Guardiola-Diaz H M, Boswell C, Seasholtz A F
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
J Biol Chem. 1994 May 20;269(20):14784-91.
Membrane depolarization is a critical element of neuronal signaling. In this study, the biochemical and molecular mechanisms involved in transcriptional regulation of the corticotropin-releasing hormone (CRH) gene by depolarization were investigated. In PC-12 cells, potassium-induced membrane depolarization increased expression of a CRH-reporter construct in a cAMP-dependent manner. This synergistic activation was mediated via calcium influx, predominantly via L-type calcium channels, and calmodulin. RNase protection assays demonstrated increased levels of CRH-reporter transcripts in stably transfected cells after treatment with cAMP and potassium, with the induced transcripts initiating at the major transcription initiation site of the human CRH gene. At the genomic level, the CRH cAMP-responsive element conferred both positive cAMP and synergistic cAMP/depolarization regulation to a heterologous promoter. Additionally, DNase I protection assays demonstrated similar nuclear protein/DNA binding profiles across the cAMP-responsive element after treatment of PC-12 cells with potassium or potassium/cAMP. These results support a model in which the protein(s) binding to the cAMP-responsive element integrates signals initiated by multiple pathways (cAMP and calcium) and transmits that integrated signal to the basal transcription machinery, resulting in increased levels of gene expression.
膜去极化是神经元信号传导的关键要素。在本研究中，对去极化介导的促肾上腺皮质激素释放激素（CRH）基因转录调控所涉及的生化和分子机制进行了研究。在PC-12细胞中，钾离子诱导的膜去极化以cAMP依赖的方式增加了CRH报告基因构建体的表达。这种协同激活是通过钙内流介导的，主要通过L型钙通道和钙调蛋白。核糖核酸酶保护试验表明，用cAMP和钾处理后，稳定转染细胞中CRH报告基因转录本水平增加，诱导的转录本起始于人CRH基因的主要转录起始位点。在基因组水平上，CRH cAMP反应元件赋予异源启动子正性cAMP和协同性cAMP/去极化调节作用。此外，DNase I保护试验表明，用钾或钾/cAMP处理PC-12细胞后，跨cAMP反应元件的核蛋白/DNA结合谱相似。这些结果支持了一种模型，即与cAMP反应元件结合的蛋白质整合了由多种途径（cAMP和钙）启动的信号，并将整合后的信号传递给基础转录机制，从而导致基因表达水平升高。
