The cAMP-responsive element in the corticotropin-releasing hormone gene mediates transcriptional regulation by depolarization

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.