Osteopontin regulates ubiquitin-proteasome degradation of STAT1

描述(申请人提供)：诱导型一氧化氮合酶(INOS)的表达是许多与脓毒症相关的全身效应的中心。INOS的表达和一氧化氮(NO)的产生改变了多种功能，包括心脏收缩、血管紧张性、肠上皮通透性和白细胞募集。利用体内和体外的小鼠内毒素血症(LPS)模型，我们先前已经证明，NO反馈通过增加骨桥蛋白(OPN)的转录来抑制自身的合成，骨桥蛋白是一种有效的iNOS表达的反式抑制因子。在这一竞争性更新中，我们建议描述OPN反馈下调iNOS转录的途径。在体内、体外和盲肠结扎穿孔(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)蛋白作用于必需的iNOS转录因子STAT1泛素化(Ub)，以实现26S蛋白酶体介导的降解并抑制STAT1依赖的iNOS表达；2)STAT1在没有SLIM的情况下不被泛素化；3)OPN缺失或SLIM缺失的小鼠的存活率显著下降，表明这一途径在脓毒症的病理生理机制中具有功能相关性。我们推测OPN通过SLIM作为E3泛素连接酶在脓毒症中作用于降解STAT1蛋白和抑制iNOS转录。我们将专注于以下特定的目标，这些目标对于确定OPN介导的脂多糖和CLP介导的脓毒症小鼠模型中STAT1降解的机制至关重要。1)我们将鉴定OPN调节的E3连接酶，它将泛素转移到STAT1，最初专注于SILM蛋白。2)我们将确定OPN在调节SLIM表达和/或激活中的作用。3)我们将证实OPN-STAT1-Ub通路在内毒素刺激和/或CLP小鼠模型中的体内相关性，该模型包括OPN缺失和瘦缺失动物。OPN在脓毒症中调节STAT1依赖蛋白表达的作用以前还没有被探索过。我们建议的研究将利用iNOS作为STAT1依赖蛋白的一个特定例子，以将OPN定义为一种独特的、迄今尚未得到充分表征的STAT1降解的反式激活剂。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：严重感染和伤害导致的死亡在美国仍然是一个严重的问题。这项研究调查了在严重感染和伤害后正常身体功能崩溃的主要参与者的监管。如果成功，我们的研究可能会防止这种崩溃。

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.