Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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).
Conclusions: :
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.
Keywords: cornea: stroma and keratocytes • wound healing