ETS Proto-oncogene 1 Transcriptionally Up-regulates the Cholangiocyte Senescence- associated Protein Cyclin-dependent Kinase Inhibitor 2A
ETS Proto-oncogene 1 Transcriptionally Up-regulates the Cholangiocyte Senescence- associated Protein Cyclin-dependent Kinase Inhibitor 2A
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DOI：
10.1074/jbc.m117.777409
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发表时间：
2017-03-24
影响因子：
4.8
通讯作者：
LaRusso, Nicholas F.
中科院分区：
文献类型：
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作者：
O'Hara, Steven P.;Splinter, Patrick L.;LaRusso, Nicholas F.
原发性硬化性胆管炎（PSC）是一种慢性纤维炎性胆管病（胆管疾病），发病机制尚不清楚。我们报道了PSC中胆管细胞衰老的显著特征，神经母细胞瘤RAS病毒癌基因同源物（NRAS）在PSC胆管细胞中被激活。此外，持续的微生物损伤（如lps）诱导细胞周期蛋白依赖性激酶抑制剂2A （CDKN2A/ p16(INK4a)）在培养的胆管细胞中以nras依赖的方式表达和衰老。然而，脂多糖诱导的胆管细胞衰老和nras依赖性CDKN2A调控的分子机制尚不清楚。通过我们的体外衰老模型，我们发现lp诱导的CDKN2A表达与ETS1 (ETS原癌基因1)mRNA增加4.5倍一致，这表明ETS1参与了CDKN2A的调节。rnai介导的ETS1抑制或基因缺失证实了这一观点，ETS1抑制或基因缺失可阻断CDKN2A表达，减少胆管细胞衰老。此外，CDKN2A启动子内预测的ets结合位点的定点突变会破坏荧光素酶报告基因的活性。RAS/ MAPK的药理抑制使ETS1和CDKN2A蛋白表达以及CDKN2A启动子驱动的荧光素酶活性降低了50%。相反，组成型活性NRAS表达诱导ETS1和CDKN2A蛋白表达，而ETS1 RNAi阻断这种增加。染色质免疫沉淀- pcr检测到lps诱导衰老后CDKN2A启动子ETS1和组蛋白3赖氨酸4三甲基化（H3K4Me3）增加。此外，phospho-ETS1在人PSC肝脏胆管细胞和Abcb4 (Mdr2)（-/-）小鼠PSC模型中表达升高。这些数据指出，ETS1和H3K4Me3是nras诱导CDKN2A表达的关键转录调控因子，因此这一调控轴可能代表PSC治疗的潜在治疗靶点。
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.