Modulation of Prostaglandins by Arsenic

描述(由申请人提供) 砷(As)是一种普遍存在的无机环境污染物。长期接触砷往往会导致外周血管疾病，以及皮肤癌、肺癌、膀胱癌和肾癌。最近的证据表明，亚砷可以刺激环氧合酶(COX-II)的表达，提示亚砷影响前列腺素的合成。研究人员发表的关于砷的研究表明，丝氨酸/苏氨酸激酶MEKK4参与了砷的信号转导。为了进一步了解砷导致其有害效应的分子机制以及MEKK4的活性如何促进砷的毒性，研究人员将重点放在表征MEKK4在血管平滑肌细胞中的活性，因为它似乎作用于COX-II的上游。这一建议的假设是前列腺素的生物合成途径受MEKK4调节，而砷通过MEKK4调节前列腺素的动态平衡来影响血管系统。具体目标是： 1.鉴定和鉴定MEKK4的酪氨酸磷酸化。我们的数据表明，Pyk2使MEKK4磷酸化。这一地点未知，将由LC-MS鉴定。然后，对该位点进行突变，并对MEKK4的催化活性进行表征，以评估该磷酸化位点的意义。 2.研究SHP-2与MEKK4相互作用的机制以及亚砷酸盐对SHP-2活性的抑制作用。SHP-2以刺激依赖的方式与MEKK4结合。细胞内钙促进MEKK4的去磷酸化，而亚砷酸盐抑制MEKK4的酪氨酸去磷酸化。 3.鉴定和鉴定MEKK4底物。候选蛋白包括MAP激酶家族的成员或将使用改进的“拴系”MEKK4方法和LC-MS鉴定的新蛋白。MEKK4可能磷酸化并调节胞浆磷脂酶A2(CPLA2)，cPLA2是参与前列腺素生物合成的关键酶，受磷酸化调控。 4.研究MEKK4调节前列腺素合成的机制。MEKK4的激酶失活突变体的功能是显性负蛋白，研究人员已经证明，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。这一结果表明，内源性MEKK4在调节前列腺素合成的途径的上游发挥作用。 这项拟议的研究将进一步了解砷诱导的信号过程的重要性，以及它是如何对周围血管系统产生不利影响并促进组织损伤的。

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.