Keratinocyte responsive element 3: analysis of a keratinocyte-specific regulatory sequence in the 230-kDa bullous pemphigoid antigen gene promoter.
Keratinocyte responsive element 3: analysis of a keratinocyte-specific regulatory sequence in the 230-kDa bullous pemphigoid antigen gene promoter.
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DOI：
10.1046/j.1523-1747.2003.12044.x
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发表时间：
2003-02
期刊：
影响因子：
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通讯作者：
Y. Matsuzaki;K. Tamai;A. Kon;D. Sawamura;J. Uitto;I. Hashimoto
中科院分区：
文献类型：
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作者：
Y. Matsuzaki;K. Tamai;A. Kon;D. Sawamura;J. Uitto;I. Hashimoto
230-kDa大疱性类天疱疮抗原基因主要（如果不是唯一的话）在表皮的基底角质形成细胞中表达。角质形成细胞反应元件3是位于人230-kDa大疱性类天疱疮抗原基因启动子的-216至-197位的顺式元件，赋予该基因组织特异性表达（Tamai等：J Biol Chem 270：7609-7614，1995）。在这项研究中，我们研究了230 kDa的大疱性类天疱疮抗原基因核心启动子的角质形成细胞反应元件3的功能特性培养的正常人角质形成细胞和正常人成纤维细胞，以及肺癌（A549），骨肉瘤（OST），胃腺癌（GT 3 TKB）细胞系的瞬时转染。修饰230-kDa大疱性类天疱疮抗原基因核心启动子/荧光素酶报告基因质粒构建体pBPL以开发一系列构建体（pKBPL-p4 KBPL），其具有角质形成细胞应答元件3的一个、两个、三个或四个串联重复的插入，并且这些质粒用于培养细胞的瞬时转染。pKBPL-p4 KBPL构建体在正常人角质形成细胞中的启动子活性相对于pBPL分别高7.6倍、15.5倍、4.6倍和2.7倍，而在其他测试的细胞系中未观察到角质形成细胞应答元件3插入的上调作用。prKBPL是一种用角质形成细胞反应元件3反向构建的质粒，在正常人角质形成细胞中基本上没有活性。在角质形成细胞反应元件3和230-kDa大疱性类天疱疮抗原基因核心启动子之间插入随机的20 bp序列导致正常人角质形成细胞中荧光素酶活性降低约40%。这些数据表明，角质形成细胞反应元件3的功能作为一个位置，拷贝数和方向依赖性顺式元件，有助于组织特异性调节的230 kDa大疱性类天疱疮抗原基因。
The 230-kDa bullous pemphigoid antigen gene is expressed primarily, if not exclusively, in basal keratinocytes of the epidermis. Keratinocyte responsive element 3, a cis-element at position -216 to -197 of the human 230-kDa bullous pemphigoid antigen gene promoter, confers tissue-specific expression to this gene (Tamai et al: J Biol Chem 270:7609-7614, 1995). In this study, we investigated the functional characteristics of keratinocyte responsive element 3 on the 230-kDa bullous pemphigoid antigen gene core promoter by transient transfections of cultured normal human keratinocytes and normal human fibroblasts, as well as of lung carcinoma (A549), osteosarcoma (OST), and gastric adenocarcinoma (GT3TKB) cell lines. A 230-kDa bullous pemphigoid antigen gene core promoter/luciferase reporter gene plasmid construct, pBPL, was modified to develop a series of constructs (pKBPL-p4KBPL), which have insertions of one, two, three, or four tandem repeats of keratinocyte responsive element 3, and these plasmids were used in transient transfections of the cultured cells. The promoter activities of pKBPL-p4KBPL constructs, relative to pBPL, in normal human keratinocytes were 7.6-, 15.5-, 4.6-, and 2.7-fold higher, respectively, whereas no upregulatory effect by keratinocyte responsive element 3 insertion was observed in other cell lines tested. prKBPL, a plasmid constructed with keratinocyte responsive element 3 in reverse orientation, showed essentially no activity in normal human keratinocytes. Insertion of a random 20 bp sequence between keratinocyte responsive element 3 and the 230-kDa bullous pemphigoid antigen gene core promoter resulted in about 40% reduction of luciferase activity in normal human keratinocytes. These data suggest that keratinocyte responsive element 3 functions as a position-, copy number-, and orientation-dependent cis-element contributing to tissue-specific regulation of the 230-kDa bullous pemphigoid antigen gene.