THE CAMP-RESPONSIVE ELEMENT IN THE CORTICOTROPIN-RELEASING HORMONE GENE MEDIATES TRANSCRIPTIONAL REGULATION BY DEPOLARIZATION

Membrane depolarization is a critical element of neuronal signaling, I n this study, the biochemical and molecular mechanisms involved in tra nscriptional regulation of the corticotropin-releasing hormone (CRH) g ene by depolarization were investigated. In PC-12 cells, potassium-ind uced membrane depolarization increased expression of a CRH-reporter co nstruct in a cAMP-dependent manner. This synergistic activation was me diated 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 treatmen t 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 simila r nuclear protein/DNA binding profiles across the cAMP-responsive elem ent after treatment of PC-12 cells with potassium or potassium/cAMP. T hese results support a model in which the protein(s) binding to the cA MP-responsive element integrates signals initiated by multiple pathway s (cAMP and calcium) and transmits that integrated signal to the basal transcription machinery, resulting in increased levels of gene expres sion.