Analysis of pathomechanism of androgenetic alopecia by investigating TGF-β1 promoter

我们利用雄激素受体（AR）表达载体和角质形成细胞（KCs）瞬时转染的真皮乳头细胞（DPCs）共培养系统，报道了雄激素对雄激素源性脱发（AGA）真皮乳头细胞（DPCs）中TGF-β1的上调。这一发现提示雄激素对TGF-β1的调控是AGA发病的关键步骤。为了探讨雄激素对TGF-β1基因表达的可能调控机制，我们分析了将TGF-β1基因上游5′区（转录起始位点上游-1362 bp）连接到荧光素酶报告基因上构建的pTGF-β1- luc载体的TGF-β1启动子活性。当pTGF-β1-Luc和AR表达载体在AGA的DPCs中共转染时，R1881将荧光素酶活性提高到10倍左右。相反，这种诱导在CV-1细胞和转化的DPCs中没有发生。因此雄激素对启动子活性的调节可能是细胞特异性的。利用AGA的DPCs对缺失的pTGF-β1-Luc载体进行分析，发现可能存在两个调控区（-1131 ~ -731和-459 ~ -323）和两个负调控区（-1326 ~ -1127和-735 ~ -459）。我们在-459 ~ -323区域发现了两个AP-1结合位点。利用包含这两个AP-1位点突变的报告载体，我们发现这些位点参与了TGF-β1启动子的抑制，而AR可以释放这种抑制。

We reported that TGF-β1 is up-regulated by androgen in dermal papilla cells (DPCs) from androgenetic alopecia (AGA) using our coculture system of DPCs transiently transfected with androgen receptor (AR) expression vector and keratinocytes (KCs). This finding suggested that the regulation of TGF-β1 by androgen is a key step in the pathogenesis of AGA. To examine the possible regulatory mechanism of TGF-β1 gene expression by androgen, we analysed TGF-β1 promoter activity of the pTGF-β1-Luc vector, which was constructed by connecting the 5' upstream region of the TGF-β1 gene (-1362 bp upstream from the transcription start site) to the luciferase reporter gene. When pTGF-β1-Luc and AR expression vector were cotransfected in DPCs from AGA, R1881 increased luciferase activity to around 10-fold. In contrast, this induction did not occur in CV-1 cells and transformed DPCs. Thus the regulation of promoter activity by androgen may be cell-specific. From analysis of deleted pTGF-β1-Luc vector using DPCs from AGA, there are two possible regulatory regions (-1131〜-731 and -459〜 -323) and two negative regulatory regions (-1326〜-1127 and -735〜-459). We found two AP-1 binding sites in the region at -459〜-323. Using the reporter vector containing the mutation at these two AP-1 sites, we found that these sites are involved in the suppression of TGF-β1 promoter and that AR can release this suppression.