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Yuanquan Yang, Hua Yang, Zheng Wang, J Mario Wolosin, Peter S. Reinach; TGF-β1 Induced Rises in Il-6 Release and α-sma Expression Depend On ROS Formation and TRPV1 Activation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1085.
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Transforming growth factor (TGF)-β1- induced increases in IL-6 release and α-smooth muscle actin (SMA) expression in corneal fibroblasts require activation of the transient receptor potential vanilloid1 (TRPV1) (Okada, Y et al American Journal of Pathology 2011). This study examined if this dependence involves increases in reactive oxygen species (ROS) production and modulation of SMAD2 phosphorylation.
Stromal fibroblasts were isolated from fresh human cadaver corneas (New York Upstate Transplant Service) and cultured following a described method. Flow cytometry assessed TGF-β1-induced ROS production. TGF-β1 and H2O2- induced Ca2+ transients were determined by image analysis, α-SMA expression and SMAD2 phosphorylation by immunoblotting and the effect of TGF-β1 on IL-6 release by ELISA.
TGF-β1 (1 ng/ml) treatment for 1 h increased ROS production by 1.5-fold. A ROS scavenger, N-acetyl-L-cysteine (10 mM), or the NADPH oxidase inhibitor, diphenylene iodonium (10 μM), abolished a TGF-β1- induced persistent SMAD2 phosphorylation (15 min and 16 h) as well as increases in α-SMA expression and IL-6 release. TGF-β1 and H2O2 (0.5 mM) induced Ca2+ transients, which were attenuated by capsazepine (CPZ, 10 µM), a selective TRPV1 antagonist. Similarly, either CPZ or withdrawal of extracellular Ca2+ inhibited TGF-β1-induced p-SMAD2 formation. TRPV1 siRNA gene silencing also suppressed TGF-β1-induced increases in SMAD2 phosphorylation, IL-6 release and α-SMA expression (vs. effects on cells transfected instead with scrambled siRNA).
TGF-β1 induced increases in α-SMA expression and IL-6 release in human corneal fibroblasts depend on ROS formation and subsequent, TRPV1-dependent, Ca2+ spikes. Increases in ROS formation driven by TGF-β1 transactivate TRPV1, which in turn contributes to SMAD2 phosphorylation.
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