Modulation of Prostaglandins by Arsenic

砷对前列腺素的调节

• 批准号: 6929694
• 负责人: RICHARD R VAILLANCOURT
• 金额: $ 30.3万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-10 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6929694
• 关键词: SDS polyacrylamide gel electrophoresis; angiotensin II; annexins; arsenic; biological signal transduction; calcium; cardiovascular system; environmental exposure; enzyme activity; immunoprecipitation; liquid chromatography mass spectrometry; mitogen activated protein kinase; phospholipase A2; phosphorylation; prostaglandin endoperoxide synthase; prostaglandins; protein kinase; protein protein interaction; site directed mutagenesis; tyrosine; western blottings

DESCRIPTION (provided by applicant) Arsenic (As) is an inorganic environmental contaminant of major concern due to its ubiquitous presence. Chronic exposure to arsenic frequently results in peripheral vascular disease, as well as skin, lung, bladder, and kidney cancer. Recent evidence demonstrates that arsenite stimulates cyclooxygenase (COX-II) expression suggesting that arsenite affects prostaglandin synthesis. The investigator's published studies with arsenic have demonstrated that the serine/threonine kinase, MEKK4, is involved in arsenic signal transduction. To further understand the molecular mechanism by which arsenic causes its deleterious effects and how the activity of MEKK4 contributes to arsenic toxicity, efforts by the investigators have focused on characterizing the activity of MEKK4 in vascular smooth muscle cells since it appears to function upstream of COX-II. The hvpothesis of this proposal is that the prostaglandin biosynthetic pathway is regulated by MEKK4 and that arsenic affects the vascular system by modulating prostaglandin homeostasis through MEKK4. The Specific Aims are to: 1. To identify and characterize tyrosine phosphorylation of MEKK4. Our data indicate that Pyk2 phosphorylates MEKK4. This site is not known and will be identified by LC-MS. Then, the site will be mutated and MEKK4 catalytic activity will be characterized to assess the significance of the phosphorylation site. 2. To characterize the mechanism by which SHP-2 interacts with MEKK4 and how arsenite inhibits SHP-2 activity. SHP-2 associates with MEKK4 in a stimulus-dependent manner. Intracellular calcium promotes dephosphorylation of MEKK4, while arsenite inhibits the tyrosine dephosphorylation of MEKK4. 3. To identify and characterize the MEKK4 substrate. Candidate proteins include members of the MAP kinase family or novel proteins that will be identified using a modification of the "tethered" MEKK4 approach and LC-MS. It is possible that MEKK4 phosphorylates and regulates cytosolic phospholipase A2 (cPLA2), a key enzyme involved in prostaglandin biosynthesis that is regulated by phosphorylation. 4. To characterize the mechanism by which MEKK4 regulates prostaglandin synthesis. The kinase-inactive mutant of MEKK4 functions as a dominant-negative protein, and the investigators have shown that expression of this protein inhibits transcription of a Cox-II promoter/luciferase chimeric plasmid. This result suggests that endogenous MEKK4 functions upstream of pathways that regulate prostaglandin synthesis. The proposed research will provide a further understanding of the importance of the arsenic-induced signaling processes and how it adversely affects the peripheral vascular system and promotes tissue injury.

描述（由申请人提供） 砷（AS）由于其无处不在，是主要关注的无机环境污染物。 长期暴露于砷经常会导致外周血管疾病以及皮肤，肺，膀胱和肾癌。 最近的证据表明，阿森特刺激环氧合酶（COX-II）表达表明砷会影响前列腺素的合成。 研究者对砷的发表研究表明，丝氨酸/苏氨酸激酶MEKK4参与砷信号转导。 为了进一步了解砷会引起其有害作用的分子机制，以及MEKK4的活性如何促进砷毒性，研究人员的努力集中在表征MEKK4在血管平滑肌细胞中的活性，因为它似乎在COX-II上游起作用。 该提议的HVPothesis是，前列腺素生物合成途径受MEKK4调节，并且砷通过通过MEKK4调节前列腺素稳态来影响血管系统。 具体目的是： 1。识别和表征MEKK4的酪氨酸磷酸化。 我们的数据表明PYK2磷酸化MEKK4。 该站点尚不清楚，并将通过LC-MS确定。 然后，将突变该位点，并将MEKK4催化活性表征以评估磷酸化位点的重要性。 2。表征SHP-2与MEKK4相互作用的机制以及砷如何抑制SHP-2活性。 SHP-2以刺激依赖性方式与MEKK4相连。 细胞内钙可促进MEKK4的去磷酸化，而砷则抑制MEKK4的酪氨酸去磷酸化。 3。识别和表征MEKK4底物。 候选蛋白包括MAP激酶家族或新型蛋白质的成员，这些成员将使用“束缚” MEKK4方法和LC-MS的修改来识别。 MEKK4可能磷酸化并调节胞质磷脂酶A2（CPLA2），这是一种参与前列腺素生物合成的键酶，受磷酸化调节。 4。表征MEKK4调节前列腺素合成的机制。 MEKK4的激酶不活性突变体是一种显性阴性蛋白，研究者表明该蛋白的表达抑制了COX-II启动子/荧光素酶嵌合质粒的转录。 该结果表明，内源性MEKK4在调节前列腺素合成的途径上游功能。 拟议的研究将进一步了解砷诱导的信号传导过程的重要性及其对外周血管系统的不利影响并促进组织损伤。