ETS Proto-oncogene 1 Transcriptionally Up-regulates the Cholangiocyte Senescence- associated Protein Cyclin-dependent Kinase Inhibitor 2A

Primary sclerosing cholangitis (PSC) is a chronic, fibroinflammatory cholangiopathy (disease of the bile ducts) of unknown pathogenesis. We reported that cholangiocyte senescence features prominently in PSC and that neuroblastoma RAS viral oncogene homolog (NRAS) is activated in PSC cholangiocytes. Additionally, persistent microbial insult (e.g. LPSs) induces cyclin-dependent kinase inhibitor 2A (CDKN2A/ p16(INK4a)) expression and senescence in cultured cholangiocytes in an NRAS-dependent manner. However, the molecular mechanisms involved in LPS-induced cholangiocyte senescence and NRAS-dependent regulation of CDKN2A remain unclear. Using our in vitro senescence model, we found that LPSinduced CDKN2A expression coincided with a 4.5-fold increase in ETS1 (ETS proto-oncogene 1) mRNA, suggesting that ETS1 is involved in regulating CDKN2A. This idea was confirmed by RNAi-mediated suppression or genetic deletion of ETS1, which blocked CDKN2A expression and reduced cholangiocyte senescence. Furthermore, site-directed mutagenesis of a predicted ETS-binding site within the CDKN2A promoter abolished luciferase reporter activity. Pharmacological inhibition of RAS/ MAPK reduced ETS1 and CDKN2A protein expression and CDKN2A promoter-driven luciferase activity by similar to 50%. In contrast, constitutively active NRAS expression induced ETS1 and CDKN2A protein expression, whereas ETS1 RNAi blocked this increase. Chromatin immunoprecipitation-PCR detected increased ETS1 and histone 3 lysine 4 trimethylation (H3K4Me3) at the CDKN2A promoter following LPS-induced senescence. Additionally, phospho-ETS1 expression was increased in cholangiocytes of human PSC livers and in the Abcb4 (Mdr2)(-/-) mouse model of PSC. These data pinpoint ETS1 and H3K4Me3 as key transcriptional regulators in NRAS-induced expression of CDKN2A, and this regulatory axis may therefore represent a potential therapeutic target for PSC treatment.