TWEAK/Fn14 Signaling Pathway Regulates Fibroblast Extracellular Matrix Synthesis In Keloid

BackgroundKeloid is a skin fibroproliferative disease characterized by excessive deposition of extracellular matrix(ECM).Its occurrence and development is related to genetic susceptibility,immune disorders,epigenetics,abnormal collagen synthesis and degradation.Numerous studies have shown that the pathogenesis of keloid and the excessive deposition of ECM-related components mainly depend on the cascade reaction of signaling pathways in fibroblasts,but the mechanism still remains unclear.Tumor necrosis factor-like weak inducer pf apoptosis(TWEAK)is a member of the tumor necrosis factor ligand superfamily,which binds to its receptor Fn14 to function.Previous studies have shown that TWEAK/Fn14 is an important pathway to mediate apoptosis,and also plays a role in cell proliferation,extracellular matrix remodeling,and inflammation.However,whether TWEAK/Fn14 signaling pathway plays a role in the development of keloid has not been further studied.ObjectiveIn this study,we intended to evaluate the expression level of the TWEAK/Fn14 signaling pathway in keloid,verify the function and role of the TWEAK/Fn14 signaling pathway in keloids,and finally clarify the molecular mechanism of the TWEAK/Fn14 signaling pathway.MethodsThe differences in the expression and localization of TWEAK and Fn14 was compared in keloids and normal skin tissues by immunohistochemical staining and immunofluorescence staining.The mRNA and protein levels of TWEAK were analyzed in keloids and normal skin tissues using qPCR and western blotting.Serum samples were Collected from patients with multiple keloids and normal individuals,and ELISA was used to analyze the expression differences of TWEAK.Primary fibroblasts were isolated to analyze mRNA and protein expression of Fn14 in keloids and normal skin tissue fibroblasts using qPCR and western blotting.The human TWEAK recombinant protein was used to stimulate keloid fibroblasts,and siRNA was used to construct Fn14 knockdown fibroblast cell line.We used qPCR,western blotting,immunofluorescence staining,CCK8,EdU,and flow cytometry to detect the effects of activation and inhibition of TWEAK/Fn14 signaling pathway on ECM-related protein synthesis,cell proliferation and apoptosis of fibroblasts.RNA-sequencing was conducted to analyze the transcription factor changes after activating and inhibiting the TWEAK/Fn14 signaling pathway in keloid fibroblasts,and the transcriptional regulation of the key transcription factor IRF1 was identified by ChIP-qPCR and dual-luciferase reporter assays.Through the review of literature and JASPAR databases,we speculated that the mediating molecule of IRF1 modulation in the TWEAK/Fn14 signaling pathway is P65,and the activation of P65 by the TWEAK/Fn14 signaling pathway is verified by qPCR,western blotting and immunofluorescence.QPCR,western blotting,and immunohistochemical staining were used to verify the expression correlation between TWEAK,Fn14,IRF1,collagen-Ⅰ and collagen-Ⅲ expression levels in keloids.ResultsThe expression of TWEAK was significantly down-regulated in keloid tissue and serum of patients,and the expression of Fnl4 receptor was significantly down-regulated in keloid fibroblasts.Human TWEAK recombinant protein treatment of keloid fibroblasts significantly reduced the expression of extracellular matrix proteins such as collagen,while Fn14 siRNA transfection of fibroblasts promoted the expression of extracellular matrix proteins such as collagen.Transcriptome sequencing showed that the addition of TWEAK recombinant protein and the knockdown of Fn14 up-regulated ERF1 and down-regulated IRF1,respectively,suggesting that IRF1 is a potent key transcription factor mediating the negative regulation of extracellular matrix in fibroblasts by TWEAK/Fn 14 signaling pathway.After knocking down IRF1 in fibroblasts,we found that the extra-cellular matrix-related proteins were significantly increased.The transcription factors of IRF1 were predicted by JASPAR database and the literature was consulted.We speculated that the up-regulation of IRF1 by TWEAK/Fn14 signaling pathway was mediated by P65.ChIP-qPCR and dual-luciferase report experiments confirmed the binding site of P65 in the promoter region of IRF1.At the same time,after knocking down P65 in fibroblasts,we found that extracellular matrix-related proteins were significantly increased.Finally,we confirmed that TWEAK,Fn14,IRF1 espressions were positively correlated with each other in keloid,and these three molecules were negatively correlated with collagen expression.ConclusionIn keloids,the TWEAK/Fn14 signaling pathway is inhibited,and the TWEAK/Fn14 signaling pathway regulates the expression of IRF1 by regulating P65 activity,thereby playing a role in the synthesis of extracellular matrix-related proteins in keloid fibroblasts,ultimately affecting the occurrence and development of keloids.This study briefly elucidates the function of TWEAK/Fn14 signaling pathway in keloids and its molecular regulation mechanism,which is of great significance for further elucidating the pathogenesis of keloids and finding new prevention and treatment targets.