Bile Acids and Biliary Fibrosis.

Biliary fibrosis is the driving pathological process in cholangiopathies such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). Cholangiopathies are also associated with cholestasis, which is the retention of biliary components, including bile acids, in the liver and blood. Cholestasis may worsen with biliary fibrosis. Furthermore, bile acid levels, composition and homeostasis are dysregulated in PBC and PSC. In fact, mounting data from animal models and human cholangiopathies suggest that bile acids play a crucial role in the pathogenesis and progression of biliary fibrosis. The identification of bile acid receptors has advanced our understanding of various signaling pathways involved in regulating cholangiocyte functions and the potential impact on biliary fibrosis. We will also briefly review recent findings linking these receptors with epigenetic regulatory mechanisms. Further detailed understanding of bile acid signaling in the pathogenesis of biliary fibrosis will uncover additional therapeutic avenues for cholangiopathies.

Long non-coding RNA ACTA2-AS1 promotes ductular reaction by interacting with the p300/ELK1 complex.

BACKGROUND & AIMS: Biliary disease is associated with a proliferative/fibrogenic ductular reaction (DR). p300 is an epigenetic regulator that acetylates lysine 27 on histone 3 (H3K27ac) and is activated during fibrosis. Long non-coding RNAs (lncRNAs) are aberrantly expressed in cholangiopathies, but little is known about how they recruit epigenetic complexes and regulate DR. We investigated epigenetic complexes, including transcription factors (TFs) and lncRNAs, contributing to p300-mediated transcription during fibrosis. METHODS: We evaluated p300 in vivo using tamoxifen-inducible, cholangiocyte-selective, p300 knockout (KO) coupled with bile duct ligation (BDL) and Mdr KO mice treated with SGC-CBP30. Primary cholangiocytes and liver tissue were analyzed for expression of Acta2-as1 lncRNA by qPCR and RNA in situ hybridization. In vitro, we performed RNA-sequencing in human cholangiocytes with a p300 inhibitor. Cholangiocytes were exposed to lipopolysaccharide (LPS) as an injury model. We confirmed formation of a p300/ELK1 complex by immunoprecipitation (IP). RNA IP was used to examine interactions between ACTA2-AS1 and p300. Chromatin IP assays were used to evaluate p300/ELK1 occupancy and p300-mediated H3K27ac. Organoids were generated from ACTA2-AS1-depleted cholangiocytes. RESULTS: BDL-induced DR and fibrosis were reduced in Krt19-CreERT/p300fl/fl mice. Similarly, Mdr KO mice were protected from DR and fibrosis after SGC-CBP30 treatment. In vitro, depletion of ACTA2-AS1 reduced expression of proliferative/fibrogenic markers, reduced LPS-induced cholangiocyte proliferation, and impaired organoid formation. ACTA2-AS1 regulated transcription by facilitating p300/ELK1 binding to the PDGFB promoter after LPS exposure. Correspondingly, LPS-induced H3K27ac was mediated by p300/ELK1 and was reduced in ACTA2-AS1-depleted cholangiocytes. CONCLUSION: Cholangiocyte-selective p300 KO or p300 inhibition attenuate DR/fibrosis in mice. ACTA2-AS1 influences recruitment of p300/ELK1 to specific promoters to drive H3K27ac and epigenetic activation of proliferative/fibrogenic genes. This suggests that cooperation between epigenetic co-activators and lncRNAs facilitates DR/fibrosis in biliary diseases. LAY SUMMARY: We identified a three-part complex containing an RNA molecule, a transcription factor, and an epigenetic enzyme. The complex is active in injured bile duct cells and contributes to activation of genes involved in proliferation and fibrosis.

Epigenomic Evaluation of Cholangiocyte Transforming Growth Factor-ß Signaling Identifies a Selective Role for Histone 3 Lysine 9 Acetylation in Biliary Fibrosis.

BACKGROUND & AIMS: Transforming growth factor ß (TGFß) upregulates cholangiocyte-derived signals that activate myofibroblasts and promote fibrosis. Using epigenomic and transcriptomic approaches, we sought to distinguish the epigenetic activation mechanisms downstream of TGFß that mediate transcription of fibrogenic signals. METHODS: Chromatin immunoprecipitation (ChIP)-seq and RNA-seq were performed to assess histone modifications and transcriptional changes following TGFß stimulation. Histone modifications and acetyltransferase occupancy were confirmed using ChIP assays. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) was used to investigate changes in chromatin accessibility. Cholangiocyte cell lines and primary cholangiocytes were used for in vitro studies. Mdr2-/- and 3,5-diethoxycarboncyl-1,4-dihydrocollidine (DDC)-fed mice were used as animal models. RESULTS: TGFß stimulation caused widespread changes in histone 3 lysine 27 acetylation (H3K27ac), and was associated with global TGFß-mediated transcription. In contrast, H3K9ac was gained in a smaller group of chromatin sites and was associated with fibrosis pathways. These pathways included overexpression of hepatic stellate cell (HSC) activators such as fibronectin 1 (FN1) and SERPINE1. The promoters of these genes showed H3K9ac enrichment following TGFß. Of the acetyltransferases responsible for H3K9ac, cholangiocytes predominantly express Lysine Acetyltransferases 2A (KAT2A). Small interfering RNA knockdown of KAT2A or H3K9ac inhibition prevented the TGFß-mediated increase in FN1 and SERPINE1. SMAD3 ChIP-seq and ATAC-seq suggested that TGFß-mediated H3K9ac occurs through SMAD signaling, which was confirmed using colocalization and genetic knockdown studies. Pharmacologic inhibition or cholangiocyte-selective deletion of Kat2a was protective in mouse models of biliary fibrosis. CONCLUSIONS: Cholangiocyte expression of HSC-activating signals occurs through SMAD-dependent, KAT2A-mediated, H3K9ac, and can be targeted to prevent biliary fibrosis.

Deregulation of Long Intergenic Non-coding RNAs in CD4+ T Cells of Lamina Propria in Crohn's Disease Through Transcriptome Profiling.

BACKGROUND: The aetiology of Crohn's disease [CD] involves immune dysregulation in a genetically susceptible individual. Genome-wide association studies [GWAS] have identified 200 loci associated with CD, ulcerative colitis, or both, most of which fall within non-coding DNA regions. Long non-coding RNAs [lncRNAs] regulate gene expression by diverse mechanisms and have been associated with disease activity in inflammatory bowel disease. However, disease-associated lncRNAs have not been characterised in pathogenic immune cell populations. METHODS: Terminal ileal samples were obtained from 22 CD patients and 13 controls. RNA from lamina propria CD4+ T cells was sequenced and long intergenic non-coding RNAs [lincRNAs] were detected. Overall expression patterns, differential expression [DE], and pathway and gene enrichment analyses were performed. Knockdown of novel lincRNAs XLOC_000261 and XLOC_000014 was performed. Expression of Th1 or Th17-associated transcription factors, T-bet and RORγt, respectively, was assessed by flow cytometry. RESULTS: A total of 6402 lincRNAs were expressed, 960 of which were novel. Unsupervised clustering and principal component analysis showed that the lincRNA expression discriminated patients from controls. A total of 1792 lincRNAs were DE, and 295 [79 novel; 216 known] mapped to 267 of 5727 DE protein-coding genes. The novel lincRNAs were enriched in inflammatory and Notch signalling pathways [p <0.05]. Furthermore, DE lincRNAs in CD patients were more frequently found in DNA regions with known inflammatory bowel disease [IBD]-associated loci. The novel lincRNA XLOC_000261 negatively regulated RORγt expression in Th17 cells. CONCLUSIONS: We describe a novel set of DE lincRNAs in CD-associated CD4+ cells and demonstrate that novel lincRNA XLOC_000261 appears to negatively regulate RORγt protein expression in Th17 cells.

Epigenetic Mechanisms of Pancreatobiliary Fibrosis.

PURPOSE OF REVIEW: The goal of this manuscript is to review the current literature related to fibrogenesis in the pancreatobiliary system and how this process contributes to pancreatic and biliary diseases. In particular, we seek to define the current state of knowledge regarding the epigenetic mechanisms that govern and regulate tissue fibrosis in these organs. A better understanding of these underlying molecular events will set the stage for future epigenetic therapeutics. RECENT FINDINGS: We highlight the significant advances that have been made in defining the pathogenesis of pancreatobiliary fibrosis as it relates to chronic pancreatitis, pancreatic cancer, and the fibro-obliterative cholangiopathies. We also review the cell types involved as well as concepts related to epithelial-mesenchymal crosstalk. Furthermore, we outline important signaling pathways (e.g., TGFß) and diverse epigenetic processes (i.e., DNA methylation, non-coding RNAs, histone modifications, and 3D chromatin remodeling) that regulate fibrogenic gene networks in these conditions. We review a growing body of scientific evidence linking epigenetic regulatory events to fibrotic disease states in the pancreas and biliary system. Advances in this understudied area will be critical toward developing epigenetic pharmacological approaches that may lead to more effective treatments for these devastating and difficult to treat disorders.

Proteasomal Degradation of Enhancer of Zeste Homologue 2 in Cholangiocytes Promotes Biliary Fibrosis.

During biliary disease, cholangiocytes become activated by various pathological stimuli, including transforming growth factor ß (TGF-ß). The result is an epigenetically regulated transcriptional program leading to a pro-fibrogenic microenvironment, activation of hepatic stellate cells (HSCs), and progression of biliary fibrosis. This study evaluated how TGF-ß signaling intersects with epigenetic machinery in cholangiocytes to support fibrogenic gene transcription. We performed RNA sequencing in cholangiocytes with or without TGF-ß. Ingenuity pathway analysis identified "HSC Activation" as the highly up-regulated pathway, including overexpression of fibronectin 1 (FN), connective tissue growth factor, and other genes. Bioinformatics identified enhancer of zeste homologue 2 (EZH2) as an epigenetic regulator of the cholangiocyte TGF-ß response. EZH2 overexpression suppressed TGF-ß-induced FN protein in vitro, suggesting FN as a direct target of EZH2-based repression. Chromatin immunoprecipitation assays identified an FN promoter element in which EZH2-mediated tri-methylation of lysine 27 on histone 3 is diminished by TGF-ß. TGF-ß also caused a 50% reduction in EZH2 protein levels. Proteasome inhibition rescued EZH2 protein and led to reduced FN production. Immunoprecipitation followed by mass spectrometry identified ubiquitin protein ligase E3 component N-recognin 4 in complex with EZH2, which was validated by western blotting in vitro. Ubiquitin mutation studies suggested K63-based ubiquitin linkage and chain elongation on EZH2 in response to TGF-ß. A deletion mutant of EZH2, lacking its N-terminal domain, abrogates both TGF-ß-stimulated EZH2 degradation and FN release. In vivo, cholangiocyte-selective knockout of EZH2 exacerbates bile duct ligation-induced fibrosis whereas MDR2-/- mice are protected from fibrosis by the proteasome inhibitor bortezomib. Conclusion: TGF-ß regulates proteasomal degradation of EZH2 through N-terminal, K63-linked ubiquitination in cholangiocytes and activates transcription of a fibrogenic gene program that supports biliary fibrosis.