Molecular Mechanisms of Cholestatic Fibrogenesis

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PROJECT SUMMARY / ABSTRACT Cholestatic fibrogenesis is a pathobiological process of the bile ducts, characterized by biliary strictures, cholestasis, and progressive peri-portal fibrosis. During biliary fibrosis, diseased cholangiocytes become highly secretory, releasing a variety of paracrine signaling molecules that subsequently activate hepatic stellate cells (HSC). Progression toward end stage disease is characterized by an exaggerated fibrogenic response to chronic injury, culminating in peri-portal deposition of matrix molecules that progresses to biliary cirrhosis. The release of paracrine, cholangiocyte-derived factors that subsequently activate hepatic stellate cells (HSC) is an early, important, and potentially reversible step in the progression of biliary fibrosis. Enhancer of zeste homologue 2 (EZH2) is a key epigenetic regulator that enzymatically mediates the tri-methylation of lysine 27 on histone 3 (H3K27me3) to silence transcription. Our novel line of investigation proposes that, in normal cholangiocytes, transcription of HSC-activating genes is homeostatically silenced by EZH2. We also propose that the p300 acetyltransferase can lead to H3K27 acetylation (H3K27ac) to initiate transcription. We have generated the following novel preliminary data: 1) RNA-seq and ChIP-seq have identified a network of cholangiocyte-derived paracrine activators of HSCs, including fibronectin (FN), that are silenced by EZH2; 2) TGF-β induces loss of silencing through proteasomal degradation of EZH2; 3) Human livers with biliary fibrosis demonstrate accumulation of p300 and H3K27ac along with peri-portal deposition of FN; 4) p300 forms a transcription factor complex downstream of TGF-β and leads to H3K27ac on the FN promoter; and 5) Genetic or pharmacologic inhibition of EZH2 in vivo leads to exaggerated fibrosis in mouse models of biliary disease. Based on this preliminary data, we propose the central hypothesis that changes in H3K27me3 and H3K27ac, epigenetically initiate fibrogenic cross talk between cholangiocytes and HSC. In Aim I, we will test the subhypothesis that EZH2 leads to homeostatic silencing of FN and other gene targets through H3K27 methylation and this process is regulated by TGF-β through proteasomal degradation of EZH2. In Aim II, we will evaluate the subhypothesis that a p300 complex downstream of TGF-β activates FN transcription through H3K27 acetylation and promotes paracrine HSC activation. In Aim III, we will investigate the subhypothesis that reciprocal regulation at H3K27 by EZH2 and p300 controls cholangiocyte FN deposition, HSC activation, and biliary fibrosis in vivo. The significance of these studies lies in the novel concept that epigenetic regulators allow specific membrane signals in cholangiocytes to re-write the histone code and generate fibrogenic paracrine molecules that subsequently promote HSC activation. In turn, interventions targeting these newly discovered pathways with epigenetic pharmacology may have the capability to prevent or reverse the extracellular matrix events that drive biliary fibrosis.