Transcriptional regulation of the nos3 gene in pulmonary myofibroblast differentiation and implications for this in pulmonary fibrosis

Abstract

Nitric oxide (NO) produced by endothelial cells via the catalytic action of nitric-oxide synthase (eNOS) represents an antifibrotic mechanism in the body. Previous studies suggest that nitric oxide (NO)-mediated signals regulates myofibroblast phenotypes and it is believed that a loss of this control may play an important role in development of pulmonary fibrosis. This work focused on the effect of specific regulators on NOS3 gene expression to elucidate the mechanisms by which nitric oxide levels are controlled in rat pulmonary myofibroblasts cells. Rat NOS3 gene promoter was cloned in front of a luciferase reporter gene and transfection assays in rat pulmonary myofibroblasts were performed and cells were treated with a variety of potential regulators of NOS3. Promoter activity of NOS3 gene, were assayed using the Dual Luciferase reporter gene assay technique. The results showed that the rat NOS3 promoter was active in the rat pulmonary myofibroblasts with the human NOS3 promoter showing little or no activity. This study confirmed that TGFβ and LPS up regulates transcriptional activity while PMA decreases NOS3 transcription.NOS3 transcriptional activity decreased in cells treated with 23187, a calcium ionophore but increased when treated with EGTA suggesting that calcium concentrations could have a potential effect on regulating NO concentrations in the cell. Treatment with L-NAME (Nw-Nitro-L-arginine methyl ester), a known NOS3 selective inhibitor had no effect on the gene expression. S-NAP (S-nitroso-Nacetylpenicillamine), a known Nitric Oxide donor suppressed NOS3 transcriptional activity. From these results it can be concluded that high concentrations of NO inhibit NOS3 activity.NOS3 is regulated by several effectors in the cell that could be targets for pharmacological agents to help in protection against pulmonary fibrosis. This work initiated a xii study to determine the functional elements involved in the transcriptional activity of the promoter by creation of deletion constructs, however this studies were not completed