Walker W H, Fucci L, Habener J F
Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute, Boston 02114, USA.
Endocrinology. 1995 Aug;136(8):3534-45. doi: 10.1210/endo.136.8.7628390.
The somatic Sertoli cells of the testis are major targets for FSH and are important for the regulation of spermatogenesis. The binding of FSH to Sertoli cells activates the cAMP-dependent protein kinase A signaling pathway, resulting in phosphorylation of the cAMP response element-binding protein (CREB), which is required to transactivate genes containing cAMP response elements (CREs). Here we show that the addition of forskolin to cultured primary Sertoli cells results in the phosphorylation of CREB within 2-5 min. Phospho-CREB levels remain elevated with continued forskolin stimulation, but fall by 60% within 5 min after the removal of forskolin. In addition, we found that 8-bromo-cAMP induces CREB RNA accumulation in the Sertoli cells. Transient transfections of primary Sertoli cells with CREB promoter-chloramphenicol acetyltransferase reporter plasmids define a conserved 300-base pair region of the CREB promoter surrounding the transcription start site that is required for both basal and cAMP-inducible expression of the CREB gene. This region of the promoter contains three Sp1-binding sites flanking the transcription initiation site and two CREs located 65 and 85 base pairs downstream of the transcription initiation site. We show that the Sp1 motifs bind Sp1 in Sertoli extracts and contribute to basal promoter activity, and that the CREs bind CREB and are essential for cAMP induction of CREB gene transcription. These findings support the model of FSH- and cAMP-mediated CREB autoregulation of its own promoter and may explain the dramatic stage-specific oscillations in Sertoli cells of CREB messenger RNA levels during the 12-day cycles of spermatogenesis in rat seminiferous tubules.
睾丸的体细胞支持细胞是促卵泡激素（FSH）的主要靶细胞，对精子发生的调节至关重要。FSH与支持细胞的结合激活了环磷酸腺苷（cAMP）依赖性蛋白激酶A信号通路，导致cAMP反应元件结合蛋白（CREB）磷酸化，而这是反式激活含有cAMP反应元件（CRE）的基因所必需的。在此我们表明，向培养的原代支持细胞中添加福斯可林会在2 - 5分钟内导致CREB磷酸化。在持续的福斯可林刺激下，磷酸化CREB水平保持升高，但在去除福斯可林后5分钟内下降60%。此外，我们发现8 - 溴 - cAMP可诱导支持细胞中CREB RNA积累。用CREB启动子 - 氯霉素乙酰转移酶报告质粒对原代支持细胞进行瞬时转染，确定了CREB启动子围绕转录起始位点的一个保守的300碱基对区域，该区域是CREB基因基础表达和cAMP诱导表达所必需的。启动子的这个区域在转录起始位点两侧包含三个Sp1结合位点，以及位于转录起始位点下游65和85个碱基对处的两个CRE。我们表明，Sp1基序在支持细胞提取物中结合Sp1并有助于基础启动子活性，并且CRE结合CREB，对于cAMP诱导的CREB基因转录至关重要。这些发现支持了FSH和cAMP介导的CREB对其自身启动子进行自动调节的模型，并可能解释了大鼠生精小管精子发生12天周期中支持细胞中CREB信使RNA水平的显著阶段特异性振荡。
