Saxlund Melissa A, Sadler-Riggleman Ingrid, Skinner Michael K
Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4231, USA.
Mol Reprod Dev. 2004 Jul;68(3):269-78. doi: 10.1002/mrd.20080.
Differentiation of Sertoli cells is marked by the presence of novel gene products such as transferrin and androgen-binding protein (ABP). Transcriptional regulation of Sertoli cell differentiation is, in part, controlled through the binding of specific transcription factors to response elements within these genes promoters. Transferrin gene expression has been shown to be regulated by the binding and interactions of basic helix-loop-helix (bHLH) and cAMP response element binding protein (CREB) to an E-box and cyclic AMP response element (CRE), respectively. Interaction between the bHLH and CREB is facilitated through subsequent binding of CREB-binding protein (CBP)/p300. The hypothesis tested in the current study is that ABP expression is regulated by a similar mechanism. The ABP promoter activation was analyzed through the use of transfection assays, site-directed mutagenesis, and electromobility shift assays (EMSA). Transient transfections of rat Sertoli cells used a reporter construct containing the proximal 619 bp of the ABP promoter. Observations suggest that cAMP and follicle stimulating hormone (FSH) upregulate the expression of ABP. Mutational studies of the three E-boxes and the CRE of the 619-bp ABP promoter indicate that all of these elements are critical for stimulation of promoter activity. EMSA revealed a weak interaction between an E-box-2 and the CRE that are overlapping in the promoter. An artificial promoter that contains only an E-box and CRE was created to further test this hypothesis. The artificial promoter was stimulated by both FSH and cAMP. Experiments with mutants of the artificial promoter demonstrate that both response elements contribute to the optimal activation of the promoter construct. The overexpression of the bHLH inhibitor Id (i.e., inhibitor of differentiation) that binds bHLH proteins and eliminates DNA binding was found to suppress hormone activation of the ABP promoter. Combined observations of the ABP promoter and artificial promoter provide insight into a common mechanism for gene regulation in differentiated Sertoli cells involving a role for both the bHLH and CREB family of transcription factors.
支持细胞的分化以新基因产物如转铁蛋白和雄激素结合蛋白（ABP）的存在为标志。支持细胞分化的转录调控部分是通过特定转录因子与这些基因启动子内的反应元件结合来控制的。转铁蛋白基因表达已被证明分别受碱性螺旋-环-螺旋（bHLH）和cAMP反应元件结合蛋白（CREB）与E盒和环磷酸腺苷反应元件（CRE）的结合及相互作用调控。bHLH和CREB之间的相互作用通过CREB结合蛋白（CBP）/p300的后续结合得以促进。本研究中检验的假设是ABP表达受类似机制调控。通过转染试验、定点诱变和电泳迁移率变动分析（EMSA）对ABP启动子激活进行了分析。大鼠支持细胞的瞬时转染使用了一个包含ABP启动子近端619 bp的报告构建体。观察结果表明cAMP和促卵泡激素（FSH）上调ABP的表达。对619 bp ABP启动子的三个E盒和CRE的突变研究表明，所有这些元件对启动子活性的刺激都至关重要。EMSA揭示了启动子中重叠的E盒-2和CRE之间存在弱相互作用。构建了一个仅包含一个E盒和CRE的人工启动子以进一步检验该假设。该人工启动子受到FSH和cAMP两者的刺激。对人工启动子突变体的实验表明，两个反应元件都有助于启动子构建体的最佳激活。发现结合bHLH蛋白并消除DNA结合的bHLH抑制剂Id（即分化抑制剂）的过表达可抑制ABP启动子的激素激活。对ABP启动子和人工启动子的综合观察为分化的支持细胞中涉及bHLH和CREB转录因子家族作用的基因调控共同机制提供了见解。
