Transcriptional regulation of the nos3 gene in pulmonary myofibroblast differentiation and implications for this in pulmonary fibrosis
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Date
2011Author
Ochwang’i, Dominic O
Type
ThesisLanguage
enMetadata
Show full item recordAbstract
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
Citation
Agriculture and Veterinary SciencesSponsorhip
University of NairobiPublisher
Department of Veterinary Anatomy and Animal Physiology