Nox4 and Duox1/2 Mediate Redox Activation of Mesenchymal Cell Migration by PDGF.

Pyotr A Tyurin-Kuzmin,Vsevolod A Tkachuk,Alexander V Vorotnikov,Eugene A Albert,Nadezhda D Zhdanovskaya,Ludmila V Ageeva,George V Sharonov,George D Sagaradze,Anna A Sukhova,Gianfranco Pintus

doi:10.1371/journal.pone.0154157

Pyotr A Tyurin-Kuzmin, Vsevolod A Tkachuk + Show 8 more

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https://doi.org/10.1371/journal.pone.0154157

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Abstract

Platelet derived growth factor (PDGF) orchestrates wound healing and tissue regeneration by regulating recruitment of the precursor mesenchymal stromal cells (MSC) and fibroblasts. PDGF stimulates generation of hydrogen peroxide that is required for cell migration, but the sources and intracellular targets of H2O2 remain obscure. Here we demonstrate sustained live responses of H2O2 to PDGF and identify PKB/Akt, but not Erk1/2, as the target for redox regulation in cultured 3T3 fibroblasts and MSC. Apocynin, cell-permeable catalase and LY294002 inhibited PDGF-induced migration and mitotic activity of these cells indicating involvement of PI3-kinase pathway and H2O2. Real-time PCR revealed Nox4 and Duox1/2 as the potential sources of H2O2. Silencing of Duox1/2 in fibroblasts or Nox4 in MSC reduced PDGF-stimulated intracellular H2O2, PKB/Akt phosphorylation and migration, but had no such effect on Erk1/2. In contrast to PDGF, EGF failed to increase cytoplasmic H2O2, phosphorylation of PKB/Akt and migration of fibroblasts and MSC, confirming the critical impact of redox signaling. We conclude that PDGF-induced migration of mesenchymal cells requires Nox4 and Duox1/2 enzymes, which mediate redox-sensitive activation of PI3-kinase pathway and PKB/Akt.

Highlights

Increased migration and proliferation of mesenchymal cells critically contributes to wound healing, tissue repair and maintenance of homeostasis [1]
epidermal growth factor (EGF) effectively activated the Erk1/2 pathway and mitotic activity in redox-independent fashion. These results show that sustained accumulation of cytoplasmic H2O2 in mesenchymal cells is a part of specific response to Platelet derived growth factor (PDGF); it provides for redox regulation of migration and mitotic activity via PI3K pathway, whereas the Erk1/2 pathway only controls mitotic activity in redox insensitive manner
We measured speed of migration of 3T3 cells transfected with Duox1/2 siRNAs and found it is approximately twice reduced as compared to that of the control cells treated with the non-targeting siRNA (Fig 6E). These results indicate that Duox1/2 mediate redox control of protein kinase B (PKB)/Akt phosphorylation and fibroblast migration stimulated by PDGF, while Nox4 is involved in the redox control of PKB/Akt phosphorylation in mesenchymal stromal cells (MSC)

Summary

Introduction

Increased migration and proliferation of mesenchymal cells critically contributes to wound healing, tissue repair and maintenance of homeostasis [1]. The inflammatory step involves oxidative stress and reactive oxygen species (ROS) [1]; it is followed by the reparatory steps mediated by mesenchymal cells such as MSC and fibroblasts [2,3]. Platelet derived growth factor (PDGF) is the major chemoattractant that guides these mesenchymal cells into injured areas where it stimulates their proliferation and extracellular matrix production [4,5].

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