Transcriptional regulation of the CRLR gene in human microvascular endothelial cells by hypoxia

Adrenomedullin is a 52 amino acid peptide that shows a remarkable range of effects on the vasculature that include inter alia, vasodilatation, regulation of permeability, inhibition of endothelial cell apoptosis, and promotion of angiogenesis. Recently the G-protein coupled receptor (GPCR) calcitonin receptor-like receptor (CRLR), and receptor activity modifying proteins (RAMPs) have become recognized as integral components of the adrenomedullin signaling system. However, mechanisms of regulation of CRLR expression are still largely unknown. This is in part due to lack of information on the gene promoter. In this study we have determined the transcriptional start of human CRLR cDNA by 5'-RACE and cloned the proximal 5'-flanking region of the gene by PCR. The 2318 bp genomic fragment contains the basal promoter of human CRLR, including potential TATA-boxes and several GC boxes. Regulatory elements binding known transcription factors, such as Sp-1, Pit-1, glucocorticoid receptor, and hypoxia-inducible factor-1 alpha (HIF-1alpha) were also identified. When cloned into reporter gene vectors, the genomic fragment showed significant promoter activity, indicating that the 5'-flanking region isolated by PCR contains the gene promoter of human CRLR. Of significance is that the cloned promoter fragments were activated by hypoxia when transfected in primary microvascular endothelial cells. Site-directed mutagenesis of the consensus hypoxia-response element (HRE) in the 5'-flanking region abolished such a response. We also demonstrated by semi-quantitative RT-PCR that transcription of the gene is activated by hypoxia in microvascular endothelial cells. In contrast, expression of RAMPs 1, 2, and 3 was unaffected by low oxygen tension. We conclude that simultaneous transcriptional up-regulation of CRLR and its ligand adrenomedullin in endothelial cells could lead to a potent survival loop and therefore might play a significant role in vascular responses to hypoxia and ischemia.