Progranulin Promotes Bleomycin-induced Skin Sclerosis By Enhancing TGF-?/Smad3 Signaling Through Up-regulation Of TGF-? Type ? Receptor

Objective:Progranulin?PGRN?is an autocrine growth factor with a variety of physiological and pathological roles.Previous reports demonstrated PGRN could increase dermal fibroblasts in wound healing and activate cancer associated fibroblasts in some cancers.Because systemic sclerosis?SSc?is a prototypical fibrosis-related disorder,we speculate that PGRN may play a role in the pathogenesis of SSc.Here,our aim was to to clarify the role and mechanism of PGRN in bleomycin?BLM?-induced model of SSc.Methods:In this study,serum from 6 patients with SSc and 5 healthy controls were collected.The expression of PGRN in serum of patients and healthy controls was detected by enzyme-linked immunosorbent assay?ELISA?.The animal model of SSc was established by subcutaneous injection of bleomycin,and the expression of PGRN in the lesion was detected by immunohistochemistry and RT-qPCR.The animal model of SSc was established in wild?WT?mice and PGRN-deficient(PGRN^-/-)mice by subcutaneous injection of bleomycin,and then the skin samples at the injection site were obtained for following experiments.The degree of skin fibrosis was assessed by pathological analysis and soluble collagen content.The degree of fibroblasts activation was analyzed by immunofluorescence and Western blotting and T?R/Smad3/CTGF pathway activation was detected by Western blotting.The animal model of SSc was established in WT and PGRN^-/-mice while treated with SIS3,a specific inhibitor of Smad3.The degree of skin fibrosis was evaluated by pathological analysis and soluble collagen content analysis in WT and PGRN^-/-mice lesions.In addition,the phosphorylation level of Smad3 was also detected.In vitro,mouse dermal fibroblasts were isolated and cultured,and the effect of exogenous PGRN on the expression of T?R I in mouse dermal fibroblasts and the level of Smad3 phosphorylation were analyzed by Western blotting.Results:We found that the level of PGRN in serum of SSc patients was elevated compared to healthy controls?p<0.05?and PGRN expression was also increased in lesions of SSc model mice?p<0.05?.In the mouse model of SSc,PGRN^-/-mice skin had thinner dermal thickness?p<0.01?,thicker fat thickness?p<0.05?,more collagen content?p<0.05?and fewer myofibroblasts?p<0.01?compared to WT mice.WB results showed that the levels of T?R??p-Smad3 and CTGF were reduced in BLM-treated PGRN^-/-mice skin compared to BLM-treated WT mice?p<0.05,p<0.001,p<0.01?,which suggested that the activation of T?R?/Smad3/CTGF signaling was decreased in BLM-treated PGRN^-/-mice.In addition,after the administration of SIS3,BLM-treated PGRN^-/-mice exhibited significantly milder skin fibrosis and almost restored to normal skin when compared with BLM-treated WT mice,and the results were consistent with the p-Smad3 level in corresponding lesional skin.In vitro,exogenous PGRN also stimulated mouse dermal fibroblasts to express T?R I and increased the level of p-Smad3?p<0.01,p<0.05?.Conclusion:This study demonstrates that PGRN plays a promoting role in the development of dermal fibrosis through the activation of the T?R I/TGF-?/Smad3 signaling.