested that in patients with RA, enhanced production of LPA by ATX and activation of LPA receptors promotes both the migration of FLS into connective tissues and the production of cytokines. These cytokines may subsequently cause the infiltration of leukocytes and exacerbate the inflammatory response in RA synovium. Targeting LPA receptors or the production of bioactive lysophospholipids by ATX may represent innovative goals for the treatment of RA.
Acknowledgements
We are grateful to Sylvie Méthot for editorial assistance. We thank Danielle Harbor for technical support in Western blot analysis, and we are grateful to Y. Xu, G. Jiang, and J. Gajewiak for antagonist and inhibitor synthesis.
ABBREVIATIONS: RA, rheumatoid arthritis; LPA, lysophosphatidic acid; FLS, fibroblast-like synoviocyte; ATX, autotaxin; IL, interleukin; TNF-, tumor necrosis factor-; MAPK, mitogen-activated protein kinase; VPC32183, (S)-phosphoric acid mono-(2-octadec-9-enoylamino-3-[4-(pyridine-2-ylmethoxy)-phenyl]-propyl) ester; 2S-OMPT, L-sn-1-O-oleoyl-2-methyl-glyceryl-3-phosphothionate; DGPP, diacylglycerol pyrophosphate; lyso-PLD, lysophospholipase D; LPC, lysophosphatidylcholine; ccPA, carbacyclic phosphatidic acid; ELISA, enzyme-linked immunosorbent assay; JNK, c-Jun NH2-terminal kinase; ATF-2, activating transcription factor-2; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-polymerase chain reaction; bp, base pair; PAGE, polyacrylamide gel electrophoresis; JGW-8, C20H39NaBrO6P; XY-44, C22H40O4PSNa; PD98059, 2'-amino-3'-methoxyflavone; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; SP600125, anthra(1,9-cd)pyrazol-6(2H)-one 1,9-pyrazoloanthrone; Y27632, N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide dihydrochloride.
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作者单位：Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUQ-CHUL (C.Z., M.T.), Départements d'Anatomie-Physiologie (M.J.F., S.G.B.) et Médecine, Facultéde Médecine (P.E.P.), Université Laval, Québec, Canada; Département de Rhumatologie et Immunologie, Centre Universitaire McGill,
【关键词】 Receptor- Activity
Nuclear receptors (NRs) are transcription factors whose activity is regulated by the binding of small lipophilic ligands, including hormones, vitamins, and metabolites. Pharmacological NR ligands serve as important therapeutic agents; for example, all-trans retinoic acid, an activating ligand for retinoic acid receptor (RAR), is used to treat leukemia. Another RAR ligand, (E)-S,S-dioxide-4-(2-(7-(heptyloxy)-3,4-dihydro-4,4-dimethyl-2H-1-benzothiopyran-6-yl)-1-propenyl)-benzoic acid (Ro 41-5253), is a potent antagonist that has been a useful and purportedly specific probe of RAR function. Here, we report that Ro 41-5253 also activates the peroxisome proliferator-activated receptor (PPAR), a master regulator of adipocyte differentiation and target of widely prescribed antidiabetic thiazolidinediones (TZDs). Ro 41-5253 enhanced differentiation of mouse and human preadipocytes and activated PPAR target genes in mature adipocytes. Like the TZDs, Ro 41-5253 also down-regulated PPAR protein expression in adipocytes. In addition, Ro 41-5253 activated the PPAR-ligand binding domain in transiently transfected HEK293T cells. These effects were not prevented by a potent RAR agonist or by depleting cells of RAR, indicating that PPAR activation was not related to RAR antagonism. Indeed, Ro 41-5253 was able to compete with TZD ligands for binding to PPAR, suggesting that Ro 41-5253 directly affects PPAR activity. These results vividly demonstrate that pharmacological NR ligands may have "off-target" effects on other NRs. Ro 41-5253 is a PPAR agonist as well as an RAR antagonist whose pleiotropic effects on NRs may signify a unique spectrum of biological responses.
The peroxisome proliferator-activated receptors (PPARs) and the retinoic acid receptor (RARs) are members of the NR superfamily of ligand-activated transcription factors (Germain et al., 2006; Michalik et al., 2006). PPAR is expressed at its highest levels in white adipose tissue and is required for adipocyte differentiation (Chawla et al., 1994; Tontonoz et al., 1994). Ligands for this receptor, the antidiabetic drugs thiazolidinediones (TZDs), were found to be high-affinity ligands for PPAR promoting adipogenesis (Lehmann et al., 1995). PPAR heterodimerizes with retinoid X receptor (RXR), and RXR ligands can both enhance or attenuate the activity of PPAR responsive genes (Yamauchi et al., 2001; Hondares et al., 2006). In contrast, RAR activation by all-trans retinoid acid (atRA), or by synthetic ligands, prevents differentiation of murine preadipocytes (Kamei et al., 1994; Schwarz et al., 1997). Unlike RXR ligands, which directly bind and activate the PPAR/RXR heterodimer, the mechanism by which RAR ligands block this activity of PPAR is less clear and is likely to be indirect (Schwarz et al., 1997).
To better understand the effects of atRA on adipocyte differentiation, we used Ro 41-5253; originally synthesized by Hoffman LaRoche, it is a specific antagonist for RAR with little affinity for RAR and RAR (Apfel et al., 1992). This compound has been widely used to dissect the role of RAR in atRA-dependent biological processes (Shang et al., 1999; Emionite et al., 2003; Higuchi et al., 2003; Engedal et al., 2004; Lu et al., 2005). We were surprised to find that Ro 41-5253 stimulated the adipogenic differentiation of mouse 3T3-L1 preadipocytes as well as a human preadipocyte cell line. This function of Ro 41-5253 is RAR-independent. In exploring potential RAR-independent mechanisms, we discovered that Ro 41-5253 is, unexpectedly, an agonist ligand for PPAR. This finding suggests, among other things, that biological studies that employed this eccentrically pleiotropic ligand may require re-interpretation.
Cell Culture and Differentiation. Reagents were obtained from Invitrogen (Carlsbad, CA) unless otherwise noted. 293T and murine 3T3-L1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (U.S. Bio-Technologies Inc., Parkerford, PA), 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were grown to confluence and induced to differentiate 2 days after confluence with media containing 0.4 µM dexamethasone, 3 µg/ml bovine insulin, and 0.25 mM 3-isobutyl-1-methylxanthine (IBMX; all from Sigma, St. Louis, MO) for 2 days and for additional 2 days in insulin only. In differentiation studies, IBMX was replaced by either pioglitazone or Ro 41-5253 for the first 4 days. Culturing and differentiation of human preadipocytes from the Simpson Golabi Behmel Syndrome (SGBS) were described elsewhere (Kim et al., 2006), and ligands were present for the first 7 days of differentiation. Oil Red-O staining was performed as described previously (Li and Lazar, 2002).
Transfections and Luciferase Assay. 3T3-L1 adipocytes and 293T cells were transfected by electroporation (Nucleofector II; Amaxa Biosystems, Gaithersburg, MD). Adipocytes were detached from culture dishes with 0.25% trypsin and 0.5 mg of collagenase/ml in phosphate-buffered saline, washed twice, resuspended in electroporation buffer (solution V; Amaxa Biosystems), mixed with 2 µgof pGl3-3xAOxPPRE plasmid, electroporated, seeded into 12-well plates, and incubated for 24 h with compounds as indicated. 293T cells were electroporated with 2 nmol of nontargeting or human smart-pool RAR oligonucleotides (Dharmacon, Lafayette, CO) and seeded into 24-well plates and used for transactivation assays 24 h later. pGal4-hPPAR-LBD, pGal4-hPPAR/-LBD, and pGal5-TK-pGL3 were transfected in 293T cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol and incubated for 24h with compounds as indicated. All transfection were normalized to cotransfected pRL-CMV and measured using the dualluciferase reporter assay (Promega, Madison, WI).
Immunoblot Analysis and Antibodies. Protein were isolated and separated in 4 to 20% SDS polyacrylamide gels (Invitrogen) and transferred to polyvinylidene difluoride membrane (Invitrogen). After incubation with the primary antibodies for PPAR (Santa Cruz Biotechnology, Santa Cruz, CA), RAR (Santa Cruz Biotechnology) or the ubiquitously expressed GTPase RAN (BD Biosciences, San Jose, CA), a secondary horseradish-conjugated antibody (Invitrogen) was added, and an enhanced chemiluminescent substrate kit (Amersham, Chalfont St. Giles, UK) was used for detection.
Quantitative Polymerase Chain Reaction. RNA was purified with the RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany). cDNA was generated using the Sprint Powerscript System (Clontech, Mountain View, CA). Primers and probes for adipose protein 2 (aP2), PPAR2, and 36B4 for normalization have been described elsewhere (Chui et al., 2005; Schupp et al., 2005). All PCR reactions were carried out using Taqman Universal Polymerase Master Mix (Applied Biosystems, Foster City, CA) and the PRISM 7900 instrument (Applied Biosystems) and were evaluated according to the standard curve method.
Scintillation Proximity Assay for the PPAR-Ligand Binding Domains. The measurement of ligand displacement was performed as described previously (Nichols et al., 1998). The radioligands were [3H]rosiglitazone for PPAR and [3H]GW2433 for PPAR and PPAR/ (Xu et al., 1999).
Statistical Analysis. Representative results of at least three independent experiments are shown. All results are expressed as mean ± S.D. of triplicates. Statistical significance was determined using either the 2-tailed Student's t test or ANOVA, as appropriate, and P < 0.05 was deemed significant.
Ro 41-5253 Was an Inducer of Adipocyte Differentiation. Ro 41-5253 was used previously in 3T3-L1 cells to block the inhibitory effect of atRA on differentiation (Kamei et al., 1994). We tested the possibility that this compound has enhancing effects on differentiation itself. 3T3-L1 and human SGBS preadipocytes were therefore induced to differentiate into fat cells by exposing them to the hormonal inducers. Using a differentiation mix devoid of IBMX diminished the grade of adipocyte differentiation (Hamm et al., 2001) (Fig. 1A). The presence of pioglitazone or Ro 41-5253 could rescue the ability of differentiation as shown in increased Oil Red O staining, PPAR2 mRNA expression in 3T3-L1 cells (Fig. 1A), and PPAR protein expression in human SGBS cells (Fig. 1B). However, pioglitazone was more efficient than Ro 41-5253 in promoting the adipocyte phenotype (Fig. 1, A and B).
Fig. 1. Ro 41-5253 induces differentiation of mouse and human preadipocytes. 3T3-L1 preadipocytes were treated with the full differentiation mix (Mix) or Mix without IBMX and supplemented with either 500 nM pioglitazone, or 500 nM Ro 41-5253. Differentiation was evaluated by phase contrast microscopy, Oil Red O staining and PPAR2 expression at day 8 after initiation of differentiation (A). Human SGBS preadipocytes were treated for 14 days with Mix without IBMX and supplemented with 500 nM ligands for the first 7 days. Protein levels of PPAR were determined and cells assessed by phase contrast microscopy. *, p < 0.05; **, p < 0.01.
Ro 41-5253 Induced aP2 Expression and Down-Regulated PPAR Protein Levels in Adipocytes. To examine whether the enhancing effects on differentiation were mediated by PPAR, we measured the expression of the PPAR target gene aP2 and PPAR protein expression after incubation with pioglitazone and Ro 41-5253 in 3T3-L1 adipocytes. PPAR levels were shown to decrease upon activation in an autoregulatory manner (Hauser et al., 2000). Ro 41-5253, like pioglitazone, significantly up-regulated aP2 mRNA and down-regulated PPAR protein expression in adipocytes. Pioglitazone had more pronounced effects than Ro 41-5253 (Fig. 2, A and B).
Fig. 2. Ro 41-5253 increases PPAR target gene expression in 3T3-L1 adipocytes and down-regulates PPAR protein. Day 8 adipocytes were incubated for 24 h with vehicle, 500 nM pioglitazone, or 500 nM Ro 41-5253. mRNA expression of aP2 (as -fold induction over vehicle treatment) and PPAR protein expression were measured (A and B). *, p < 0.05.
Ro 41-5253 Activated Endogenous PPAR Activity. Consistent with the effects of Ro 41-5253 on adipocyte differentiation and aP2 expression, Ro 41-5253 increased the activity of endogenous PPAR on a transfected AOx-PPAR response element (PPRE) in 3T3-L1 adipocytes (Fig. 3).
Fig. 3. Ro 41-5253 increases endogenous PPAR activity in adipocytes. 3T3-L1 adipocytes were electroporated with either empty vector (pGL3) or a PPAR-response element containing reporter (pGL3-3xAOx-PPRE) and incubated for 24h with vehicle and 500 nM pioglitazone or 500 nM Ro 41-5253 and assayed for luciferase activity. Data represent the -fold induction of luciferase activity over vehicle treatment of the PPRE-containing plasmid. *, p < 0.05; **, p < 0.01.
Ro 41-5253 Activated the PPAR-LBD but Not the PPAR/-LBD. 3T3-L1 adipocytes express both PPAR and PPAR/ (Yan et al., 2007). We therefore investigated whether Ro 41-5253 was able to directly activate the PPAR or /-LBDs. We transfected the Gal4-PPAR and /-LBDs and the corresponding reporter in 293T cells and incubated with increasing concentrations of pioglitazone or the PPAR/ agonist GW 610742 (Sznaidman et al., 2003; van der Veen et al., 2005) and Ro 41-5253.
The RAR antagonist potently activated the PPAR-LBD but with much less efficiency than pioglitazone (Fig. 4A). Although the concentrations necessary for half-maximal activation (EC50) for both ligands are in the same range, the maximal activation of Ro 41-5253 over vehicle-treated cells was less than 30% of the activation of pioglitazone (Table 1). The full activation of the PPAR-LBD induced by 1 µM pioglitazone was consistently attenuated by cotreatment with increasing concentrations of Ro 41-5253 (Fig. 4C). This strongly suggests that pioglitazone and Ro 41-5253 both act via the same PPAR activating mechanism. On the contrary, Ro 41-5253 could not activate the PPAR/-LBD (Fig. 4B). We noticed a slight decrease of the basal PPAR/-LBD activity with high concentrations of Ro 41-5253. Accordingly, there was a reduction in the full PPAR/-LBD activation induced by 20 nM GW 610742 by cotreatment with 10 µMRo 41-5253 to 53% (data not shown).
Fig. 4. Ro 41-5253 partially activates the PPAR-LBD but not the PPAR/-LBD and competes with TZD for activation. 293T cells were transiently transfected with pGal4-hPPAR_DEF or pGAL-hPPAR/_DEF and the pGal5-Tk-pGL3 reporter followed by stimulation with pioglitazone or GW 610742 (black squares in A and B) or Ro 41-5253 (red circles) as indicated for 24 h. Data represent the -fold induction of luciferase activity over vehicle treatment for activation of PPAR (A) or PPAR/ (B). Transfected cells were incubated with 1 µM pioglitazone and increasing concentrations of Ro 41-5253 for 24 h and assayed for luciferase activity (C).
TABLE 1 EC50 and maximal activation of the human PPAR-LBD in transient transfections
Dose response curves from the transiently transfected 293T cells with the pGal4-hPPAR_DEF and pGal5-Tk-pGL3 reporter (Fig. 4) were used to calculate values for the EC50. The maximal activation represents the maximal relative activation of Ro 41-5253 in comparison to pioglitazone (=100%).
Ro 41-5253 Activated the PPAR-LBD Independent of RAR. We next addressed the question of whether RAR was involved in the specific PPAR-activating property of Ro 41-5253 using a pharmacological approach. 293T cells were transfected with the Gal4-RAR-LBD and the corresponding reporter and titrated for activating/repressing concentrations of Ro 41-5253, and the RAR agonist AM-580. The strong repression elicited by 500 nM Ro 41-5253 was completely abolished by coincubation with 5 µM AM-580 (Fig. 5A, compare the Ro 41-5253 and the Ro 41-5253 + AM-580 repression). The same concentration of the RAR agonist AM-580 had little effect of the pioglitazone-as well as Ro 41-5253-induced activation of the PPAR-LBD (Fig. 5B), showing that RAR antagonism is not necessary for the activation of the PPAR-LBD by Ro 41-5253.
Fig. 5. Ro 41-5253 activates the PPAR-LBD in presence of the RAR agonist AM-580. 293T cells were transiently transfected with the pGal4-hRAR_DEF (A) or pGal4-hPPAR_DEF (B) and pGal5-Tk-pGL3 reporter. Cells were treated with 500 nM Ro 41-5253, 5 µM AM-580 (a RAR agonist), or both for 24 h. Data represent the -fold repression over vehicle-treated cells (A). Activation the PPAR-LBD was measured after 24-h incubation with 0.03, 0.3, and 3 µM concentrations of either pioglitazone or Ro 41-5253 in the presence of vehicle or 5 µM AM-580. Data represent the -fold induction over vehicle treated cells (B). **, p < 0.01.
To provide further evidence for the RAR-independent mechanism, we depleted RAR in 293T cells (Fig. 6A, compare protein levels of cells electroporated with either siControl or siRAR). This depletion did not prevent the activation of the PPAR-LBD by Ro 41-5253, proving that RAR is not involved in the PPAR activation (Fig. 5B). On the contrary, it slightly increased the efficiency by which Ro 41-5253 activated the PPAR-LBD.
Fig. 6. Ro 41-5253 activates the PPAR-LBD in cells depleted of RAR. Reduction of RAR protein levels by electroporating siRNA control or siRNA RAR in 293T cells (A). After 24 h, cells were transfected with pGal4-hPPAR_DEF and pGal5-Tk-pGL3 and incubated for another 24 h with 3 µM Ro 41-5253. Data are shown as -fold induction over vehicle-treated cells (B). *, p < 0.05.
Ro 41-5253 Competes with Specific Ligands for Direct Binding to PPAR. Because RAR was nonrelevant for the PPAR activation, we questioned whether Ro 41-5253 directly interacts with the PPAR protein. We therefore measured the competition with specific radiolabeled ligands to human PPAR-LBDs by a scintillation proximity assay and calculated the concentrations for half-maximal displacement. Ro 41-5253 was able to bind to PPAR and PPAR/ with IC50 values in the low micromolar range (Table 2).
TABLE 2 IC50 of Ro 41-5253 in displacing specific 3H-labeled ligands from hsPPAR isoforms
Ro 41-5253 displaces specific ligands from hsPPAR-LBDs as measured by a scintillation proximity assay. Data represent mean IC50 values of three independent experiments.
In this study, we have shown that Ro 41-5253 is a strong inducer of adipogenesis in mouse and human preadipocytes. We provide evidence that Ro 41-5253 can bind and activate PPAR, the master regulator of adipogenesis. On the other hand, Ro 41-5253 could bind PPAR/ but was not able to activate the PPAR/-LBD Gal4 fusion protein. Using pharmacological and biochemical interventions, we can exclude the involvement of RAR in the PPAR activating property of Ro 41-5253. Thus, Ro 41-5253 is not only an antagonist for RAR but also an agonist for PPAR.
Although the potency of Ro 41-5253 was comparable with pioglitazone (Table 1), its efficiency of promoting lipid accumulation, target gene expression, and activation of the PPAR-LBD was consistently lower than by using pioglitazone, a full agonist (Figs. 2A and 4A). Although Ro 41-5253 seems to bind the PPAR-LBD in a manner similar to that of TZDs, TZD-induced activation could be diminished with increasing concentrations of Ro 41-5253, which is the traditional definition of partial agonist behavior. The exact mechanism for this partial agonism of PPAR by Ro 41-5253 may involve selective or reduced interaction with NR coactivators as has been shown for other partial agonists, such as N-(9-Fluorenylmethoxycarbonyl)-L-leucine (Rocchi et al., 2001), MCC-555 (Reginato et al., 1998) and certain angiotensin receptor blockers (Schupp et al., 2005).
No known endogenous ligand for NRs has both RAR-antagonizing and PPAR-activating properties. However, it is intriguing to think of the existence of endogenous pleiotropic ligands, considering the functional antagonism of RAR and PPAR for instance during adipogenesis (Chawla and Lazar, 1994; Xue et al., 1996). Furthermore, Ro 41-5253, chemically derived from atRA, has no obvious similarity with any synthetic PPAR activator. On the other hand, it shares structural elements with arachidonic metabolites, such as prostaglandin J2, that have been shown to activate PPAR (Forman et al., 1995; Kliewer et al., 1995; Yu et al., 1995).
Finally, the unexpectedly pleiotropic effects of Ro 41-5253 that we have uncovered indicate that the caution must be applied to the interpretation of effects elicited by Ro 41-5253 and previously attributed to specific RAR antagonism. This caution pertains not only to studies of adipogenesis (Kamei et al., 1994) but also to many other cell types. For example, several studies have used Ro 41-5253 as an ostensibly specific probe of RAR function in breast cancer (Shang et al., 1999; Schneider et al., 2000; Emionite et al., 2004; Lu et al., 2005; Toma et al., 2005), where PPAR is expressed at significant levels, not only in cell lines such as MCF-7, MDA-MB-231. or ZR-75.1 (Kilgore et al., 1997; Nwankwo and Robbins, 2001; James et al., 2003) but also in primary and metastatic breast adenocarcinomas (Kilgore et al., 1997; Mueller et al., 1998). Ro 41-5253 was able to bind / in the low micromolar range, which adds to the concerns about overinterpretation of its effects as RAR-specific in biological systems. Unfortunately, to our knowledge, no other RAR antagonist is commercially available.
Acknowledgements
We thank Dr. M. Wabitsch (Pediatric Endocrinology, University of Ulm, Germany) for the SGBS preadipocyte cell line.
ABBREVIATIONS: PPAR, peroxisome proliferator-activated receptor; RAR, retinoic acid receptor; TZD, thiazolidinedione; RXR, retinoid X receptor; NR, nuclear receptors; atRA, all-trans retinoid acid; Ro 41-5253, (E)-S,S-dioxide-4-(2-(7-(heptyloxy)-3,4-dihydro-4,4-dimethyl-2H-1-benzothiopyran-6-yl)-1-propenyl)-benzoic acid; IBMX, 3-isobutyl-1-methylxanthine; SGBS, Simpson Golabi Behmel Syndrome; LBD, ligand binding domain; aP2, adipose protein 2; GW2433, 2-(4-{3-[1-[2-(2-chloro-6-fluoro-phenyl)-ethyl]-3-(2,3-dichloro-phenyl)-ureido]-propyl}-phenoxy)-2-methyl-propionic acid; PPRE, peroxisome proliferator-activated receptor response element; AM-580, 4-[(5,5,8,8-tetramethyl6,7-dihydronaphthalene-2-carbonyl)amino]benzoic acid.
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作者单位：Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine and Department of Genetics, and the Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania (M.S., J.C.C., R.J.K., A.N.B., M.A.L); Division of Endocrinology,
【关键词】 Nordihydroguaiaretic
Nordihydroguaiaretic acid (NDGA), a well known lipoxygenase inhibitor, actually has pleiotropic effects on cells, which include cell proliferation, apoptosis, differentiation, and chemotaxis. We and others have shown previously that this compound causes Golgi disassembly by an unknown mechanism. In this study, we show that, in parallel with Golgi disassembly, NDGA induces the accumulation of the microtubule minus-end-directed motor dynein-dynactin complex at the centrosome, where microtubules minus-ends lie. Concomitant with this accumulation, dynein-dynactin-interacting proteins, such as ZW10 and EB1, were also redistributed to the centrosomal region. In cells where microtubules were depolymerized by nocodazole, NDGA promoted the formation of filaments consisting of dynein-dynactin and its interacting proteins, suggesting that it stimulates the association of these proteins in an ordered, not random, manner. Loss of dynactin function abolished not only NDGA-induced redistribution in intact cells but also filament formation in nocodazole-treated cells. The latter finding implies that dynactin is a key molecule for the association between dynein-dynactin and its interacting proteins. In mitotic cells, NDGA induced robust accumulation of dyneindynactin and its interacting proteins at the spindle poles. These results taken together suggest that NDGA perturbs membrane traffic by affecting the function of the microtubule motor dynein-dynactin complex and its auxiliary proteins. To our knowledge, NDGA is the first case of a reagent that can modulate dynein-dynactin-related processes.
Nordihydroguaiaretic acid (NDGA) is a drug that affects a wide variety of cellular processes, including growth factor- and tumor necrosis factor-induced signal transduction (Domin et al., 1994; Lee et al., 2003; West et al., 2004), leukocyte chemotaxis (Goetzl, 1980), myoblast cell differentiation (Ito et al., 2005), cancer cell proliferation (Avis et al., 1996; McDonald et al., 2001; Seufferlein et al., 2002; Youngren et al., 2005), and viral proliferation in infected cells (Gnabre et al., 1995). It can also induce nitric-oxide synthase expression (Ramasamy et al., 1999), regulate calcium channel activity (Korn and Horn, 1990; Huang et al., 2004), and inhibit growth of -amyloid (1-40) protofibril (Moss et al., 2004). Although many of the effects of NDGA on cellular events seem to be ascribable to its action as a lipoxygenase inhibitor or an antioxidant, some are obviously peculiar to the action of NDGA (Korn and Horn, 1990; Lee et al., 2003; Huang et al., 2004; Ito et al., 2005). The anticancer activity of NDGA may be due in part to its inhibition of protein kinase C and receptor tyrosine kinases (Domin et al., 1994; Youngren et al., 2005).
We have shown for the first time that NDGA perturbs intracellular membrane traffic. NDGA inhibits the vesicle-mediated transport of vesicular stomatitis virus-encoded glycoprotein both within the Golgi apparatus in vitro (Tagaya et al., 1993) and from the endoplasmic reticulum (ER) to the Golgi in vivo (Tagaya et al., 1996). Later studies demonstrated that NDGA induces Golgi disassembly (Yamaguchi et al., 1997), which leads Golgi components to be redistributed to the ER (Drecktrah et al., 1998; Fujiwara et al., 1998a). In addition to the secretory pathway, this compound blocks the endocytic pathway in human dendritic cells in a manner independent of inhibition of lipoxygenases and prevention of reactive oxygen species formation (Ramoner et al., 1998), raising the possibility that NDGA affects the machinery generally required for vesicular transport but not that for specific transport processes. In this context, the finding of Nakamura et al. (2003) that showed that NDGA is capable of stabilizing microtubules (MTs) is worth noting, because they generally participate in membrane transport along the secretory and endocytic pathways. However, the relationship between the two NDGA-induced effects, MT stabilization and transport defect, is totally unknown.
We have recently demonstrated that, in interphase cells, ZW10 is present in the ER membrane, as well as in the cytosol, and plays a role in membrane traffic between the ER and Golgi (Hirose et al., 2004). ZW10 was originally characterized as a kinetochore-associated component that interacts with dynamitin (Starr et al., 1998), a subunit of dynactin that provides a link between the MT minus-enddirected motor dynein and cargo molecules. In the course of our study on ZW10, we found that NDGA induces the accumulation of ZW10 at the centrosome, where MT minus-ends lie. To better understand the mechanism for NDGA-induced Golgi disassembly, we examined in detail how NDGA induces ZW10 redistribution. We found that NDGA affects dynein-dynactin such that this motor associates more tightly with ZW10. The enhanced association may allow a long-range movement of dynein-dynactin and its interacting proteins toward the centrosome, which leads to imbalance in membrane traffic, thereby causing Golgi disassembly.
Antibodies. Monoclonal antibodies (Abs) against dynamitin, EB1 and p150Glued were obtained from Transduction Laboratory. Monoclonal Abs against dynein intermediate chain (IC) and -tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Accurate Chemical & Scientific (Westbury, NY), respectively. The preparation and sources of other Abs were described previously (Yoshimori et al., 1988; Hirose et al., 2004).
Chemicals. Ascorbic acid, N-acetyl cysteine, -tocopherol, propidium iodide, and paclitaxel (Taxol) were obtained from Wako Pure Chemicals (Osaka, Japan). Nocodazole (Noc) and 5,8,11,14-eicosatetraynoic acid were obtained from Sigma (St. Louis, MO). NDGA was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA) and freshly dissolved in dimethyl sulfoxide (DMSO) before use. When cells were incubated with these reagents, fetal calf serum was omitted from culture medium. Transferrin (Tf)-fluorescein isothiocyanate (FITC) was purchased from Invitrogen (Carlsbad, CA).
Plasmid, Cell Culture, and Transfection. The full-length cDNA of dynamitin was inserted into pFLAG-CMV2. HeLa cells were cultured in Eagle's minimum essential medium supplemented with 50 IU/ml penicillin, 50 µg/ml streptomycin, and 10% fetal calf serum. Transfection of cells with plasmids was performed according to the manufacturer's protocol using LipofectAMINE PLUS reagent (Invitrogen). Double thymidine block was performed as described by Chan et al. (2000).
Immunoprecipitation and Immunofluorescence. Preparation of cell lysates and immunoprecipitation were performed as described previously (Hirose et al., 2004). Immunofluorescence microscopy was performed as described previously (Hirose et al., 2004). Unless otherwise stated, cells were fixed with methanol at -20°C for 5 min.
Semi-Intact Cells. To express ZW10-GFP, Tet-on inducible ZW10-GFP expression cells (Hirose et al., 2004) were incubated with 1.0 µg/ml doxycycline for 48 h. The cells were washed twice with permeabilization buffer (25 mM HEPES-KOH, pH 7.4, 115 mM potassium acetate, 2.5 mM MgCl2, 2 mM EGTA, and 1 mg/ml glucose), and then permeabilized with 40 µg/ml digitonin in the same buffer at 0°C for 5 min. After washing with permeabilization buffer twice, the cells were incubated in 1 ml of the reaction mixture at 32°C for 1 h. The reaction mixture contained permeabilization buffer plus an ATP-regenerating system (8 mM creatine phosphate, 1 mM ATP, and 50 µg/ml creatine kinase) and 2.5 mg/ml rat liver cytosol.
RNA Interference. Duplex RNAs for targeting were ZW10 (102) (5'-AAGGGTGAGGTGTGCAATATG-3') and p150Glued (207) (5'-TGATGGAACTGTTCAAGGC-3'). They were purchased from Japan Bioservice (Asaka, Japan). RNA interference experiments were conducted as described previously (Hirose et al., 2004)
Uptake and Recycling of Tf. Uptake and recycling experiments were conducted as described previously (Hirose et al., 2004) with a slight modification.
NDGA Caused Redistribution of ZW10 to the Centrosomal Region. Upon incubation of HeLa cells with 30 µM NDGA for 1 h, ZW10 was redistributed from the ER to the perinuclear area in parallel with dispersion of a Golgi marker protein, GM130 (Fig. 1A, +NDGA, top and middle rows). The perinuclear region where ZW10 was accumulated was marked by -tubulin, a centrosome marker (bottom row).
Fig. 1. Characterization of NDGA-induced ZW10 redistribution. A, HeLa cells were treated for 1 h with 0.1% DMSO (Vehicle) or 30 µM NDGA (+NDGA) and then double stained with Abs against ZW10 and GM130 (top and middle rows) or -tubulin (bottom row). N denotes the position of the nucleus. Scale bar, 5 µm. B, HeLa cells were treated with 0.1% DMSO (Vehicle) for 1 h, 20 µM -tocopherol for 5 h, 1 mM ascorbic acid for 5 h, 1mM N-acetyl cysteine (NAC) for 5 h, 20 µM 5,8,11,14-eicosatetraynoic acid (ETYA) for 3 h, or 10 µg/ml paclitaxel for 2 h. Fetal calf serum was omitted during incubation with these reagents. The cells were stained with an Ab against ZW10. Scale bar, 5 µm. C, requirement of energy and cytosolic factor(s) for ZW10 redistribution. Digitonin-permeabilized HeLa cells expressing ZW10-GFP were incubated at 32°C for 60 min without or with 30 µM NDGA in the presence of cytosol (Cy) and/or an ATP-regenerating system (ATP). Arrows indicate the position of centrosomal ZW10-GFP. Scale bar, 5 µm. The quantitative results are shown on the right. Error bars represent the S.E.M. for three experiments.
To gain insight into the mechanism for NDGA-induced ZW10 redistribution, we first examined whether this redistribution is caused by scavenging of reactive oxygen species or inhibiting lipoxygenases. Cells were incubated with an antioxidant (-tocopherol, ascorbic acid, or N-acetyl cysteine) or a lipoxygenase inhibitor (5,8,11,14-eicosatetraynoic acid) for a prolonged time, and then the distribution of ZW10 was analyzed. As shown in Fig. 1B, none of the reagents induced the redistribution of ZW10 to the centrosomal region. In addition, paclitaxel, a well known MT-stabilizing reagent, did not cause ZW10 redistribution. These results indicate that ZW10 redistribution to the centrosomal region was not due to the prevention of reactive oxygen species formation, inhibition of lipoxygenases, or stabilization of MTs.
To determine whether energy and/or cytosolic factors are necessary for NDGA-induced ZW10 redistribution, we used cells expressing ZW10-GFP. ZW10-GFP-expressing cells were permeabilized to remove the cytosol (including cytosolic ZW10-GFP), and then NDGA, together with ATP and rat liver cytosol, was added. As shown in Fig. 1C, ZW10 was translocated to the centrosomal region only when all components were present, implying that ATP and cytosolic component(s) are required for ZW10 redistribution. In addition, this result confirmed that the ER-associated form of ZW10 is translocated.
ZW10 Redistribution Was Driven by Dynein-Dynactin. Given that ZW10 interacts with dynamitin (Starr et al., 1998), a subunit of dynactin, the most straightforward interpretation of the results described above is that NDGA induces ZW10 redistribution to the centrosome by facilitating its minus-end-directed movement driven by the MT motor dynein-dynactin complex. This idea was supported by the observation that a subunit of the dynactin, p150Glued, and dynein IC also accumulated at the centrosomal region upon NDGA treatment (Fig. 2A, +NDGA).
Fig. 2. Dynactin function is required for NDGA-induced ZW10 redistribution. A, HeLa cells were treated with 0.1% DMSO (Vehicle) or 30 µM NDGA (+NDGA) for 1 h and then double-stained with Abs against ZW10 and p150Glued or dynein IC. Scale bar, 5 µm. B, HeLa cells were transfected with the plasmid for FLAG-dynamitin or DsRed. After 24 h, the cells were treated with 30 µM NDGA for 1 h and then double stained with Abs against ZW10 and FLAG (upper row) or only stained for ZW10 (lower row). Although methanol treatment failed to fix most of the expressed DsRed, DsRed-expressing cells were recognizable because of the presence of DsRed remnants. Scale bar, 5 µm. The quantitative results are shown on the right. Error bars represent the S.E.M. for three experiments. C, HeLa cells were transfected without (Mock) or with p150Glued (207) or ZW10 (102). At 72 h after transfection, the cells were solubilized in phosphate-buffered saline with 0.5% SDS and analyzed by immunoblotting. Alternatively, the transfected cells were incubated for 1 h with 0.1% DMSO (Vehicle) or 30 µM NDGA (+NDGA) and then double-stained with Abs against p150Glued and ZW10. Scale bar, 5 µm.
To verify that dynein-dynactin mediates the NDGA-induced movement of ZW10 to the centrosomal region, we first examined the effect of overexpression of dynamitin. Overexpression of dynamitin is known to block dynein-dynactinmediated processes by disassembling the dynactin complex (Burkhardt et al., 1997). The plasmid for FLAG-dynamitin or, as a control, DsRed, was transfected into HeLa cells; 24 h after transfection, the cells were treated with NDGA. As shown in Fig. 2B, NDGA-induced ZW10 redistribution was almost completely inhibited by dynamitin overexpression but not by DsRed overexpression.
Next, we examined the effect of knockdown of p150Glued on NDGA-induced ZW10 redistribution. HeLa cells were transfected with a short interfering RNA named p150Glued (207) or ZW10 (102) and incubated for 72 h. Immunoblotting revealed that the expression levels of p150Glued and ZW10 were markedly reduced (Fig. 2C). In p150Glued-depleted cells, ZW10 did not accumulate at the centrosomal region upon NDGA treatment [Fig. 2C, p150Glued (207)], suggesting that dynactin function might be required for NDGA-induced ZW10 redistribution. It should be noted that depletion of ZW10 did not affect the accumulation of p150Glued at the centrosome [Fig. 2C, ZW10 (102)]. This implies that dynactin is required for the redistribution of ZW10 but not vice versa.
NDGA Seemed to Stimulate the Association of Dynactin with ZW10. Consistent with the idea that NDGA-induced redistribution of ZW10 is mediated by dynein-dynactin, ZW10 was not accumulated at the centrosomal region when MTs were depolymerized by Noc before NDGA treatment. It is noteworthy that, in Noc- and NDGA-treated cells, ZW10 exhibited filamentous structures at the cell periphery. These filaments were positive for the dynactin subunit p150Glued (Fig. 3A) but negative for MTs (data not shown). The formation of these filaments could imply that NDGA stimulates the association between ZW10 and dynactin in a manner that forms an ordered structure. To test whether NDGA influences the association between these proteins, we performed immunoprecipitation using an anti-ZW10 Ab. As shown in Fig. 3A, the amounts of p150Glued and dynein IC coprecipitated with ZW10 were increased in the presence of NDGA, whereas -tubulin, which was used as a negative control, was not coprecipitated with ZW10 regardless of whether NDGA was present or not.
Fig. 3. Dynactin is required for ZW10 filament formation induced by NDGA. A, HeLa cells were pretreated with 10 µg/ml Noc for 1 h to depolymerize MTs. NDGA (30 µM) was added, and then the cells were incubated for 1 h and double-stained with Abs against ZW10 and p150Glued. The boxed area is shown on an expanded scale. Scale bar, 5 µm. For an immunoprecipitation experiment, 293T cells were incubated for 1 h with 0.1% DMSO (Vehicle) or 30 µM NDGA (+NDGA), or pretreated with 10 µg/ml Noc for 1 h followed by incubation with 10 µg/ml Noc plus 30 µM NDGA for 1 h (+Noc/NDGA). Cell lysates were prepared, immunoprecipitated with an anti-ZW10 Ab and analyzed by immunoblotting with the indicated Abs. *, immunoglobulin heavy chain. B, p150Glued or ZW10 was knocked down as described in the legend to Fig. 2C. The cells were incubated with 10 µg/ml Noc for 90 min, and then with 10 µg/ml Noc plus 30 µM NDGA for 1 h. The cells were stained with Abs against p150Glued and ZW10. Arrowheads indicate the position of typical filamentous structures. Scale bar, 5 µm.
To obtain evidence that dynactin is required for the formation of ZW10-positive filaments, the expression of p150Glued was suppressed by RNA interference, and then NDGA was added to cells with depolymerized MTs. As shown in Fig. 3B, no obvious ZW10 filaments were observed in p150Glued-depleted cells (middle row), whereas p150Glued-positive filamentous structures were detected in ZW10-depleted cells (bottom row).
NDGA Induced Centrosomal Accumulation of EB1. Is the action of NDGA specific for ZW10? Alternatively, do other dynactin-interacting proteins also undergo redistribution in the presence of NDGA? To address this question, we investigated the effect of NDGA on the localization of EB1, a MT plus-end tracking protein that is known to interact directly with p150Glued (Berrueta et al., 1999). Without NDGA treatment, EB1 displayed a "comet tail" pattern, representing its predominant association with the growing ends of MTs (Fig. 4, Vehicle). Upon incubation of cells with NDGA, EB1 as well as ZW10 accumulated at the centrosomal region (Fig. 4, +NDGA). As observed for ZW10, NDGA-induced EB1 redistribution was prevented by dynamitin overexpression or depletion of p150Glued and the formation of EB1 filaments observed in Noc-treated cells was dependent on the presence of p150Glued (data not shown).
Fig. 4. Distribution of EB1 in NDGA-treated cells. HeLa cells were treated for 1 h with 0.1% DMSO (Vehicle) or 30 µM NDGA (+NDGA) and then double-stained with Abs against EB1 and ZW10. Scale bar, 5 µm.
NDGA Induced Robust Accumulation of Dynein-Dynactin and Its Interacting Proteins at the Spindle Poles. During mitosis, dynein-dynactin and its interacting proteins, including ZW10 and EB1, participate in the organization of spindles, spindle checkpoint, and segregation of chromosomes (Chan et al., 2000; Green et al., 2005). We investigated whether the distributions of dynein-dynactin and its interacting proteins in mitotic cells are affected by NDGA, as observed in interphase cells. Incubation of mitotic cells with NDGA resulted in robust accumulation of these proteins at the spindle poles (Fig. 5A +NDGA). It was remarkable that almost all cells treated with NDGA displayed a metaphase-like pattern. Moreover, a significant fraction of NDGA-treated cells showed aberrant chromosome distribution. In addition to being localized at the metaphase plate, chromosomes were localized at or near spindle poles (Fig. 5B, +NDGA) or outside of the spindles (data not shown).
Fig. 5. Effect of NDGA on the distribution of dynein-dynactin and its interacting proteins in mitotic cells. HeLa cells were synchronized by double thymidine block. At 6 h after washing out thymidine, the cells were incubated for 1 h with 0.1% DMSO (Vehicle) or 30 µM NDGA (+NDGA) and then double-stained with the indicated Abs (A) or with ZW10 and propidium iodide (PI) (B). Scale bar, 5 µm. The percentage of cells with abnormal chromosome alignment was scored. Error bars represent the S.E.M. for four experiments.
Effect of NDGA on the Distribution of Tf Receptor. Dynein-dynactin is present on endosomes (Habermann et al., 2000) and endocytosis of Tf is blocked by disruption of dynein-dynactin function (Burkhardt et al., 1997). Because NDGA inhibits the endocytic pathway (Ramoner et al., 1998), we examined whether the localization of TfR is affected by NDGA. In control cells, TfR was distributed throughout the cytoplasm with some concentration in the perinuclear region, which may represent recycling endosomes (Fig. 6A, Vehicle). Upon incubation with NDGA, TfR accumulated in the perinuclear region with a marked loss of peripheral localization (Fig. 6A, +NDGA). Consistent with a previous study (Ramoner et al., 1998), uptake of Tf-FITC was blocked in the presence of NDGA (Fig. 6B, +NDGA). Furthermore, recycling of Tf-FITC (i.e., release of incorporated Tf-FITC into the medium) was also blocked by NDGA, and the Tf-FITC remained colocalized with TfR in the perinuclear region (Fig. 6C, +NDGA). This phenotype is in marked contrast to that in cells overexpressing dynamitin. In dynamitin-overexpressing cells, uptake of Tf occurs but its movement to the cell center is blocked (Burkhardt et al., 1997).
Fig. 6. Redistribution of TfR by NDGA. A, HeLa cells were incubated for 1 h with 0.1% DMSO (Vehicle) or 30 µM NDGA (+NDGA), fixed with 4% paraformaldehyde at room temperature for 20 min and double-stained with Abs against TfR and a Golgi marker, syntaxin 5 (Syn 5). Scale bar, 5 µm. B, HeLa cells were preincubated in the absence of fetal calf serum for 1 h to remove endogenous Tf, and then 0.1% DMSO (Vehicle) or 30 µM NDGA (+NDGA) was added. After a 1-h incubation, Tf-FITC was added to a final concentration of 25 µg/ml, and the incubation was continued for another 1 h. The cells were stained with an Ab against TfR. C, HeLa cells were incubated with 25 µg/ml Tf-FITC for 1 h to allow the uptake of ligand. The cells were washed and incubated with 0.1% DMSO (Vehicle) or 30 µM NDGA (+NDGA). After a 1-h incubation, the cells fixed and stained with an Ab against TfR. D, HeLa cells were synchronized by double thymidine block. At 6 h after washing out thymidine, the cells were treated for 1 h with 0.1% DMSO (Vehicle) or 30 µM NDGA (+NDGA) and then double stained with Abs against TfR and -tubulin. Scale bar, 5 µm.
Uptake and recycling of Tf are arrested during mitosis (Sager et al., 1984). We were interested in whether TfR distribution in mitotic cells was affected by NDGA. In mitotic cells, TfR was found to be distributed diffusely throughout the cell with some concentration at the spindle poles (Fig. 6D, Vehicle). In marked contrast to the case of interphase cells, addition of NDGA did not significantly affect the localization of TfR (Fig. 6D, +NDGA). This may imply that the connection between TfR-containing endosomes and dynein-dynactin is regulated in a cell cycle-dependent manner. Indeed, previous work demonstrated that dynein-dynactin detaches from membranes in metaphase Xenopus laevis egg extracts (Niclas et al., 1996).
In the present study, we demonstrated that NDGA induces the accumulation of dynein-dynactin at the centrosome in interphase cells and at the spindle poles in mitotic cells. Concomitant with the movement of dynein-dynactin, dynactin-interacting proteins, such as ZW10 and EB1, were also transported, depending on cell cycle, to the centrosomal region or the spindle poles. Upon NDGA treatment, TfR was also redistributed to the centrosomal region in interphase cells. Loss of dynactin function caused by disassembly of dynactin by dynamitin overexpression or depletion of the dynactin subunit p150Glued abrogated the NDGA-induced movement of dynactin-interacting components to the centrosomal region, whereas depletion of ZW10 had no effect on NDGA-induced redistribution of dynein-dynactin. These results suggest that NDGA can affect dynein-dynactin function and facilitate processes mediated by this motor.
The Mechanism for NDGA Action on Dynein-dynactin and Its Interacting Proteins. How can NDGA induce the movement of dynein-dynactin and its interacting proteins to the centrosome? The phenotype of cells with depolymerized MTs provides a clue to understanding the mechanism for this NDGA action. When MTs were depolymerized, NDGA promoted the formation of filaments comprising dynein-dynactin, ZW10, and EB1. The filament formation in the absence of intact MTs most likely reflects the enhanced association between dynein-dynactin and its interacting proteins. Perhaps the NDGA-induced association of these proteins is highly ordered because NDGA treatment of Noc-treated cells did not induce aggregation, which is a hallmark of nonspecific protein-protein interactions. The result of immunoprecipitation analysis supported the view that NDGA strengthens the association between dynein-dynactin and ZW10. Given that dynactin functions not only as a cargo adaptor but also as a factor conferring dynein processivity (Culver-Hanlon et al., 2006), it is tempting to speculate that the enhanced association between dynein-dynactin and its interacting proteins allows a long-range movement of these proteins toward the minus-end of MTs, leading to their accumulation at the centrosomal region.
Mechanism of Golgi Disassembly and Endocytosis Inhibition by NDGA. Our results suggest that the Golgi disassembly and blockage of endocytosis induced by NDGA are due to the excessive stimulation of dynein-dynactin-mediated processes. This view can explain the finding by Fujiwara et al. (1998b) that, upon NDGA treatment, a marker for the ER-Golgi intermediate compartment, ERGIC-53, rapidly moves to the perinuclear, centrosomal region before Golgi enzymes to be redistributed to the ER. This movement of ERGIC-53 is probably coupled to the movement of dyneindynactin to the centrosomal region. The ER-Golgi intermediate compartment coalescences with the Golgi to form aggregated membrane structures, which may fuse directly with proximal ER membranes (Fujiwara et al., 2003). In the case of endocytosis, TfR-containing endosomes, in association with dynein-dynactin, accumulate at the centrosomal region, leading to a deficiency of TfR at the plasma membrane.
Based on the observation that activation of trimeric GTP-binding proteins prevents NDGA-induced Golgi disassembly, we previously suggested that this reagent affects the function of trimeric GTP-binding proteins (Yamaguchi et al., 1997). However, as dynein-dynactin and its interacting proteins accumulated at the centrosomal region upon NDGA treatment irrespective of the presence or absence of aluminum fluoride (data not shown), the previous interpretation should be modified. Perhaps, activation of GTP-binding proteins stabilizes Golgi membranes by extensively recruiting peripheral protein complexes to the membranes so that lateral movement of Golgi membrane proteins is constrained (Cole et al., 1996). The stabilized Golgi apparatus can maintain its structure even when large amounts of dynein-dynactin and its interacting proteins accumulate at the centrosomal region.
NDGA as a Drug to Regulate MT Stability and MT-related Processes. NDGA, unlike paclitaxel, does not promote MT polymerization, although it stabilizes MTs (Nakamura et al., 2003). Based on the result of an indirect measurement, Nakamura et al. (2003) suggested that NDGA prevents MT depolymerization by directly binding to tubulin. However, no tubulin was found in the filaments formed upon NDGA treatment of Noc-treated cells, suggesting that the target for NDGA is, at the very least, not limited to tubulin. Because plus-end tracking proteins, such as EB1 and dynactin, play a role in regulating MT dynamics (Carvalho et al., 2003), it is possible that the NDGA, in addition to its direct binding to tubulin, indirectly stabilizes MTs by regulating the function of plus-end tracking proteins.
The action of NDGA on MTs and/or their associated proteins may provide insight into the action of NDGA, not as a lipoxygenase inhibitor or an antioxidant. Lee et al. (2003) reported that NDGA, but not other antioxidants, inhibits transforming growth factor- activity by blocking the phosphorylation and nuclear translocation of Smad2. This effect of NDGA can be explained by the fact that Smad2 binds to MTs (Dong et al., 2000). NDGA may block transforming growth factor- activity by stabilizing MTs or affecting MT dynamics. Indeed, depolymerization of MTs by Noc was found to induce the phosphorylation and nuclear translocation of Smad2 (Dong et al., 2000). Because many transcription factors and protein kinases interact with MTs (Gundersen and Cook, 1999), NDGA may influence transcription and signal transduction by affecting the stability of MTs and/or dynein-dynactin function.
MT-stabilizing reagents such as paclitaxel have been successfully used in the treatment of solid tumors (Bergstralh and Ting, 2006). The suppression of MT dynamics disrupts the mitotic spindle, halting the cell cycle at the metaphaseanaphase and eventually leading to apoptosis (Yvon et al., 1999). In the presence of NDGA, the mitotic spindle seemed not to be substantially disrupted. The different effects of paclitaxel and NDGA on the mitotic spindle are consistent with the observations that NDGA does not affect the radiation of MTs originating from the centrosome in interphase cells, whereas paclitaxel perturbs MT array (Nakamura et al., 2003). The misalignment of chromosomes induced by NDGA might be due to premature removal of spindle checkpoint proteins, such as ZW10, from kinetochores. Although the mechanisms of stabilization of MTs by paclitaxel and NDGA are probably different, the stabilization of MT by NDGA also seems to halt the cell cycle at the metaphaseanaphase. This may explain why NDGA causes apoptosis in several different tumor xenografts (Avis et al., 1996; Seufferlein et al., 2002).
In summary, we have disclosed a novel action of NDGA (i.e., stimulation of processes mediated by the MT motor dynein-dynactin complex). The anticancer and other drug activities of NDGA should be investigated in the light of MT-related processes.
ABBREVIATIONS: NDGA, nordihydroguaiaretic acid; ER, endoplasmic reticulum; MT, microtubule; Ab, antibody; IC, intermediate chain; Noc, Nocodazole; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; Tf, Transferrin; TfR, Tf receptor.
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作者单位：School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan (K.A., K.T., M.T.); Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan (T.Y.); and Department of Biochemistry, University of Bristol, School of Medical Scie
【关键词】 Agonist properties
Agonist properties of the P2X7 receptor (P2X7R) differ strikingly from other P2X receptors in two main ways: high concentrations of ATP (> 100 µM) are required to activate the receptor, and the ATP analog 2',3'-O-(4-benzoyl-benzoyl)ATP (BzATP) is both more potent than ATP and evokes a higher maximum current. However, there are striking species differences in these properties. We sought to exploit the large differences in ATP and BzATP responses between rat and mouse P2X7R to delineate regions or specific residues that may be responsible for the unique actions of these agonists at the P2X7R. We measured membrane currents in response to ATP and BzATP at wild-type rat and mouse P2X7R, at chimeric P2X7Rs, and at mouse P2X7Rs bearing point mutations. Wild-type rat P2X7R was 10 times more sensitive to ATP and 100 times more sensitive to BzATP than wild-type mouse P2X7R. We found that agonist EC50 values were determined solely by the ectodomain of the P2X7R. Two segments (residues 115-136 and 282-288), when transposed together, converted mouse sensitivities to those of rat. Point mutations through these regions revealed a single residue, asparagine284, in the rat P2X7R that fully accounted for the 10-fold difference in ATP sensitivity, whereas the 100-fold difference in BzATP sensitivity required the transfer of both Lys127 and Asn284 from rat to mouse. Thus, single amino acid differences between species can account for large changes in agonist effectiveness and differentiate between the two widely used agonists at P2X7 receptors.
The P2X7 receptors belong to a family of cation-permeable membrane proteins gated by extracellular ATP. They can be distinguished from other family members (P2X1-P2X6)by several properties. First, receptor activation is followed in several seconds by the appearance of a permeation pathway that allows passage of molecules up to 900 Da (North, 2002). Second, activation by extracellular ATP rapidly engages a series of cytoskeletal and mitochondrial alterations, which include actin/-tubulin rearrangements, phosphatidylserine translocation, mitochondrial swelling and loss of mitochondrial membrane potential, and membrane blebbing (MacKenzie et al., 2001, 2005; Le Feuvre et al., 2002; Morelli et al., 2003; Verhoef et al., 2003; Pfeiffer et al., 2004; Elliott et al., 2005; Ferrari et al., 2006). Third, the P2X7 receptor in immune cells of monocyte/macrophage lineage becomes upregulated and functionally active in response to inflammatory stimuli (Guerra et al., 2003; Ferrari et al., 2006); its activation there engages cascades that culminate in processing and release of interleukin-1, release of tumor necrosis factor , and activation of nuclear factor-B (North, 2002; Ferrari et al., 2006). Finally, studies using mice in which the P2X7 receptor has been deleted further support a role in inflammatory processes (Solle et al., 2001; Labasi et al., 2002; Chessell et al., 2005).
P2X7 receptors can also be readily distinguished from other family members when membrane ionic current is measured directly. First, they are more potently inhibited by extracellular calcium and/or magnesium. Second, they are unusually insensitive to ATP; the EC50 value (>300 µM) is approximately 100-fold greater than for other P2X receptors (North, 2002). Third, the ATP analog 2',3'-O-(4-benzoyl-benzoyl)ATP (BzATP) is considerably more potent that ATP itself at P2X7 receptors, whereas at other P2X receptors it is less potent (North, 2002; Baraldi et al., 2004). Fourth, certain antagonists are selective for the P2X7 receptor, although few have been studied at the primary effect of membrane current (Humphreys et al., 1998; Jiang et al., 2000).
There are marked species differences in agonist and antagonist pharmacology for P2X7 receptors. The effectiveness of BzATP relative to ATP, which often has been used as the primary distinguishing feature of the P2X7 receptor, is not equally reliable among species (Surprenant et al., 1996; Chessell et al., 1998a,b; Hibell et al., 2000; Young et al., 2006). For example, isoquinolone derivatives such as KN-62 and KN-04 block human P2X7 receptors with low nanomolar affinity but are without effect at rodent P2X7 receptors, even at high micromolar concentrations (Humphreys et al., 1998; Baraldi et al., 2004). Conversely, Brilliant Blue G is 20 times more potent at rat than human P2X7 receptors (Jiang et al., 2000).
Fig. 1. Alignment of rat and mouse P2X7R sequences and design of experimental approach. A, amino acid differences between species are shown in red boldface type; transmembrane domains indicated by black lines; boxed residues (115-136 and 282-288) show regions of lowest conservation in the ectodomain. B, schematic representation of chimeras where red and black represent rat and mouse P2X7R sequence, respectively, and are shown in this, and all subsequent figures, as rat P2X7R residues inserted into mouse P2X7R background.
The differences in properties between the P2X7 receptors of different species are important for several reasons. First, quite different polymorphisms have been described in the human and mouse P2X7 receptors (Adriouch et al., 2002; Gu et al., 2004; Cabrini et al., 2005; Shemon et al., 2005); these may affect their properties in different ways. Second, pharmacological characterizations and structure-function studies of P2X7 receptors have mainly been obtained from heterologous expression of the rat ortholog; such studies will be of limited value if properties of agonists and antagonists are very different from human. Third, increasing the use of P2X7 receptor knockout mice as animal models of disease (e.g., neuropathic and inflammatory pain, Chessell et al., 2005; osteopenia, Li et al., 2005; and joint inflammation, Labasi et al., 2002) drives the need for a more precise knowledge of the properties of this receptor. In general, information concerning residues involved in agonist action at P2X receptors is extensive for P2X1, P2X2, and P2X4 receptors but minimal for P2X7 receptors (North, 2002; Vial et al., 2004; Zemkova et al., 2004).
The purpose of the present study was to identify residues in P2X7 receptors that may be involved in agonist action. We recently noticed that the sensitivity to BzATP was different between rat and mouse P2X7 receptors (Young et al., 2006). Of the 595 amino acids of the P2X7 receptors, 88 are different between mouse and rat (Fig. 1A). In the present study, we have investigated which of these might be responsible for the differences in effectiveness of ATP and BzATP. We have identified two residues in the ectodomain of P2X7R that can account for the differential agonist sensitivities: residue 127, which primarily influences BzATP affinity; and residue 284, which can fully account for ATP sensitivity.
Cell Culture, Transfection, and Site-Directed Mutagenesis. Both rat and mouse P2X7 constructs (Surprenant et al., 1996; Chessell et al., 1998b) were subcloned in the same expression vector background (pcDNA3; Invitrogen, Paisley, UK) and bore C-terminal glutamic acid-glutamic acid epitope tags (EYMPME) to allow detection of protein expression by Western blotting. The 3'- and 5'-noncoding regions of the mouse P2X7 construct was engineered to be identical with that of the rat P2X7 construct to eliminate any differences in expression as a result of noncoding sequences. Point mutations were generated from the above constructs using the PCR overlap extension method and Accuzyme proof-reading DNA polymerase (Bioline, London, UK). Single chimeras (Fig. 1B) were produced using 21-nucleotide synthetic oligonucleotides designed with an inframe 9-nucleotide 5'-adapter tail to introduce overlapping sequences to fuse chimeras between rat and mouse P2X7R sequences. These oligonucleotides were used in combination with the T7 sense and BGH antisense oligonucleotides annealing in the pcDNA3 expression vector sequence. Overlapping amplification products were purified from a 1% agarose gel electrophoresis and used in combination for a second PCR amplification using the T7 sense and BGH antisense oligonucleotides. Three consecutive overlapping PCR amplifications were necessary to produce double chimeras (Fig. 1B). Final T7/BGH amplified products were double-digested with HindIII and XbaI and replaced back in the HindIII-XbaI positions of the original vector. A high concentration of template vector in combination with a proof-reading DNA polymerase and a low number of cycling steps were used in all amplification reactions to minimize random mutations. All subcloned products were confirmed by sequencing (CEQ 2000 Dye Terminator; Beckman Coulter, Fullerton, CA), and protein expression was verified by Western blotting.
Human embryonic kidney 293 cells were transiently transfected using Lipofectamine 2000 (Invitrogen, Paisley, UK). Cells were plated onto 13-mm glass coverslips and maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum and 2 mM L-glutamine at 37°C in a humidified 5% CO2 incubator. We were concerned that differences in expression levels between constructs (Young et al., 2006) might change the pharmacological properties of the currents. Reducing the rat P2X7R cDNA concentration by 10-fold (from the standard 1to0.1 µg/ml) resulted in approximately 50% reduction in maximum currents to ATP or BzATP without a significant change in agonist EC50 values (Fig. 2A). Therefore, in all subsequent experiments we used the standard (1 µg/ml cDNA) concentration for all transfections.
Fig. 2. Comparison of agonist actions at wild-type rat and mouse P2X7R. A, representative currents recorded from cells expressing rat or mouse P2X7Rin response to increasing concentrations of ATP or BzATP as indicated. B, summary of all experiments as illustrated in A for wild-type rat P2X7R for cells in which standard cDNA concentration (1 µg/ml, filled symbols) or 10-fold lower concentration (0.1 µg/ml, open symbols) was used for transfection. C, agonist concentration-response curves obtained for wild-type mouse P2X7R(1 µg/ml cDNA transfected). D, mean ratio of maximum current amplitudes to BzATP relative to ATP shown for rat and mouse P2X7R; these were similar for both species and for low and high expression levels. E, mean ratio of EC50 values of BzATP relative to ATP; low or high expression of rat P2X7R showed the same approximate 35-fold difference, whereas mouse P2X7R showed only 4-fold difference.
Protein Solubilization, Deglycosylation, and Western Blotting. Confluent cells were washed with phosphate-buffered saline and pelleted. Cell pellets were lysed in phosphate-buffered saline containing 1% Triton X-100 and antiproteases (Complete; Roche, Lewes, UK) for 1 h at 4°C followed by centrifugation at 16,000g for 10 min to pellet debris. Total protein samples were removed and assayed for protein content using the Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hemel Hempstead, UK). SDS-polyacrylamide gel electrophoresis sample buffer was added, and the samples were boiled for 2 min at 100°C to denature the protein. Where appropriate, deglycosylation was performed by incubating protein samples (100 µg) for 1 h at 37°C with 500 units of PNGase F (New England Biolabs, Herts, UK) according to the manufacturer's instructions. Samples were separated on 8% polyacrylamide gels according to standard methods and transferred to polyvinylidene difluoride membranes. Western blotting was performed according to standard protocols, and proteins were visualized using anti glutamic acid-glutamic acid primary antibody (Bethyl Laboratories, Cambridge, UK) and horseradish peroxidase-conjugated secondary antibody (Dako UK Ltd., Ely, UK), both at 1:2000 dilution, followed by detection using the ECL-plus kit (Amersham, Bucks, UK) and Kodak Bio-Max MS film (Sigma, Poole, Dorset, UK).
Electrophysiological Recordings. Whole-cell recordings were made 24 to 48 h after transfection using an EPC9 patchclamp amplifier (HEKA Electronik, Lambrecht, Germany). Membrane potential was held at -60 mV. Recording pipettes (5-7 M) were pulled from borosilicate glass (World Precision Instruments, Sarasota, FL) and filled with an intracellular solution that consisted of 145 mM NaCl, 10 mM EGTA, and 10 mM HEPES. The external solution contained 147 mM NaCl, 10 mM HEPES, 13 mM glucose, 2 mM KCl, 2 mM CaCl2, and 1 mM MgCl2. Agonists were applied in divalent-free solution; cells were otherwise super fused with normal external solution. Osmolarity and pH values of all solutions were 295 to 310 mOsM and 7.3, respectively. All experiments were performed at room temperature. Agonists were applied using a RSC 200 fast-flow delivery system (BioLogic Science Instruments, Grenoble, France). Agonists were applied for 5- or 10-s duration to obtain steady-state responses. Concentration-response curves to ATP and BzATP were obtained by first obtaining a maximum response to agonist, because marked run-up of response was observed at both rat and mouse P2X7 receptors, (Surprenant et al., 1996; Chessell et al., 1998b; Young et al., 2006), and then either applying decreasing or increasing concentrations. In either case, similar curves were obtained, provided that a maximum response had been obtained beforehand. Concentration-response curves were plotted using KaleidaGraph (Abelbeck/Synergy Software, Reading, PA) and Prism version 3.0a software (GraphPad Software Inc., San Diego, CA) using the Hill equation provided in Prism.
Comparison of Mouse and Rat P2X7 Receptors. Although all P2X7Rs are potently inhibited by extracellular divalent cations, there is a significant species difference with Mg2+ and Ca2+ being approximately 10-fold more potent to inhibit human than rat P2X7Rs (Surprenant et al., 1996; Rassendren et al., 1997). In preliminary experiments, we found that mouse P2X7R was also more sensitive (by approximately 5-fold) to inhibition by Mg2+ and Ca2+ than was rat P2X7R (data not shown). Therefore, all agonist responses were recorded in the divalent-free cation solution to rule out possible contributions of differential divalent cation sensitivity to agonist concentration responses. We first compared ATP and BzATP concentration-response curves from wild-type rat and mouse P2X7Rs using equal amounts of cDNA for transfection and using a 10-fold lower concentration of rP2X7R cDNA, which we have found previously results in similar protein expression of rat and mouse P2X7Rs (Young et al., 2006). Typical currents recorded from cells transfected with equal cDNA concentrations are shown in Fig. 2A, and results from all experiments are shown in Fig. 2, B and C. Reducing the rP2X7R cDNA concentration by 10-fold resulted in approximately 50% reduction in maximum currents to ATP or BzATP (Fig. 2B) without a significant change in agonist EC50 values or BzATP/ATP maximum current ratio or EC50 ratio (Fig. 2, D and E). The maximum agonist-evoked currents recorded from cells transfected with mP2X7R were approximately the same as those recorded from cells transfected with the 10-fold lower rP2X7R cDNA concentration (Fig. 2, B and C). In agreement with earlier studies on human and rat P2X7Rs (Surprenant et al., 1996; Wiley et al., 1998; Hibell et al., 2000), we found ATP to be a partial agonist relative to BzATP, with maximum BzATP-evoked currents that were 30 to 45% greater than maximum ATP-evoked currents at both rat and mouse P2X7R (Fig. 2D). EC50 values for BzATP and ATP at the rat P2X7R (3.6 and 123 µM, respectively) were several-fold lower than at the mouse P2X7R (285 and 936 µM) (Table 1). These values yield a striking difference in the BzATP/ATP EC50 ratio, which was 34 at the rP2X7R but only 3.3 at the mouse P2X7R (Fig. 2E).
TABLE 1 EC50 values for key P2X7 constructs used in this study
Introducing Segments of Rat P2X7R into Mouse P2X7R. Rat and mouse P2X7R sequences are 84% identical with 88 specific amino acid differences; most of the nonconservative differences are found in two distinct regions of the ectodomain or in the intracellular C-terminal domain (Fig. 1A). Of the 22 nonconserved amino acids in the ectodomain, 8 are found in the region encompassing residues 115 to 136, 4 are found between residues 282 and 288, and the remaining half are scattered throughout the extracellular loop (Fig. 1A). We therefore made a series of chimeric constructs depicted in Fig. 2B to examine the effects of transposing rat ectodomain, C terminus, and residues 115 to 136 and 282 to 288 (alone and in combination) into the mouse P2X7R on agonist-evoked responses.
Neither agonist concentration-response curves nor agonist-evoked kinetics was altered by transposing rat intracellular C terminus onto mouse P2X7R or vice versa (Fig. 3, A and C). Transposition of rat ectodomain onto mouse P2X7R resulted in ATP and BzATP EC50 values that were the same as for wild-type rat P2X7R (Fig. 3C), but deactivation kinetics were several-fold slower (Fig. 3B). No responses were recorded from cells in which the mouse ectodomain was transposed into the rat P2X7R. The chimeric mouse P2X7R containing residues 115 to 136 of rat P2X7R resulted in a pronounced leftward shift of the BzATP concentration-response curve without any alteration in the ATP-evoked responses (Fig. 4A and Table 1). When residues 282 to 288 of rat P2X7R were inserted into mouse P2X7R, there was a small leftward shift in the BzATP concentration-response curve but a large leftward shift in the ATP concentration response (Fig. 4B and Table 1). Substitution of both regions of rat P2X7R into mouse P2X7R resulted in agonist-evoked concentration-response curves and EC50 values that were not significantly different from wild-type rat P2X7R (Fig. 4C and Table 1).
Fig. 3. The ectodomain is solely responsible for agonist potency at P2X7R. A and B, examples of currents recorded from rat P2X7R containing mouse C terminus (A) and from mouse P2X7R containing rat ectodomain (B). Note the prolonged deactivation kinetics upon removal of BzATP at the mouse P2X7R containing rat ectodomain (B); no other chimeric or mutant receptor showed altered kinetics of onset or offset. C, summary of EC50 values for ATP and BzATP obtained from chimeric receptors; note difference in x-axis scale for BzATP to better depict the 100-fold difference between rat and mouse receptors.
Fig. 4. Small subregions in the ectodomain account for differences between mouse and rat sensitivity to ATP and BzATP. A to C, concentration-response curves for ATP (left graphs) and BzATP (right graphs) from wild-type rat P2X7R (red-filled circles), mouse P2X7R(), and chimeric receptors (). A, when residues 115 to 136 from rat P2X7R were inserted into mouse P2X7R, no change in ATP-evoked responses occurred, whereas BzATP response showed a 10-fold leftward shift. B, insertion of rat P2X7R residues 282 to 288 resulted in a significant leftward shift in both agonist concentration-response curves with the most pronounced effect occurring for the ATP dose-response curve. C, insertion of both these regions of rat P2X7R into mouse P2X7R resulted in ATP concentration response that was slightly shifted to the left of wild-type rat P2X7R and a BzATP response that was not different from wild-type rat P2X7R.
Introducing Single Amino Acids of Rat P2X7R into Mouse P2X7R. We next substituted individually each rat P2X7R residue in these regions into the mouse receptor and examined agonist responses in these mutant receptors. ATP-evoked responses were not different from wild-type mouse P2X7R for any mutation except at residue 284, at which substitution of asparagine (rat P2X7R) for aspartate (mouse P2X7R) resulted in ATP concentration response and EC50 value that was not significantly different from wild-type rat P2X7R (Fig. 5A and Table 1). In contrast, the sensitivity of the mouse receptor to BzATP was clearly increased over wild-type by substitutions of the equivalent rat residue at several positions. These were D284N (p < 0.01), A127K (p < 0.01), S130H (p < 0.1), R134G (p < 0.05), and K136I (p < 0.05). The largest difference was seen for mouse P2X7[A127K], in which case the EC50 value for BzATP (80 µM) was intermediate between that for the rat (3 µM) and mouse (285 µM) receptor (Fig. 5B and Table 1).
Fig. 5. Asparagine284 solely governs ATP potency differential whereas lysine127 with Asp284 largely governs BzATP potency differential at P2X7R. Histograms of ATP (A) and BzATP (B) EC50 values obtained from mouse P2X7R carrying point mutations in the regions between 115 to 136 and 282 to 288. In each case, the residue substituted was that found in wild-type rat P2X7R. Stippled bars indicate values significantly different from wild-type mP2X7R. A, ATP EC50 value at mP2X7[D284N] was shifted 10-fold to the left of wild-type mP2X7R and was not significantly different from wild-type rat P2X7R, whereas the BzATP EC50 value was shifted only 1.6-fold to the left at this mutation. B, mutations at residues 127, 130, 134, and 136 also shifted the BzATP EC50 value significantly to the left, with A127K mutation showing the largest effect. The mouse P2X7[A127K/D284N] double mutation yielded agonist EC50 values that were not significantly different from wild-type rat P2X7R. C, histograms of BzATP/ATP EC50 ratios for chimeric and mutant receptors as indicated. Ratios were similar to wild-type rat P2X7R only when both A127K and D284N mutations were present.
The double substitution fully converted the mouse receptor (P2X7[A128K,D284N]) to the sensitivity of the rat receptor with respect to both ATP and BzATP EC50 (Fig. 5A and Table 1) and was the only construct BzATP/ATP potency ratio was the same as that for wild-type rat P2X7R (Fig. 5C). However, we noticed that at this receptor BzATP was not able to produce as great a maximal current as ATP, which is the opposite for either of the wild-type receptors. Maximum BzATP-evoked currents were approximately 30% greater than maximum ATP-evoked currents at both mouse and rat P2X7R (Fig. 2D), but in the mouse P2X7[A128K,D284N] receptor, the ATP-evoked currents were 36 ± 5% (n = 5) greater than the maximal currents evoked by BzATP.
Involvement of N-Glycosylation. The substitution of Asp284 with asparagine at the mouse P2X7R generates a potential N-glycosylation acceptor sequence. The wild-type rat P2X7R also contains a similar potential acceptor sequence at this position (NESL). Because it has been well-demonstrated at other P2XRs that adding N-glycosylation sites significantly increases, whereas removing N-glycosylation sites decreases, protein expression (Newbolt et al., 1998; Torres et al., 1998; Rettinger et al., 2000; Chaumont et al., 2004), we assayed protein expression in wild-type, chimeric, and mP2X7D284N receptor and asked whether this site was, indeed, glycosylated. As found previously (Young et al., 2006), transfection with equal concentrations of rat and mouse P2X7R cDNA yielded protein level ratios of approximately 3:1 (Fig. 6A). The presence of rat P2X7R ectodomain, but not N terminus, C terminus, or transmembrane domains, yielded protein levels not significantly different from wild-type rat P2X7R, whereas the presence of mouse ectodomain yielded low protein expression equivalent to wild-type mouse P2X7R (Fig. 6A). We then removed N-glycan chains from wild-type mouse and rat P2X7R and mouse P2X7-D284N receptor using PNGase F and examined molecular mass by SDS-polyacrylamide gel electrophoresis. Wild-type rat P2X7R, mouse P2X7-D284N receptor, and mouse P2X7R bands were detected at 78, 78, and 75 kDa, respectively (Fig. 6B). After PNGase F treatment, all receptors were detected at approximately 65 kDa (Fig. 6B). This result is consistent with Asn284 being glycosylated in the wild-type rat receptor and the mouse P2X7[D284N] receptor.
Fig. 6. Protein expression and N-linked glycosylation at mouse and rat P2X7Rs. A, total protein expression of constructs as illustrated. Equal protein (10 µg) was loaded per lane and confirmed by comparing -actin levels. P2X7R protein was detected using the anti-glutamic acid-glutamic acid epitope tag antibody. The ectodomain, but not transmembrane or intracellular domains, determined levels of P2X7R protein expression. B, Western blot of native and deglycosylated receptors. Both rat P2X7R and mouse P2X7R-D284N exhibited higher molecular mass bands (78 kDa) than mouse P2X7R (75 kDa). Treatment with PNGase F gave rise to products of the same lower mass of approximately 65 kDa, with an additional lower mass band (at 60 kDa) that may be due to protein degradation. C, schematic indicating the two important residues conferring ATP (284N) and BzATP (127K + 284N) sensitivity at rat and mouse P2X7Rs.
The sequence relatedness between the mouse and rat P2X7 receptors (84.9%) is substantially less than for the other P2X receptors (for 1 through 6, 97.5, 97.2, 99.2, 94.7, 94.5, and 92.6% identity, respectively). This may underlie the large species difference in agonist sensitivity, which have generally not been described for other (homomeric) members of the P2X receptor family. The differences are somewhat clustered, and in two parts of the ectodomain, the identity is only 68% (115-136) and 43% (282-288) (Fig. 1A, boxes). Unlike other P2X receptors, in which mutations in transmembrane domains have been demonstrated to significantly alter ATP concentration-response curves (Haines et al., 2001), we found agonist potency to be determined solely by the ectodomain of the P2X7 receptor. We then identified two amino acids that are not conserved in any of the other P2X receptors, one in each of these ectodomain segments, that could account for the differential ATP/BzATP agonist sensitivity at the P2X7 receptor.
The first main finding of the present study is that, of all the differences between rat and mouse P2X7 receptor ectodomain residues, a single amino acid is largely responsible for the difference in sensitivity to ATP. Thus, introducing asparagine in place of aspartate at position 284 in the mouse P2X7R changed the EC50 value from 936 to 146 µM, which is close to the value for the wild-type rat receptor (123 µM, Table 1). The change in sensitivity associated with the aspartate-to-asparagine substitution (from -O- to -NH2) is striking. The asparagine is situated within a sequence commonly found at sites of N-linked glycosylation (N-X-S; NESL in rat P2X7; N-glycosylation acceptor sequence in mouse P2X7[D284N]). We found evidence that the mutated mouse P2X7[D284N] receptor was glycosylated at this position, because the molecular mass was approximately 3 kDa higher than the wild-type mouse receptor. The mass corresponded to that of the wild-type rat receptor (78 kDa). We note that the wild-type rat receptor and mouse receptor both carry the same five potential N-linked glycosylation sites (Asn74, Asn100, Asn106, Asn187, Asn241), whereas only rat P2X7R carries a sixth (Asn284) glycosylation site. Thus, the wild-type rat P2X7R and the mouse P2X7[D284N] mutated receptor each carry the same N-linked glycosylation sites. In both cases, treatment with PNGase F reduced the molecular mass to approximately 65 kDa, which is similar to the calculated molecular mass (68.5 kDa) of the receptor. The large effect that this aspartate-to-asparagine substitution has on the potency of ATP could plausibly indicate that the attached sugar moiety participates directly in ATP binding. An alternative explanation is that the glycan impedes the conformational change leading from binding to gating; this seems less likely because it is the form of the P2X7 receptor with the attached sugar at this position (mouse P2X7[D284N] or rat P2X7) that is more sensitive to ATP than the form without it (mouse P2X7). Rettinger et al. (2000) have found previously that, in the case of the P2X1 receptor, glycosylation affects the potency of ATP. Although the difference in potency was small at the P2X1 receptor (approximately 3-fold), it was also the form of the receptor without attached sugar (P2X1[N210Q]) that was less sensitive. In the P2X1 receptor, the asparagine was situated close to the center of the ectodomain (Asn210 of rat P2X1; Roberts and Evans, 2004). The subject asparagine of the present study, at position 284, is situated in region that is poorly conserved among P2X receptors. Thus, the present results indicate that it plays a key role in determining the unique response to ATP observed at the P2X7 receptor.
The second main finding of the present work was that the sensitivity to BzATP was almost 100-fold different between rat and mouse receptors, 10 times greater than the difference for ATP, and this BzATP differential was largely influenced by residue 127 (lysine in rat and alanine in mouse P2X7R). The residue Asp284 also had an effect on the BzATP sensitivity because substitution of aspartic acid for asparagine at this position did increase the potency of BzATP, but by only approximately 3-fold (Table 1). A much greater further increase of approximately 30-fold was observed when the additional A127K mutation was introduced into the mouse receptor. The observation that the effects of ATP and BzATP are differentially affected by two point mutations might imply that the residues are involved directly in agonist binding rather than in the subsequent gating conformational changes (which might be expected to be more in common for distinct agonists). If this is the case, then one might ask why an alanine-to-lysine substitution might affect the action of BzATP but not ATP. The obvious difference between the two agonists is the presence of the two (O-linked) aromatic moieties at the 2'(3') position. One might speculate that the cationic lysine residue in the binding site might interact with the electron cloud on one or another of the aromatic rings, providing a contribution to binding energy that would be unique to BzATP.
Irrespective of the detailed mechanism of the differences between ATP and BzATP, the present results provide a stark qualitative reminder of the criticality of species differences in ATP receptor pharmacology. Important differences in antagonist effectiveness have been reported for P2X1 receptors (chick versus human: Soto et al., 2003), P2X4 receptors (human versus rat: Buell et al., 1996; Soto et al., 1996), and for P2X7 receptors (human versus rat: Humphreys et al., 1998; Jiang et al., 2000; Baraldi et al., 2004; and mammalian versus nonmammalian: Lopez-Castejon et al., 2007). However, differences among species in agonist sensitivity have not been widely described. The findings therefore suggest that caution should be exercised in cross-species extrapolation on studies on P2X7 receptors, in which BzATP is in very widespread use as an experimental agonist.
Acknowledgements
We thank E. Martin and L. Collinson for cell and molecular biology technical support.
ABBREVIATIONS: BzATP, 2'-3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate; PCR, polymerase chain reaction; KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine; KN-04, N-(1-(p-(5-isoquinolinesulfonyl)benzyl)-2-(4-phenylpiperazinyl)ethyl)-5-isoquinolinesulfonamide.
1 Current affiliation: Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.
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作者单位：Department of Biomedical Science, University of Sheffield, Sheffield, United Kingdom
