Dai Xiuju, Sayama Koji, Shirakata Yuji, Tokumaru Sho, Yang Lujun, Tohyama Mikiko, Hirakawa Satoshi, Hanakawa Yasushi, Hashimoto Koji
Department of Dermatology, Ehime University School of Medicine, Ehime, Japan.
J Dermatol Sci. 2008 Apr;50(1):53-60. doi: 10.1016/j.jdermsci.2007.10.011.
1 alpha,25-Dihydroxyvitamin D3 (1 alpha,25(OH)2D3), the active form of vitamin D, suppresses keratinocyte proliferation, promotes keratinocyte differentiation, and induces involucrin expression. Peroxisome proliferation-activated receptors (PPARs) are ligand-activated transcription factors. It has been reported that PPARs stimulate keratinocyte differentiation and regulate the expression of differentiation molecules.
Keratinocytes treated with 1 alpha,25(OH)2D3 induced PPAR gamma, which was followed by increased involucrin expression. In this study, we investigated whether PPAR gamma is involved in the 1 alpha,25(OH)2D3-induced involucrin expression in human keratinocytes.
Subconfluent keratinocytes were treated with 10(-7)M 1 alpha,25(OH)2D3 for the indicated times, and PPAR and involucrin mRNA expression were determined by real-time RT-PCR. The levels of PPARs, involucrin, p38, and phospho-p38 proteins were assayed by Western blotting, and the DNA binding activities of PPAR gamma and AP-1 were investigated by electrophoretic mobility shift assays (EMSA). To examine the role of PPAR gamma in 1 alpha,25(OH)2D3 responses, recombinant adenovirus carrying a dominant-negative form of PPAR gamma (Axdn-PPAR gamma) was constructed and transfected into keratinocytes. The p38 inhibitor SB203580 was added to the cultures to evaluate the involvement of p38 in involucrin expression.
1 alpha,25(OH)2D3 induced PPAR gamma expression and stimulated PPAR gamma activity. The introduction of dn-PPAR gamma inhibited the expression of involucrin mRNA and protein induced by 1 alpha,25(OH)2D3, and suppressed AP-1 DNA binding activity. 1 alpha,25(OH)2D3 also triggered the phosphorylation of p38, which contributes to involucrin induction. Moreover, dn-PPAR gamma prevented the 1 alpha,25(OH)2D3-induced phosphorylation of p38.
These results suggest that PPAR gamma regulates involucrin expression by controlling the AP-1 signal and p38 activation in 1 alpha,25(OH)2D3-induced keratinocyte differentiation.
1α,25 - 二羟基维生素D3（1α,25(OH)2D3）是维生素D的活性形式，可抑制角质形成细胞增殖，促进角质形成细胞分化，并诱导兜甲蛋白表达。过氧化物酶体增殖物激活受体（PPARs）是配体激活的转录因子。据报道，PPARs可刺激角质形成细胞分化并调节分化分子的表达。
用1α,25(OH)2D3处理角质形成细胞可诱导PPARγ表达，随后兜甲蛋白表达增加。在本研究中，我们调查了PPARγ是否参与1α,25(OH)2D3诱导的人角质形成细胞中兜甲蛋白的表达。
将亚汇合的角质形成细胞用10(-7)M 1α,25(OH)2D3处理指定时间，通过实时RT-PCR测定PPAR和兜甲蛋白mRNA表达。通过蛋白质印迹法检测PPARs、兜甲蛋白、p38和磷酸化p38蛋白的水平，并通过电泳迁移率变动分析（EMSA）研究PPARγ和AP-1的DNA结合活性。为了研究PPARγ在1α,25(OH)2D3反应中的作用，构建携带PPARγ显性负性形式的重组腺病毒（Axdn-PPARγ）并转染到角质形成细胞中。向培养物中加入p38抑制剂SB203580以评估p38在兜甲蛋白表达中的作用。
1α,25(OH)2D3诱导PPARγ表达并刺激PPARγ活性。导入显性负性PPARγ可抑制1α,25(OH)2D3诱导的兜甲蛋白mRNA和蛋白表达，并抑制AP-1 DNA结合活性。1α,25(OH)2D3还可触发p38的磷酸化，这有助于兜甲蛋白的诱导。此外，显性负性PPARγ可阻止1α,25(OH)2D3诱导的p38磷酸化。
这些结果表明PPARγ在1α,25(OH)2D3诱导的角质形成细胞分化中通过控制AP-1信号和p38激活来调节兜甲蛋白表达。
