Phorbol ester and calcium regulation of corticotrophin-releasing factor receptor 1 expression in a neuronal cell line

We have previously demonstrated that corticotrophin-releasing factor receptor 1 (CRF-R1) mRNA levels can be down-regulated via activation of the cyclic AMP pathway in CATH.a cells, a neuronal cell line. In this study, we show evidence for down-regulation of CRF-R1 mRNA levels via activation of the protein kinase C (PKC) and calcium second messenger pathways. Incubation of CATH.a cells with phorbol 12-myristate 13-acetate (PMA), an activator of PKC, resulted in a time- and concentration-dependent down-regulation of CRF-R1 mRNA levels. Pretreatment with the inactive phorbol ester 4alpha-phorbol failed to influence significantly CRF-R1 mRNA levels. Incubation with carbachol, a cholinergic agonist known to activate PKC and increase intracellular calcium levels via phosphatidylinositol breakdown, also down-regulated CRF-R1 mRNA levels. Intracellular calcium levels were directly increased using A23187, a calcium ionophore, and thapsigargin, a calcium-ATPase inhibitor. Elevation of intracellular calcium content using either A23187 or thapsigargin significantly down-regulated levels of CRF-R1 mRNA. Furthermore, chelation of calcium with EGTA or blockade of voltage-dependent calcium channels with nifedipine inhibited agonist-mediated down-regulation of CRF-R1 mRNA levels. These results indicate that activation of PKC or calcium signal transduction pathways is sufficient to cause down-regulation of CRF-R1 mRNA levels and that calcium is required for agonist-mediated down-regulation of this receptor.