Osteopontin regulates ubiquitin-proteasome degradation of STAT1

描述（由申请人提供）：诱导型一氧化氮合酶（iNOS）的表达是与脓毒症相关的许多全身效应的核心。iNOS的表达和一氧化氮（NO）的产生改变多种功能，包括心脏收缩力、血管舒缩张力、肠上皮通透性和白细胞募集。利用体内和体外小鼠内毒素血症（LPS）模型，我们之前已经证明，NO反馈通过增加骨桥蛋白（OPN）的转录来抑制其自身的合成，骨桥蛋白是一种有效的iNOS表达反式抑制因子。在这种竞争性更新中，我们提出表征OPN反馈下调iNOS转录的途径。在体内、离体和体外LPS和盲肠结扎穿刺（CLP）介导的脓毒症小鼠模型中，我们的研究表明：1) OPN与stat相互作用的LIM （SLIM）蛋白一起作用，使必需的iNOS转录因子STAT1泛素化（Ub），用于26s蛋白酶体介导的降解，抑制STAT1依赖性iNOS的表达；2)在没有SLIM的情况下，STAT1不会泛素化；3)OPN缺失或SLIM缺失小鼠的存活率显著降低，表明该途径在脓毒症病理生理中的功能相关性。我们假设OPN通过SLIM作为E3泛素连接酶来降解STAT1蛋白并抑制脓毒症中的iNOS转录。我们将重点关注以下具体目标，这些目标对于确定LPS和CLP介导的脓毒症小鼠模型中OPN介导的STAT1降解的机制至关重要。1)我们将确定opn调控的将泛素转移到STAT1的E3连接酶，最初将重点放在SLIM蛋白上。2)我们将明确OPN在调节SLIM表达和/或激活中的作用。3)我们将确认OPN- stat1 - ub通路在LPS刺激和/或CLP小鼠模型中的体内相关性，这些模型包括OPN缺失和SLIM缺失的动物。在脓毒症中，OPN在STAT1依赖性蛋白表达调控中的作用尚未被探索。我们提出的研究将利用iNOS作为STAT1依赖蛋白的具体例子，将OPN定义为STAT1降解的独特且尚未被充分表征的反式激活因子。这一调控途径的特征可以确定感染性休克治疗的潜在调控靶点。公共卫生相关性：严重感染和损伤导致的死亡在美国仍然是一个关键问题。本研究探讨了在严重感染和损伤后正常身体功能崩溃的主要参与者的调节。如果成功，我们的研究可能会导致预防这种崩溃。

DESCRIPTION (provided by applicant): Expression of inducible nitric oxide synthase (iNOS) is central to many of the systemic effects associated with sepsis. iNOS expression and nitric oxide (NO) production alter multiple functions, including cardiac contractility, vasomotor tone, intestinal epithelial permeability, and leukocyte recruitment. Utilizing both in vivo and in vitro murine models of endotoxemia (LPS), we have previously demonstrated that NO feedback inhibits its own synthesis by increasing transcription of osteopontin (OPN), a potent trans-repressor of iNOS expression. In this competitive renewal, we propose to characterize the pathway by which OPN feeds back to downregulate iNOS transcription. In in vivo, ex vivo, and in vitro murine models of LPS- and cecal ligation and puncture (CLP) mediated sepsis, our studies show that: 1) OPN acts with STAT-interacting LIM (SLIM) protein to ubiquitinate (Ub) an essential iNOS transcription factor, STAT1, for 26s proteasome mediated degradation and inhibit STAT1 dependent iNOS expression, 2) STAT1 is not ubiquitinated in the absence of SLIM, and 3) survival of OPN null or SLIM null mice is significantly decreased indicating the functional relevance of this pathway in the pathophysiology of sepsis. We hypothesize that OPN acts through SLIM as an E3 ubiquitin ligase to degrade STAT1 protein and inhibit iNOS transcription in sepsis. We will focus on the following specific aims which are critical to defining the mechanisms underlying OPN mediated STAT1 degradation in murine models of LPS and CLP mediated sepsis. 1) We will identify the OPN-regulated E3 ligase which transfers ubiquitin to STAT1, focusing initially on SLIM protein. 2) We will define the role of OPN in regulating expression and/or activation of SLIM. 3) We will confirm in vivo relevance of the OPN-STAT1-Ub pathway in murine models of LPS stimulation and/or CLP that incorporate OPN null and SLIM null animals. The role of OPN in the regulation of STAT1 dependent protein expression in sepsis has not been previously explored. Our proposed studies will utilize iNOS as a specific example of a STAT1 dependent protein to define OPN as a unique and as yet, poorly characterized, trans-activator of STAT1 degradation. Characterization of this regulatory pathway may identify potential regulatory targets for therapy in septic shock. PUBLIC HEALTH RELEVANCE: Death as the result of severe infection and injury remains a critical problem in the U.S. This research examines the regulation of a major player in the breakdown of normal bodily functions following major infection and injury. If successful, our studies may result in prevention of this breakdown.