ETS proto-oncogene 1 transcriptionally up-regulates the cholangiocyte senescence-associated protein cyclin-dependent kinase inhibitor 2A

Steven P. O'Hara, Patrick L. Splinter, Christy E. Trussoni, Maria J. Lorenzo Pisarello, Lorena Loarca, Noah S. Splinter, Bryce F. Schutte, Nicholas F La Russo

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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 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 ∼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.

Original languageEnglish (US)
Pages (from-to)4833-4846
Number of pages14
JournalJournal of Biological Chemistry
Volume292
Issue number12
DOIs
StatePublished - Mar 24 2017

Fingerprint

Cyclin-Dependent Kinase Inhibitor p16
Sclerosing Cholangitis
Proto-Oncogenes
Up-Regulation
Luciferases
Proteins
Mutagenesis
RNA Interference
Liver
Histones
Ducts
Lysine
Chromatin
Bile Duct Diseases
Binding Sites
Genetic Suppression
Messenger RNA
Chromatin Immunoprecipitation
Site-Directed Mutagenesis
Neuroblastoma

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

ETS proto-oncogene 1 transcriptionally up-regulates the cholangiocyte senescence-associated protein cyclin-dependent kinase inhibitor 2A. / O'Hara, Steven P.; Splinter, Patrick L.; Trussoni, Christy E.; Lorenzo Pisarello, Maria J.; Loarca, Lorena; Splinter, Noah S.; Schutte, Bryce F.; La Russo, Nicholas F.

In: Journal of Biological Chemistry, Vol. 292, No. 12, 24.03.2017, p. 4833-4846.

Research output: Contribution to journalArticle

O'Hara, Steven P. ; Splinter, Patrick L. ; Trussoni, Christy E. ; Lorenzo Pisarello, Maria J. ; Loarca, Lorena ; Splinter, Noah S. ; Schutte, Bryce F. ; La Russo, Nicholas F. / ETS proto-oncogene 1 transcriptionally up-regulates the cholangiocyte senescence-associated protein cyclin-dependent kinase inhibitor 2A. In: Journal of Biological Chemistry. 2017 ; Vol. 292, No. 12. pp. 4833-4846.
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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 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 ∼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.",
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AU - Splinter, Patrick L.

AU - Trussoni, Christy E.

AU - Lorenzo Pisarello, Maria J.

AU - Loarca, Lorena

AU - Splinter, Noah S.

AU - Schutte, Bryce F.

AU - La Russo, Nicholas F

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