The Mechanism Of Smurf2Regulating Inhibitory Smad7of TGF-β1Signaling Pathway In Hypertrophic Scar

Background During wound healing, excessive proliferation of fibroblasts and a large numberof secretion of extracellular matrix resulting from imbalance of synthesis and degradationpromotes the formation of hypertrophic scars. Transforming growth factor β1(TGF-β1) plays acritical role in this pathological process. Smads proteins are key intracellular signaling effectorsfor TGF-β1and the signal transduction subject to a widely complex and precise regulation atdifferent levels. Smad7, the inhibitory Smad protein, is one of major inhibitory regulators ofTGF-β1/Smad signaling pathway.Ubiquitin proteasome pathway (Ubiquitin Proteasome Pathway, UPP) is an important way oftargeted degradation of proteins and associated with a wide range of human biological processesincluding cell cycle, DNA repair, signal transduction, transcriptional regulation, phagocytosisand so on. The dysfunction of ubiquitin proteasome pathway is involved in a variety of humandiseases. Ubiquitin proteasome pathway consists of ubiquitin, a cascade of enzymes including anubiquitin-activating enzyme E1, an ubiquitin-conjugating enzyme E2and an ubiquitin ligase E3and26S proteasome. E3ubiquitin ligases are crucial in the selective recognition of targetproteins and also function in subsequent protein degradation by the26S proteasomes, which playa key role in the ubiquitin-mediated proteolytic.In TGF-β1/Smad signaling pathway, E3ubiquitin ligases induce the ubiquitination anddegradation of Smads proteins, TGF-βreceptors and so on, to regulate TGF-β1signaling.Abnormalities in E3ubiquitin ligases that control components of TGF-β1signaling may lead tothe development and progression of various diseases. Smurf2(Smad ubiquitination regulatoryfactor2) is HECT (homologous to the E6-accessory protein C-terminus)-type E3ubiquitinligases that regulates TGF-β1signaling. Recent reports have implicated aberrant expression ofSmurf2is involved in a variety of fibrotic diseases by ubiquitination and degradation of Smad7protein, transcriptional corepressors c-Ski (Sloan-Kettering Institute proto-oncogene) and SnoN (Ski-related novel gene N) to lead to the alterations of TGF-βsignaling and the development offibrotic diseases. Recently, we found that hypertrophic scar fibroblasts exhibited increasedSmurf2protein and mRNA levels compared with normal fibroblasts, and silencing theexpression of Smurf2in hypertrophic scar fibroblasts dramatically reduced TGF-β1production,inhibited TGF-β1-induced a-SMA expression and inhibited TGF-β1-induced collagen Isynthesis. Our results suggest that the enhanced expression of Smurf2is involved in theprogression of hypertrophic scarring. It remains to be unclarified which kind of signal moleculesdoes Smurf2target for degradation to regulate TGF-β1signaling in hypertrophic scars. Thisstudy aims to demonstrate that Smurf2, an E3ubiquitin ligase, promotes the formation ofhypertrophic scar through enhancing the degradation of Smad7, a negative regulator ofTGF-β1/Smad signaling pathway.Objective To investigate the mechanism of Smurf2regulating inhibitory Smad7of TGF-β1signaling pathway in hypertrophic scar fibroblasts.Methods (1) Hypertrophic scar fibroblasts and normal skin fibroblasts were cultured in vitro,and lysed and analyzed by Western Blot to detect the protein expression of Smad7.(2)Hypertrophic scar fibroblasts and normal skin fibroblasts were treated with exogenous TGF-β1at concentration of10ng/ml for5min,15min,30min,1h,2h and12h, and the expression ofSmad7protein and mRNA were detected by Western Blot and RT-PCR.(3) Lysates collectedfrom hypertrophic scar fibroblasts and normal skin fibroblasts were mixed with theubiquitination mixture, and incubated for0-6h at37°C, then the reaction mixtures were subjectedto Western Blot to detect the degradation of Smad7protein by using anti-Smad7antibody.(4) Invitro degradation assay of endogenous Smad7was also performed for6h, with or withoutaddition of1×proteasome inhibitor mixture (50μM MG132/50μM MG115) to investigate theinhibition of degradation for endogenous Smad7.(5) Lysates collected from hypertrophic scarfibroblasts were subjected to Smurf2immunoprecipitation(IP), and followed by Smad7WesternBlot and Smurf2Western Blot to investigate the interaction of Smurf2with Smad7.(6) Aftersilencing Smurf2gene expression by small interference RNA(siRNA), the expression of Smad7protein were observed by Western blot in hypertrophic scar fibroblasts with TGF-β1stimulation. Results (1) There was no significant difference in the level of Smad7protein betweenhypertrophic scar fibroblasts and normal skin fibroblasts.(2) With the exogenous TGF-β1stimulation, Smad7mRNA and protein gradually increased in a time-dependent fashion innormal skin fibroblasts, while in the hypertrophic scar fibroblasts, the expression of Smad7mRNA was increased in a time-dependent, but there was no difference in the level of Smad7protein with and without TGF-β1stimulation.(3) Degradation of Smad7protein wasprogressed in lysates collected from hypertrophic scar fibroblasts, while no significant reductionin Smad7protein was noted in lysates collected from normal skin fibroblasts.(4) Enhanceddegradation of Smad7in hypertrophic scar fibroblasts was prevented by the addition of theproteasome inhibitors MG132/MG115, which indicated the enhancement ofproteasome-mediated degradation of Smad7in hypertrophic scar fibroblasts.(5) In hypertrophicscar fibroblasts, the Smurf2-Smad7complex was noted, which indicated interaction of Smurf2with Smad7in hypertrophic scar fibroblasts.(6) In the hypertrophic scar fibroblasts, the Smad7protein was not increased with TGF-β1stimulation, but was increased with the stimulation ofTGF-β1after silencing Smurf2gene expression.Conclusion Our results suggest that the non-increasing of Smad7protein in the hypertrophicscar fibroblasts with TGF-β1stimulation might result from ubiquitin degradation mediated bySmurf2, and which may be involved in the progression of hypertrophic scarring.