PROJECT SUMMARY / ABSTRACT Cirrhotic stage liver disease is the 11th leading cause of mortality in the US, and no treatment exists for late- stage disease other than liver transplantation. Thus, the overall objective of this proposal is to elucidate novel mechanisms that drive the release of fibrogenic signals leading to liver fibrosis progression and to guide the development of potential treatment strategies. Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs). Our preliminary data in primary human and mouse HSCs as well as in vivo demonstrate that 1. platelet-derived growth factor B (PDGF) induces metabolic reprogramming by increasing glycolysis; 2. PDGF- mediated glycolysis increase the transcriptional activation mark, histone 3 lysine 9 acetylation (H3K9ac) on the promoter region of vesicle trafficking-related Ras-related protein Rab (RAB) genes; 3. glycolysis promotes EV release and enrichment with fibrogenic proteins; and 4. in vivo glycolysis inhibition by HSC-selective hexokinase 2 (HK2) deletion abrogates liver fibrosis. We have utilized our novel findings to generate the CENTRAL HYPOTHESIS of the current proposal that PDGF-mediated glycolysis in HSCs induces fibrogenic EV release through H3K9ac-dependent transcriptional upregulation of RABs to amplify liver fibrosis. We will employ sophisticated cellular and animal models, including in vitro and in vivo utilization of dCas-KRAB model, in vivo HSC-specific HK2 deletion model as well as acetyl-coA-deficient HSCs, to investigate the following integrated, yet independent aims. In Aim 1, we will test the hypothesis that PDGF increases glycolysis through lysine-deficient kinase 1 (WNK1) phosphorylation to mediate glucose transporter 1 (GLUT1) translocation to the plasma membrane. We will uncover the kinase signaling leading to glycolysis in HSCs by: a. studying how PDGF increases glycolysis through phosphorylation of WNK1, a novel PDGF downstream lysine-deficient kinase; and b. investigating how WNK1 phosphorylation promotes GLUT1 translocation to the plasma membrane to increase glycolysis, which represents a new mechanism in HSCs. In Aim 2, we will test the hypothesis that glycolysis leads to EV release by upregulating the transcription of RABs through acetyl coenzyme A (acetyl-CoA)-mediated H3K9ac. We will dissect how glycolysis drives epigenetic regulation of vesicle trafficking gene program to control EV release by: a. studying how PDGF promotes the accumulation of the metabolite acetyl-CoA to increase H3K9ac; and b. examining how H3K9ac promotes RAB transcription to induces EV release. In Aim 3, we will test the hypothesis that HSC-specific glycolysis and subsequent epigenetic regulation of fibrogenic EV release amplifies in vivo liver fibrosis. We will investigate the mechanism of in vivo liver fibrosis amplification by: a. studying how glycolysis-mediated EVs amplify liver fibrosis by targeting HSCs; and b. investigating how the disruption of epigenetic r...