Induction of disease-associated keratin 16 gene expression by epidermal growth factor is regulated through cooperation of transcription factors Sp1 and c-Jun
Induction of disease-associated keratin 16 gene expression by epidermal growth factor is regulated through cooperation of transcription factors Sp1 and c-Jun
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DOI：
10.1074/jbc.m302630200
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发表时间：
2003-11-14
影响因子：
4.8
通讯作者：
Chang, WC
中科院分区：
文献类型：
--
作者：
Wang, YN;Chang, WC
角蛋白16的过度表达已经在那些以过度增殖为特征的皮肤病如银屑病的角质形成细胞中观察到。因此，角蛋白16通常被称为疾病相关角蛋白。在本研究中，我们发现，表皮生长因子（EGF）增加角蛋白16 mRNA的表达和蛋白质合成的时间依赖性的方式在HaCaT细胞。报告基因分析显示EGF反应区在-162至-114 bp的范围内。通过定点突变破坏角蛋白16启动子的Sp1位点（-127至-122 bp）和AP 1位点（-148至-142 bp）显著抑制EGF诱导的角蛋白16启动子活性。此外，角蛋白16基因表达诱导Ras活化也调节以同样的方式作为EGF的反应。在HaCaT和SL 2细胞中，Sp1与启动子的Sp1位点直接相互作用，而与Sp1寡核苷酸沉淀的c-Jun和c-Fos则是由于Sp1和AP 1蛋白之间的相互作用。此外，共转染实验表明，Sp1与c-Jun协同作用，激活角蛋白16。共激活因子p300/CBP可以与Sp1和c-Jun协同激活角蛋白16启动子，而EGF诱导的启动子激活被病毒癌蛋白E1 A阻断。两者合计，这些结果表明，Sp1和AP 1位点的必需启动子区域是至关重要的EGF反应，和Sp1表现出功能合作与c-Jun和辅激活因子p300/CBP在驱动EGF诱导的角蛋白16基因表达的转录调控。
Overexpression of keratin 16 has been observed in keratinocytes in those skin diseases characterized by hyperproliferation such as psoriasis. Therefore, keratin 16 is usually referred to as a disease-associated keratin. In the present study, we found that epidermal growth factor (EGF) increased the expression of keratin 16 mRNA and protein synthesis in a time-dependent manner in HaCaT cells. Reporter assays revealed that the EGF response region was in the range of -162 to -114 bp. Disruption of the Sp1 site (-127 to -122 bp) and the AP1 site (-148 to -142 bp) of the keratin 16 promoter by site-directed mutagenesis significantly inhibited keratin 16 promoter activity induced by EGF. Furthermore, keratin 16 gene expression induced by Ras activation was also regulated in the same manner as the EGF response. By using the DNA affinity precipitation assay in HaCaT and SL2 cells, Sp1 directly interacted with the Sp1 site of the promoter, and c-Jun and c-Fos precipitated with the Sp1 oligonucleotide was attributable to the interaction between the Sp1 and AP1 proteins. Moreover, cotransfection assays revealed that Sp1 acted synergistically with c-Jun to activate keratin 16. The coactivators p300/CBP could collaborate with Sp1 and c-Jun in the activation of keratin 16 promoter, and EGF-induced promoter activation was blocked by the viral oncoprotein E1A. Taken together, these results suggest that Sp1 and AP1 sites in the essential promoter region are critical for EGF response, and Sp1 showed a functional cooperation with c-Jun and coactivators p300/CBP in driving the transcriptional regulation of EGF-induced keratin 16 gene expression.