【作者】 苏金；

【导师】 药立波； 刘云才； 刘新平；

【作者基本信息】 第四军医大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 DDR2 (Discoidin Domain Receptor 2)属于受体型蛋白酪氨酸激酶,其配体为天然纤维型胶原(I, II和III型)。胶原与DDR2的结合能使其发生酪氨酸磷酸化,从而介导其下游的信号转导。我们实验室已有的研究表明,DDR2在体外培养的RA成纤维样滑膜细胞中高表达。RA的主要病理特征为血管翳的形成和滑膜组织的过度增殖并贴附于关节软骨,而关节软骨的主要胶原成份为II型胶原,这就为DDR2与其配体的结合并介导相关信号转导过程提供了空间上的可能。由于DDR2的主要功能是增加细胞中MMPs的表达。因此,我们认为在RA病人的关节中可能存在这样的病理过程:异常的炎症反应和滑膜功能的异常上调了滑膜细胞中DDR2的表达水平,同时滑膜的过度增殖为DDR2与胶原配体的相互作用提供了生理基础,使DDR2发生持续活化并过分泌MMPs侵蚀破坏软骨结构。在MMPs家族成员中,胶原酶是天然胶原降解的限速酶。RA滑膜细胞主要分泌胶原酶1(MMP-1)和胶原酶3(MMP-13)。而MMP-13对II型胶原的降解能力是MMP-1的5~10倍,此外,MMP-13还能降解关节软骨中的蛋白聚糖。因此,DDR2是否调控RA滑膜细胞过表达MMP-13及其调控机制成为本研究的出发点。我们首先体外分离了RA滑膜组织,经原代培养生长至四代的成纤维样滑膜胞用于实验。以II型胶原刺激RA滑膜细胞48小时后,收集细胞并分别分析MMP-13和MMP-1的mRNA和蛋白表达水平。结果显示,II型胶原刺激同时上调MM-1和MMP-13的mRNA和蛋白水平,然而,MMP-13被上调的幅度显著大于MMP-1,前者mRNA上调了8倍左右,而后者只上调了两倍。提示,II型胶原主要诱导滑膜细胞表达MMP-13。进一步通过逆转录病毒介导的方式,在小鼠成纤维样滑膜细胞中过表达DDR2,显著增加了滑膜细胞中II型胶原诱导的MMP-13的表达水平,明确了DDR2与MMP-13在滑膜细胞中表达的相关性。与原代RA滑膜细胞相比,永生化的RA滑膜细胞系MH7A几乎失去受II型胶原诱导表达MMP-13的能力。而根据文献报道,此细胞系与原代培养的RA滑膜细胞主要的表型差别是,细胞中ERK MAPK的组成型活化和细胞增殖速率的增加。据此,我们分析ERK MAPK可能参与了RA滑膜细胞中II型胶原诱导的MMP-13的表达。进一步用研究发现,ERK MAPK通路的特异性抑制剂PD98059能阻断DDR2诱导的MMP-13的表达和转录。此外,过表达DDR2显著增加了细胞中ERK MAPK的磷酸化水平,上述实验证据充分说明了ERK MAPK为DDR2下游的信号转导分子之一,并参与诱导MMP-13的表达。为了进一步探索DDR2介导MMP-13表达的分子机制,我们构建了MMP-13启动子报告基因,发现过表达野生型和组成型活化的DDR2分子(将人IgG1Fc段与无信号肽的DDR2序列进行融合,并连入分泌型蛋白表达载体中)都能上调MMP-13启动子报告基因的活性,且后者的调控活性强于前者,然而激酶区突变的DDR2分子则不具备这种调控作用,明确了DDR2对MMP-13启动子活性呈现活化依赖的上调作用。我们对MMP-13启动子区序列进行截短突变分析,发现将近端的Runx-2结合位点突变后,启动子报告基因失去了受DDR2诱导的活性,进一步共表达DDR2和Runx-2,发现它们能协同增加MMP-13的启动子活性,提示Runx-2可能受DDR2活化的调控并参与MMP-13的转录。然而,Runx-2基因主要表达于成骨细胞和骨骼肌细胞中,是调控骨形成和骨骼肌功能的重要转录因子之一。目前,还没有Runx-2在滑膜组织中表达的相关报道。我们通过免疫组织化学检测到了Runx-2蛋白在RA滑膜组织的表达,发现RA炎症条件下与软骨相接触的滑膜衬里层Runx-2表达水平较滑膜下层高,这与DDR2和MMP-13在RA滑膜组织中的表达模式一致。此外,在滑膜细胞中过表达Runx-2,显著增加滑膜细胞中II型胶原诱导的MMP-13的表达。上述结果充分提示了Runx-2可能受DDR2活化的调控上调RA滑膜细胞中MMP-13的表达。细胞表面RTK的活性是受到严格的控制和调节的,这对于维持细胞内稳态有着非常重要的意义。Cbl家族的蛋白作为泛素连接酶E3能促进多种RTK偶联泛素标签并走向降解的途径,是负向调控RTK功能的重要机制之一。那么,作为RTK的DDR2是否能受Cbl蛋白的泛素化调节呢?如果活化的DDR2可以被Cbl蛋白负向调控,那么这类泛素连接酶E3是否能降低滑膜细胞中DDR2介导的MMP-13的表达呢?基于上述疑问,我们首先检测了Cbl和Cbl-b对DDR2泛素化水平的影响。体内体外泛素化实验都显示,Cbl-b能显著增加活化DDR2的泛素化水平,而Cbl蛋白则不具备这种能力,并且Cbl-b对DDR2泛素化的调控依赖于其泛素连接酶的活性。脉冲追踪实验也显示,Cbl-b对DDR2泛素化的调控使其走向了依赖蛋白酶体的降解途径。为了明确Cbl-b对DDR2的泛素化调控是否降低滑膜细胞中胶原诱导的MMP-13的表达,我们分析了Cbl-b- / -小鼠滑膜细胞中II型胶原诱导的MMP-13表达量。结果显示, Cbl-b基因缺失上调了滑膜细胞中II型胶原诱导的MMP-13的表达量和胶原酶活性。我们关于Cbl-b负调控DDR2并降低滑膜细胞中MMP-13表达水平的研究很好的解释了Cbl-b - / -小鼠以II型胶原诱导关节炎后,滑膜组织呈现更严重的侵蚀破坏软骨的这一表型。更多还原

【Abstract】 Discoidin domain receptor 1 (DDR1) and 2 (DDR2) are receptor tyrosine kinases (RTKs) that bind different types of collagen as their ligands. We previously reported that DDR2 was highly expressed and activated in Rheumatoid Arthritis (RA) fibroblast-like synoviocytes (FLSs). RA is characterized by synovial hyperplasia and progressive joint destruction. The adhesion of synovial tissue with joint cartilage in RA renders the possibility that DDR2 binds to collagen II which is the main component of cartilage. As the main function of DDR2 is to upregulate the expression levels of MMPs, we speculated that“collagen II—DDR2—MMPs”pathway may occur and promotes the cartilage erosion in RA.Among the members of MMP family, interstitial collagenase (MMP-1) and collagenase-3 (MMP-13) play critical roles because they cleave type I, II and III collagens at a specific site generating 3/4 N-terminal and 1/4 C-terminal fragments, which rapidly denature at physiologic temperature and become susceptible to degradation by other MMPs. MMP-13 is of special interest with regard to cartilage degradation due to its preference to type II collagen in articular cartilage. In addition, MMP-13 is efficient in cleaving minor cartilage type IX and X collagen as well as aggrecan. Notably, the expression of MMP-13 was detected in synovial fibroblasts in RA, particularly at sites of synovial invasion into cartilage. Therefore, the present study focused on regulation of MMP-13 by DDR2 in synovial fibroblasts.We performed in vitro experiments to confirm the effects of culturing synovial fibroblasts of RA at passage 4 on type II collagen-coated plate on MMP-13 and MMP-1 expression. Real time PCR analysis demonstrated that MMP-13 mRNA level in synovial fibroblasts was elevated, to a similar extent, approximately 7-fold by native collagen II, while mRNA expression of MMP-1 was only slightly increased in synovial fibroblasts cultured on collagen-coated plate with 2-fold induction. Four synovial fibroblast cultures obtained from RA patients were included in this study. The induction of MMP-13 and MMP-1 protein in the culturing media by collagen II were comparable to that of their mRNA. Therefore, the expression of MMP-13 and MMP-1 is differentially regulated by collagen II in RA FLSs. To confirm the induction of MMP-13 production by DDR2, we expressed wild type DDR2 by retrovirus infection in murine synovial fibroblasts. As indicated, MMP-13 expression was dramatically increased by the overexpression of DDR2 in synoviocytes cutured on native type II collagen-coated plate.It is notable that rheumatoid MH7A cells exhibited dramatically decreased MMP-13 induction after treatment with collagen II, however, induction of MMP-13 production by collagen II was appropriately 8-fold in the primary cultured FLSs from RA patients. It is known that ERK is constitutively activated in MH7A cells thus this phenomenon indicates that ERK activation may contribute to collagen-induced MMP-13 expression in RA FLSs. To test the role of MEK/ERK cascade in DDR2—MMP-13 signaling, experiments were performed to make sure that whether PD98059 was able to block DDR2-induced MMP-13 expression. It was shown that MMP-13 mRNA and protein levels induced by collagen II can be decreased by PD98059 treatment. The enhancement of MMP-13 promoter activity upon DDR2 over-expression was reduced dramatically in the presence of PD98059. ERK1/2 phosphorylation in wild type DDR2-transfected MC3T3-E1 cells increased. However, collagen-induced ERK phosphoylation in the kinase-dead DDR2 mutant (K608E) transfected cells decreased compared with that in the mock transfection cells. These results indicated that ERK MAPK were involved in DDR2-induced MMP-13 expression.We further investigated whether activation of DDR2 was able to up-regulate MMP-13 promoter activity. The ectopic expression of wild-type DDR2 led to a 2.5-fold increase in MMP-13 promoter activity compared with the vector control. The kinase-dead DDR2 mutant (K608E) had no effect on MMP-13 promoter activity. When a constitutively active form DDR2 (FcDDR2) was introduced into the cells, it yielded a much greater increase of MMP-13 promoter activity compared with other constructs. To identify the responsive regions of the MMP-13 promoter to DDR2 signaling, serial deletion and mutant constructs of the 5’-flanking region of the human MMP-13 gene as well as DDR2 were transiently co-transfected into 293T cells. Constructs containing a mutated Runx-2 site exhibited 1.5-fold reduced DDR2 induction and no change for basal reporter expression. FcDDR2 and Runx2 were co-transfected with the reporter gene, a synergistic increase in reporter activity was observed. These experiments demonstrated that Runx-2 is required for DDR2-mediated induction of MMP-13. Runx-2 expression was also confirmed in RA synovial tissue by immunohistochemistry. It was demonstrated that Runx-2 was highly expressed in the synovial lining which is coincident with DDR2 and MMP-13 expression pattern in RA. Furthermore, collagen-induced MMP-13 expression was augmented by overexpression of Runx-2 mediated by retrovirus infection in murine synovial fibroblasts. In general, Runx-2 might be a key transcriptional modulator involved in DDR2-induced MMP-13 expression in RA synovial fibroblasts.RTK is tightly controlled in order to keep the normal function of the cells. Members of Cbl family functions as E3 ubiquitin ligases to facilitate the ubiquitination and degradation of multiple RTKs. We were wondering whether DDR2 might be a target for Cbl proteins. It was shown that Cbl-b but not Cbl could promote the ubiquitination of DDR2 in a collagen dependent manner. Pulse chase experiment demonstrated that Cbl-b promotes the degradation of DDR2. Synovial fibroblasts from Cbl-b- / - mice were shown to expressed more MMP-13 mRNA and protein upon collagen II treatment compared with cells from wild mice. Our above observation accounts for a more severe phenotype of synovial tissue from Cbl-b- / - mice upon induction of CIA(collagen-induced arthritis).更多还原