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William J. O'Brien, Tom Heimann, Deborah Conklyn, Farhan Rizvi; TGF-β Regulation of NADPH Oxidase in Human Corneal Stromal Fibroblasts/Myofibroblasts. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2045.
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We hypothesize that TGF-β regulates NADPH oxidase expression and superoxide production in human corneal stromal fibroblasts(HCSF)/myofibroblasts, an event believed to be critical in the conversion of fibroblasts to myofibroblasts.
Cultures of HCSF were established from donor corneas following collagenase digestion of the stroma after removal of the epithelium and endothelium. Cells were recovered and grown in DMEM or DMEM/F12 containing no serum or low serum. Cells were plated in DMEM containing 5% defined FBS and 10 ng/ml TGF-β or a mixture of the pro-inflammatory cytokines; IL1-β, TNF-α and rHuIFN-γ. At various intervals post-cytokine treatment, cells were harvested and gene expression assessed by qRT-PCR and western blotting. NADPH oxidase was assayed as superoxide dismutase (SOD) inhibitable NADPH dependent conversion of dihydroethidium to 2-hydroxyeithidium. Knockdown of NADPH oxidase was achieved by treatment of cells with 40 nM NOX4 siRNA SMARTpools.
TGF-β treatment of HCSF induced the expression of the NOX4 containing isoform of NADPH oxidase as determined by qRT-PCR (5 to 30 fold). Membranous NADPH oxidase activity was increased by TGF-β treatment (81.4 ± 10.4 to 107.6 ± 18.3 RFU/min/mg). Superoxide production was NADPH dependent and DPI inhibitable but not inhibitable by rotenone, allopurinol or L-NAME. Treatment of HCSF with the mixture of pro-inflammatory cytokines decreased the amount of NOX4 transcripts (2 to 3 fold) and the production of superoxide (e.g. 81.4 ± 10.4 to 42.2 ± 17.9 RFU/min/mg). TGF-β increased α-sma expression as measured by RT-qPCR ( >5 fold) and by western blots relative to β-actin (4 to 8 fold). The mixture of pro-inflammatory cytokines increased the levels of NOS2 (40 to 230 fold) and COX2 (9 to 22 fold). Treatment of TGF-β treated cultures with NOX4 specific siRNA reduced the expression of NOX4 (30 to 90%), superoxide production, and α-sma expression.
The data demonstrate coordinate up-regulation of the NOX4 isoform of NADPH oxidase and α-sma by TGF-β in HCSF. The transition from the fibroblastic to myofibroblastic phenotype appears regulated by NOX4 dependent superoxide production.
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