Post-transcriptional regulation of CRLR expression during hypoxia.

Adrenomedullin and CGRP are two potent vasodilator peptides, and their receptors are formed by heterodimerization of the CRLR and a RAMP molecule. Hypoxia is associated with many diseases of the cardiovascular system. It was recently shown that the human CRLR gene promoter contains an HIF-1alpha regulatory element, and that CRLR mRNA was increased by hypoxia in human endothelial cells. In the present work, we have assessed the effect of hypoxia on CRLR expression both in vivo and in vitro using two different experimental models. We have also investigated the effect of hypoxia on RAMP expression. (1) We analyzed the effects of a chronic hypobaric hypoxia on rat ventricle expression of RAMPs and CRLR. (2) Acute hypoxia was studied in human vascular smooth cells from coronary artery (CASMC) exposed for 6h to 2% O(2). RT-PCR was used to analyze the mRNA expression, and protein levels were determined by Western blotting. A sharp increase in HIF-1alpha protein levels was induced by hypoxia in CASMC, and 3.5-fold rise of the CRLR protein occurred after 1h of hypoxia in face of unchanged mRNA levels. The CRLR mRNA levels were only elevated later. A clear decrease of the CRLR protein level occurred after 3 and 6h of hypoxia. Thus, acute hypoxia in CASMC induced a rapid change of the CRLR protein amount independently of changes in the CRLR mRNA. This finding suggested a major post-transcriptional effect of hypoxia on CRLR expression in CASMC. RAMP2 and adrenomedullin mRNAs were increased after 4h, but no change was observed for RAMP1. Chronic hypoxia in rats enhanced both mRNA and protein levels of the three RAMPs and CRLR in right and left ventricles. Together, our in vivo and in vitro data suggested that hypoxia up-regulates both adrenomedullin and its receptor (CRLR/RAMP2) to enhance the signaling at the target cell.