Nox4 modulates collagen production stimulated by transforming growth factor β1 in vivo and in vitro

The synthesis of extracellular matrix including collagen during wound healing responses involves signaling via reactive oxygen species (ROS). We hypothesized that NADPH oxidase isoform Nox4 facilitates the stimulatory effects of the profibrotic cytokine transforming growth factor (TGF) beta(1) on collagen production in vitro and in vivo. TGF beta(1) stimulated collagen synthesis and hydrogen peroxide generation in mouse cardiac fibroblasts, and both responses were attenuated by a scavenger of superoxide and hydrogen peroxide (EUK-134). Furthermore, by expressing a dominant negative form of Nox4 (Adv-Nox4(Delta NADPH)) in fibroblasts, TGF beta(1)-induced hydrogen peroxide production and collagen production were abrogated, suggesting that Nox4-dependent ROS are important for TGF beta(1) signaling in collagen production. This was confirmed by the inhibitory effect of an adenovirus carrying siRNA targeting Nox4 (Adv-Nox4i) on TGF beta(1)-induced collagen synthesis and expression of activated myofibroblasts marker smooth muscle alpha actin. Finally we used a mouse model of subcutaneous sponge implant to examine the role of Nox4 in the local stimulatory effects of TGF beta(1) on collagen accumulation in vivo. TGF beta(1)-induced collagen accumulation was significantly reduced when the sponges were instilled with Adv-Nox4(Delta NADPH). In conclusion, Nox4 acts as an intermediary in the signaling of TGF beta(1) to facilitate collagen synthesis. (C) 2012 Elsevier Inc. All rights reserved.