Mentored Clinical Scientist Research Career Development (K08) research grant

项目摘要 本提案的总体目标是确定与肝细胞损伤相关的脂毒性机制。 非酒精性脂肪性肝炎(NASH)是最常见的儿科肝病，伴有肝脏炎症。 NASH的特征是循环饱和游离脂肪酸水平升高S，肝细胞 脂毒性和巨噬细胞介导的肝脏炎症。肝细胞脂肪毒性和肝损伤在一定程度上是 由SFA及其胞内代谢物溶血磷脂酰胆碱(LPC)诱导。然而，蜂窝和 肝细胞脂肪毒性与巨噬细胞相关性肝脏炎症的分子机制是 未定义。新出现的数据表明，S在肝细胞脂毒应激过程中释放的细胞外小泡(EV)为 细胞间通讯的重要媒介。在已公布的和初步的实验中，我们有 发现，在与脂毒介体孵育的肝细胞中：i)应激激酶混合谱系激酶 (MLK)3促进C-X-C基序配体10(CXCL10)的信号转导和激活 转录(STAT)1依赖的机制；II)依赖MLK3的c-jun氨基末端激酶(JNK)的激活 促进富含CXCL10的EVS的释放；以及iii)富含CXCL10的EVS激活巨噬细胞 趋化性。基于这些新奇的观察，我们已经形成了 提出在肝细胞脂肪毒性过程中，活化的MLK3介导趋化EVS的释放。 诱导CXCL10表达和刺激CXCL10促进巨噬细胞相关性肝脏炎症 分类并投放到新形成的电动汽车中。我们建议使用电流和互补、分子、 生物化学和细胞生物学方法来验证这一假说。我们的以下独立具体目标 将检验三个综合假说。首先，我们将直接测试假设MLK3激活在 肝细胞脂毒性通过丝裂原活化蛋白激酶(MAPK)传递促进CXCL10的诱导 导致STAT1磷酸化的模块，以及ii)通过STAT1依赖的直接转录激活 CXCL10。其次，我们将检验假设，在肝细胞脂肪毒性过程中，MLK3诱导CXCL10 释放到EVS中i)通过JNK促进的将CXCL10分选成EVS，以及ii)通过JNK依赖的形成， 从肝细胞转运和释放特定的EV亚群。第三，使用NASH的动物模型， 我们将检验这样的假设：1)缺乏CXCL10或其受体的小鼠肝脏炎症减轻 CXCR3，以及ii)MLK3药理抑制对肝损伤有保护作用。我们已经建立了 研究脂肪毒性、MLK3和CXCL10信号和EV生物学所需的细胞和动物模型。这 该提议在技术和概念上都是创新的，因为它寻求整合分子机制 潜在的肝细胞损伤与肝脏炎症，并将肝脏病理生理学与纳米药物联系起来。这 研究将促进我们对MLK3激活与肝脏炎症之间的信号通路的理解， 并有可能确定新的治疗策略来预防或逆转人类NASH的肝损伤。

PROJECT ABSTRACT The OVERALL OBJECTIVES of this proposal are to define the lipotoxic mechanisms linking hepatocyte injury with hepatic inflammation in nonalcoholic steatohepatitis (NASH), the most common pediatric liver disease. NASH is characterized by elevated levels of circulating saturated free fatty acids (SFA)s, hepatocyte lipotoxicity and macrophage-mediated liver inflammation. Hepatocyte lipotoxicity and liver injury are, in part, induced by SFAs and their intracellular metabolite lysophosphatidyl choline (LPC). However, the cellular and molecular mechanisms linking hepatocyte lipotoxicity to macrophage-associated liver inflammation are undefined. Emerging data implicate extracellular vesicles (EV)s released during hepatocyte lipotoxic stress as important mediators of cell-to-cell communication. In published and preliminary experiments, we have discovered that, in hepatocytes incubated with lipotoxic mediators: i) the stress kinase mixed lineage kinase (MLK)3 promotes the induction of C-X-C motif ligand 10 (CXCL10) by a signal transducer and activator of transcription (STAT)1-dependent mechanism; ii) MLK3-dependent c-Jun N-terminal Kinase (JNK) activation promotes the release of CXCL10-enriched EVs; and iii) CXCL10-enriched EVs activate macrophage chemotaxis. Based on these novel observations, we have formulated the CENTRAL HYPOTHESIS of the proposal that during hepatocyte lipotoxicity, activated MLK3 mediates the release of chemotactic EVs, therby promoting macrophage-associated liver inflammation by inducing CXCL10 expression and stimulating CXCL10 sorting and release into newly formed EVs. We propose to employ current and complementary, molecular, biochemical and cell biological approaches to test this hypothesis. Our following independent SPECIFIC AIMS will test three integrated hypotheses. FIRST, we will directly test the hypothesis that MLK3 activation during hepatocyte lipotoxicity promotes CXCL10 induction i) by a mitogen activated protein kinase (MAPK) relay module resulting in STAT1 phosphorylation, and ii) by a direct STAT1-dependent transcriptional activation of CXCL10. SECOND, we will test the hypothesis that during hepatocyte lipotoxicity, MLK3 induces CXCL10 release into EVs i) by JNK-facilitated sorting of CXCL10 into EVs, and ii) by JNK-dependent formation, transport and release of a specific EV subpopulation from hepatocytes.Third, using an animal model of NASH, we will test the hypothesis that i) liver inflammation is attenuated in mice that lack CXCL10 or its receptor CXCR3, and ii) MLK3 pharmacological inhibition is protective against liver injury. We have established the requisite cell and animal models to study lipotoxicity, MLK3 and CXCL10 signaling and EV biology. This proposal is technically and conceptually innovative, as it seeks to integrate the molecular mechanisms underlying hepatocyte injury with liver inflammation, and links hepatic pathophysiology with nanomedicine. This research will advance our understanding of the signaling pathway linking MLK3 activation to liver inflammation, and has the potential to identify new therapeutic strategies to prevent or reverse liver injury in human NASH.