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

基本信息

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
项目类别：
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

项目摘要

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确定与肝细胞损伤相关的脂毒性机制伴有非酒精性脂肪性肝炎 (NASH) 中的肝脏炎症，这是最常见的小儿肝脏疾病。 NASH 的特点是循环饱和游离脂肪酸 (SFA) 水平升高、肝细胞脂毒性和巨噬细胞介导的肝脏炎症。肝细胞脂毒性和肝损伤部分是，由 SFA 及其细胞内代谢物溶血磷脂酰胆碱 (LPC) 诱导。然而，蜂窝和将肝细胞脂毒性与巨噬细胞相关肝脏炎症联系起来的分子机制是不明确的。新数据表明，肝细胞脂毒性应激期间释放的细胞外囊泡 (EV) 细胞间通讯的重要介质。在已发表的和初步的实验中，我们有发现，在与脂毒性介质一起孵育的肝细胞中： i) 应激激酶混合谱系激酶 (MLK)3 通过信号转导器和激活剂促进 C-X-C 基序配体 10 (CXCL10) 的诱导转录（STAT）1依赖机制； ii) MLK3 依赖性 c-Jun N 末端激酶 (JNK) 激活促进富含 CXCL10 的电动汽车的发布； iii) 富含 CXCL10 的 EV 激活巨噬细胞趋化性。基于这些新颖的观察，我们提出了中心假设认为在肝细胞脂毒性过程中，激活的 MLK3 介导趋化 EV 的释放，从而通过诱导 CXCL10 表达和刺激 CXCL10 促进巨噬细胞相关的肝脏炎症分类并释放到新形成的电动汽车中。我们建议采用当前和互补的分子、生物化学和细胞生物学方法来检验这一假设。我们以下独立的具体目标将测试三个综合假设。首先，我们将直接检验 MLK3 激活期间的假设肝细胞脂毒性通过丝裂原激活蛋白激酶 (MAPK) 中继促进 CXCL10 诱导 i) 导致 STAT1 磷酸化的模块，以及 ii) 通过直接 STAT1 依赖性转录激活 CXCL10。其次，我们将检验以下假设：在肝细胞脂毒性期间，MLK3 诱导 CXCL10 i) 通过 JNK 促进 CXCL10 分选到 EV 中，以及 ii) 通过 JNK 依赖性形成，释放到 EV 中，从肝细胞转运和释放特定的 EV 亚群。第三，使用 NASH 动物模型，我们将检验以下假设：i) 缺乏 CXCL10 或其受体的小鼠肝脏炎症减弱 CXCR3 和 ii) MLK3 药理学抑制可预防肝损伤。我们已经建立了研究脂毒性、MLK3 和 CXCL10 信号传导以及 EV 生物学所必需的细胞和动物模型。这该提案在技术和概念上都具有创新性，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医学联系起来。这研究将加深我们对 MLK3 激活与肝脏炎症之间信号通路的理解，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。