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）是许多肿瘤的核心。 与败血症相关的全身效应。iNOS表达和一氧化氮（NO）产生改变了多个 功能，包括心肌收缩力、血管紧张素、肠上皮通透性和白细胞 招聘利用LPS刺激的体内和体外小鼠模型，我们先前已经 表明NO反馈通过增加骨桥蛋白（OPN）的转录来抑制其自身的合成， iNOS表达的有效反式阻遏物。在这次竞争性更新中，我们建议将 OPN通过其作用下调iNOS转录的途径。使用体外和体内小鼠模型， LPS刺激和/或盲肠结扎穿孔（CLP）介导的脓毒症，我们的初步数据表明， OPN增加STAT 1泛素化和随后的26 s蛋白酶体介导的STAT 1降解，以抑制 STAT 1依赖性iNOS启动子活性、转录和蛋白表达。STAT 1是一种重要的 LPS的激活剂和/或促炎性丝氨酸介导的iNOS转录。我们的研究表明OPN是 一种新的调节激活的STAT 1降解的情况下，LPS刺激。OPN的作用 STAT 1依赖性蛋白表达的调节以前没有被探索过。我们假设 OPN加速泛蛋白（Ub）依赖性STAT 1蛋白降解以抑制iNOS转录。我们将 重点关注以下特定目标，这些目标对于定义OPN介导的潜在机制至关重要 LPS和CLP介导的脓毒症小鼠模型中的STAT 1降解。1)我们将确定OPN监管的 E3连接酶，将泛素转移到STAT 1，最初集中在STAT相互作用LIM（SLIM）蛋白。2)我们 将定义OPN在调节SLIM（或适当的E3连接酶）的表达和/或激活中的作用。第三章 我们将在LPS刺激和/或LPS刺激的小鼠模型中证实OPN-STAT 1-Ub通路的体内相关性。 CLP包括OPN无效和SLIM无效动物。我们提出的研究将利用iNOS作为一种特异性 STAT 1依赖性蛋白的一个例子，将OPN定义为一种独特的、迄今为止表征不佳的反式激活因子 STAT 1降解。这种调节途径的表征可以识别潜在的调节途径。 治疗感染性休克的靶点。

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.