ADAMTS-7 And ADAMTS-12: Novel Mediators Of Chondrogenesis

ADAMTS-7 and ADAMTS-12,metalloproteinases that belongs to ADAMTS family, are important for the degradation of cartilage extracellular matrix proteins and their levels are significantly levated in arthritis.The ADAMTS(a disintegrin and metalloproteinase with thrombospondin type 1 motifs) family consists of secreted zinc metalloproteinases with a precisely ordered modular organization that includes at least one thrombospondin typeâ… repeat.Important functions have been established for several members of the ADAMTS family.ADAMTS-1,ADAMTS-4,ADAMTS-5, ADAMTS-8,ADAMTS-9,ADAMTS-16 and ADAMTS-18 degrade aggrecan and ADAMTS-5 plays a primary role in aggrecan loss in murine arthritis.ADAMTS-7 and ADAMTS-12 share the same domain organization and form a subgroup with unique properties within ADAMTS family.Our previously reports demonstrate that ADAMTS-7 and ADAMTS-12 directly associate with and degrade cartilage oligomeric matrix protein,COMP),a prominent noncollagenous component of cartilage.In addition,Alpha-2-Macroglobulin inhibits their degradation of COMP. Recent report revealed that ADAMTS-12 also degraded aggrecan.In this study we report that ADAMTS-7 and ADAMTS-12 are strongly upregulated during chondrogenesis and demonstrates the temporal and spatial expression pattern during skeletal development.The current study focused on the roles of ADAMTS-7 and ADAMTS-12 in chondrogenesis as well as the molecular mechanism involved. ADAMTS-7 and ADAMTS-12 proteins were highly induced in the course of chondrogenesis.Real-time PCR for measurements of ADAMTS-7 and ADAMTS-12 showed that the level of ADAMTS-7 and ADAMTS-12 mRNAs were relatively low until day 5,and at day 7 it tripled and thereafter remained at high levels during the late differential stage.In this study we found ADAMTS-7 and ADAMTS-12 inhibited chondrocyte differentiation and endochondral bone formation.ADAMTS-7 potently inhibits chondrocyte differentiation and endochondral bone formation,and this inhibition depends on the proteolytic activity of ADAMTS-7.The cysteine-rich domain of ADAMTS-7 is required for its interaction with extracellular matrix and cell surface localization,and the C-terminal four thrombospondin motifs is necessary for its full proteolytic activity and inhibition of chondrocyte differentiation. ADAMTS-7 appears to be a potent negative regulator of chondrocyte differentiation and endochondral bone growth,and its inhibitory activities strictly depend on its enzymatic activities,since its point mutant lacking enzymatic activity completely lost these inhibitions.ADAMTS-7 is composed of multiple functional domains,including a prodomain,a catalytic domain,a disintegrin domain,a thrombospondin motif,a cysteine-rich domain,a spacer-1 domain,three thrombospondin motifs,a spacer-2 domain,and a C-terminal four thrombospondin motifs.In addition to its C-terminal four thrombospondin motifs known to bind to substrates,including COMP,the role of individual domain in regulating the biochemical properties of ADAMTS-7 remains unknown.To address this issue,we generated series of domain deletion mutants of ADAMTS-7 and found that The CRD is required for ADAMTS-7 binding to the cell surface and ECM in chondrocytes.Interestingly,this CRD-dependent localization appears to be cell type-specific,since ADAMTS-7 and its deletion mutants are predominately localized in the cytoplasm of Cos-7 cells.In addition,the enzymatic activities of ADAMTS-7 are also precisely regulated by its non-catalytic domains, specially its substrate-capturing C-terminal four thrombospondin motifs and an inhibitory spacer-2 domain.ADAMTS-7 and ADAMTS-12 are important targets of PTHrP signaling,since(1) PTHrP induces ADAMTS-7 and ADAMTS-12,(2) ADAMTS-7 and ADAMTS-12 are hardly detectable in PTHrP-/- growth plate chondrocytes,and(3) knocking down ADAMTS-7/ADAMTS-12 expression or blocking ADAMTS-7/ADAMTS-12 activities almost abolishes PTHrP-mediated inhibition of chondrocyte hypertrophy and endochondral bone growth.ADAMTS-7 associates with Granulin-epithelin precursor(GEP),a novel chondrogenic growth factor.Multiple signaling pathways are involved in endochondral ossification in epiphyseal growth plate.Our studies demonstrating that 1) overexpressing ADAMTS-7 and ADAMTS-12 enhanced the expression of PTHrP whereas inhibited IHH,2) PTHrP induced ADAMTS-7 and ADAMTS-12 expression in the course of chondrogenesis in vitro,and 3) ADAMTS-7 and ADAMTS-12 expression strictly depend on PTHrP in the growth plate chondrocytes in mice.These findings suggest that there exists a positive feedback loop between ADAMTS-7,ADAMTS-12 and PTHrP signaling in the course of chondrogenesis.In addition,ADAMTS-7 and ADAMTS-12 appear to be a crucial downstream molecule of PTHrP based on the facts that 1) repression of ADAMTS-7/ADAMTS-12 via siRNA approach or blocking ADAMTS-7/ ADAMTS-12 activities using its blocking antibodies almost abolished PTHrP-mediated inhibition of Col X expression,but reexpression of ADAMTS-7/ ADAMTS-12 restored PTHrP action;and 2) ADAMTS-7/ADAMTS-12 blocking antibody totally neutralized the inhibition of chondrocyte hypertrophy,mineralization, and bone growth by PTHrP.Furthermore,similar to PTHrP that is known to stimulate chondrocyte proliferation,ADAMTS-7 was also found to increase chondrocyte proliferation and its stimulation on cell proliferation largely depends on its substrate-binding C-terminal four thrombospondin motifs.In addition,ADAMTS-7 acts as a new GEP-convertase and neutralizes GEP-stimulated endochondral bone formation.Collectively,these findings demonstrate that ADAMTS-7,an important downstream molecule of PTHrP signaling, negatively regulates endochondral bone formation via associating with and inactivating GEP chondrogenic growth factor.GEP is a growth factor implicated in tissue regeneration,tumorigenesis,and inflammation.GEP was previously shown to associate with and be processed by elastase.Elastase digests GEP exclusively in the intergranulin linkers,resulting in the generation of granulin peptides;whereas the secretory leukocyte protease inhibitor(SLPI) blocks this proteolysis either by directly binding to elastase or by sequestering granulin peptides from the enzyme.Recent report reveled that proteinase 3 associated with and processed GEP,and proteinase 3 and elastase enhance neutrophil-dependent inflammation by eliminating the anti-inflammatory activity of GEP.Here we present evidences showing that GEP associates with ADAMTS-7 metalloproteinase in chondrocytes,and ADAMTS-7 is able to convert GEP into its processed fragments;in addition,ADAMTS-7-inhibits chondrocyte differentiation and endochondral bone formation probably via inactivating chondrogenic activity of GEP.Our previous report showing that 1) ADAMTS-7 binds to and degrades COMP,and 2) COMP interacts with GEP and potentiates GEP-stimulated chondrocyte functions,together with present study revealing the interaction between ADAMTS-7 and GEP,indicate that ADAMTS-7, GEP and COMP form a protein-protein interaction network in regulating chondrocyte functions.It remains to determine how the interaction network among ADAMTS-7 metallproteinase,GEP growth factor and COMP extracellular matrix molecule acts in concert in regulating chondrocyte differentiation and endochondral ossification.The study in this paper provides novel insights into the role of ADAMTS-7 and ADAMTS-12,novel mediators in PTHrP pathway,in regulating chondrocyte differentiation and endochondral bone formation,and sheds light on the molecular mechanism by which ADAMTS-7 adversely regulates chondrogenesis,i.e. ADAMTS-7 metalloproteinase associates with GEP chondrogenic grow factor,and eliminates GEP-stimulated chondrogenesis via conversion of GEP into its processed fragments.In addition,this study also provides us with potential molecule targets for treatment of cartilage disorders and arthritic conditions.