Transcriptional regulation of Rat Endothelial Nitric Oxide Promoter in Pulmonary

Abstract

Nitric oxide levels may exert control on the persistence of pulmonary myofibroblasts cells in pulmonary fibrosis. This study examined the regulation of NO levels by transcription factors that influence the endothelial nitric oxide synthase (eNOS3) gene expression. Using a pGL3-Basic vector plasmid DNA, rat and human eNOS3 gene promoters were inserted upstream of a luciferase reporter gene and cloned in competent E. coli cells (DH5α). Transfection assays were performed and the cells treated with potential regulators of eNOS3 gene. Promoter activity of eNOS3 gene was assayed using the Dual Luciferase reporter gene assay. The results indicated that the rat NOS3 promoter was active in the cells with the human NOS3 promoter showing little or no activity.The results demonstrated that transforming growth factor-β ,EGTA and lipopolysaccharide up regulated transcriptional activity while Phorbol 12-myristate 13- acetate, 23187 and S-nitroso-N-acetylpenicillamine, suppressed eNOS3 transcriptional activity. Treatment with Nw-Nitro-L-arginine methyl ester, had no effect on the gene expression. The results of this study demonstrates that high concentrations of Nitric Oxide (NO) inhibit NOS3 gene activity hence an enhanced expression of eNOS in response to pharmacological interventions using some transcriptional factors from these study could provide protection against interstitial pulmonary.