Background: We reported that cholangiocyte senescence is a pathogenic feature of Primary Sclerosing Cholangitis (PSC) and is regulated by the transcription factor, ETS1. We also showed that Histone 3 Lysine 27 is acetylated at senescence-associated loci. The bromodomain and extra-terminal domain (BET) proteins are epigenetic readers that 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 promote cholangiocyte senescence. Methods: We performed immunofluorescence to assess cholangiocyte expression of two BET proteins, BRD2 and BRD4, in tissue from PSC patients and PSC mouse models (Mdr2-/- and DDC-fed). Using Normal Human Cholangiocytes (NHC), NHC induced to senescence (senNHC), and PSC patient-derived cholangiocytes (PDC), we assessed BRD2 & 4 expression (qPCR and immunoblotting), senescence (SA-βGal staining and p21 immunoblot), fibroinflammatory secretome (multiplex array) and expression profile (qPCR), and apoptosis (Caspase 3/7 assay) following BET protein inhibition (JQ1) or RNAi depletion of BRD2 & 4. We assessed BET protein interaction with ETS1 in senNHC [immunoprecipitation (IP)] and in PSC patient tissue [proximity ligation assay (PLA)]. We also assessed the effects of JQ1 on liver fibrosis [Sirius red staining, hydroxyproline assay, smooth muscle actin (αSMA) immunoblot] and inflammatory gene expression (qPCR) in PSC mouse models. Results: PSC patient and PSC mouse model tissues exhibited increased cholangiocyte BRD2 & 4 protein (~5x) compared to non-disease controls. BRD2 & 4 protein was increased in senNHC (~2x) and BRD2 protein was increased in PDC (~2x) relative to NHC. JQ1 reduced senNHC and PDC SA-βGal and p21 protein expression (~80%), and inflammatory/fibrotic protein secretion and mRNA expression (>50%). Additionally, JQ1 induced apoptosis in senNHC and PDC, while RNAi depletion of BRD2 diminished cholangiocyte senescence in senNHC. ETS1 interaction with BRD2 was increased in senNHC (IP, ~10x) compared to NHC, and in cholangiocytes of PSC patient tissues (PLA, ~10x) compared to non-disease controls. JQ1 reduced liver Ccl2, and Ccl20 mRNA expression (>50%), Sirius red positivity, hydroxyproline, and αSMA (>50%) in the DDC 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.
Kang JH, Splinter P, Trussoni C, Pirius NE, Guicciardi ME, Krishnan A, et al. [Ozturk NB]. BET protein complexes with ETS1 and mediates the senescence-associated secretory and apoptotic resistant phenotypes of senescent cholangiocytes. Hepatology. 2022 Oct;76(S1):S164-S165. doi:10.1002/hep.32697.