Calcineurin/NFAT-dependent regulation of 230-kDa bullous pemphigoid antigen (BPAG1) gene expression in normal human epidermal keratinocytes
Calcineurin/NFAT-dependent regulation of 230-kDa bullous pemphigoid antigen (BPAG1) gene expression in normal human epidermal keratinocytes
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DOI：
10.1016/j.jdermsci.2008.01.006
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发表时间：
2008-07-01
影响因子：
4.6
通讯作者：
Sawamura, Daisuke
中科院分区：
文献类型：
--
作者：
Aizu, Takayuki;Tamai, Katsuto;Sawamura, Daisuke
背景与目的：环孢菌素A（CsA）被广泛用于治疗炎症性皮肤病，如寻常性银屑病。CsA的治疗作用被认为是通过其对病变皮肤中浸润淋巴细胞的免疫抑制作用介导的。CsA还抑制表皮角质形成细胞增殖，表明对角质形成细胞的直接生物作用。在这里，我们测试的假设，CsA可以调节表皮角质形成细胞的活化T细胞的核因子（NFAT）的表达。我们还调查了是否角质形成细胞特异性基因表达的修改CsA通过NFAT活性与分化induction.Methods：RT-PCR使用总RNA提取培养的正常人表皮角质形成细胞（NHEK），正常人真皮成纤维细胞（NHDF），和正常人表皮黑色素细胞（NHEM）检测NFAT异构分子。瞬时转染NHEK与230 kDa的大疱性类天疱疮抗原（BPAG 1）启动子/荧光素酶报告基因和荧光素酶测定进行检查CsA对BPAG 1基因的启动子活性的影响。结果：RT-PCR结果显示NFAT的5种亚型均表达于NHEK细胞中。CsA处理可下调NHEK中NFAT 1、NFAT 2、BPAG 1和外皮蛋白的mRNA表达水平。荧光素酶测定表明CsA抑制启动子活性。EMSA与NFAT共识探针确定在BPAG 1启动子区域表现出特异性结合活性的核蛋白的表皮keratinocyte.Conclusion：如前所述，我们的研究结果表明，表皮角质形成细胞具有钙调神经磷酸酶/NFAT系统，这是抑制CsA。此外，数据表明，CsA可能通过BPAG 1启动子区的NFAT共有顺式元件下调BPAG 1基因的表达。这种转录调控系统可能参与角质形成细胞分化和增殖的调控。(C)2008年日本皮肤病研究学会。由Elsevier爱尔兰有限公司出版。保留所有权利。
Background and objective: Cyclosporin A (CsA) is utilized widely for treatment of inflammatory skin diseases, such as psoriasis vulgaris. The therapeutic effects of CsA are thought to be mediated by its immunosuppressive action on infiltrating lymphocytes in the lesional skin. CsA also inhibits epidermal keratinocyte proliferation, suggesting a direct biological action on keratinocytes. Here we tested the hypothesis that CsA can modulate the expression of the nuclear factor of activated T-cell (NFAT) in epidermal keratinocytes. We also investigated whether the keratinocyte-specific gene expression is modified by CsA through NFAT activity in association with differentiation induction.Methods: RT-PCR was performed using total RNAs extracted from cultured normal human epidermal keratinocytes (NHEK), normal human dermal fibroblasts (NHDF), and normal human epidermal melanocytes (NHEM) for detecting NFAT isomolecules. Transient transfections of NHEK with a 230-kDa bullous pemphigoid antigen (BPAG1) promoter/luciferase reporter gene and the luciferase assay were conducted for examining the effect of CsA on the promoter activity of the BPAG1 gene. Etectrophoretic gel mobility shift assays (EMSA) with probes containing NFAT consensus sequences for analyzing the binding activities of the nuclear proteins extracted from NHEK.Results: RT-PCR revealed expression of all of the five isoforms of NFAT in the cell lines examined. The mRNA expression levels of NFAT1, NFAT2, BPAG1, and involucrin were downregulated by CsA treatment in NHEK. The luciferase assay indicated suppression of the promoter activity by CsA. EMSA with NFAT consensus probes identified in the BPAG1 promoter region demonstrated specific binding activity in the nuclear proteins of epidermal keratinocytes.Conclusion: As reported previously, our results indicate that epidermal keratinocytes possess calcineurin/NFAT system, which is suppressed by CsA. In addition, the data suggest that CsA can downregulate the BPAG1 gene expression perhaps via the NFAT consensus cis-elements in the BPAG1 promoter region. Such transcriptional regulatory system might be involved in the regulation of keratinocyte differentiation and proliferation. (C) 2008 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.