The Epigenetic Reader, Bromodomain Containing 2, Mediates Cholangiocyte Senescence via Interaction With ETS Proto-Oncogene 1.

BACKGROUND & AIMS: We reported that cholangiocyte senescence, regulated by the transcription factor ETS proto-oncogene 1 (ETS1), is a pathogenic feature of primary sclerosing cholangitis (PSC). Furthermore, histone 3 lysine 27 is acetylated at senescence-associated loci. The epigenetic readers, bromodomain and extra-terminal domain (BET) proteins, bind acetylated histones, recruit transcription factors, and drive gene expression. Thus, we tested the hypothesis that BET proteins interact with ETS1 to drive gene expression and cholangiocyte senescence. METHODS: We performed immunofluorescence for BET proteins (BRD2 and 4) in liver tissue from liver tissue from PSC patients and a mouse PSC model. Using normal human cholangiocytes (NHCs), NHCs experimentally induced to senescence (NHCsen), and PSC patient-derived cholangiocytes (PSCDCs), we assessed senescence, fibroinflammatory secretome, and apoptosis after BET inhibition or RNA interference depletion. We assessed BET interaction with ETS1 in NHCsen and tissues from PSC patient, and the effects of BET inhibitors on liver fibrosis, senescence, and inflammatory gene expression in mouse models. RESULTS: Tissue from patients with PSC and a mouse PSC model exhibited increased cholangiocyte BRD2 and 4 protein (∼5×) compared with controls without disease. NHCsen exhibited increased BRD2 and 4 (∼2×), whereas PSCDCs exhibited increased BRD2 protein (∼2×) relative to NHC. BET inhibition in NHCsen and PSCDCs reduced senescence markers and inhibited the fibroinflammatory secretome. ETS1 interacted with BRD2 in NHCsen, and BRD2 depletion diminished NHCsen p21 expression. BET inhibitors reduced senescence, fibroinflammatory gene expression, and fibrosis in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine-fed and Mdr2-/- mouse models. CONCLUSION: Our data suggest that BRD2 is an essential mediator of the senescent cholangiocyte phenotype and is a potential therapeutic target for patients with PSC.

Autophagy promotes hepatic cystogenesis in polycystic liver disease by depletion of cholangiocyte ciliogenic proteins.

BACKGROUNDS AND AIMS: Polycystic liver disease (PLD) is characterized by defective cholangiocyte cilia that regulate progressive growth of hepatic cysts. Because formation of primary cilia is influenced by autophagy through degradation of proteins involved in ciliogenesis, we hypothesized that ciliary defects in PLD cholangiocytes (PLDCs) originate from autophagy-mediated depletion of ciliogenic proteins ADP-ribosylation factor-like protein 3 (ARL3) and ADP-ribosylation factor-like protein 13B (ARL13B) and ARL-dependent mislocation of a ciliary-localized bile acid receptor, Takeda G-protein-coupled receptor 5 (TGR5), the activation of which enhances hepatic cystogenesis (HCG). The aims here were to determine whether: (1) ciliogenesis is impaired in PLDC, is associated with increased autophagy, and involves autophagy-mediated depletion of ARL3 and ARL13B; (2) depletion of ARL3 and ARL13B in PLDC cilia impacts ciliary localization of TGR5; and (3) pharmacological inhibition of autophagy re-establishes cholangiocyte cilia and ciliary localization of ARL3, ARL3B, and TGR5 and reduces HCG. APPROACH AND RESULTS: By using liver tissue from healthy persons and patients with PLD, in vitro and in vivo models of PLD, and in vitro models of ciliogenesis, we demonstrated that, in PLDCs: ciliogenesis is impaired; autophagy is enhanced; ARL3 and ARL13B are ubiquitinated by HDAC6, depleted in cilia, and present in autophagosomes; depletion of ARL3 and ARL13B impacts ciliary localization of TGR5; and pharmacological inhibition of autophagy with mefloquine and verteporfin re-establishes cholangiocyte cilia and ciliary localization of ARL3, ARL13B, and TGR5 and reduces HCG. CONCLUSIONS: The intersection between autophagy, defective cholangiocyte cilia, and enhanced HCG contributes to PLD progression and can be considered a target for therapeutic interventions.

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.

Genetic or pharmacological reduction of cholangiocyte senescence improves inflammation and fibrosis in the Mdr2 -/- mouse.

BACKGROUND & AIMS: Cholangiocyte senescence is important in the pathogenesis of primary sclerosing cholangitis (PSC). We found that CDKN2A (p16), a cyclin-dependent kinase inhibitor and mediator of senescence, was increased in cholangiocytes of patients with PSC and from a PSC mouse model (multidrug resistance 2; Mdr2 -/-). Given that recent data suggest that a reduction of senescent cells is beneficial in different diseases, we hypothesised that inhibition of cholangiocyte senescence would ameliorate disease in Mdr2 -/- mice. METHODS: We used 2 novel genetic murine models to reduce cholangiocyte senescence: (i) p16Ink4a apoptosis through targeted activation of caspase (INK-ATTAC)xMdr2 -/-, in which the dimerizing molecule AP20187 promotes selective apoptotic removal of p16-expressing cells; and (ii) mice deficient in both p16 and Mdr2. Mdr2 -/- mice were also treated with fisetin, a flavonoid molecule that selectively kills senescent cells. p16, p21, and inflammatory markers (tumour necrosis factor [TNF]-α, IL-1ß, and monocyte chemoattractant protein-1 [MCP-1]) were measured by PCR, and hepatic fibrosis via a hydroxyproline assay and Sirius red staining. RESULTS: AP20187 treatment reduced p16 and p21 expression by ~35% and ~70% (p >0.05), respectively. Expression of inflammatory markers (TNF-α, IL-1ß, and MCP-1) decreased (by 60%, 40%, and 60%, respectively), and fibrosis was reduced by ~60% (p >0.05). Similarly, p16 -/- xMdr2 -/- mice exhibited reduced p21 expression (70%), decreased expression of TNF-α, IL-1ß (60%), and MCP-1 (65%) and reduced fibrosis (~50%) (p >0.05) compared with Mdr2 -/- mice. Fisetin treatment reduced expression of p16 and p21 (80% and 90%, respectively), TNF-α (50%), IL-1ß (50%), MCP-1 (70%), and fibrosis (60%) (p >0.05). CONCLUSIONS: Our data support a pathophysiological role of cholangiocyte senescence in the progression of PSC, and that targeted removal of senescent cholangiocytes is a plausible therapeutic approach. LAY SUMMARY: Primary sclerosing cholangitis is a fibroinflammatory, incurable biliary disease. We previously reported that biliary epithelial cell senescence (cell-cycle arrest and hypersecretion of profibrotic molecules) is an important phenotype in primary sclerosing cholangitis. Herein, we demonstrate that reducing the number of senescent cholangiocytes leads to a reduction in the expression of inflammatory, fibrotic, and senescence markers associated with the disease.