The human urocortin 2 gene is regulated by hypoxia : identification of a hypoxia-responsive element in the 3'-flanking region

Ucn2 (urocortin 2) has been shown to exert potent beneficial effects in the cardiovascular system, including inhibition of apoptosis, improvement of cardiomyocyte contractility and decrease of oxidative stress. The mechanisms that contribute to the regulation of hUcn2 (human Ucn2) expression in cardiovascular pathologies are not known. In the present study, we analysed the mechanism by which hypoxia, a major stimulus in ischaemic heart disease, regulates Ucn2 gene expression. Hypoxia and CPX (ciclopirox olamine), which prevents proteolytic degradation of HIF (hypoxia-inducible factor), significantly increased hUcn2 mRNA levels in TE-671 cells. Gene silencing of endogenous HIF1alpha abolishes this increase. Hypoxia and CPX activated a luciferase-linked fragment of the 3'FLR (3'-flanking region) of the hUcn2 gene containing two putative HREs (hypoxia-response elements), HRE1 and HRE2. Site-directed mutagenesis experiments demonstrated that HRE1 is required for HIF1alpha-dependent luciferase activation. This activation was conserved in constructs with the 3'FLR fragment placed upstream of the luciferase gene, indicating an enhancer function for HRE1. Competition assays revealed direct binding between HRE1 and HIF1alpha. Regulation of Ucn2 by hypoxia was confirmed in rat neonatal cardiomyocytes and in cardiac-derived H9c2 cells transfected with constructs of the 3'FLR of the hUcn2 gene. In conclusion, our study demonstrates that hypoxia induces hUcn2 expression via a specific HRE in the 3'FLR of the hUcn2 gene, which interacts with the transcription factor HIF1alpha. Hypoxia-mediated stimulation of cardioprotective Ucn2 may help to preserve cardiac function and prevent apoptosis in ischaemic conditions in the heart.