Osteopontin regulates ubiquitin-proteasome degradation of STAT1

巨噬细胞和实质细胞表达诱导型一氧化氮合酶(INOS)是许多 与败血症相关的全身性影响。一氧化氮合酶表达和一氧化氮(NO)的产生改变 功能，包括心脏收缩、血管舒缩、肠上皮通透性和白细胞 招聘。利用体内和体外小鼠的内毒素刺激模型，我们以前已经 研究表明，NO反馈通过增加骨桥蛋白(OPN)的转录来抑制自身的合成。 强大的iNOS表达反式抑制因子。在这场竞争性更新中，我们建议将 骨桥蛋白下调iNOS转录的途径。使用体外和体内的小鼠模型 内毒素刺激和/或盲肠结扎和穿孔(CLP)介导的脓毒症，我们的初步数据表明 OPN促进STAT1泛素化和随后的26S蛋白酶体介导的STAT1降解以抑制 STAT1依赖的iNOS启动子活性、转录和蛋白表达。STAT1是一个必不可少的 内毒素激活剂和/或促炎细胞因子介导的iNOS转录。我们的研究表明OPN是 一种新的内毒素刺激下激活的STAT1降解调节因子。OPN在网络中的作用 以前还没有人探索过对STAT1依赖蛋白表达的调控。我们假设 OPN加速依赖泛素(Ub)的STAT1蛋白的降解，从而抑制iNOS的转录。我们会 重点关注以下具体目标，这些目标对于定义OPN中介的基础机制至关重要 脂多糖和CLP介导的脓毒症小鼠模型中STAT1的降解。1)我们将识别受OPN监管的 E3连接酶，将泛素转移到STAT1，最初专注于STAT相互作用的LIM(SLIM)蛋白。2)我们 将确定OPN在调节SLIM(或适当的E3连接酶)表达和/或激活中的作用。3) 我们将确认OPN-STAT1-Ub通路在内毒素刺激和/或小鼠模型中的体内相关性 合并了OPN零和瘦零动物的CLP。我们提议的研究将利用iNOS作为一种特定的 一个STAT1依赖蛋白的例子，它将OPN定义为一种独特的、迄今特征不佳的反式激活因子 STAT1的降解。对这一调控途径的表征可以识别潜在的调控 感染性休克的治疗靶点。

Macrophage and parenchymal cell 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 LPS stimulation, 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 acts to downregulate iNOS transcription. Using in vitro and in vivo murine models of LPS stimulation and/or cecal ligation and puncture (CLP) mediated sepsis, our preliminary data indicate that OPN increases STAT1 ubiquitination and subsequent 26s proteasome mediated STAT1 degradation to inhibit STAT1 dependent iNOS promoter activity, transcription, and protein expression. STAT1 is an essential activator of LPS and/or pro-inflammatory cytokine-mediated iNOS transcription. Our studies indicate OPN to be a novel regulator of activated STAT1 degradation in the context of LPS stimulation. The role of OPN in the regulation of STAT1 dependent protein expression has not been previously explored. We hypothesize that OPN accelerates ubiquitin (Ub)-dependent STAT1 protein degradation to inhibit iNOS transcription. 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 STAT-interacting LIM (SLIM) protein. 2) We will define the role of OPN in regulating expression and/or activation of SLIM (or the appropriate E3 ligase). 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. 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, transactivator of STAT1 degradation. Characterization of this regulatory pathway may identify potential regulatory targets for therapy in septic shock.