Role Of DDR2 In Pathogenesis Of Rheumatoid Arthritis

Rheumatoid Arthritis (RA) is an autoimmune disease manifesting as chronic and symmetric arthritis. Many factors are involved in the pathogenesis of RA including immune cells, cytokines and proteases. DDR2 is a recently identified receptor tyrosine kinase. Its extracellular discoidin domain could recognize and bind collagen. DDR2 plays important roles in cell proliferation, differentiation, invasion and metastasis. Previous study showed that DDR2 was overexpressed in synovial fibroblast cells in RA, and could induce secretion of MMP-1. MMP-1 is a matrix metalloproteinase, which plays important roles in RA by degrading extracellular matrix and cause damage to cartilage, ligament and bone. MMP-13 is another kind of important matrix metalloproteinase in RA pathogenesis, which has been shown to be more potent than MMP-1 in degrading collagen and cartilage. Whether DDR2 could induce MMP-13 in RA is unknown. Therefore, this study focused on the regulation of MMP-13 by DDR2. ObjectivesTo study the regulation of MMP-13 by DDR2 and elucidate the possible signaling pathway.Material and Methods1. Expression of DDR2 in mouse model of RA1.1 Mouse modelC57BL/6J mice were used. Mice were given: (1) chicken collage II (i. sc); (2) mBSA+IL-1β; (3) TNF- or IL-1β to induce arthritis phenotype. Excised knees were fixed in 4% phosphate-buffered formalin and decalcified in 10% EDTA. The tissues were then dehydrated in a gradient of alcohols, paraffin-embedded, sectioned, mounted on glass slides, and stained with hematoxylin and eosin to confirm the success of the model.1.2 Immunohistochemical staining of DDR2Immunohistochemical staining was performed to investigate the expression of DDR2 in the cartilage and synovium of the mouse arthritis model. 2. Construction of eukaryotic expression vectors(1) Total RNA was extracted from NIH3T3 cells. RT-PCR was performed to amplify the full-length DDR2 gene and truncated DDR2. PCR products were cloned into pMD18T vector and sequenced. Afterwards, the PCR products were subcloned into pcDNA3.1(+) to construct the eukaryotic vectors: pcDNA3.1-DDR2-FLAG pcDNA3.1-ttDDR2-FLAG. The eukaryotic vectors were verified by enzyme digestion; (2) Using cDNA from NIH3T3 cells and human B cells, PCR was performed to construct the fusion gene of human IgGl Fc fragment and DDR2. The fusion gene was subcloned into pMkit-Neo vector; (3) Using mutant primers, mutated DDR2 at 608aa and 471aa was amplified by PCR and subcloned into pcDNA3.1; (4) Lipofectamine 2000 was used to transiently transfect the COS-7 cells with these eukaryotic expression vectors, and Western Blot was used to detect the expression of the introduced gene; (5) Immunoprecipitation and Western Blot were used to detect the expression and autophosphorylation of FcDDR2. 3 Regulation of MMP-13 by DDR2 3.1 Effect of DDR2 on the expression of MMP-13(1) Type II collagen was added into the media of MC3T3-E1 cells.Immunoprecipitation and Western Blot was used to detect the phosphorylation of DDR2; Western Blot was also used to detect the expression of MMP-13. (2) MC3T3-E1 cells were transiently transfected with DDR2 and FcDDR2 and control empty vector. MMP-13 was detected by Western Blot. (3) MC3T3-E1 was transiently transfected with DDR2, ttDDR2 or mDDR2, and collagen was added before Western Blot for MMP-13. 3.2 Effect of DDR2 on the activity of MMP-13(1)MC3T3-E1 was transiently transfected with DDR2 or FcDDR2. Conditioned medium was collected for the detection. (2)MC3T3-E1 was transfected with DDR2, ttDDR2 or mDDR2 and treated with collagen. Conditioned medium was collected for the detection. (3)Samples were loaded onto the SDS-PAGE electrophoresis, and gelatin zymography was performed. 4 Effects of DDR2 on the mobility and invasiveness of the cells(1) MC3T3-E1 cells were transiently transfected with DDR2 or FcDDR2. Gradient serum concentrations were applied to test the mobility of the cells using transwell chamber assay. (2)Transwell chambers precoated with collagen gel were used to examine the invasiveness of the different transfected cells. (3)Broad spectrum MMP inhibitor GM6001 was added to the culture media of DDR2 transfected cells to investigate its effect on cell invasiveness caused of DDR2. 5. Possible mechanisms of MMP-13 regulated by DDR25.1 Promoter region amplification and reporter gene vectors constructionGenomic DNA from MC3T3-E1 was used as template to amplify the -984~+125s -665~+125^ -189~+125^ -62-H25 fragments of MMP-13 promoter and cloned into the pGL3-basic vector (named as promoter K promoter2> promoter3 and promoter4 respectively) o5.2 Reporter gene assay(1) DDR2, FcDDR2, ttDDR2 or mDDR2 were co-transfected into HeLa cells with promoter 1 by Fugene 6, and dual luciferase reporter assay was performed to detect the reporter fluorescence directed by MMP-13 promoter. (2) FcDDR2 was co-transfected into HeLa cells with promoter 1 -. promoter2>promoter3 or promoter4 to examine the regulatory effects of FcDDR2 on different forms of promoter.5.3 Bioinformatic analysisMinimal promoter region that could be regulated by DDR2 was analyzed using the Transfac database to identify the potential transcription factors involved. The websites are as follows:http://transfac.gbf.de/TRANSFAC http://www.gene-regulation.com5.4 Identification of signaling pathway(1)MC3T3-E1 cells were transiently transfected with DDR2. (2) IP and Western Blot was used to detect the phosphorylated DDR2 and ERK under the condition of the collagen. (3)Effect of MAPK inhibitor PD98059 on promoter activity regulated by DDR2 was examined by reporter gene assay.Results1. Establishment of mouse RA model and detection of DDR2 expressionHE staining suggested that chicken collagen II induced mild arthritis (score 1.0 + 0.9). Histological changes were mildly swelling soft tissue and mild inflammatory infiltration. mBSA+IL-1 3 (9.67 + 2.9) induced severe inflammation. Histology of the joint tissue included synovial membrane edema and hyperplasia, inflammatory infiltration, exudation in the articular cavity, and subcartilage bone resorption. TNF- a group were also mild in inflammation response, showing mild inflammatory infiltration (0.8 + 0.75); IL-1J3 was shown to be potent in inducing arthritis, causing similar inflammation response with mBSA+IL-1 P (4.28 ±2.83).Immunohistochemical staining showed that normal synovial cells and cartilage cells expressed low level of DDR2; Hyperplasic synovial membrane and cartilage cells in mBSA+IL-1 P and IL-lp expressed a significantly higher level of DDR2. Collagen and TNF-a injection cause only a weak increase in DDR2 expression. 2. Construction of eukaryotic expression vectorsSequencing suggested that the PCR products of DDR2, ttDDR2 andFcDDR2 were properly encoded. Nucleotides corresponding to 608 and 471 amino acid were mutated successfully in mDDR2. pcDNA3.1-DDR2-Fl^XK pcDNA3.1-ttDDR2-FlJ\X^ pcDNA3.1-mDDR2-FLAG> pMkit-FcDDR2-FLAG were verified by enzyme digestion.While transfected into the COS-7 cells, all the eukaryotic expression vector could produce fusion proteins recognized by Western Blot with anti-FLAG antibody, suggesting that all of them could be transcribed and expressed by the cell. IP and Western Blot suggested that FcDDR2 could be folded into properly tertiary structure, and the intracellular domain of DDR2 could be autophosphorylated.3. Expression regulation of MMP-13 by DDR2Western Blot suggested that collagen could induce the phosphorylation of DDR2 and increased expression of MMP-13 in dependent of collagen dosage. Transfecting cells with DDR2 or FcDDR2 caused overexpression of MMP-13. Presence of collagen induced MMP-13 increase in cells transfected with DDR2 but not in the cells with ttDDR2 and mDDR2.Gelatin zymography assay suggested that introduction of DDR2 and FcDDR2 upregulated the activity of MMP-13 and the effect of FcDDR2 was stronger. When treated with collagen, cells transfected with DDR2 showed stronger MMP-13 activity, while ttDDR2 and mDDR2 transfectants failed to respond to collagen stimulation a4. Effects of DDR2 on cell mobility and invasivenessUsing transwell chambers, we found that DDR2 and FcDDR2 could enhance the mobility and invasiveness of the cells. Broad spectrum MMP inhibitor GM6001 could partially reverse the effect of DDR2, suggesting that regulation of MMP expression by DDR2 was involved in this procedure.5. Possible regulatory mechanisms of DDR2 on MMP-135.1 Amplification of MMP-13 promoter, reporter gene vector construction and reporter gene assay(l)Fragments corresponding to MMP-13 promoter -984~+125, -665-+125, -189—1-125 and -62- +125 sequences were successfully amplified byPCR and confirmed by sequencing. Then they were subcloned into pGL3-Basic and verified by enzyme digestion. (2)Reporter gene assay suggested that the activity of MMP-13 promoter -984-H-125 was enhanced by DDR2 and FcDDR2. FcDDR2 had a stronger effect. ttDDR2 and mDDR2 did not show any influence on the promoter activity. (3)Co-transfection of FcDDR2 and individual promoter sequences showed that FcDDR2 enhanced transcription of -984—(-125, -665—(-125 and -189-+-125 promoters, but had a dramatically weaker effect on -62—f-125 promoter. This result suggested that -189 promoter is maybe the minimal promoter region that is regulated by DDR2.5.2 Bioinformatic analysis of the promoter sequenceAnalyzing the -189~-62 sequence of MMP-13 promoter revealed 3 potential Spl binding sites, indicating that Spl might be a major transcription factor activated by DDR2.5.3 Downstream signaling pathway of DDR2(1) Western Blot showed that collagen treatment of cell transfected with DDR2 induced stronger ERK phosphorylation than its empty vector control counterpart, suggesting that ERK might be the downstream target of DDR2. (2) Using reporter gene assay, we found that MAPK inhibitor PD98059 could block the effect of FcDDR2 on the MMP-13 promoter activity, giving further evidence for the hypothesis that DDR2 regulated MMP-13 expression by MAPK/ERK pathway.Conclusions1. In this study, we successfully established mouse RA model by injection of mBSA+IL-lp or IL-lp\ Pathological features of this model are: synovial membrane edema, synovial cell hyperplasia, inflammatory cell infiltration, and bone resorption. Immunohistochemical staining found that DDR2 is upregulated in hyperplastic synovial cells and cartilage cells, suggesting that DDR2 is involved in RA pathogenesis.2. Binding of collagen to DDR2 increased the phosphorylation level of...