The transcription factor ETS1 promotes apoptosis resistance of senescent cholangiocytes by epigenetically up-regulating the apoptosis suppressor BCL2L1

Primary sclerosing cholangitis (PSC) is an idiopathic, progressive cholangiopathy. Cholangiocyte senescence is important in PSC pathogenesis, and we have previously reported that senescence is regulated by the transcription factor ETS proto-oncogene 1 (ETS1) and associated with overexpression of BCL2 like 1 (BCL2L1 or BCL-xL), an anti-apoptotic BCL2-family member. Here, we further explored the mechanisms regulating BCL-xL-mediated, apoptosis resistance in senescent cholangiocytes and uncovered that ETS1 and the histone acetyltransferase E1A-binding protein P300 (EP300 or p300) both promote BCL-xL transcription. Using immunofluorescence, we found that BCL-xL protein expression is increased both in cholangiocytes of livers from individuals with PSC and a mouse model of PSC. Using an in vitro model of lipopolysaccharide-induced senescence in normal human cholangiocytes (NHCs), we found increased BCL-xL mRNA and protein levels, and ChIP-PCRs indicated increased occupancy of ETS1, p300, and histone 3 Lys-27 acetylation (H3K27Ac) at the BCL-xL promoter. Using co-immunoprecipitation and proximity ligation assays, we further demonstrate that ETS1 and p300 physically interact in senescent but not control NHCs. Additionally, mutagenesis of predicted ETS1-binding sites within the BCL-xL promoter blocked luciferase reporter activity, and CRISPR/Cas9-mediated genetic deletion of ETS1 reduced senescence-associated BCL-xL expression. In senescent NHCs, TRAIL-mediated apoptosis was reduced ∼70%, and ETS1 deletion or RNAi-mediated BCL-xL suppression increased apoptosis. Overall, our results suggest that ETS1 and p300 promote senescent cholangiocyte resistance to apoptosis by modifying chromatin and inducing BCL-xL expression. These findings reveal ETS1 as a central regulator of both cholangiocyte senescence and the associated apoptosis-resistant phenotype. Primary sclerosing cholangitis (PSC) is an idiopathic, progressive cholangiopathy. Cholangiocyte senescence is important in PSC pathogenesis, and we have previously reported that senescence is regulated by the transcription factor ETS proto-oncogene 1 (ETS1) and associated with overexpression of BCL2 like 1 (BCL2L1 or BCL-xL), an anti-apoptotic BCL2-family member. Here, we further explored the mechanisms regulating BCL-xL-mediated, apoptosis resistance in senescent cholangiocytes and uncovered that ETS1 and the histone acetyltransferase E1A-binding protein P300 (EP300 or p300) both promote BCL-xL transcription. Using immunofluorescence, we found that BCL-xL protein expression is increased both in cholangiocytes of livers from individuals with PSC and a mouse model of PSC. Using an in vitro model of lipopolysaccharide-induced senescence in normal human cholangiocytes (NHCs), we found increased BCL-xL mRNA and protein levels, and ChIP-PCRs indicated increased occupancy of ETS1, p300, and histone 3 Lys-27 acetylation (H3K27Ac) at the BCL-xL promoter. Using co-immunoprecipitation and proximity ligation assays, we further demonstrate that ETS1 and p300 physically interact in senescent but not control NHCs. Additionally, mutagenesis of predicted ETS1-binding sites within the BCL-xL promoter blocked luciferase reporter activity, and CRISPR/Cas9-mediated genetic deletion of ETS1 reduced senescence-associated BCL-xL expression. In senescent NHCs, TRAIL-mediated apoptosis was reduced ∼70%, and ETS1 deletion or RNAi-mediated BCL-xL suppression increased apoptosis. Overall, our results suggest that ETS1 and p300 promote senescent cholangiocyte resistance to apoptosis by modifying chromatin and inducing BCL-xL expression. These findings reveal ETS1 as a central regulator of both cholangiocyte senescence and the associated apoptosis-resistant phenotype. Bile ducts are lined by epithelial cells, termed cholangiocytes, that transport and modify primary bile as it transits from the liver to the small intestine. Primary sclerosing cholangitis (PSC) 3The abbreviations used are: PSCprimary sclerosing cholangitisEtsV-Ets avian erythroblastosis virus E26 oncogene homolog 1EP300E1A-binding protein p300BCL2L1BCL2-like 1SASPsenescence-associated secretory phenotypeLPSlipopolysaccharideH3K27Achistone 3 lysine 27 acetylationSA-β-galsenescence-associated β-galactosidasePLAproximity ligation assayqPCRquantitative PCRPCNAproliferating cell nuclear antigenβ-galβ-galactosidasefmkfluoromethyl ketonePOLR2RNA polymerase 2DAPI4′,6-diamidino-2-phenylindoleEVempty vectorFLfull-lengthOEoverexpressionshRNAshort hairpin RNAMOMPmitochondrial outer-membrane permeabilizationBCL2B cell lymphoma 2CREBcAMP-response element-binding proteinNHCnormal human cholangiocyteSDMsite-directed mutagenesisHAThypoxanthine/aminopterin/thymidine mediumIPimmunoprecipitationLPS-ISLPS-induced senescenceCtrlcontrol. is an idiopathic, progressive fibro-inflammatory disease whereby inflammation and fibrotic stricturing ultimately destroy these intra- and/or extrahepatic bile ducts (1Lazaridis K.N. 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Our recent studies have implicated cholangiocyte senescence as an etiologic feature of PSC and a potential therapeutic target. primary sclerosing cholangitis V-Ets avian erythroblastosis virus E26 oncogene homolog 1 E1A-binding protein p300 BCL2-like 1 senescence-associated secretory phenotype lipopolysaccharide histone 3 lysine 27 acetylation senescence-associated β-galactosidase proximity ligation assay quantitative PCR proliferating cell nuclear antigen β-galactosidase fluoromethyl ketone RNA polymerase 2 4′,6-diamidino-2-phenylindole empty vector full-length overexpression short hairpin RNA mitochondrial outer-membrane permeabilization B cell lymphoma 2 cAMP-response element-binding protein normal human cholangiocyte site-directed mutagenesis hypoxanthine/aminopterin/thymidine medium immunoprecipitation LPS-induced senescence control. The senescent cell fate is characterized by permanent withdrawal from the cell cycle in phase G1 or G2 (4Campisi J. d'Adda di Fagagna F. 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Our data also support that the interaction of a RAS responsive transcription factor, ETS1, with the promoter elements of p16INK4a is required for p16INK4a expression and the senescent cholangiocyte cell fate in both LPS-induced and NRAS overexpression culture models (18O'Hara S.P. Splinter P.L. Trussoni C.E. Pisarello M.J. Loarca L. Splinter N.S. Schutte B.F. LaRusso N.F. ETS proto-oncogene 1 transcriptionally up-regulates the cholangiocyte senescence-associated protein cyclin-dependent kinase inhibitor 2A.J. Biol. Chem. 2017; 292 (28184004): 4833-484610.1074/jbc.M117.777409Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The relevance of these in vitro observations was supported by in vivo data showing that phospho-ETS1 protein expression was increased in cholangiocytes of both human PSC liver samples and in the ABC subfamily B member 4 genetic knockout (Abcb4−/−; also known as multidrug-resistant 2 (Mdr2−/−) mouse, an animal model of PSC (18O'Hara S.P. Splinter P.L. Trussoni C.E. Pisarello M.J. Loarca L. Splinter N.S. Schutte B.F. LaRusso N.F. ETS proto-oncogene 1 transcriptionally up-regulates the cholangiocyte senescence-associated protein cyclin-dependent kinase inhibitor 2A.J. Biol. Chem. 2017; 292 (28184004): 4833-484610.1074/jbc.M117.777409Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Although these data enhanced our understanding of the molecular mechanisms of cholangiocyte senescence, they did not address other phenotypic features of senescent cholangiocytes. Senescence is frequently associated with resistance to apoptosis, which may account for the persistence of senescent cells in tissues and associated deleterious consequences (19Wang E. Senescent human fibroblasts resist programmed cell death, and failure to suppress bcl2 is involved.Cancer Res. 1995; 55 (7757977): 2284-2292PubMed Google Scholar, 20Yosef R. Pilpel N. Tokarsky-Amiel R. Biran A. Ovadya Y. Cohen S. Vadai E. Dassa L. Shahar E. 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This family includes the mitochondrial pore forming effector proteins, BAK and BAX, as well as pro-apoptotic activators and anti-apoptotic mediators, the balance of which determines cell survival or death (22Adams J.M. Cory S. Bcl-2-regulated apoptosis: mechanism and therapeutic potential.Curr. Opin. Immunol. 2007; 19 (17629468): 488-49610.1016/j.coi.2007.05.004Crossref PubMed Scopus (521) Google Scholar, 23Wang C. Youle R.J. The role of mitochondria in apoptosis.Annu. Rev. Genet. 2009; 43 (19659442): 95-11810.1146/annurev-genet-102108-134850Crossref PubMed Scopus (1243) Google Scholar). We recently demonstrated in vitro that the anti-apoptotic protein, BCL2L1 (BCL-xL), is up-regulated in senescent cholangiocytes, and pharmacological inhibition of BCL-xL with the small molecule inhibitor, A1331852, selectively kills cultured senescent cholangiocytes. Moreover, pharmacological inhibition of BCL-xL in the Mdr2−/− mouse diminished the number of senescent cholangiocytes and decreased liver fibrosis (24Moncsek A. Al-Suraih M.S. Trussoni C.E. O’Hara S.P. Splinter P.L. Zuber C. Patsenker E. Valli P.V. Fingas C.D. Weber A. Zhu Y. Tchkonia T. Kirkland J.L. Gores G.J. Müllhaupt B. LaRusso N.F. Mertens J.C. Targeting senescent cholangiocytes and activated fibroblasts with B-cell lymphoma-extra large inhibitors ameliorates fibrosis in multidrug resistance 2 gene knockout (Mdr2(−/−)) mice.Hepatology. 2018; 67 (28802066): 247-25910.1002/hep.29464Crossref PubMed Scopus (73) Google Scholar). Although ETS1 has been implicated in promoting the expression of prosurvival proteins and resistance to apoptosis (25Li R. Pei H. Watson D.K. Papas T.S. EAP1/Daxx interacts with ETS1 and represses transcriptional activation of ETS1 target genes.Oncogene. 2000; 19 (10698492): 745-75310.1038/sj.onc.1203385Crossref PubMed Scopus (158) Google Scholar, 26Yu Z. Shah D.M. Curcumin down-regulates Ets-1 and Bcl-2 expression in human endometrial carcinoma HEC-1-A cells.Gynecol. Oncol. 2007; 106 (17590421): 541-54810.1016/j.ygyno.2007.05.024Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), whether ETS1 promotes apoptosis resistance of senescent cells in general, and of senescent cholangiocytes in particular is unclear and is the focus of our work here. Our collective data suggest that ETS1 not only promotes cholangiocyte senescence via the up-regulation of p16INK4a, but also drives the expression of BCL-xL via the recruitment of the chromatin remodeling histone acetyltransferase, p300. These novel results provide mechanistic insight into senescent cholangiocyte apoptosis resistance, and suggest a potential pathophysiological role in the development and progression of PSC and perhaps other diseases. Moreover, pharmacologic targeting of this pathway may provide a new therapeutic strategy for PSC and other conditions where apoptosis-resistant senescent cells likely contribute to disease progression. We previously published that BCL-xL inhibition in the Mdr2−/− mouse model of PSC-depleted senescent cholangiocyte number and improved fibrosis. To extend this observation, we assessed BCL-xL protein expression by immunofluorescent confocal microscopy and confirmed that cholangiocytes from nondiseased human liver (normal control) express very little BCL-xL protein, whereas cholangiocytes from PSC patient liver tissue expressed increased BCL-xL (Fig. 1A). Quantitation of fluorescent intensity was performed and confirmed increased BCL-xL expression (∼5-fold) in PSC cholangiocytes compared with normal control cholangiocytes (Fig. 1B). Given that Mdr2−/− mice exhibit increased senescent cholangiocytes (14Tabibian J.H. O'Hara S.P. Splinter P.L. Trussoni C.E. LaRusso N.F. Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.Hepatology. 2014; 59 (24390753): 2263-227510.1002/hep.26993Crossref PubMed Scopus (161) Google Scholar) and that BCL-xL inhibition improves fibrosis (24Moncsek A. Al-Suraih M.S. Trussoni C.E. O’Hara S.P. Splinter P.L. Zuber C. Patsenker E. Valli P.V. Fingas C.D. Weber A. Zhu Y. Tchkonia T. Kirkland J.L. Gores G.J. Müllhaupt B. LaRusso N.F. Mertens J.C. Targeting senescent cholangiocytes and activated fibroblasts with B-cell lymphoma-extra large inhibitors ameliorates fibrosis in multidrug resistance 2 gene knockout (Mdr2(−/−)) mice.Hepatology. 2018; 67 (28802066): 247-25910.1002/hep.29464Crossref PubMed Scopus (73) Google Scholar), we next assessed cholangiocyte BCL-xL expression in wildtype (WT) C57bl6 and Mdr2−/− mouse liver tissue. As with human specimens, BCL-xL was minimally expressed in cholangiocytes from WT mice, whereas expression was increased ∼3-fold in Mdr2−/− mice (Fig. 1, C and D). Thus, these in vivo data confirm and extend our previous work by demonstrating up-regulated expression of the prosurvival protein, BCL-xL, in cholangiocytes in samples of liver from PSC patients and the Mdr2−/− mouse. The anti-apoptotic Bcl-2 proteins may be up-regulated in senescent cells and prevent senescent cell death (20Yosef R. Pilpel N. Tokarsky-Amiel R. Biran A. Ovadya Y. Cohen S. Vadai E. Dassa L. Shahar E. Condiotti R. Ben-Porath I. Krizhanovsky V. Directed elimination of senescent cells by inhibition of BCL-W and BCL-XL.Nat. Commun. 2016; 7 (27048913): 1119010.1038/ncomms11190Crossref PubMed Scopus (495) Google Scholar). In an effort to assess the expression of potential pro- and anti-apoptotic mediators in senescent cholangiocytes, we utilized our culture model of LPS-induced cholangiocyte senescence (14Tabibian J.H. O'Hara S.P. Splinter P.L. Trussoni C.E. LaRusso N.F. Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.Hepatology. 2014; 59 (24390753): 2263-227510.1002/hep.26993Crossref PubMed Scopus (161) Google Scholar). Quantitative PCR (qPCR) demonstrated a selective increase in BCL-xL mRNA (∼8-fold), in senescent versus nonsenescent controls (Fig. 2A); other anti-apoptotic BCL2 family members were not affected. We further found that the pro-apoptotic effector, BAK1, increased ∼2-fold in senescent cholangiocytes, whereas the proapoptotic activator, Noxa, decreased (Fig. 2A). Western blotting analyses confirmed the selective up-regulation of BCL-xL and BAK1, as well as the decreased expression of BAX and NOXA in senescent cholangiocytes (Fig. 2B). To confirm the induction of cholangiocyte senescence in our culture model, we performed qPCR and Western blotting on senescence-associated gene products. qPCR confirmed that both CDKN2A (p16) and CDKN1A (p21) were up-regulated in our culture system, whereas Western blotting confirmed the up-regulation of CDKN2A, CDKN1A as well as the senescence-associated DNA damage histone mark, γH2A.X (Fig. 2C). The proliferation-associated gene product, proliferating cell nuclear antigen (PCNA) also decreased (Fig. 2C). Furthermore, and as demonstrated by us previously (18O'Hara S.P. Splinter P.L. Trussoni C.E. Pisarello M.J. Loarca L. Splinter N.S. Schutte B.F. LaRusso N.F. ETS proto-oncogene 1 transcriptionally up-regulates the cholangiocyte senescence-associated protein cyclin-dependent kinase inhibitor 2A.J. Biol. Chem. 2017; 292 (28184004): 4833-484610.1074/jbc.M117.777409Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), ETS1 is up-regulated in senescent cholangiocytes (Fig. 2C), whereas pharmacologic inhibition of Ras/MAPK prevents ETS1 up-regulation and also diminishes BCL-xL mRNA and protein up-regulation in our senescent cholangiocyte culture model (Fig. S1). Collectively, these data suggest that the mitochondrial apoptotic pathway in senescent cholangiocytes is primed for mitochondrial cell death via up-regulation of the pro-apoptotic effector protein, BAK1, and support that the increased expression of the anti-apoptotic mediator, BCL-xL, may be essential to sequester BAK1 and confer the apoptosis-resistant phenotype of senescent cholangiocytes. We previously demonstrated that the Ras-responsive transcription factor, ETS1, promoted CDKN21 (p16) expression and cholangiocyte senescence following LPS treatment (18O'Hara S.P. Splinter P.L. Trussoni C.E. Pisarello M.J. Loarca L. Splinter N.S. Schutte B.F. LaRusso N.F. ETS proto-oncogene 1 transcriptionally up-regulates the cholangiocyte senescence-associated protein cyclin-dependent kinase inhibitor 2A.J. Biol. Chem. 2017; 292 (28184004): 4833-484610.1074/jbc.M117.777409Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). We therefore asked whether the expression of BCL-xL dynamically correlated with the induction of cholangiocyte senescence and ETS1 expression in our experimentally-induced senescent cholangiocytes. Western blotting demonstrated that BCL-xL up-regulation occurred following 6 days of repeated LPS treatment, which correlates with ETS1 up-regulation and the induction of senescence as demonstrated with the induction of CDKN2A and CDKN1A (Fig. 3A). To directly assess whether the up-regulated expression of BCL-xL correlated with apoptosis resistance and senescence, we utilized our cell culture model of induced senescence and assessed apoptosis by measuring activation of caspase-3/7 and senescence by β-galactosidase positivity. Although untreated normal human cholangiocytes (NHCs) exhibited minimal apoptosis, we found that exposure to LPS induced cultured NHC apoptosis at days 1–4, but decreased on days 6–10 (Fig. 3B). Moreover, and as demonstrated previously (14Tabibian J.H. O'Hara S.P. Splinter P.L. Trussoni C.E. LaRusso N.F. Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.Hepatology. 2014; 59 (24390753): 2263-227510.1002/hep.26993Crossref PubMed Scopus (161) Google Scholar), β-gal activity increased at 6 days post-repeated LPS treatment, and persisted through 10 days post-repeated LPS treatment (Fig. 3C). To confirm the apoptotic-resistant phenotype we treated nonsenescent or induced senescent NHC with TRAIL. Although TRAIL treatment activates the extrinsic (death receptor-mediated) apoptotic pathway, activation of effector caspases (such as caspase-3/7) in liver epithelia (i.e. hepatocytes and cholangiocytes) requires the engagement of the mitochondrial apoptotic pathway (27Guicciardi M.E. Malhi H. Mott J.L. Gores G.J. Apoptosis and necrosis in the liver.Compr. Physiol. 2013; 3 (23720337): 977-1010Crossref PubMed Scopus (254) Google Scholar, 28Kurita S. Mott J.L. Cazanave S.C. Fingas C.D. Guicciardi M.E. Bronk S.F. Roberts L.R. Fernandez-Zapico M.E. Gores G.J. Hedgehog inhibition promotes a switch from type II to type I cell death receptor signaling in cancer cells.PLoS ONE. 2011; 6 (21483830): e1833010.1371/journal.pone.0018330Crossref PubMed Scopus (23) Google Scholar). We found TRAIL-induced apoptosis in nonsenescent control NHC, but not in senescent NHC and that the induction of apoptosis in nonsenescent NHC was prevented by the caspase inhibitor Z-VAD-fmk (Fig. 3, D and E). Senescence detection remained elevated in LPS-IS cholangiocytes in both the presence and absence of TRAIL and the presence and absence of Z-VAD-FMK (Fig. 3F). Together, these data continue to support an essential role of ETS1 in cholangiocyte senescence and demonstrate further that BCL-xL up-regulation correlates with the expression of ETS1, senescence induction, and the apoptosis-resistance phenotype. Anti-apoptotic Bcl-2 proteins (e.g. BCL-xL) either inhibit the pro-apoptotic effectors (BAK and BAX) via direct binding of their activated form or by sequestering “activator” BH3-only proteins (e.g. BIM, NOXA) and preventing their activation of BAK and BAX (22Adams J.M. Cory S. Bcl-2-regulated apoptosis: mechanism and therapeutic potential.Curr. Opin. Immunol. 2007; 19 (17629468): 488-49610.1016/j.coi.2007.05.004Crossref PubMed Scopus (521) Google Scholar). We therefore assessed whether the anti-apoptotic protein BCL-xL directly interacted with apoptotic effectors or activators in senescent cholangiocytes. We immunoprecipitated BCL-xL from control and senescent cholangiocytes and performed immunoblotting for apoptosis activators and effectors. We found that BCL-xL formed an immunoprecipitable complex with BAK and BAX, but not BIM or NOXA (Fig. 4A). It is proposed that BCL-xL interacts with activated BAK and/or BAX, blocks oligomerization of these proapoptotic molecules, and thus prevents apoptosis (29Kale J. Osterlund E.J. Andrews D.W. BCL-2 family proteins: changing partners in the dance towards death.Cell Death Differ. 2018; 25 (29149100): 65-8010.1038/cdd.2017.186Crossref PubMed Scopus (709) Google Scholar). Therefore, we next immunoprecipitated activated BAK and BAX and immunoblotted for BCL-xL. We detected BCL-xL only in senescent cholangiocytes further supporting that active BAK and BAX interact with BCL-xL in senescent cholangiocytes (Fig. 4B). Finally, to determine whether the antiapoptotic protein BCL-xL interacts with BAX and BAK in human PSC liver tissue, we performed proximity ligation assays on formalin-fixed paraffin-embedded tissue. This fluorescence-based approach suggests increased BAK and BAX interaction with BCL-xL in PSC cholangiocytes (Fig. 4, C and D). Together, these in vitro results support that up-regulated BCL-xL directly interacts with active BAK and BAX in senescent cholangiocytes and that this interaction is a likely mechanism for the apoptosis-resistant phenotype of senescent cholangiocytes. Furthermore, the data supports that this process occurs in cholangiocytes from PSC liver tissue. The DNA elements upstream of the BCL-xL transcriptional start site were analyzed for potential ETS1-binding sites and also analyzed for potential epigenetic regulation (i.e. histone acetylation marks). To perform these in silico analyses, we used the Encyclopedia of DNA Elements (ENCODE) annotation data (30Rosenbloom K.R. Armstrong J. Barber G.P. Casper J. Clawson H. Diekhans M. Dreszer T.R. Fujita P.A. Guruvadoo L. Haeussler M. Harte R.A. Heitner S. Hickey G. Hinrichs A.S. Hubley R. et al.The UCSC genome browser database: 2015 update.Nucleic Acids Res. 2015; 43 (25428374): D670-D68110.1093/nar/gku1177Crossref PubMed Scopus (674) Google Scholar) through the