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.
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