The Role And Molecular Mechanism Of ADAMTS-7in The Pathogenesis Of Inflammatory Arthritis In Mice

Rheumatoid arthritis (RA) is a chronic systemic inflammatory diseasecharacterized by inflammation of multiple joints and destruction of cartilage and bone.It is a common disorder and occurs in0.5-1%of the adult population in the world,mostly developed in women. Although the cause of RA is unknown, proteinases andcytokines produced in play an important role in the destruction of articular cartilageand subchondral bones. The ADAMTS (a disintegrin and metalloproteinase withthrombospondin motifs) proteases are a distinct group of zinc metalloproteinasescomprising19members, which are non-membrane-bound enzymes that interact withcomponents of the extracellular matrix (ECM) including procollagen, hyalectans andcartilage oligomeric matrix protein to cause their degradation. The ADAMTS familycan be subdivided into four classes based on their structural and functional similaritiessuch as the proteoglycanases, the procollagen-n-peptidases, von Willebrand cleavingfactor (ADAMTS-13) and the other ADAMTS proteases (including ADAMTS-6,-7,-10,-12and-16to-19). These enzymes plays an important role in the turnover ofextracellular matrix proteins in various tissues and their altered regulation has beenimplicated in a number of inflammatory pathological conditions such as cancer,rheumatoid arthritis and atherosclerosis. ADAMTS-7shared a similar domainorganization and structure with ADAMTS-12and formed a subgroup within theADAMTS family. ADAMTS-7is expressed in bone, cartilage, synovium, tendon andligament. ADAMTS-7did not cleave aggrecan or versican and directly associatedwith degrade COMP which have been observed in RA patients. Further investigationdemonstrated that four C-terminal thrombospondin repeats of ADAMTS-7wererequired for interaction with COMP. Although the physiological function ofADAMTS-7had been reported that ADAMTS-7as an important target of PTHrPsignaling and inhibits chondrocyte differentiation and endochondral bone formation.The expression of ADAMTS-7was increased in synovium tissue from RA and OA patients. In surgically-induced OA model, the overexpression of ADAMTS-7accelerates the progression of osteoarthritis. But which role of ADAMTS-7participated in the pathogenesis of rhematoid arthritis is still not well understood.To further characterize ADAMTS-7in CIA mice, we established the CIA model inDBA/1J mice and investigated the alteration of ADAMTS-7expression during theprogression of disease by the immunohistochemistry. The results demonstrated thatthe expression level of ADAMTS-7in collagen induced atthritis significantlyincreased with the deepening of inflammation. Therefore, ADAMTS-7was involvedin the CIA development.To further investigate the effect of ADAMTS-7on CIA incidence and severity,collagen induced arthritis animal model was established in ADAMTS-7transgenic(TG) and wild type littermates mice. The results indicated that arthritis onset wasaccelerated for4weeks in TG mice, while arthritis in wild type mice appeared on5weeks after first immunization and second booster injection. On weeks10, theincidence of TG mice was89%, in contrast, only33%wild type mice developedarthritis. Severe joints inflammation were observed in TG mice by visual examinationincluding marked swelling and erythema of the forepaws and hindpaws encompassingthe wrist and ankle and extended distally through the limb and digits14weeks aftercollagen induction. Only less inflammation were observed in wild type mice. Theresults of radiographic examination and three-dimensional micro-CT imagingdemonstrated that significant bone erosions and destruction in the joints wereobserved in TG group as compared to control after collagen induction. To determinewhether the clinical arthritis severity was associated with histological changes, wecompared paw joint sections between wild type and TG mice after collagen induction.The results demonstrated that TG mice mice had a statistically significant increase insynovitis (P <0.001), pannus (P <0.001) and erosion score (P <0.001), asignificantly more loss of safranin O staining in paraffin sections of joints (P <0.01),and more TRAP-positive osteoclast-like cells presented on focal bone erosions anddeveloped severe bone erosions, compared with wild type mice. These findingssuggest that overexpression of ADAMTS-7accelerates and increased the incidence and severity of collagen-induced arthritis in mice.The alteration of metalloproteinases and degradation of extracellular matrix playan important role in the progression of inflammatory arthritis. To determine whetherthe severity of CIA in TG mice is associated with the expression of aggrecan, COMPand MMP-13, we performed real-time PCR, ELISA and immunohistochemistrymethods to detect the expression of these genes. The results revealed that theoverexpression of ADAMTS-7in CIA mice upregulated MMP-13level, enhanceddegradation of COMP and aggrecan and accerlerated progression of collagen-inducedarthritis.To investigate the effect of ADAMTS-7overexpression on inflammatorycytokines production, we detected the expression of TNF-α and IL-17using real-timePCR, ELISA and flow cytometry (FACS). These finding suggest that ADAMTS-7overexpression enhances TNF-α and IL-17expression in arthritic TG mice aftercollagen induction. To gain further insight into the possible mechanism ofADAMTS-7in collagen-induced arthritis, we transfected ADAMTS-7expressingplasmid and control plasmid into Raw264.7cell lines and then treated with TNF-αand detected TNF-α-induced signaling pathways such as NF-κB and p38activationusing western blotting. Our results demonstrated that overexpression of ADAMTS-7accelerated activation of TNF-α-induced p38MAPK pathways and TNF-α-inducedphosphorylation of IκB and its degradation.Taken together, these findings suggest that ADAMTS-7is induced in the courseof collagen-induced arthritis development, and ADAMTS-7overexpressionaccelerates the onset and increases the severity of CIA in TG mice. Arthritic TG micedisplayed significantly higher clinical and histological scores. Overexpression ofADAMTS-7enhances TNF-α-induced NF-κB activation and p38MAPKphosphorylation. We concluded that TNF-α and IL-17are two mediators involved indestruction of cartilage and bone in TG mice after collagen induction. In summary,our results demonstrate that the importance of ADAMTS-7in modulating rheumatoidarthritis, and ADAMTS-7will become a promising target for prevention andtreatment of bone destruction in inflammatory arthritis. The role of ADAMTS-18in the development of cartilage and boneADAMTS is a recently described family of zinc-dependent proteases which playimportant roles in a variety of normal and pathological conditions, including bonedevelopment and arthritis. ADAMTS-18belongs to the four subgroup of ADAMTSfamily members, its structure is similar to ADAMTS-16. ADAMTS-18has a lowerability to degrade aggrecan in vitro, and its effect needs a high concentration ofADAMTS-18. At present, the role of ADAMTS-18in the development of cartilageand bone is not clear. To determine the role of ADAMTS-18in the development ofcartilage and bone, immunohistochemistry method was performed to evaluate thetemporal and spatial pattern of ADAMTS-18expression during skeletal development.The results revealed that stronger expression of ADAMTS-18in hypertrophychondrocytes of growth plate in postnatal mice. Growth retardation was evident by2weeks of age and still present at12weeks. To examine the effect of ADAMTS-18deficiency on growth plate, the paraffin sections of proximal tibia were prepared inADAMTS-18-deficient mice and wild-type mice. The results demonstrated thatADAMTS-18deficiency alters the architecture of growth plate in mice, and the cellnumber of prehypertrophy chondrocytes in growth plate was decreased and thehypertrophic zone thickness was also reduced. The results of micro-CT showedADAMTS-18deficiency alters the volume and thickness of trabecular bone. Tofurther investigate the possible mechanism of ADAMTS-18in regulation of skeletaldevelopment, real-time PCR was performed to detect the differential gene expressionin cartilage from ADAMTS-18-deficient mice and wild-type mice. The resultsshowed that the gene expression of ColⅡ, CoIX, MMP13, Sox5and Runx2weresignificantly decreased in ADAMTS-18-deficient mice compared with wild-typemice.In conclusion, our results demonstrate that ADAMTS-18was highly expressed inhypertrophy chondrocytes in growth plate of mice, and growth retardation wasevident in postnatal mice from2weeks to adult mice age. ADAMTS-18contributessignificantly in cartilage development, especially plays an important role in differentiation of chondrocytes into hypertrophy chondrocytes. The function ofADAMTS-18in skeletal development is possibly associated with regulation of Runx2transcription factors.