The Role And Regulating Mechanism Of SUMO Proteins In Keloids Through SUMOylation Of Transforming Growth Factor-Beta/Smad Signaling Pathway

Aims:Keloids are hyperproliferative responses of skin caused by dermal injuries.It can lead to continuous collagen synthesis in the dermis and subcutaneous tissues,and can grow beyond the original wound boundary and invade adjacent structures,which can cause appearance deformities and dysfunction.Due to the unclear pathogenesis,despite the great variety of treatments,keloids are still difficult to cure and have high recurrence.There are multiple types of post-translational modifications(PTMs)of proteins in cells,which are essential for maintaining the normal biological functions of the target proteins.Small ubiquitin-related modifiers(SUMO)can conduct SUMOylation of the substrates,and the destruction of its intracellular homeostasis is related to tumors and fibrotic diseases.The pathological nature of keloids is the excessive deposition of extracellular matrix(ECM)caused by the abnormal proliferation of fibroblasts,and transforming growth factor-beta(TGF-β)is the key keloid-promoting factor.TGF-β can activate the downstream Smad pathway to regulate the expression of target genes,and ultimately promote the proliferation,migration and ECM synthesis of human keloid fibroblasts(HKFs).Previous studies have suggested the SUMOylation of TGF-β/Smad played an important role in modulating its function.To further explore the pathogenesis of keloids and find new therapeutic targets,here we investigated the expression level of SUMO proteins in keloids and its impact on HKFs phenotypes in Section 1,then studied the influence of SUMOylation modification on HKFs and keloids in vitro and in vivo in Section 2,and finally explored the molecular mechanisms by which SUMO1 caused phenotypic changes in HKFs through its modulation of TGF-β/Smad pathway in Section 3.Methods:In Section 1,keloids and normal skin tissues and primary fibroblasts were obtained,and immunohistochemistry,immunofluorescence,q RT-PCR and Western Blot were used to compare the protein and m RNA expression of SUMO-related proteins.The expression of SUMO1 was down-regulated by transfection of small interfering RNA that can silence SUMO1 and up-regulated by construction of SUMO1-overexpressed stable transgenic fibroblasts,then CCK-8,Ed U,flow cytometry,wound-healing and Transwell assay were used to detect the effect of SUMO1 on the phenotypic changes of fibroblasts such as proliferation,apoptosis,migration and invasion.In Section 2,Ginkgolic acid(GA),an inhibitor of SUMOylation,was used in HKFs to down-regulate the level of SUMOylation modification,and the above-mentioned methods in Section 1were used to explore the phenotypic changes as well;GA were then used to treat keloid in an animal model that implanted human keloid tissues into immunodeficient mice,and by measuring the weights,volumes and immunohistochemical staining of treated masses to detect the expression of collagen I,collagen III,α-SMA and the deposition of ECM,we further explored the effect of inhibiting SUMOylation on keloids in vivo.In Section 3,SUMO1 expression were alternated by the aforementioned methods in HKFs,and Western Blot and q RT-PCR were applied to detect the expression changes of TGF-β/Smad pathway proteins and downstream effector molecules.At the same time,the dual-luciferase reporter assay was applied to detect the activity of transcriptional activation of Smad.Finally,the protein interaction between SUMO1 and Smad4 was explored by protein Co-immunoprecipitation(Co-IP)and confocal laser scanning,which can show the effects of Smad4 protein stability and subcellular distributions by SUMOylation.Results:The results of Section 1 showed the m RNA and protein levels of SUMO-related proteins in keloid tissues and primary fibroblasts were higher than that in normal skins,especially SUMO1.SUMO1 was located in the nucleus mainly,and down-regulation of SUMO1 can effectively inhibit the proliferation,migration and invasion of HKFs and promote apoptosis,while overexpression of SUMO1 can reverse those changes.In Section 2,GA could inhibit the proliferation,migration and invasion of HKFs in vitro,promote apoptosis,and also eliminate the stimulation that overexpressed SUMO1 brought to HKFs.Intralesional injection of GA could reduce the volume and weight of keloid tissues in animal model,at the same time down-regulating the expression of collegan I,collegan III,α-SMA and alleviating ECM deposition.As for Section 3,SUMO1 reduction can inhibit TGF-β/Smad pathway activation,down-regulate the levels of Smad4 protein and phosphorylated Smad2/3,and reduce the transcriptional activity of SBE and expression of downstream effector molecules,while increasing SUMO1 can carry out the opposite changes.SUMO1 and Smad4 were co-localized in the nucleus,and by directly binding with each other,SUMOylation enhanced the stability of Smad4 protein and regulated its subncellular distribution.Conclusions:First,the expression of SUMO-related proteins in keloids were higher than normal skins and SUMO1 can promote the cell viabilities and functions of HKFs.Second,SUMO1 functioned mainly through SUMOylation,and the reduction of SUMOylation can inhibit keloid in vitro and in vivo.Last,SUMO1 can enhance TGF-β/Smad signaling,stimulate transcriptions and increase the expression of downstream effector molecules by modifying Smad4 protein.In summary,SUMO can promote pathogenesis and development of keloids by modifying TGF-β/Smad signaling pathway.