The Study Of Decorin (DCN) Effect On The Growth Of Rat Mesangial Cells And DCN Degradation Via Ubiquitin-proteasome Pathway

Decorin (DCN) is a leucine-rich proteoglycan secreted by glomerular mesangial cells (MC), and plays an important role in the regulation of cell growth, synthesization and composition of extracellular matrix (ECM). The over-expression of DCN can inhibit cell growth, protein level of TGF-β1 and mRNA level of collagen type IV and TIMP-2 in cultured mesangial cells in vitro, suggesting DCN might be a promising therapeutic target for treating mesangial proliferative glomerulonephritis and glomerulosclerosis. Recently, more and more studies indicated that DCN can suppress cell growth and induce apoptosis in various tumor cells. Its mechanism may involve the down-regulation of epidermal growth factor receptor (EGFR) and the up-regulation of endogenous cyclin-dependent kinase inhibitor p21.However, the inhibitory effect of DCN on cell proliferation was reported inconsistently in non-tumor cells. Up to now, the divarications still exist on whether DCN could induce apoptosis and suppress cell growth in non-tumor cells and whether EGFR plays a key role in its mechanism. Our previous work has demonstrated that DCN gene over-expression can inhibit cell growth of cultured MC in vitro. However, the effect of DCN on apoptosis and the role of EGFR in inhibitory effect of DCN on the growth of MCs remain unknown.Because of DCN's diverse bioactivities especially its antagonized effect on the MC proliferation and ECM synthesization, it is evident that the regulation of DCN stability and activity is of great physiological and pathological importance. Recent researches on modulation of DCN metabolization have shown that DCN might be degradated by ubiquitin-proteasome pathway (UPP). Since the inhibitor of proteasome MG132 could increase the level of cytoplasmic DCN in ovarian tumors, it is supposed that the degradation of intracellular DCN may be regulated by UPP. It is now appreciated that UPP plays a significant role in regulating turnover and function of different proteins involved in several cellular processes. It is recognized that the level and function of proteins could be reduced or enhanced via controlling this pathway, which consequently leads to a change of cellular activities. Therefore, it is important to explore the mechanisms of intracellular DCN degradation. However, there still lack direct evidences on roles of ubiquitination in the stability of intracellular DCN and factors that control this process.Ubiquitin-proteasome pathway is also regulated by a reversible process involving many deubiquitinating enzymes, which may recognize and stabilize target protein specifically. Ubiquitin-specific processing protease 2-69 (USP2-69) is an important member of ubiquitin-specific processing proteases (USPs) family. It is reported that USP2-69, highly expressed in kidney, is relative to cell proliferation and apoptosis. Our previous studies showed that the potential relationship between the up-expression of USP2-69 and mesangial cell proliferation. As we know, DCN is also relative to cell growth. Therefore we speculate that USP2-69 might be a deubiquitination enzyme close to DCN, and can influence the stability and activity of DCN by regulating ubiquitination and degradation of DCN.Consequently, in this study we use gene transfection, RNA interference, co-immunoprecipitation and other techniques to observe the effect of DCN on cell growth and apoptosis in cultured MC and the related mechanisms; to clarify the degradation pathway of intracellular DCN, factors that control the process, and possible effects of the process on the function of DCN; to study the interaction existed between USP2-69 and DCN and the effect of USP2-69 on ubiquitination and level of intracellular DCN. And the purpose of this work is to find out a new orientation for treating the glomerular diseases which is characterized with proliferation of MC and glomerulosclerosis. PartⅠEffect of DCN on the growth and apoptosis of cultured rat MCObjectives To observe the effect of DCN on cell growth and apoptosis in cultured rat MC and their related mechanisms.Methods pcDNA3.1A-DCN plasmid was purfied and identified, then transfected into cultured rat MC, and positive clones stably expressing DCN (MC/DCN) were selected. DCN siRNA was used for blocking DCN expression in MC/DCN. RT-PCR and Western blot were used to analyze gene transcription and protein expression of DCN. Apoptosis and cell growth of MCs were assayed by flow cytometry and Hoechst staining. Expressions of active caspase-3, EGFR, p21 and TGF-β1 were analyzed using Western blot.Results After treatment with the enzymes of EcoRⅠand XbaⅠ, the pcDNA3.1A-DCN plasmid was cut into two fragments whose lengths were 5400bp (vector) and 1068bp (DCN gene), respectively. The over-expression of DCN in MC increased mRNA and protein level of DCN, arrested cells in G0/G1 phase and induced down-regulation of EGFR and up-expression of p21. DCN transfection also induced apoptosis of MC and elevated the protein level of active caspase-3. In addition, the expression of TGF-β1 was significantly inhibited in MC/DCN. DCN-siRNA transfection remarkably blocked the expression of DCN and reversed the down-regulatory effects of DCN on MC's proliferation.Conclusions A positive clone (D19) over-expressed DCN gene was established by cationic lipid-mediated transfection. In cultured rat MC, DCN could induce apoptosis by activating caspase-3 and inhibit cell growth by down-regulating EGFR and up-regulating p21 in vitro. DCN also effectively down-regulates protein expression of TGF-β1.PartⅡUbiquitin-proteasome pathway mediates the degradation of intracellular DCN in MCObjectives To investigate the degradation pathway of intracellular DCN in MC and possible effects of the process on the function of DCN.Methods Co-immunoprecipitation (Co-IP) was used to detect whether DCN was ubiquitinated in MC. Co-IP and Western blot were used to analyze the ubiquitination and protein level of DCN in MC, which was treated with MG132, NH4Cl or tunicamycin, respectively. Time-course western blot was used to analyze the half-life of DCN in MC, which was treated with MG-132, tunicamycin or/and cycloheximide, respectively. After treating with MG132 or tunicamycin, RT-PCR was used to analyze the mRNA level of DCN and ColⅣ, Western blot was used to analyze the protein level of DCN and TGF-β1 and flow cytometry was used to analyze the cell cycle of MCs.Results Ubiquitinated DCN was found in normal MC. After treating with the proteasome inhibitor MG132, the content of DCN and its ubiquitinated form accumulated and displayed a prolonged half-life, accompanied by decreased TGF-(31 protein expression, reduced collagenⅣmRNA level and inhibition of MCs proliferation in MC. Lysosome inhibitor NH4Cl had no effect on the content of DCN and its ubiquitinated form. After treating with the N-glycosylation inhibitor tunicamycin, ubiquitination and degradation of DCN increased and displayed a shortened half-life, accompanied by increased TGF-β1 protein expression.Conclusions In cultured rat MC, the degradation of DCN is mediated via ubiquitin-proteasome pathway (UPP). Suppression of N-linked oligosaccharide synthesization of DCN could induce the ubiquitination and degradation of intracellular DCN. The regulation of DCN degradation via UPP can affect its functions which include the influence on the expression of ColⅣmRNA and TGF-β1 protein and the growth of MC.PartⅢEffect of USP2-69 on the ubiquitination and degradation of intracellular DCN in MCObjectives To clarify the effects of USP2-69 on regulating the ubiquitination and degradation of DCN. Methods Western blot were used to analyze protein expression of USP2-69 in rat MC, hepatocytes (BRL-3A) and podocytes (GEC).Co-IP and confocal microscopy were used to analyze the interaction between USP2-69 and DCN. pRK5-USP2-69-HA eukaryon expression plasmid was purfied and identified, then transfected into MC. Western blot were used to analyze protein expression of HA,USP2-69 and DCN. Co-IP was used to analyze the ubiquitination of DCN.Results The protein expression of USP2-69 was higher in MC than in BRL-3A and GEC. Then we confirmed a novel physical interaction between USP2-69 and DCN, and a co-localization in cytoplasm as well. After treatment with the enzymes of BamH I and Hind III, the pRK5-USP2-69-HA plasmid was cut into two fragments whose lengths were 4600bp (vector) and 1970bp (USP2-69 gene), respectively. Transfected with pRK5-USP2-69-HA plasmid induced protein expressions of HA which represented extrinsic USP2-69, increased protein level of USP2-69 and DCN, and an obvious decrease of ubiquitinated DCN in MC.Conclusions In cultured rat MC, a physical interaction and a co-localization in cytoplasm exist between USP2-69 and DCN. USP2-69 could decrease the ubiquitinated form of DCN and increase its protein expression.