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Bondi CD, Manickam N, Lee DY, Block K, Gorin Y, Abboud HE, Barnes JL: NAD (P) H oxidase mediates TGF-beta1-induced activation of kidney myofibroblasts. Pulm Pharmacol Ther. 2009 Dec;22(6):487-91. Epub 2009 Apr 22.
TGF-beta1 expression closely associates with activation and conversion of fibroblasts to a myofibroblast phenotype and synthesis of an alternatively spliced cellular fibronectin variant, Fn-ED-A. Reactive oxygen species (ROS), such as superoxide, which is a product of NAD (P) H oxidase, also promote the transition of fibroblasts to myofibroblasts, but whether these two pathways are interrelated is unknown. Here, we examined a role for NAD (P) H oxidase-derived ROS in TGF-beta1-induced activation of rat kidney fibroblasts and expression of alpha-smooth muscle actin (alpha-SMA) and Fn-ED-A. In vitro, TGF-beta1 stimulated formation of abundant stress fibers and increased expression of both alpha-SMA and Fn-ED-A. In addition, TGF-beta1 increased both the activity of NADPH oxidase and expression of Nox2 and Nox4, homologs of the NAD (P) H oxidase family, indicating that this growth factor induces production of ROS. Small interfering RNA targeted against Nox4 markedly inhibited TGF-beta1-induced stimulation of NADPH oxidase activity and reduced alpha-SMA and Fn-ED-A expression. Inhibition of TGF-beta1 receptor 1 blocked Smad3 phosphorylation; reduced TGF-beta1-enhanced NADPH oxidase activity; and decreased expression of Nox4, alpha-SMA, and Fn-ED-A. Diphenyleneiodonium, an inhibitor of flavin-containing enzymes such as the Nox oxidases, had no effect on TGF-beta1-induced Smad3 but reduced both alpha-SMA and Fn-ED-A protein expression. The Smad3 inhibitor SIS3 reduced NADPH oxidase activity, Nox4 expression, and blocked alpha-SMA and Fn-ED-A, indicating that stimulation of myofibroblast activation by ROS is downstream of Smad3. In addition, TGF-beta1 stimulated phosphorylation of extracellular signal-regulated kinase (ERK1/2), and this was inhibited by blocking TGF-beta1 receptor 1, Smad3, or the Nox oxidases; ERK1/2 activation increased alpha-SMA and Fn-ED-A. Taken together, these results suggest that TGF-beta1-induced conversion of fibroblasts to a myofibroblast phenotype involves a signaling cascade through Smad3, NAD (P) H oxidase, and ERK1/2. |
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