The role of NOX4 in skin fibroblasts

Differentiation of fibroblasts to myofibroblasts is governed by the transforming growth factor beta (TGF-β) through a mechanism involving redox signaling and generation of reactive oxygen species (ROS). Myofibroblasts synthesize proteins of the extracellular matrix and display a contractile phenotype. Myofibroblasts are predominant contributors of wound healing and several pathological states, including fibrotic diseases and cancer. Inhibition of the ROS-generating enzyme NADPH oxidase 4 (NOX4) has been proposed to mitigate fibroblast to myofibroblast differentiation and to offer a therapeutic option for the treatment of fibrotic diseases. In this study, we addressed the role of NOX4 in physiological wound healing and in TGF-β-induced myofibroblast differentiation. We explored the phenotypic changes induced by TGF-β in primary skin fibroblasts isolated from Nox4-deficient mice by immunofluorescence, Western blotting and RNA sequencing. Mice deficient for Cyba, the gene coding for p22phox, a key subunit of NOX4 were used for confirmatory experiments as well as human primary skin fibroblasts. In vivo, the wound healing was similar in wild-type and Nox4-deficient mice. In vitro, despite a strong upregulation following TGF-β treatment, Nox4 did not influence skin myofibroblast differentiation. Nevertheless, up-regulation of the mitochondrial protein Ucp2 and the stress-response protein Hddc3, as well as down-regulation of Islr were observed in Nox4-deficient fibroblasts. Altogether, we provide extensive evidence challenging a profibrotic role of NOX4 in skin fibroblasts and show that Nox4 regulates Ucp2 and Hddc3 expression, suggesting the presence of a so far undescribed redox crosstalk between NOX4 and redox homeostasis in fibroblasts. WT and NOX4 KO primary mouse skin fibroblasts were stimulated with 10ng/ml TGF-beta 2 in three biological replicates, total 12 samples were analyzed