ETS1
Protein C-ets-1 (p54) [EWSR2]
Publications[править]
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 [i]BCL-xL[/i] 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 [i]in vitro[/i] 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 [i]BCL-xL[/i] 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 [i]BCL-xL[/i] promoter blocked luciferase reporter activity, and CRISPR/Cas9-mediated genetic deletion of [i]ETS1[/i] 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.
MeSH Terms
- ATP Binding Cassette Transporter, Subfamily B
- Animals
- Apoptosis
- Cellular Senescence
- Hepatocytes
- Humans
- Lipopolysaccharides
- Liver
- Mice
- Proto-Oncogene Protein c-ets-1
- Transcription Factors
- bcl-X Protein
Keywords
- BCL2 like 1 (BCL2L1)
- apoptosis
- cholangiocyte
- chromatin modification
- epigenetics
- gene expression
- primary sclerosing cholangitis (PSC)
- senescence
- transcription factor
Senescent cells have lost their capacity for proliferation and manifest as irreversibly in cell cycle arrest. Many membrane receptors, including G protein-coupled receptors (GPCRs), initiate a variety of intracellular signaling cascades modulating cell division and potentially play roles in triggering cellular senescence response. GPCR kinases (GRKs) belong to a family of serine/threonine kinases. Although their role in homologous desensitization of activated GPCRs is well established, the involvement of the kinases in cell proliferation is still largely unknown. In this study, we isolated GRK4-GFP expressing HEK293 cells by fluorescence-activated cell sorting (FACS) and found that the ectopic expression of GRK4 halted cell proliferation. Cells expressing GRK4 (GRK4( )) demonstrated cell cycle G1/G0 phase arrest, accompanied with significant increase of senescence-associated-β-galactosidase (SA-β-Gal) activity. Expression profiling analysis of 78 senescence-related genes by qRT-PCR showed a total of 17 genes significantly changed in GRK4( ) cells (≥ 2 fold, p < 0.05). Among these, 9 genes - AKT1, p16 , p27 , p19 , IGFBP3, MAPK14, PLAU, THBS1, TP73 - were up-regulated, while 8 genes, Cyclin A2, Cyclin D1, CDK2, CDK6, ETS1, NBN, RB1, SIRT1, were down-regulated. The increase in cyclin-dependent kinase inhibitors (p16, p27) and p38 MAPK proteins (MAPK14) was validated by immunoblotting. Neither p53 nor p21 protein was detectable, suggesting no p53 activation in the HEK293 cells. These results unveil a novel function of GRK4 on triggering a p53-independent cellular senescence, which involves an intricate signaling network.
MeSH Terms
- Cell Division
- Cell Line, Tumor
- Cell Proliferation
- Cellular Senescence
- Flow Cytometry
- G-Protein-Coupled Receptor Kinase 4
- Gene Expression Profiling
- Gene Expression Regulation
- HEK293 Cells
- Humans
- MCF-7 Cells
- Transcriptome
- Tumor Suppressor Protein p53
Keywords
- Cellular senescence
- G protein-coupled receptor kinase 4
- Gene expression profiling
- p53-independent senescence
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 ([i]e.g.[/i] LPSs) induces cyclin-dependent kinase inhibitor 2A ([[CDKN2A]]/p16 ) 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 [i]in vitro[/i] senescence model, we found that LPS-induced [i][[CDKN2A]][/i] expression coincided with a 4.5-fold increase in [i]ETS1[/i] ([i]ETS proto-oncogene 1[/i]) mRNA, suggesting that ETS1 is involved in regulating [i][[CDKN2A]][/i] 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 [i][[CDKN2A]][/i] promoter abolished luciferase reporter activity. Pharmacological inhibition of RAS/MAPK reduced ETS1 and [[CDKN2A]] protein expression and [i][[CDKN2A]][/i] 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 [i][[CDKN2A]][/i] promoter following LPS-induced senescence. Additionally, phospho-ETS1 expression was increased in cholangiocytes of human PSC livers and in the [i]Abcb4[/i] ([i]Mdr2[/i]) mouse model of PSC. These data pinpoint ETS1 and H3K4Me3 as key transcriptional regulators in NRAS-induced expression of [i][[CDKN2A]][/i], and this regulatory axis may therefore represent a potential therapeutic target for PSC treatment.
MeSH Terms
- Animals
- Cell Line
- Cellular Senescence
- Cholangitis, Sclerosing
- Cyclin-Dependent Kinase Inhibitor p16
- Humans
- Lipopolysaccharides
- Liver
- Mice
- Proto-Oncogene Protein c-ets-1
- RNA, Messenger
- Transcriptional Activation
- Up-Regulation
Keywords
- CDKN2A
- Cholangiocytes
- ETS1
- cell signaling
- epigenetics
- epithelial cell
- senescence
- transcription