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William J. O'Brien, Tom Heimann, Farhan Rizvi, Deborah Conklyn; Antagonistic Interactions of Interferon- and TGF-β1 within Human Corneal Stromal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3576.
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We proposed to determine the cellular signaling pathways and the mechanism(s) by which TGF-β1 and IFN-γ regulate transcription of selected genes in human corneal stromal fibroblasts(HCSF).
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 and 5% FBS. To study the regulation of transcription and signaling, cells were plated in DMEM containing 5% defined FBS, serum starved and treated with TGF-β1 and/or pro-inflammatory cytokines; IL1-β, TNF-α and rHuIFN-γ. At various intervals post-treatment, cells were harvested and gene expression was assessed by real-time RT-PCR and western blotting. The genes of interest included NOX NADPH oxidases(NOXs 1,4 and 5), p22phox, cyclooxygenase 2 (COX2), α-smooth muscle actin(α-SMA), nitric oxide synthase 2 (NOS2) and collagen type1, alpha 2(COL1α2).
TGF-β1 significantly up-regulated transcription of NOX4 (20 to 380 fold)(p= 0.03) and marginally up-regulated transcription of α-SMA(2 to 4 fold)(p=0.01) and COL1α2(2 to 3 fold)(p=0.01) while having little effect on NOX1,NOX5, p22phox, NOS2 and COX2 (≤ 2 fold) as determined by real-time RT PCR when the data was analyzed by the Pfaffl equation and associated statistics. The mixture of pro-inflammatory cytokines as well as low molecular weight inhibitors of TGF-β1 receptor function significantly (p=0.01)reduced steady state levels NOX4 transcripts induced by TGF-β1. Antagonism of TGF-β1 signaling by IFN-γ or the mixture of cytokines was not Smad7 dependent. Mixtures of IL1-β, TNF-α and rHuIFN-γ up-regulated the steady state mRNA pools of NOS2 (20 to 66 fold)(p=0.01),COX 2(5 to 13 fold) and NOX1(p=0.001) while significantly down regulatating NOX 4(p=0.001). This mixture was significantly more effective at reducing the effects of TGF-β1 than any of the cytokines alone. INF-γ significantly increased the phosphorylation of p38 MAP kinase 1hr post-treatment and increased the steady state amounts of the regulatory protein YB-1.
Steady state pools of NOX4 mRNA were significantly up-regulated in a time and dose dependent fashion by TGF β1. IFN-γ and other proinflammatory cytokines antagonized the regulatory activity of TGF β1 but not through Smad signaling.
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