Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: cornea: basic science • differentiation • cytokines/chemokines