Identification and characterization of the minimal androgen-regulated kidney-specific kidney androgen-regulated protein gene promoter
Identification and characterization of the minimal androgen-regulated kidney-specific kidney androgen-regulated protein gene promoter
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DOI：
10.1111/j.1745-7270.2008.00482.x
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发表时间：
2008-12-01
影响因子：
3.7
通讯作者：
Li, Suxia
中科院分区：
文献类型：
--
作者：
Fan, Liqiang;Hardy, Dianne O.;Li, Suxia
肾脏雄激素调节蛋白(KAP)基因是组织特异性的，在小鼠肾脏近端小管细胞(PTCs)中受雄激素调控。在本研究中，我们旨在寻找最小的PTC特异性雄激素调节的KAP启动子，并分析其雄激素反应元件(ARES)。在负鼠肾(OK)PTCs中检测了Kap1542启动子/荧光素酶构建体在15 nM双氢睾酮(DHT)存在或不存在的情况下的缺失序列。Kap1542和Kap637活性低，没有雄激素诱导；Kap224的基础活性是Kap1542的4-5倍，但只被DHT轻微诱导。Kap147的基本活性是Kap1542的2-3倍，并可被DHT诱导4-6倍。Kap77取消了基础启动子活性，但仍被DHT诱导。结果表明，在体外，Kap147是一个最小的雄激素调节启动子。不同细胞的瞬时转染表明，Kap147特异性地启动了报告基因在PTCs中的表达。序列分析发现两个潜在的ARE位于Kap147的-124和-39位。突变分析表明，在OK细胞中，只有ARE-124参与雄激素反应。电泳迁移率改变分析也证实了-124与雄激素受体特异性结合。总之，我们定义了最小的Kap147启动子，它可能是研究肾脏PTC特异性表达和体内导致雄激素特异性反应的分子机制的良好模型。
The kidney androgen-regulated protein (Kap) gene is tissue specific and regulated by androgen in mouse kidney proximal tubule cells (PTCs). In the present study, we aimed to identify the minimal PTC-specific androgen-regulated Kap promoter and analyze its androgen response elements (AREs). A deletion series of the Kap1542 promoter/luciferase constructs were assayed in opossum kidney (OK) PTCs in the presence or absence of 15 nM dihydrotestosterone (DHT). Kap1542 and Kap637 had low activity and no androgen induction; Kap224 had a basal activity that was 4- to 5-fold higher than that of Kap1542, but was only slightly induced by DHT. Kap147 had a basal activity that was 2- to 3-fold higher than that of Kap1542 and was induced by DHT 4- to 6-fold. Kap77 abolished basal promoter activity but was still induced by DHT. Results showed that, in vitro, Kap147 was a minimal androgen-regulated promoter. Transient transfection in different cells demonstrated that Kap147 specifically initiated reporter gene expression in PTCs. Sequence analysis revealed two potential AREs located at positions -124 and -39 of Kap147. Mutational assays showed that only the ARE at -124 was involved in androgen response in OK cells. Electrophoretic mobility shift assay also verified -124 ARE bound specifically to androgen receptor. In conclusion, we defined the minimal Kap147 promoter that may be a good model for the study of kidney PTC-specific expression and molecular mechanisms that lead to an androgen-specific responsiveness in vivo.