Biochemical and Biophysical Research Communications
Nox4 modulates collagen production stimulated by transforming growth factor β1 in vivo and in vitro
Highlights
► We examined the role of Nox4 on collagen accumulation in vivo and vitro. ► TGFβ1-induced collagen accumulation in sponges cotreated with Adv-Nox4ΔNADPH implanted in mice. ► Suppressing Nox4 reduced collagen accumulation in vivo. ► Inhibiting Nox4 gene expression and ROS release reduced collagen synthesis in vitro. ► Nox4 acts as an intermediary effector to facilitate TGFβ-induced collagen synthesis.
Introduction
The synthesis of extracellular matrix is crucial for tissue repair and remodeling during normal wound healing responses. On the other hand, extensive accumulation of matrix proteins in fibrotic scars during pathological conditions such as pressure overload, myocardial infarction and lung inflammation tends to compromise normal tissue functions [1], [2]. Fibroblasts and activated myofibroblasts identified in scarred tissues are responsible for matrix protein production [3], [4]. Matrix protein collagen is predominantly found in fibrotic tissues and its synthesis is modulated by a profibrotic cytokine transforming growth factor β (TGFβ) [1]. Indeed an exogenous application of TGFβ to incisional wounds accelerated collagen production and improved healing efficiency in rats [5]. In a clinical situation, there was a concomitant increase in gene expression of TGFβ and collagen 1 in endomyocardial biopsies from patients with dilated cardiomyopathy [6] while augmented expression of TGF β1 and its receptor was observed in human cardiac hypertrophy [7]. The intervention to block TGFβ might be considered as a strategy for suppressing matrix protein synthesis, but this would have undesired systemic side effects due to the multiple activity of TGFβ [8]. TGFβ has been shown to modulate a variety of biological and pathological responses such as vasculogenesis [9], inflammation [10] and cancer progression [11]. Therefore further characterization of the downstream signaling pathway of TGFβ in generation of matrix proteins might provide an alternative anti-fibrotic strategy for this large group of fibrotic disorders.
Recently, the generation of ROS from NADPH oxidase has been implicated in TGFβ1-induced collagen and fibronectin expression in cultured mouse [12] and human [13] cardiac fibroblasts and human lung fibroblasts [2]. Liu et al. [14] recently showed the inhibitory effect of oral administration of a NADPH oxidase inhibitor apocynin on pressure overload-induced collagen deposition and TGFβ expression in the myocardium of rabbits. Others demonstrated a reduction in interstitial fibrosis in the myocardium of Nox2 type NADPH oxidase-deficient mice following myocardial infarction [15] or pressure overload [16] in comparison to wildtypes. Targeted deletion of NADPH oxidase isoform Nox4 with tracheal administration of siRNA also suppressed bleomycin-induced lung fibrosis in mice [2]. However the precise role of NADPH oxidase in mediating TGFβ-induced collagen production is poorly defined. We therefore hypothesized that TGFβ-induced collagen synthesis is dependent on NADPH oxidase isoform Nox4-derived ROS generation in cultured mouse cardiac fibroblasts. We then used a murine model of sponge implant [17] to investigate whether an intervention targeting NADPH oxidase isoform Nox4 affects the local stimulatory effects of TGF β1 on collagen accumulation in vivo.
Section snippets
Cell culture
Cardiac fibroblasts were isolated from male mice (C57B/L6, 16–20 weeks old) and cultured and characterized as previously described [18]. Briefly, freshly excised mouse cardiac ventricles were minced and digested in serum free Dulbecco’s Modified Eagle Medium (DMEM from Invitrogen, USA) containing collagenase I (1 mg/mL, Sigma–Aldrich, Australia) and trypsin (2 mg/mL, Sigma–Aldrich) at 37 °C. The supernatant was transferred to culture medium (DMEM supplemented with 10% fetal calf serum, penicillin
Profibrotic effect of TGFβ1 is mediated by ROS in cardiac fibroblasts
TGF-β1 (10 ng/mL) significantly increased collagen 1 mRNA level (Fig. 1A) and the release of newly synthesized collagen in the media (Fig. 1B). Such response was accompanied by an increase in H2O2 generation (Fig. 1C). To clarify whether TGFβ1 effects were mediated via TGFβ/activin signaling, we co-treated the cells with TGFβ1 and the inhibitor of TGFβ type 1 receptor/ALK5 (SB431542, 10 μM). SB431542 inhibited the stimulatory effects of TGFβ1 on collagen 1 mRNA expression (Fig. 1A), soluble
Discussion
Excessive accumulation of collagen in tissues during wound healing can be deleterious affecting normal tissue functions in diseases such as myocardial infarction [24]. In the present study, we demonstrated that Nox4 is an effector that facilitates TGFβ1-induced collagen production in cultured cardiac fibroblasts. Furthermore, we showed for the first time that suppressing Nox4 with Adv-Nox4ΔNADPH attenuated the stimulatory effect of TGFβ1 on collagen accumulation in sponges in mice. Given the
Acknowledgments
We thanked Professor John F Keaney from the University of Massachusetts Medical School, USA for providing Adv-Nox4i and Dr Barry Goldstein from Thomas Jefferson University, Philadelphia, USA for providing Adv-Nox4ΔNADPH. HMP is supported by Post-Doc Fellowship (PF 11 M 6093) from National Heart Foundation Australia. F.J. is supported by a grant from Natural Science Foundation of China (Grant Number 81070164). The O’Brien Institute acknowledges the Victorian State Government’s Department of
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