Mechanism Investigation Of DDR2-MMP-13 Signalling And Their Potential Roles In The Cartilage Destruction Of RA

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).