Abstract
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/p16INK4a) 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 LPS-induced 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 ∼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.
Highlights
Primary sclerosing cholangitis (PSC) is a chronic, fibroinflammatory cholangiopathy of unknown pathogenesis
We showed that neuroblastoma RAS viral oncogene homolog (NRAS) activation and CDKN2A mRNA is increased in cholangiocytes of PSC livers, supporting a role for the RAS/MAPK signaling axis and CDKN2A expression in cholangiocyte senescence in this disease
Using an in vitro model of stress-induced cholangiocyte senescence, we demonstrated that: (i) prolonged LPS treatment of cholangiocytes increased ETS1, but not ETS2, expression and phosphorylation; (ii) overexpression of constitutively active NRAS promoted CDKN2A expression and cholangiocyte senescence in an ETS1-dependent manner; (iii) in prolonged LPS-treated cells, pharmacologic inhibition of RAS/
Summary
Primary sclerosing cholangitis (PSC) is a chronic, fibroinflammatory cholangiopathy (disease of the bile ducts) of unknown pathogenesis. The precise molecular mechanisms regulating insult-induced cholangiocyte senescence remain ambiguous It is known, that the ETS1/2 transcription factors are effectors of the RAS/MAPK signaling pathway [17,18,19], and both ETS1 and ETS2 have been implicated in CDKN2A expression and the induction of senescence [20]. Our collective data suggest that during the process of cholangiocyte senescence, the RAS/MAPK pathway induces expression and phosphorylation of ETS1, but not ETS2, promotes CDKN2A transcription via loss of repressive chromatin marks (i.e. histone 3 lysine 27 trimethylation (H3K27me3)) and the induction of permissive chromatin remodeling (H3K4me3) These novel results provide mechanistic insight into insultinduced cholangiocyte senescence, a cellular state with a potential pathophysiological role in the development and progression of PSC and other diseases. A greater understanding of these pathways may provide new therapeutic strategies for PSC and perhaps other conditions where senescent cells likely contribute to disease progression
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