Id2 protein is selectively upregulated by UVB in primary, but not in immortalized human keratinocytes and inhibits differentiation
Id2 protein is selectively upregulated by UVB in primary, but not in immortalized human keratinocytes and inhibits differentiation
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DOI：
10.1038/sj.onc.1208709
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发表时间：
2005-08-18
期刊：
影响因子：
8
通讯作者：
Rosenthal, DS
中科院分区：
文献类型：
--
作者：
Simbulan-Rosenthal, CM;Trabosh, V;Rosenthal, DS
太阳紫外线B（UVB）在多阶段皮肤癌发生模型中既是引发剂又是促进剂。我们发现，而UVB诱导人乳头瘤病毒16 E6/7永生化角质形成细胞凋亡，它抑制人包皮角质形成细胞（HFK）的分化标志物。这种差异反应的潜在机制进行了研究，DNA微阵列，这表明，UVB改变了三个人的分化/DNA结合（Id）蛋白，包括一类的螺旋-环-螺旋家族的转录因子参与增殖，分化，凋亡和癌变的抑制剂的表达。这些结果进行了验证，通过RT-PCR和免疫印迹分析的控制和UVB照射的原代和永生化的角质形成细胞。而Id 1在两种细胞类型中下调，Id 2表达在原代HFK中上调，但不永生化细胞。与此相反，Id 3的表达显着增加，只有在永生化细胞。Id 2对UVB应答的差异表达模式在含有该基因的5'调控区的报告构建体中重现。Id 2启动子活性增加，在HFK中，而不是在永生化细胞中响应UVB。为了鉴定Id 2启动子中介导UVB在HFK中的转录激活的调控元件，进行启动子缺失/突变分析。缺失分析表明，反式激活涉及一个166 bp的区域上游的Id 2转录起始位点，是独立的c-Myc。在-120处的共有E 26（ETS）结合位点似乎介导Id 2的UVB转录激活，因为该位点处的点突变完全废除了该应答。染色质免疫沉淀和电泳迁移率变动分析证实，Id 2启动子与已知的Id 2启动子（ETS）结合因子Erg 1/2和Fli 1相互作用，但不与c-Myc;这种相互作用增强UVB曝光后。与UVB暴露的影响相似，原发性HFK中Id 2蛋白的异位表达导致分化抑制，如终末分化标记物角蛋白K1水平降低和外皮蛋白交联抑制所示。通过小干扰RNA减少Id 2表达减弱了UVB诱导的这些细胞分化抑制。这些结果表明，UVB诱导的原发性HFK的分化抑制至少部分是由于Id 2的上调，并且UVB上调Id 2可能通过阻止其正常分化程序而使角质形成细胞易于致癌。
Solar ultraviolet B (UVB) acts as both an initiator and promoter in models of multistage skin carcinogenesis. We found that, whereas UVB induces apoptosis in human papillomavirus-16 E6/7-immortalized keratinocytes, it inhibits markers of differentiation in human foreskin keratinocytes (HFK). Potential mechanisms for this differential response were examined by DNA microarray, which revealed that UVB alters the expression of three of the four human inhibitor of differentiation/DNA binding (Id) proteins that comprise a class of helix-loop-helix family of transcription factors involved in proliferation, differentiation, apoptosis, and carcinogenesis. These results were verified by RT-PCR and immunoblot analysis of control and UVB-irradiated primary and immortalized keratinocytes. Whereas Id1 was down-regulated in both cell types, Id2 expression was upregulated in primary HFK, but not immortalized cells. In contrast, Id3 expression was significantly increased only in immortalized cells. The differential expression pattern of Id2 in response to UVB was recapitulated in reporter constructs containing the 5' regulatory regions of this gene. Id2 promoter activity increased in response to UVB in HFK, but not in immortalized cells. To identify the regulatory elements in the Id2 promoter that mediate transcriptional activation by UVB in HFK, promoter deletion/mutation analysis was performed. Deletion analysis revealed that transactivation involves a 166 bp region immediately upstream to the Id2 transcriptional start site and is independent of c-Myc. The consensus E twenty-six (ETS) binding site at -120 appears to mediate UVB transcriptional activation of Id2 because point mutations at this site completely abrogated this response. Chromatin immunoprecipitation and electrophoretic mobility-shift assays verified that the Id2 promoter interacts with known Id2 promoter (ETS) binding factors Erg1/2 and Fli1, but not with c-Myc; and this interaction is enhanced after UVB exposure. Similar to the effects of UVB exposure, ectopic expression of Id2 protein in primary HFK resulted in inhibition of differentiation, as shown by decreased levels of the terminal differentiation marker keratin K1 and inhibition of involucrin crosslinking. Reduction of Id2 expression by small interfering RNAs attenuated the UVB-induced inhibition of differentiation in these cells. These results suggest that UVB-induced inhibition of differentiation of primary HFK is at least, in part, due to the upregulation of Id2, and that upregulation of Id2 by UVB might predispose keratinocytes to carcinogenesis by preventing their normal differentiation program.