Establishment of hypoxia inducible factor-1alpha overexpressing cells that produce endothelin-1

Cardiomyocytes produce endothelin-1. Upregulation of endothelin-1 is induced under hypoxic conditions. It has been reported that tissue hypoxia induces transcriptional factor hypoxia inducible factor-1alpha (HIF-1alpha) expression. It has also been reported that cellular hypoxia leads to HIF-1alpha activation. Previously, we reported that HIF-1alpha bound to the site in the ET-1 gene promoter region. HIF-1alpha is a master transcriptional factor that controls transcriptional activation of a number of genes responsive to cellular hypoxia, including endothelin-1, vascular endothelial growth factor, erythropoietin and glycolytic enzymes. We aimed to establish the HIF-1alpha-overexpressing cardiomyocytes. Because it has been reported that HIF-1alpha is destroyed by the ubiquitin proteasome pathway under normoxic conditions, we constructed the point-mutated HIF-1alpha that was exchanged at 564 proline with alanine. The point-mutated HIF-1alpha is not destroyed by ubiquitin proteasome, because ubiquitin ligase cannot bind to the point-mutated area. This mutated HIF-1alpha was transfected to rat cardiomyocytes. Using the protein extraction from the mutated HIF- 1alpha transfected cells under normoxic conditions, western blot methods with anti-HIF-1alpha antibody showed that the band was detected at 120 kDa, which is of identical size to HIF-1alpha. In the normoxic conditions of the control cardiomyocyte cells, the band was undetectable. The endothelin-1 mRNA was significantly altered in HIF-1alpha-overexpressing cells. These results indicate that we have established the cells overexpressing-HIF-1alpha, and that these cells produce endothelin-1 mRNA.