Osteopontin regulates ubiquitin-proteasome degradation of STAT1

诱导型一氧化氮合酶（iNOS）的表达是许多全身性效应的核心， 败血症iNOS表达和一氧化氮（NO）产生改变多种功能，包括心肌收缩力， 血管紧张素、肠上皮通透性和白细胞募集。利用体内和体外 在小鼠内毒素血症（LPS）模型中，我们先前已经证明NO反馈抑制其自身的 通过增加骨桥蛋白（OPN）的转录来合成，骨桥蛋白是iNOS表达的有效反式阻遏物。在这 竞争性更新，我们建议的特点，通过OPN反馈下调iNOS的途径 转录。LPS和盲肠结扎穿孔（CLP）的体内、离体和体外小鼠模型 我们的研究表明：1）OPN与STAT相互作用的LIM（SLIM）蛋白共同作用， (Ub)一种必需的iNOS转录因子STAT 1，用于26 s蛋白酶体介导的降解和抑制 STAT 1依赖的iNOS表达，2）STAT 1在SLIM不存在时不被泛素化，3） OPN缺失或SLIM缺失小鼠显著减少，表明该途径在免疫缺陷中的功能相关性。 脓毒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STAT 1蛋白并抑制iNOS转录。我们将重点关注以下具体问题 目的是确定OPN介导的小鼠STAT 1降解的潜在机制， LPS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STAT 1，最初专注于SLIM蛋白。2)我们将明确骨桥蛋白在调控表达中的作用， 和/或SLIM的激活。3)我们将证实OPN-STAT 1-Ub通路在小鼠中的体内相关性。 LPS刺激和/或CLP的模型，其包括OPN无效和SLIM无效动物。OPN的作用 以前没有研究过脓毒症中STAT 1依赖性蛋白表达的调节。我们提出的 研究将利用iNOS作为STAT 1依赖性蛋白的具体实例，以将OPN定义为一种独特的， 到目前为止，STAT 1降解的反式激活剂的特征还很差。 这种调控途径的特征可能会确定潜在的调控目标，治疗感染性休克。

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.