Mentored Clinical Scientist Research Career Development (K08) research grant

指导临床科学家研究职业发展（K08）研究补助金

• 批准号: 9212291
• 负责人: Samar Ibrahim
• 金额: $ 16.7万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-11 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9212291
• 关键词: Address; Animal Model; Attenuated; Binding; Biochemical; Biogenesis; Biological; Biology; CXC Chemokines; CXC chemokine receptor 3; CXCR3 gene; Cell Communication; Cell membrane; Cell model; Cells; Chemotaxis; Child; Childhood; Choline; Clinical; Data; Disease; Functional disorder; Hepatic; Hepatocyte; Human; Incubated; Inflammation; Injury; Knock-out; Leukocytes; Ligands; Link; Lipids; Liver Fibrosis; Liver diseases; MAPK8 gene; Mediating; Mediator of activation protein; Membrane; Mentors; Metabolic syndrome; Mitogen-Activated Protein Kinases; Modeling; Molecular; Multivesicular Body; Mus; N-terminal; Nonesterified Fatty Acids; Nutritional; Obesity; Pharmacology; Phosphorylation; Phosphotransferases; Process; Publishing; Recruitment Activity; Research; Research Project Grants; Role; STAT1 gene; STAT1 protein; Scientist; Signal Pathway; Signal Transduction; Sorting - Cell Movement; Stress; Testing; Transcriptional Activation; base; career development; exosome; experimental study; extracellular vesicles; innovation; liver inflammation; liver injury; macrophage; microvesicles; mixed lineage kinase 3; mouse model; nanomedicine; nanoparticle; new therapeutic target; non-alcoholic fatty liver; nonalcoholic steatohepatitis; novel; novel therapeutic intervention; prevent; trafficking; vesicular release

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.

项目摘要 该提案的总体目标是定义连接肝细胞损伤的脂肪毒性机制 非酒精性脂肪性肝炎（NASH）中的肝发炎是最常见的儿科肝病。 NASH的特征是循环饱和游离脂肪酸（SFA）S，肝细胞的水平升高 脂肪毒性和巨噬细胞介导的肝炎。肝细胞脂肪毒性和肝损伤部分是 由SFA及其细胞内代谢物溶磷脂酰胆碱（LPC）诱导。但是，细胞和 将肝细胞脂毒性与巨噬细胞相关的肝脏炎症联系起来的分子机制是 不明确的。新兴数据暗示肝细胞脂肪毒性应激期间释放的细胞外囊泡（EV）作为 细胞间通信的重要介体。在发布和初步实验中，我们有 发现，在与脂肪毒性介质一起孵育的肝细胞中：i）应力激酶混合谱系激酶 （MLK）3通过信号传感器和激活因子的诱导C-X-C基序配体10（CXCL10）的诱导 转录（STAT）1依赖性机制； ii）MLK3依赖性C-JUN N末端激酶（JNK）激活 促进CXCL10富含电动汽车的释放； iii）富含CXCL10的EV激活巨噬细胞 趋化性。基于这些新颖的观察，我们提出了核心的假设 提议在肝细胞脂肪毒性期间激活的MLK3介导趋化电动汽车的释放 通过诱导CXCL10表达并刺激CXCL10，促进与巨噬细胞相关的肝脏炎症 分类并释放成新形成的电动汽车。我们建议采用电流和互补的分子， 生化和细胞生物学方法检验该假设。我们以下独立的特定目标 将检验三个综合假设。首先，我们将直接检验以下假设：MLK3在 肝细胞脂毒性通过有丝分裂原活化蛋白激酶（MAPK）继电器促进CXCL10诱导i） 导致STAT1磷酸化的模块，II）通过直接依赖STAT1的转录激活 CXCL10。其次，我们将检验以下假设：在肝细胞脂肪毒性期间，MLK3诱导CXCL10 将CXCL10的JNK-FACILITAID STROTIT释放到EV中，而II）由JNK依赖性形成， 使用纳什动物模型从肝细胞运输和释放特定的EV亚群。 我们将检验以下假设：I）缺乏CXCL10或其受体的小鼠肝脏炎症已减弱 CXCR3和II）MLK3药理学抑制作用可防止肝损伤。我们已经建立了 必要的细胞和动物模型，以研究脂肪毒性，MLK3和CXCL10信号传导和EV生物学。这 提案在技术上和概念上都是创新的，因为它试图整合分子机制 肝炎的基础肝细胞损伤，并将肝病生理学与纳米医学联系起来。这 研究将促进我们对将MLK3激活与肝脏炎症联系起来的信号通路的理解， 并有潜力确定新的治疗策略，以预防或逆转人类NASH的肝损伤。