Modulation of Prostaglandins by Arsenic

砷对前列腺素的调节

基本信息

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
项目类别：
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

项目摘要

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) 因其普遍存在而成为备受关注的无机环境污染物。长期接触砷经常会导致周围血管疾病以及皮肤癌、肺癌、膀胱癌和肾癌。最近的证据表明亚砷酸盐刺激环氧合酶 (COX-II) 表达，表明亚砷酸盐影响前列腺素合成。研究人员发表的砷研究表明，丝氨酸/苏氨酸激酶 MEKK4 参与砷信号转导。为了进一步了解砷造成有害影响的分子机制以及 MEKK4 的活性如何导致砷毒性，研究人员的工作重点是表征 MEKK4 在血管平滑肌细胞中的活性，因为它似乎在 COX- 的上游发挥作用。二. 该提议的假设是前列腺素生物合成途径受 MEKK4 调节，砷通过 MEKK4 调节前列腺素稳态来影响血管系统。具体目标是： 1. 鉴定和表征 MEKK4 的酪氨酸磷酸化。我们的数据表明 Pyk2 磷酸化 MEKK4。该位点未知，将通过 LC-MS 进行识别。然后，该位点将被突变，并对 MEKK4 催化活性进行表征，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亚砷酸盐如何抑制SHP-2活性。 SHP-2 以刺激依赖性方式与 MEKK4 结合。细胞内钙促进MEKK4去磷酸化，而亚砷酸盐抑制MEKK4酪氨酸去磷酸化。 3. 识别和表征 MEKK4 底物。候选蛋白质包括 MAP 激酶家族的成员或将使用“束缚”MEKK4 方法和 LC-MS 的改进来鉴定的新型蛋白质。 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关键酶，受磷酸化调节。 4. 表征MEKK4调节前列腺素合成的机制。 MEKK4 的激酶失活突变体充当显性失活蛋白，研究人员已经表明该蛋白的表达会抑制 Cox-II 启动子/荧光素酶嵌合质粒的转录。这一结果表明内源性 MEKK4 在调节前列腺素合成的途径上游发挥作用。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以及它如何对周围血管系统产生不利影响并促进组织损伤。