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Tissue injury/hypoxia and oxidative stress induced-extracellular adenosine triphosphate (ATP) can act as damage-associated molecular pattern molecules, which initiate inflammatory response. Our objective was to elucidate the role of extracellular ATP in skin fibrosis in systemic sclerosis (SSc). We identified that hypoxia enhanced ATP release and that extracellular ATP enhanced IL-6 production more significantly in SSc fibroblasts than in normal fibroblasts. There were no significant differences of P2X and P2Y receptor expression levels between normal and SSc fibroblasts. Nonselective P2 receptor antagonist and selective P2Y2receptor antagonists, kaempferol and AR-C118925XX, significantly inhibited ATP-induced IL-6 production and phosphorylation of p38 in SSc fibroblasts. ATP-induced IL-6 production was significantly inhibited by p38 inhibitors, SB203580, and doramapimod. Collagen type I production in SSc fibroblasts by ATP-induced IL-6/IL-6 receptor trans-signaling was inhibited by kaempferol and SB203580. The amount of ATP in bleomycin-treated skin was increased, and administration of AR-C118925XX significantly inhibited bleomycin-induced dermal fibrosis in mice. These results suggest that vasculopathy-induced hypoxia and oxidative stress might enhance ATP release in the dermis in SSc and that extracellular ATP-induced phosphorylation of p38 via P2Y2receptor might enhance IL-6 and collagen type I production in SSc fibroblasts. P2Y2receptor antagonist therapy could be a treatment for skin sclerosis in patients with SSc.

Ischemia-reperfusion (I/R) injury is a key player in the pathogeneses of pressure ulcer formation. Our previous work demonstrated that inducing transcription factor sex-determining region Y-box 2 (SOX2) promotes cutaneous wound healing via epidermal growth factor receptor (EGFR) signaling pathway enhancement. However, its protective effect on cutaneous I/R injury was not well characterized. We aimed to assess the role of SOX2 in cutaneous I/R injury and the tissue-protective effect of SOX2 induction in keratinocytes in cutaneous I/R injury. SOX2 was transiently expressed in keratinocytes following cutaneous I/R injury. Ulcer formation was significantly suppressed in keratinocytes-specific SOX2-overexpressing mice. SOX2 in skin keratinocytes significantly suppressed the infiltrating inflammatory cells, apoptotic cells, vascular damage, and hypoxic areas in cutaneous I/R injury. Oxidative stress-induced mRNA levels of inflammatory cytokines expression were suppressed, and antioxidant stress factors and amphiregulin (AREG) was elevated by SOX2 induction in skin keratinocytes. Recombinant (r) AREG administration suppressed pressure ulcer development following cutaneous I/R injury in mice and suppressed oxidative stress-induced reactive oxygen species production and apoptosis in vitro. These findings support that SOX2 in keratinocytes might regulate cutaneous I/R injury via AREG production, resulting in oxidative stress suppression. rAREG can be a potential therapeutic agent for cutaneous I/R injury.