NADPH oxidase 4 is dispensable for skin myofibroblast differentiation and wound healing
Aleksandra Malgorzata Siedlar,
Tamara Seredenina,
Anna Faivre,
Yves Cambet,
Marie-José Stasia,
Dominik André-Lévigne,
Marie-Luce Bochaton-Piallat,
Brigitte Pittet-Cuénod,
Sophie de Seigneux,
Karl-Heinz Krause,
Ali Modarressi,
Vincent Jaquet
Affiliations
Aleksandra Malgorzata Siedlar
Division of Plastic, Reconstructive and Aesthetic Surgery, Geneva University Hospitals, University of Geneva Faculty of Medicine, Geneva, Switzerland; Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, Switzerland
Tamara Seredenina
Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, Switzerland
Anna Faivre
Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland
Yves Cambet
READS Unit, Faculty of Medicine, University of Geneva, Geneva, Switzerland
Marie-José Stasia
Université Grenoble Alpes, CEA, CNRS, IBS, F-38044, Grenoble, France
Dominik André-Lévigne
Division of Plastic, Reconstructive and Aesthetic Surgery, Geneva University Hospitals, University of Geneva Faculty of Medicine, Geneva, Switzerland
Marie-Luce Bochaton-Piallat
Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, Switzerland
Brigitte Pittet-Cuénod
Division of Plastic, Reconstructive and Aesthetic Surgery, Geneva University Hospitals, University of Geneva Faculty of Medicine, Geneva, Switzerland
Sophie de Seigneux
Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland; Service and Laboratory of Nephrology, Department of Internal Medicine Specialties and of Physiology and Metabolism, University and University Hospital of Geneva, Geneva, Switzerland
Karl-Heinz Krause
Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, Switzerland
Ali Modarressi
Division of Plastic, Reconstructive and Aesthetic Surgery, Geneva University Hospitals, University of Geneva Faculty of Medicine, Geneva, Switzerland
Vincent Jaquet
Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, Switzerland; READS Unit, Faculty of Medicine, University of Geneva, Geneva, Switzerland; Corresponding author: Head of READS Unit, Department of Pathology & Immunology, Centre Médical Universitaire, Rue Michel Servet 1, 1211 Genève 4, Switzerland
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 (ECM) 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 although a putative NOX4 inhibitor GKT137831 and a flavoprotein inhibitor diphenylene iodonium mitigated this mechanism. Transcriptomic analysis revealed upregulation of the mitochondrial protein Ucp2 and the stress-response protein Hddc3 in Nox4-deficient fibroblasts, which had however no impact on fibroblast bioenergetics. Altogether, we provide extensive evidence that NOX4 is dispensable for wound healing and skin fibroblast to myofibroblast differentiation, and suggest that another H2O2-generating flavoprotein drives this mechanism.
