S-Nitrosylation-mediated coupling of DJ-1 with PTEN induces PI3K/AKT/mTOR pathway-dependent keloid formation

Background:Keloids are aberrant dermal wound healing characterized by invasive growth,extracellular matrix deposition,cytokine overexpression and easy recurrence.Many factors have been implicated as pathological causes of keloids,particularly hyperactive inflammation,tension alignment and genetic predisposition.S-Nitrosylation(SNO),a unique form of protein modification,is associated with the local inflammatory response but its function in excessive fibrosis and keloid formation remains unknown.We aimed to discover the association between protein SNO and keloid formation.Methods:Normal and keloid fibroblasts were isolated from collected normal skin and keloid tissues.The obtained fibroblasts were cultured in DMEM supplemented with 10%fetal bovine serum and 1%penicillin/streptomycin.The effects of DJ-1 on cell proliferation,apoptosis,migration and invasion,and on the expression of proteins were assayed.TurboID-based proximity labelling and liquid chromatography-mass spectrometry were conducted to explore the potential targets of DJ-1.Biotin-switch assays and transnitrosylation reactions were used to detect protein SNO.Quantitative data were compared by two-tailed Student’s t test.Results:We found that DJ-1 served as an essential positive modulator to facilitate keloid cell proliferation,migration and invasion.A higher S-nitrosylated DJ-1(SNO-DJ-1)level was observed in keloids,and the effect of DJ-1 on keloids was dependent on SNO of the Cys106 residue of the DJ-1 protein.SNO-DJ-1 was found to increase the level of phosphatase and tensin homolog(PTEN)S-nitrosylated at its Cys136 residue via transnitrosylation in keloids,thus diminishing the phosphatase activity of PTEN and activating the PI3K/AKT/mTOR pathway.Furthermore,Cys106-mutant DJ-1 is refractory to SNO and abrogates DJ-1-PTEN coupling and the SNO of the PTEN protein,thus repressing the PI3K/AKT/mTOR pathway and alleviating keloid formation.Importantly,the biological effect of DJ-1 in keloids is dependent on the SNO-DJ-1/SNO-PTEN/PI3K/AKT/mTOR axis.Conclusions:For the first time,this study demonstrated the effect of transnitrosylation from DJ-1 to PTEN on promoting keloid formation via the PI3K/AKT/mTOR signaling pathway,suggesting that SNO of DJ-1 may be a novel therapeutic target for keloid treatment.

Melatonin influences the biological characteristics of keloid fibroblasts through the Erk and Smad signalling pathways

Background:Keloids are abnormal fibrous hyperplasias that are difficult to treat.Melatonin can be used to inhibit the development of certain fibrotic diseases but has never been used to treat keloids.We aimed to discover the effects and mechanisms of melatonin in keloid fibroblasts(KFs).Methods:Flow cytometry,CCK-8 assays,western blotting,wound-healing assays,transwell assays,collagen gel contraction assays and immunofluorescence assays were applied to demonstrate the effects and mechanisms of melatonin in fibroblasts derived from normal skin,hypertrophic scars and keloids.The therapeutic potential of the combination of melatonin and 5-fluorouracil(5-FU)was investigated in KFs.Results:Melatonin significantly promoted cell apoptosis and inhibited cell proliferation,migration and invasion,contractile capability and collagen production in KFs.Further mechanistic studies demonstrated that melatonin could inhibit the cAMP/PKA/Erk and Smad pathways through the membrane receptor MT2 to alter the biological characteristics of KFs.Moreover,the combination of melatonin and 5-FU remarkably promoted cell apoptosis and inhibited cell migration and invasion,contractile capability and collagen production in KFs.Furthermore,5-FU suppressed the phosphorylation of Akt,mTOR,Smad3 and Erk,and melatonin in combination with 5-FU markedly suppressed the activation of the Akt,Erk and Smad pathways.Conclusions:Collectively,melatonin may inhibit the Erk and Smad pathways through the mem-brane receptor MT2 to alter the cell functions of KFs,while combination with 5-FU could exert even more inhibitory effects in KFs through simultaneous suppression of multiple signalling pathways.