Osteopontin regulates ubiquitin-proteasome degradation of STAT1

诱导型一氧化氮合酶(INOS)的表达是许多与以下相关的全身效应的中心 败血症。一氧化氮合酶的表达和一氧化氮(NO)的产生改变了多种功能，包括心脏的收缩能力， 血管舒张性、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，我们先前已经证明，没有反馈会抑制其自身 通过增加骨桥蛋白(OPN)的转录合成，骨桥蛋白是一种有效的iNOS表达的反式抑制因子。在这 竞争性更新，我们建议描述OPN反馈下调iNOS的途径 抄写。内毒素和盲肠结扎穿孔(CLP)的体内、外和体外小鼠模型 我们的研究表明：1)OPN与STAT相互作用的LIM(SLIM)蛋白作用于泛素化 (UB)一种重要的iNOS转录因子STAT1，用于26S蛋白酶体介导的降解和抑制 STAT1依赖的iNOS表达，2)在没有SLIM的情况下，STAT1不是泛素化的，以及3)存活 OPN缺失或苗条缺失的小鼠显著减少，表明这一途径在 败血症的病理生理学。我们假设OPN通过SLIM作为E3泛素连接酶发挥作用 在脓毒症中降解STAT1蛋白并抑制iNOS转录。我们将重点关注以下具体内容 对确定OPN介导的小鼠STAT1降解机制至关重要的AIMS 内毒素和CLP介导的脓毒症模型。1)我们将鉴定OPN调节的E3连接酶 泛素到STAT1，最初专注于纤细蛋白质。2)我们将确定OPN在调控表达中的作用 和/或激活SLIM。3)我们将确认OPN-STAT1-Ub通路在小鼠体内的相关性 内毒素刺激和/或CLP模型，包括骨桥蛋白缺失和纤细缺失动物。OPN在网络中的作用 在脓毒症中对STAT1依赖蛋白表达的调控以前还没有被探索过。我们的建议 研究将利用iNOS作为STAT1依赖蛋白的特定例子来定义OPN为独特的和 到目前为止，STAT1降解的反式激活剂的特性还不是很好。 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。

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.