【作者】 吴慧娟；

【导师】 张志刚；

【作者基本信息】 复旦大学 ， 病理学， 2010， 博士

【摘要】 饰胶蛋白聚糖(decorin, DCN)是一种可由肾小球系膜细胞(mesangial cell,MC)分泌、富含亮氨酸的蛋白聚糖,在调控细胞生长及细胞外基质(extracellular matrix, ECM)合成、组成等方面都发挥重要的作用。DCN的过表达可抑制体外培养MC的生长及其TGF-β1蛋白的合成,并可影响其TIMP-2 mRNA的表达和IV型胶原的合成,因此,DCN是一个很有前景的防治肾小球肾炎及阻止肾小球硬化的细胞因子。近年来研究进一步发现,DCN还可抑制多种肿瘤细胞的生长,并有促进肿瘤细胞凋亡的作用,其机制可能与EGFR蛋白表达的下调及p21蛋白表达的上调相关。但DCN的这一作用,在非肿瘤细胞中的报道不尽相同,尽管近年有研究发现DCN能够上调p21的表达,但其对非肿瘤细胞是否具有抑生长、促凋亡作用,以及是否与EGFR表达下调有关等问题仍存在分歧。本课题组在前期实验中,已证实过表达DCN可抑制体外培养MC的生长,但有关DCN能否促进MC凋亡以及其抑制MC生长是否通过EGFR表达改变,目前尚不清楚。由于DCN具有重要的生物学效应,尤其在拮抗肾小球肾炎MC增生和ECM合成过程中起重要作用,故在实验中如何增加DCN的稳定性,并增强其生物活性,被视为对DCN进行深入研究的重点。近年来,一些学者在对DCN蛋白活性调节的研究中,发现DCN的降解代谢可能经泛素-蛋白酶体途径(ubiquitin -proteasome pathway, UPP)介导,有学者在对卵巢肿瘤的研究中,应用蛋白酶体抑制剂MG132处理后,发现该肿瘤细胞的DCN蛋白总量升高,提示DCN的降解可能是通过泛素化调节的。UPP是一个参与细胞内蛋白质降解过程,并影响细胞生理功能的重要系统,已有许多研究表明,其对很多种细胞的生理活动起重要调节作用。但至今,仍没有直接证据表明DCN在细胞内是通过UPP而发生降解及其影响因素。因此,明确MC内DCN降解与UPP的关系,并探索通过调控该途径可否达到增强或减弱DCN蛋白的活性、继而影响肾MC活动的目的,是本课题研究的一个重要出发点。有研究表明,UPP是一个可逆的过程,即细胞内还同时存在一些特异性的去泛素化蛋白酶,后者能阻碍靶蛋白的降解而对其形成负反馈调节。泛素特异性修饰酶2-69(ubiquitin-specific processing protease 2-69, USP2-69)为泛素特异性修饰酶家族(ubiquitin-specific processing proteases, USPs)中的一个重要成员,不仅已被证实在肾组织内呈现高表达,而且还是一个与细胞增殖和凋亡相关的去泛素化酶。本课题组前期实验证实,USP2-69表达上调与肾小球肾炎病变中MC增生有一定的相关性,而DCN对MC的生长有抑制作用,因此,我们设想USP2-69有可能是一种与DCN表达密切相关的去泛素化酶,可能依赖其对蛋白降解水平的调节而影响DCN的稳定性及活性。因此,本课题采用基因转染、RNA干扰和免疫共沉淀等技术,观察DCN对MC生长和凋亡的影响及其相关机制；明确MC内DCN的降解途径及其影响因素,以及调控该途径对DCN功能的影响；观察MC内USP2-69和DCN的结合及定位,以及USP2-69对DCN的泛素化和细胞内DCN蛋白水平的影响,以期为治疗以系膜增生为特征的肾小球疾病寻找新的作用靶点而提供实验依据。第一部分DCN对大鼠肾MC生长及凋亡的影响目的探讨DCN对大鼠肾MC生长及凋亡的影响及其可能机制。方法纯化pcDNA3.1A-DCN真核表达质粒及酶切鉴定；将pcDNA3.1A-DCN质粒稳定转染MC,并用DCN siRNA双链寡核糖核苷酸对其进行干扰。再分别采用RT-PCR和Western blot方法,检测DCN的基因转录和蛋白表达；应用流式细胞仪和Hoechst 33258染色法分别检测细胞周期和凋亡；用Westernblot方法,分别检测活性caspase-3、EGFR、p21和TGF-β1的蛋白表达。结果纯化的pcDNA3.1A-DCN质粒经过EcoRⅠ及Xba I双酶切后得到约5400bp大小的pcDNA3.1A条带和1068bp大小的DCN目的基因条带。稳定转染pcDNA3.1A-DCN质粒可上调MC内DCN的mRNA和蛋白表达；使G0/G1期的细胞数明显增多,S期的细胞数减少,提示MC生长受到抑制,且p2l蛋白表达升高,EGFR蛋白表达降低；同时细胞凋亡率明显升高,且活性caspase-3蛋白表达升高；TGF-β1的蛋白表达被明显抑制。转染DCN siRNA可以下调MC内DCN的蛋白表达,使G0/G1期的细胞数减少,S期的细胞数增多,且p21蛋白表达降低,EGFR蛋白表达升高；同时细胞凋亡率降低,且活性caspase-3降低；TGF-β1的蛋白表达明显升高。小结经脂质体介导法成功获得稳定高表达DCN基因的MC株(D19)。DCN可以通过活化caspase-3参与调控MC的凋亡,并能通过对EGFR和p21蛋白表达的影响,参与调控MC的生长,且DCN可有效调节MC内TGF-β1的蛋白表达。第二部分泛素-蛋白酶体途径介导MC内DCN的降解过程目的明确大鼠肾MC内DCN的降解途径以及调控该途径对DCN功能的影响方法应用免疫共沉淀的方法在MC内检测DCN是否与泛素蛋白相结合；使用蛋白酶体抑制剂MG132或溶酶体抑制剂NH4C1处理MC,应用免疫共沉淀检测泛素化DCN水平及Western blot检测细胞内DCN蛋白总量；分别使用MG132和/或蛋白合成抑制剂cycloheximide(CHX)处理MC,用Time-coursewestern blot法检测DCN半衰期；使用N-糖链抑制剂tunicamycin处理MC,应用免疫共沉淀检测泛素化DCN水平及Western blot检测细胞内DCN蛋白总量;使用tunicamycin和/或CHX处理MC,用Time-course western blot检测DCN半衰期；使用MG132或tunicamycin处理MC,用RT-PCR检测DCN和Col IV的mRNA表达,Western blot检测DCN和TGF-β1的蛋白表达,流式细胞仪检测细胞周期。结果MC内DCN与泛素蛋白相结合,使用MG132可以显著提高MC内DCN的泛素化水平和蛋白总量,而应用NH4C1对DCN的泛素化水平和蛋白总量无明显作用。使用CHX单独处理的MC内DCN的半衰期约为12h,联合使用MG132后,DCN降解被阻断,其半衰期明显延长。使用tunicamycin可以提高细胞内DCN的泛素化水平,促进DCN降解而降低DCN蛋白总量,以及使DCN的半衰期明显缩短,约为6h。经MG132处理后,伴随MC内DCN蛋白表达升高,Col IV的mRNA表达降低,TGF-β1蛋白表达降低,同时Go/G1期的细胞数明显增多；而经tunicamycin处理后,伴随MC内DCN蛋白表达降低,TGF-β1蛋白表达则显著升高。小结大鼠肾MC内DCN主要通过泛素-蛋白酶体途径降解,DCN N-糖链的合成抑制可促进其泛素化和降解,调控DCN的泛素化降解可影响Col IV的mRNA表达、TGF-β1的蛋白表达和MC生长。第三部分USP2-69对MC内DCN泛素化降解过程的调节目的明确USP2-69对DCN泛素化降解过程的影响。方法采用Western blot法检测大鼠MC、肝细胞株(BRL-3A)和足细胞(GEC)内USP2-69的表达；分别采用免疫共沉淀和激光共聚焦显微镜观察方法,检测MC内USP2-69是否与DCN结合,并观察两者在细胞内的定位；纯化pRK5-USP2-69-HA真核表达质粒及酶切鉴定；将pRK5-USP2-69-HA质粒瞬时转染MC,应用Western blot的方法,分别检测HA、USP2-69和DCN的蛋白表达,用免疫共沉淀的方法检测MC内DCN的泛素化水平。结果在MC内USP2-69的蛋白表达高于BRL-3A和GEC。MC内USP2-69可与DCN相互结合,且两者在胞质内共定位。纯化的pRK5-USP2-69-HA质粒经过BamHⅠ及HindⅢ双酶切后得到约4600bp大小的pRK5条带和1970bp大小的USP2-69目的基因条带。转染pRK5-USP2-69-HA质粒的MC内可检测到代表外源性USP2-69表达的HA蛋白,以及USP2-69和DCN蛋白表达的升高,同时DCN的泛素化水平明显降低。小结大鼠肾MC内USP2-69可与DCN相结合,及两者在胞质内共定位,USP2-69能够降低DCN的泛素化水平及提高MC内DCN蛋白总量。更多还原

【Abstract】 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.更多还原