NAD(P)H Oxidase Mediates TGF-β1–Induced Activation of Kidney Myofibroblasts

Abstract

TGF-␤1 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-␤1-induced activation of rat kidney fibroblasts and expression of ␣-smooth muscle actin (␣-SMA) and Fn-ED-A. In vitro, TGF-␤1 stimulated formation of abundant stress fibers and increased expression of both ␣-SMA and Fn-ED-A. In addition, TGF-␤1 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-␤1-induced stimulation of NADPH oxidase activity and reduced ␣-SMA and Fn-ED-A expression. Inhibition of TGF-␤1 receptor 1 blocked Smad3 phosphorylation; reduced TGF-␤1-enhanced NADPH oxidase activity; and decreased expression of Nox4, ␣-SMA, and Fn-ED-A. Diphenyleneiodonium, an inhibitor of flavin-containing enzymes such as the Nox oxidases, had no effect on TGF-␤1-induced Smad3 but reduced both ␣-SMA and Fn-ED-A protein expression. The Smad3 inhibitor SIS3 reduced NADPH oxidase activity, Nox4 expression, and blocked ␣-SMA and Fn-ED-A, indicating that stimulation of myofibroblast activation by ROS is downstream of Smad3. In addition, TGF-␤1 stimulated phosphorylation of extracellular signal-regulated kinase (ERK1/2), and this was inhibited by blocking TGF-␤1 receptor 1, Smad3, or the Nox oxidases; ERK1/2 activation increased ␣-SMA and Fn-ED-A. Taken together, these results suggest that TGF-␤1-induced conversion of fibroblasts to a myofibroblast phenotype involves a signaling cascade through Smad3, NAD(P)H oxidase, and ERK1/2.

Transforming Growth Factor β1-induced Apoptosis in Podocytes via the Extracellular Signal-regulated Kinase-Mammalian Target of Rapamycin Complex 1-NADPH Oxidase 4 Axis

“…In another study, sustained activation of Smad3 by TGF-␤1 suppresses deptor expression to activate mTORC1 and mTORC2 in a similar cell type (39). In renal fibroblasts, Smad3-Nox4-induced ROS generation phosphorylates ERK1/2, leading to myofibroblastic transformation (56). In podocytes, Smad3 phosphorylation has been reported previously to regulate ERK1/2 activation (49).…”

Myocardin-related Transcription Factor Regulates Nox4 Protein Expression

“…There is strong evidence that Nox4 is up-regulated during and is required for fibroblast-myofibroblast transition in vitro (19,(21)(22)(23). Moreover, Nox4 down-regulation by siRNA or antisense oligonucleotides proved to be protective in a lung fibrosis model (22) and reduced matrix deposition in diabetic kidney disease (DKD) (27), whereas new Nox1/4-selective pharmacological inhibitors exerted potent kidney-protective and antifibrotic effects in DKD models (28,29).…”

“…NADPH Oxidase Activity and Nox4 Expression Are Necessary for the Myogenic Shift of the Epithelium-In non-epithelial progenitors, in which TGF␤ is sufficient for myofibroblast transition, ROS production was shown to be necessary for the induction of key myofibroblast features, including increased contractility and SMA expression (19,21,22). To determine whether ROS are also essential for double hit-provoked epithelial-myofibroblast transition, we initially tested the effect of the general NADPH oxidase inhibitor VAS2870 (Fig.…”

Renal Allograft Fibrosis: Biology and Therapeutic Targets

“…These cells exhibit a remarkable phenotypic plasticity and functional overlap. They exert various functions depending on their phenotype and pathological context which can result in proor anti-fibrotic effects or have no effects at all (23,(31)(32)(33)(34)(35)(36)(37)(38)(39)(40). Apart from a number of chemokines driving their influx and activation such as CX3CR1 (28,30,31,41), several molecules also play an important role including galectin-3 (42,43), the receptor for macrophage colony-stimulating factor (44), the Wnt pathway ligand Wnt7b (45) and interleukin 10 (46).…”

“…Several molecules were proposed to be involved in the profibrotic switch of fibroblasts (or pericytes) during renal fibrosis, including the Wnt signaling pathway (e.g., Wnt4, LRP-6, and DKK-1) (183,184), oxidative stress and NADPH oxidase (39,88,185), integrins including aV integrin (186), Rheb/mTORC1 signaling (187) as well as lipid mediators, including prostaglandins and their receptors like EP4 (188,189), prostacyclins (190), or lipoxins (191,192).…”