The Role Of Sumoylation In Regulation Of TGF-β/Smad Signaling Pathway In Glomerular Mesangial Cells Induced By High Glucose

Diabetic nephropathy （DN） is a special and serious microvascularcomplications of diabetes. The main pathological characteristics are glomerularsclerosis and renal tubular interstitial fibrosis in DN later stage. Transforminggrowth factor-β(TGF-β) is a key mediator of fibrogenesis, and TGF-β/Smadpathway is an important signaling pathway which mediates renal fibrosis indiabetic nephropathy. It has been recognized that TGF-β signaling pathwaymodulated by post-translational modifications such as phosphorylation andubiquitination, recent studies have shown that SUMOylation involved in theregulation of TGF-β signaling pathway. Studies showed that small ubiquitinrelated modifier (SUMO) and its specific enzymes regulate TGF-β pathway bymodifying some important signaling molecules such as: type I TGF-β receptor（TβRI) and Smad4in this pathway. Since the SUMOylation plays animportant role in regulating TGF-β signaling pathway which is the key pathwayinduced fibrosis, it is worth to study the role of SUMOylation in regulation ofTGF-β signaling in diabetic nephropathy. Therefore, we investigate theexpression of SUMO and Smad4in Rat Glomerular Mesangial Cells (GMCs)induced by high glucose, and the interaction between SUMO and Smad4toexplore the role and mechanism of sumoylation in regulation of TGF-βsignaling pathway in diabetic nephropathy. Methods: Rat GMCs were cultured and divided into5groups: normal glucose group (NC, culture mediumcontains5.6mmol/L glucose);10mmol/L glucose group (HG10);20mmol/Lglucose group (HG20);30mmol/L glucose group (HG30); osmotic pressuregroup (OP, culture medium contains5.6mmol/L glucose+24.4mmol/Lmannitol). The cells in every group were stimulated and cultured for6h,12hand24h. the expression of SUMO、 Smad4and fibronectin(FN) was measuredby Western Blot and（or） RT-PCR; the interaction and colocalization betweenSUMO and smad4were detected by co-immunoprecipitation andimmunofluorescence confocal laser microscopy respectively. Results:Compared with normal group, the expression of SUMO1、SUMO2/3and smad4were increased in high glucose groups in a dose-dependent manner(p<0.05), theexpression were increased gradually at12h,24h(p<0.05),but were not changedsignificantly at6h. There was no significant difference of SUMO1and Smad4between OP and NC group (P>0.05), while the expression of SUMO2/3inOP group was higher than that in NC group, but weaker than that in highglucose groups (p<0.05). There was no expression of SUMO4in GMCs.Analyses of co-immunoprecipitation and immunofluorescence confocal laserscanning showed that smad4interacted and colocalized with SUMO2/3inevery group and the sumolyation (SUMO2/3) of smad4in high glucose groupwas strongly enhanced compared with normal group(p<0.05). Compared withnormal group, the expression of SUMO1、 SUMO2/3and FN mRNA wereincreased in high glucose groups in a dose-dependent manner(p<0.05), the mRNA expression were increased gradually at12h,24h(p<0.05). Conclusion:1. High glucose can increase the expression of SUMO and FN in rat GMCs,indicating that SUMO may play an important role in diabetic nephropathy.2.Sumolyation of samd4by SUMO2/3is involved in the regulation of TGF-βsignaling in diabetic nephropathy.