| Objective:Cyclin G2（CCNG2）is a noncanonical cyclin that is categorized in the same family as cyclin I and cyclin G1.A large body of evidence indicates that cyclin G2 is an important contributor in the development of cancer;the expression of cyclin G2 is downregulated in cancers.Results of our preliminary study suggested that cyclin G2 can inhibit canonical Wnt signaling and participates in the regulation of bone metabolism.The canonical Wnt signaling pathway is highly conserved and is essential for tumor occurrence,fetal development,and cell fate decisions;Wnt signaling also controls physiologic and pathologic processes,such as angiogenesis,inflammatory reactions,and fibrosis.High-glucose induction activates the canonical Wnt signaling pathway in glomerular podocytes and mesangial cells and causes excessive apoptosis of intrinsic renal cells.Suppression of canonical Wnt signaling is known to help improve proteinuria and renal fibrosis in patients with type 1 diabetes.Diabetic nephropathy（DN）,one of the most severe diabetic complications,has become the leading cause of end-stage renal disease.Pathologic features of DN include renal glomerular hypertrophy,thickening of the glomerular and tubular cell membranes and of the basilar membrane,extracellular matrix accumulation,and eventual tubular interstitial fibrosis and glomerulosclerosis.The clinical outcome of DN involves progressive,irreversible renal dysfunction and ultimately renal failure.Several cytokines and signaling pathways participate in the development of DN.Wnt signaling can be involved in multiple renal injury and renal interstitial fibrosis.Renal interstitial fibrosis is the main pathological basis of DN,therefore canonical Wnt signaling may have a key role in the occurrence and development of DN.In this study,we investigated the regulatory participation of cyclin G2 in pathologic processes of DN in vitro and in vivo by means of several molecular biologic techniques.We found that cyclin G2 contributes to the occurrence and development of glomerulosclerosis and renal interstitial fibrosis in DN by regulating canonical Wnt signaling.Our results revealing the new biological function of cyclin G2 involved in the development of the abnormal glucose metabolism.Methods:1.The effects of cyclin G2 over-expression on the expression of tubular interstitial fibrosis and glomerulosclerosis related proteins and canonical Wnt signaling pathway-related proteins in HMC cells and in HK-2 cells.We overexpressed cyclin G2 in HMC cells and in HK-2 cells using lentiviral particles,extracted total protein of cells to detected the expression of tubular interstitial fibrosis and glomerulosclerosis related proteins and canonical Wnt signaling pathway-related proteins by western blot.2.Cyclin G2 influences the expression of tubular interstitial fibrosis and glomerulosclerosis related proteins by regulating canonical Wnt signaling.We activated the canonical Wnt signaling pathway using the GSK3βinhibitor CHIR99021 for 72h,hen extracted total protein of cells to detected the expression level change of tubular interstitial fibrosis and glomerulosclerosis related proteins and canonical Wnt signaling pathway-related proteins by cyclin G2 overexpressed.3.Cyclin G2 interacts with Dpr1 and influences the level of CK1-mediated phosphorylation of Dpr1.We applied coimmunoprecipitation and a Duolink in situ PLA to validate that Dpr1 interacts with cyclin G2 in HMC cells and HK-2 cells;We overexpression of cyclin G2 in HMC cells and HK-2 to detected Dpr1 phosphorylation using an anti-phosphoserine/threonine antibody after coimmunoprecipitation with an anti-Dpr1antibody;Using a Duolink in situ PLA,we detected binding between Dpr1 and CK1after overexpression of cyclin G2 in HMC cells and HK-2 cells;Using acoimmunoprecipitation,we detected phosphorylation level of Dpr1 by CK1 after overexpression of cyclin G2 or CK1 in HMC cells and HK-2 cells;Using acoimmunoprecipitation and western blot,we detected phosphorylation level of Dpr1and canonical Wnt signaling pathway-related proteins in HMC cells and HK-2 cells after co-transfected with cyclin G2 expression vector,wild type or mutant Dpr1expression vector.4.Cyclin G2 has a negative regulatory effect on up-regulation of high glucose-induced canonical Wnt signaling pathway proteins and fibrosis-related proteins in vitro.We overexpressed cyclin G2 in HMC cells and in HK-2 cells using lentiviral particles and cultured cells in high glucose environment or low glucose environment for 72h,then extracted total protein of cells to detected the expression of tubular interstitial fibrosis and glomerulosclerosis related proteins and canonical Wnt signaling pathway-related proteins by western blot.5.Effect of cyclin G2 knockout on proteinuria,renal inflammation and renal injury in STZ-induced DN mice.We examined cyclin G2 expression in renal tissues of STZ-induced DN mice by means of immunohistochemisty and western blot.Urinary creatinine and urinary albumin were detected by kit using 24 hrs urine from STZ-induced DN WT mice and DN cyclin G2 knockout mice.The severity of glomerulosclerosis was ascertained by PAS staining.We also evaluated collagen fibers in the renal cortex by Masson’s trichrome staining.6.Effects of cyclin G2 knockout on tubular interstitial fibrosis and glomerulosclerosis related proteins and canonical Wnt signaling pathway-related proteins in STZ-induced DN mice.Immunohistochemistry and western blot were used to detect the changes of tubular interstitial fibrosis and glomerulosclerosis related proteins and canonical Wnt signaling pathway in WT DN mice and cyclin G2 knockout DN mice.Results:1.Cyclin G2 inhibits tubular interstitial fibrosis and glomerulosclerosis related proteins proteins and canonical Wnt signaling pathway proteins expression in vitro.The expression of tubular interstitial fibrosis and glomerulosclerosis related proteins and canonical Wnt signaling pathway-related protein was detected by western blotting in HMC cells and HK-2 cells after overexpression cyclin G2.Compared with the control group（LV-GFP）,the levels of N-cadherin,vimentin,ET-1,FN,and Collagen IV were significantly downregulated in cells with cyclin G2overexpression（p<0.05）;E-cadherin expression was upregulated in these cells（p<0.05）.We also found that ectopic expression of cyclin G2 inhibited the expression ofβ-catenin—the key regulator of the canonical Wnt signaling pathway—and of its targets,cyclin D1,c-Myc,and MMP7.Overexpression of cyclin G2 also up-regulated the expression of phosphorylated（p）-β-catenin（Ser33/37/Thr41）and down-regulated the expression of p-GSK3β（Ser9）.Therefore,cyclin G2 can inhibit the expression of tubular interstitial fibrosis and glomerulosclerosis related proteins and canonical Wnt signaling pathway in HMC cells and in HK-2 cells.2.Cyclin G2 influences the expression of tubular interstitial fibrosis and glomerulosclerosis related proteins by regulating canonical Wnt signaling in vitro.We activated the canonical Wnt signaling pathway using the GSK3βinhibitor CHIR99021.Before CHIR99021 addition,cyclin G2 overexpression inhibited the expression of proteins associated with canonical Wnt signaling and tubular interstitial fibrosis and glomerulosclerosis related proteins.After addition of CHIR99021,cyclin G2–mediated inhibition of Wnt signaling factors was abolished;moreover,downregulation of tubular interstitial fibrosis and glomerulosclerosis related proteins and upregulation of E-cadherin were diminished.Therefore,cyclin G2 regulates the expression of tubular interstitial fibrosis and glomerulosclerosis related proteins via canonical Wnt signaling in HMC cells and HK-2 cells.3.Mechanism research of cyclin G2 regulating canonical Wnt signaling pathway canonical.We applied coimmunoprecipitation and a Duolink in situ PLA to validate that Dpr1 interacts with cyclin G2 in HMC cells and HK-2 cells.We found that cyclin G2could inhibit the expression of Dvl2,β-catenin,and cyclin D1;this inhibition Dvl was blocked by Dpr1 silencing.These results indicated that cyclin G2 binding with Dpr1and suppressing the canonical Wnt signaling pathway.Overexpression of cyclin G2 in HMC cells and HK-2 cells resulted in a reduced level of phosphorylated Dpr1.Using a Duolink in situ PLA,we detected a reduction in binding between Dpr1 and CK1after overexpression of cyclin G2 in HMC cells and HK-2 cells.After overexpression of CK1,the phosphorylation level of Dpr1 was upregulated.However,after overexpression of CK1 and cyclin G2 simultaneously,this level was decreased.4.Cyclin G2 inhibits high glucose-induced tubular interstitial fibrosis and glomerulosclerosis related proteins and canonical Wnt signaling pathway-related protein expression in vitro.We cultured HMC cells and HK-2 cells in high-glucose conditions for 72 hours,the results of western blot suggested that,compared with the low-glucose control group,the levels of the tubular interstitial fibrosis and glomerulosclerosis related proteins N-cadherin,ET-1,FN,vimentin,and Collagen IV were upregulated,whereas E-cadherin was downregulated.Overexpression of cyclin G2 suppressed the high-glucose–induced upregulation of N-cadherin,ET-1,FN,vimentin,and Collagen IV.Immunofluorescence assay findings showed that cyclin G2 could inhibit the upregulation of N-cadherin induced by high glucose.When cells were cultured in high glucose,the levels ofβ-catenin,cyclin D1,c-Myc,and MMP7 were induced;overexpression of cyclin G2 in these cells abolished this effect.5.A deficiency of cyclin G2 increases albuminuria and yields glomerular basement membrane accumulation,increased renal cortical fibrosis and other pathological changes in DN mice.Immunohistochemisty and western blot results indicated that cyclin G2expression was significantly decreased in DN WT mice,compared with WT mice.The 24-hour urinary albumin levels and the UACR were significantly increased in STZ-induced DN Ccng2-/-mice,compared with DN WT mice.PAS staining results indicated that the glomerular basement membrane index was significantly increased in DN Ccng2-/-mice,compared with DN WT mice.Masson’s trichrome staining results indicated that the highest proportion of fibrotic area was found in renal tissues from the DN Ccng2-/-group.Therefore,a lack of cyclin G2 may yield an increase in proteinuria and more severe renal injury and renal fibrosis in DN mice.6.Cyclin G2 knockout affects proteinuria and renal injury in DN mice.We assessed protein expression levels of Wnt signaling factors in the renal tissues of DN Ccng2-/-mice and of DN WT mice.We found that the levels of fibrosis-related proteins and canonical Wnt signaling pathway-related proteins in DN Ccng2-/-mice were substantially higher than those of DN WT mice.Conclusion:1.Cyclin G2 influences the expression of tubular interstitial fibrosis and glomerulosclerosis related proteins by regulating canonical Wnt signaling in vitro.2.Cyclin G2 inhibits tubular interstitial fibrosis and glomerulosclerosis related proteins expression in vitro.3.Cyclin G2 inhibits tubular interstitial fibrosis and glomerulosclerosis related proteins and canonical Wnt signaling pathway-related protein expression,and functions to prevent renal fibrosis and relieve renal injury in vivo. |
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