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

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/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 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.

Keywords: CDKN2A; Cholangiocytes; ETS1; cell signaling; epigenetics; epithelial cell; senescence; transcription.

FIGURE 1.

Persistent LPS treatment of cholangiocytes increases ETS1 and CDKN2A expression. A, CDKN2A, ETS1, and ETS2 mRNA expression was assessed at 1, 2, 6, and 10 days. By day 10, CDKN2A and ETS1 were significantly increased (∼9- and ∼5-fold compared with no LPS control cells), but ETS2 remained unchanged. B, total ETS1 and phospho-ETS1 protein were increased following 10-day treatment of LPS (compared with control), whereas total and phospho-ETS2 protein did not change following persistent LPS treatment. C, quantitation of ETS Western blotting (n = 4). D, RNAi-mediated (shRNA) depletion of ETS1 suppresses LPS-induced CDKN2A expression and cholangiocyte senescence. Transfection with an ETS1-shRNA suppressed LPS-mediated ETS1 and CDKN2A protein expression. E, ETS1-shRNA suppressed LPS-induced senescence by ∼7-fold compared with empty vector controls as assessed by SA-β-gal detection. SA-β-gal data are expressed as the percentage of SA-β-gal-positive cells per total cell counts (n = 3). Ctrl, control.

FIGURE 2.

Genetic deletion (CRISPR-Cas9 double nickase) of ETS1 suppresses LPS-induced CDKN2A expression and cholangiocyte senescence. A and B, ETS1 expression was prevented in NHC cells by cotransfection with the CRISPR-Cas9 double nickase system plasmids, and the cells were maintained in culture in the presence or absence of LPS for 10 days. Phospho-ETS1, ETS1, and CDKN2A expression was not detected in these cells in the presence or absence of LPS. Accordingly, no increase in cholangiocyte senescence was detected by SA-β-gal assay. Reconstitution of ETS1 expression via transfection with an ETS1-GFP expression construct promoted phospho-ETS1 detection, promoted CDKN2A expression, and increased SA-β-gal detection in both the presence and absence of LPS. Reconstitution of ETS1-deficient cells with forced expression of a mutant ETS1 lacking the transactivation domain (ETS1-Trans) resulted in increased phospho-ETS1 detection in the presence and absence of LPS yet prevented CDKN2A expression and associated cholangiocyte senescence (SA-β-gal detection). Additionally, reconstitution of ETS1-deficient cells with forced expression of a non-phosphorylatable mutant ETS1 (T38A) prevented phospho-ETS1 detection, CDKN2A expression, and cholangiocyte senescence. C and D, forced overexpression of CDKN2A in ETS1-deficient NHCs promotes CDKN2A detection in the presence and absence of LPS and increases cholangiocyte senescence (SA-β-gal detection). The Western blots shown are representative from three separate experiments. SA-β-gal data are expressed as the percentages of SA-β-gal-positive cells per total cell counts (n = 4). E, the expression of IL6 and IL8 was assessed by qPCR in NHCs and NHCs depleted of ETS1 cultured in the presence or absence of LPS. NHCs cultured in the presence of LPS exhibited ∼2-fold increase in IL6 and IL8 mRNA expression compared with NHCs not treated with LPS (Ctrl). ETS1-deficient cells cultured in the absence of LPS exhibited an increase in both IL6 (>2-fold) and IL8 (>3-fold) mRNA compared with Ctrl NHCs. ETS1-deficient cells cultured in the presence of LPS also exhibited an increase in IL6 (>3-fold) and IL8 (>3-fold) compared with Ctrl NHCs. The data represent fold change ± standard deviation compared with NHCs cultured in the absence of LPS (n = 3). F, the expression of IL6 and IL8 was assessed by ELISA in NHCs and NHCs depleted of ETS1 cultured in the presence or absence of LPS. NHCs cultured in the presence of LPS exhibited an increase in IL6 and IL8 expression (4- and 2.5-fold, respectively) compared with control NHCs. ETS1-deficient cells cultured in the absence of LPS exhibited an increase in both IL6 (∼6-fold) and IL8 (∼3-fold) compared with Ctrl NHCs. ETS1-depleted cells cultured in the presence of LPS also exhibited an increase in IL6 (∼15-fold) and IL8 (∼6-fold) compared with control NHCs. The data are presented as mean pmol/ml ± standard deviation (n = 3). Ctrl, control.

FIGURE 3.

Expression of constitutively active NRAS promotes ETS1 and CDKN2A expression. A, stable transfection with the NRAS 12D demonstrates increased activation of NRAS in the NHC cells. Western blotting analysis of RBD-GST pulldowns suggests greater amounts of activated NRAS in the NRAS 12D transfected cells compared with the control non-transfected (Ctrl) or empty vector (EV) control. Total protein was detected by Ponceau Red staining as a loading control. B, the NRAS 12D coding sequence was subcloned into a cumate-inducible expression vector for regulated expression of constitutive active NRAS. Activated NRAS was induced by administration of cumate for 0, 4, or 8 days. Very little phospho-ERK1/2, ETS1, and CDKN2A was detected at day 0 or in day 4 and 8 cells not induced to overexpress activated NRAS (days 4 and 8 minus cumate). Phospho-ERK 1/2, ETS1, and CDKN2A expression increased from days 4 to 8 in the presence of cumate (i.e. activated NRAS). C, Western blotting quantitation (densitometry) demonstrates the time-dependent increase in phospho-ERK1/2 to ERK1/2 ratio as well and ETS1 and CDKN2A to β-actin (ACTB) ratio (n = 3). D, SA-β-gal assays demonstrate that the induced expression of activated NRAS increases cholangiocyte senescence. In the absence of cumate, no increase in cholangiocyte senescence was observed at days 4 and 8. In contrast, cholangiocyte senescence is increased in cells treated with cumate for 4 or 8 days (i.e. activated NRAS overexpression). SA-β-gal data are expressed as the percentages of SA-β-gal-positive cells per total cell counts (n = 3). E, ETS1 shRNA prevents NRAS-dependent up-regulation of CDKN2A expression. The ETS1 shRNA and an NRAS 12D expression construct (pCGN NRAS 12D) were cotransfected into NHC cells. Western blotting demonstrates that in the absence of the ETS1 shRNA, constitutive active NRAS promotes the up-regulation of CDKN2A compared with control NHCs (Ctrl) with or without the ETS1 shRNA and pCGN empty vector transfected cells (EV Ctrl) without the ETS1 shRNA. The activated NRAS-dependent up-regulation of CDKN2A is blocked in the presence of the ETS1 shRNA. F, Western blotting quantitation (densitometry) of the CDKN2A to β-actin (ACTB) ratio demonstrates that RNAi depletion of ETS1 (shRNA) prevents NRAS 12D-dependent up-regulation of CDKN2A (n = 3).

FIGURE 4.

Pharmacologic inhibitors of Ras (manumycin A)/MAPK (UO126 or PD98059) block LPS-induced ERK phosphorylation and Ets1 and CDKN2A expression. A, phospho-ERK1/2 is increased in persistent LPS-treated cells; this increase is blocked in the presence of both Ras (manumycin A) and MEK inhibitors (UO126 and PD98059). B, densitometry of the phospho-ERK1/2 and total ERK 1/2 blots demonstrate a significant increase of phospho-ERK 1/2 following persistent LPS treatment compared with control cells (no LPS) and a suppression of this increase in the presence of the RAS and MEK inhibitors (n = 3). C, total ETS1 and CDKN2A protein expression is increased in persistent LPS-treated cells; this increase is reduced when the cells are treated with manumycin A, UO126, or PD98059 as determined by immunoblot analysis. D, densitometry of the ETS1 immunoblots demonstrate a significant increase of Ets1 protein following LPS treatment compared with control cells (no LPS). In addition, treatment with manumycin A, UO126, or PD98059 resulted in a significant ∼50% decrease of Ets1 protein following LPS treatment compared with LPS alone (n = 4). E, densitometry of the CDKN2A immunoblots demonstrates a significant increase of CDKN2A protein following LPS treatment compared with control cells (no LPS) consistent with previous observations. In addition, treatment with manumycin A, UO126, or PD98059 resulted in a significant ∼50% decrease of CDKN2A protein following LPS treatment compared with LPS alone (n = 5). Ctrl, control; Man, manumycin A; PD, PD98059.

FIGURE 5.

NRAS and Ets1 drive CDKN2A promoter-induced luciferase expression. A, three CDKN2A promoter-driven luciferase constructs were generated: Full-length (FL, 820 bp, including 2 ETS1 putative binding sites, truncation 1 (T1, lacking the distal ETS1 putative binding site), and truncation 2 (T2, lacking both ETS1 putative binding sites). None of the constructs promoted luciferase expression above empty vector (EV) control in the absence of LPS. Persistent LPS treatment induced luciferase expression from the full-length construct only (n = 6). B, treatment with RAS/ERK inhibitors on day 9 of the 10-day LPS treatment blocked reporter luciferase expression. LPS treatment in the absence of inhibitors resulted in a 4.5-fold increase of CDKN2A promoter luciferase expression. In contrast, RAS (manumycin A) and ERK (U0126 and PD98059) inhibition prevented LPS-induced reporter luciferase expression (∼50% reduction) (n = 7). C, overexpression of ETS1 resulted in a ∼3.5-fold increase in reporter luciferase expression. This increase is diminished by ∼50% when cotransfected with an ETS1-shRNA. Furthermore, pCGN-NRAS 12D overexpression increased reporter luciferase expression by ∼4-fold, and this increase was suppressed (∼40%) by cotransfection with an ETS1-shRNA (n = 7). D, site-directed mutagenesis was performed to eliminate the distal (Δ1), proximal (Δ2), or both (Δ1/2) ETS1 binding sites, and dual luciferase assays were again performed. Luciferase activity was not increased above control EV for any of the constructs in the absence of LPS. Persistent LPS treatment induced luciferase activity in cells transfected with both the full-length (FL) and mutated proximal predicted ETS1 sites (Δ2). Luciferase detection was diminished in persistent LPS-treated cells transfected with either the Δ1 or Δ1,2 constructs, suggesting that the distal ETS1 site is required for LPS-induced CDKN2A expression (n = 6). Ctrl, control; Man, manumycin A; PD, PD98059.

FIGURE 6.

CHIP-PCR confirms LPS-induced ETS1 binding and chromatin remodeling within the CDKN2A promoter. A, schematic representation of the CDKN2A promoter located at chromosome 9p21. Directly upstream of the transcriptional start site, two putative ETS binding sites were identified in the genomic DNA (positions −518 and −279). PCR primers were designed to detect protein interactions or chromatin modification at this region or at the core promoter region (POLR2 amplicon) by performing ChIP-PCR. B, using genomic DNA from 10-day control or 10-day LPS-treated NHCs, we performed ChIP-PCR. ChIP-PCR demonstrates that ETS1 and Ser(P)-2 POLR2 occupy the CDKN2A promoter following persistent LPS treatment but not in the control (no LPS) cells. Additionally an accumulation of H3K4Me3 occurs at the promoter, whereas H3K27me3 is decreased after LPS treatment, suggesting a shift from repressive to permissive chromatin following persistent LPS treatment. C, quantitative ChIP PCR demonstrates significant increases in ETS1 (∼8-fold), Ser(P)-2 POLR2 (∼4-fold), and H3K4me3 (∼4-fold) at the CDKN2A locus, whereas H3K27me3 is diminished to control Ig levels. Each quantitative ChIP PCR was performed on three independent experiments. D, inhibition of the H3K4 histone methyltransferase, KMT2A, suppresses persistent LPS-induced CDKN2A expression. Quantitative PCR was performed on cells cultured in the presence or absence of persistent LPS and the presence or absence of the KMT2A inhibitors WDR50103 or OIC9429. Persistent LPS increased CDKN2A expression ∼7-fold. However, the KMT2A inhibitors blocked persistent LPS-induced CDKN2A expression ∼80% (n = 3). E, Western blotting was performed on cells cultured in the presence or absence of LPS and in the presence or absence of the KMT2A inhibitors. The KMT2A inhibitors blocked persistent LPS-induced CDKN2A protein expression (representative Western blot from an n = 3). Ctrl, control.

FIGURE 7.

p-ETS1 is expressed in vivo in both human PSC and in a murine model of PSC (Abcb4 (Mdr2)^−/− mice). A, representative confocal images for Dapi (blue), p-ETS1 (red), and CDKN2A mRNA (green) in cholangiocytes of normal control and PSC human liver samples. Cholangiocyte p-ETS1 is increased and exhibits predominant nuclear localization in PSC cholangiocytes compared with normal patient control samples. B, semiquantitative analysis of fluorescence intensity demonstrated increased p-ETS1 (∼3.5-fold) in PSC cholangiocytes. Fluorescence intensity was measured in no fewer than five bile ducts from three normal human control tissue samples and three PSC patient samples. C, representative confocal images for Dapi (blue), p-Ets1 (red), and Cdkn2a mRNA (green) in cholangiocytes of wild type FVBN and the Abcb4^−/− mouse model of PSC. Again, cholangiocyte p-Ets1 is increased and exhibits predominant nuclear localization in Abcb4^−/− mouse livers compared with wild type (FVBN) control. Additionally, Cdkn2a mRNA detection is increased in cholangiocytes of the Abcb4^−/− mouse. D, semiquantitative analysis of fluorescence intensity demonstrates increased p-Ets1 (∼10-fold) in Abcb4^−/− cholangiocytes compared with FVBN control cholangiocytes. Fluorescence intensity (arbitrary units) was normalized to FVBN control and is presented as mean fold change ± standard deviation. Fluorescence intensity was measured in no fewer than five bile ducts from three FVBN control and three Abcb4^−/− mice.

FIGURE 8.

Conceptual framework of CDKN2A expression during persistent LPS senescence induction. Persistent cholangiocyte stress results in the activation of NRAS and subsequently the MAPK/ERK signaling pathway. ETS1 is up-regulated, is phosphorylated, and translocates to the nucleus. In the nucleus, epigenetic histone modification (e.g. H3K4me3) allows p-ETS1 to bind to the CDKN2A promoter, resulting in CDKN2A transcription and, ultimately, cellular senescence. Pharmacologic inhibition of RAS/MEK/ERK (manumycin A, PD98059, or UO126) and suppression of ETS1 (ETS1-shRNA and genome editing) prevents CDKN2A transcription and senescence.