ADAMTS-9 in Mouse Cartilage Has Aggrecanase Activity That Is Distinct from ADAMTS-4 and ADAMTS-5.

A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 are the principal aggrecanases in mice and humans; however, mice lacking the catalytic domain of both enzymes (TS-4/5∆cat) have no skeletal phenotype, suggesting there is an alternative aggrecanase for modulating normal growth and development in these mice. We previously identified aggrecanase activity that (a) cleaved at E↓G rather than E↓A bonds in the aggrecan core protein, and (b) was upregulated by retinoic acid but not IL-1α. The present study aimed to identify the alternative aggrecanase. Femoral head cartilage explants from TS-4/5∆cat mice were stimulated with IL-1α or retinoic acid and total RNA was analysed by microarray. In addition to ADAMTS-5 and matrix metalloproteinase (MMP)-13, which are not candidates for the novel aggrecanase, the microarray analyses identified MMP-11, calpain-5 and ADAMTS-9 as candidate aggrecanases upregulated by retinoic acid. When calpain-5 and MMP-11 failed to meet subsequent criteria, ADAMTS-9 emerged as the most likely candidate for the novel aggrecanase. Immunohistochemistry revealed ADAMTS-9 expression throughout the mouse growth plate and strong expression, particularly in the proliferative zone of the TS-4/5-∆cat mice. In conclusion, ADAMTS-9 has a novel specificity for aggrecan, cleaving primarily at E↓G rather than E↓A bonds in mouse cartilage. ADAMTS-9 might have more important roles in normal skeletal development compared with ADAMTS-4 and ADAMTS-5, which have key roles in joint pathology.

Wide bandwidth nanomechanical assessment of murine cartilage reveals protection of aggrecan knock-in mice from joint-overuse.

Aggrecan loss in human and animal cartilage precedes clinical symptoms of osteoarthritis, suggesting that aggrecan loss is an initiating step in cartilage pathology. Characterizing early stages of cartilage degeneration caused by aging and overuse is important in the search for therapeutics. In this study, atomic force microscopy (AFM)-based force-displacement micromechanics, AFM-based wide bandwidth nanomechanics (nanodynamic), and histologic assessments were used to study changes in distal femur cartilage of wildtype mice and mice in which the aggrecan interglobular domain was mutated to make the cartilage aggrecanase-resistant. Half the animals were subjected to voluntary running-wheel exercise of varying durations. Wildtype mice at three selected age groups were compared. While histological assessment was not sensitive enough to capture any statistically significant changes in these relatively young populations of mice, micromechanical assessment captured changes in the quasi-equilibrium structural-elastic behavior of the cartilage matrix. Additionally, nanodynamic assessment captured changes in the fluid-solid poroelastic behavior and the high frequency stiffness of the tissue, which proved to be the most sensitive assessment of changes in cartilage associated with aging and joint-overuse. In wildtype mice, aging caused softening of the cartilage tissue at the microscale and at the nanoscale. Softening with increased animal age was found at high loading rates (frequencies), suggesting an increase in hydraulic permeability, with implications for loss of function pertinent to running and impact-injury. Running caused substantial changes in fluid-solid interactions in aggrecanase-resistant mice, suggestive of tissue degradation. However, higher nanodynamic stiffness magnitude and lower hydraulic permeability was observed in running aggrecanase-resistant mice compared to running wildtype controls at the same age, thereby suggesting protection from joint-overuse.

A Disintegrin and Metalloproteinase with Thrombospondin Motifs-5 (ADAMTS-5) Forms Catalytically Active Oligomers.

The metalloproteinase ADAMTS-5 (A disintegrin and metalloproteinase with thrombospondin motifs) degrades aggrecan, a proteoglycan essential for cartilage structure and function. ADAMTS-5 is the major aggrecanase in mouse cartilage, and is also likely to be the major aggrecanase in humans. ADAMTS-5 is a multidomain enzyme, but the function of the C-terminal ancillary domains is poorly understood. We show that mutant ADAMTS-5 lacking the catalytic domain, but with a full suite of ancillary domains inhibits wild type ADAMTS activity, in vitro and in vivo, in a dominant-negative manner. The data suggest that mutant ADAMTS-5 binds to wild type ADAMTS-5; thus we tested the hypothesis that ADAMTS-5 associates to form oligomers. Co-elution, competition, and in situ PLA experiments using full-length and truncated recombinant ADAMTS-5 confirmed that ADAMTS-5 molecules interact, and showed that the catalytic and disintegrin-like domains support these intermolecular interactions. Cross-linking experiments revealed that recombinant ADAMTS-5 formed large, reduction-sensitive oligomers with a nominal molecular mass of ∼ 400 kDa. The oligomers were unimolecular and proteolytically active. ADAMTS-5 truncates comprising the disintegrin and/or catalytic domains were able to competitively block full-length ADAMTS-5-mediated aggrecan cleavage, measured by production of the G1-EGE(373) neoepitope. These results show that ADAMTS-5 oligomerization is required for full aggrecanase activity, and they provide evidence that blocking oligomerization inhibits ADAMTS-5 activity. The data identify the surface provided by the catalytic and disintegrin-like domains of ADAMTS-5 as a legitimate target for the design of aggrecanase inhibitors.

High-bandwidth AFM-based rheology is a sensitive indicator of early cartilage aggrecan degradation relevant to mouse models of osteoarthritis.

Murine models of osteoarthritis (OA) and post-traumatic OA have been widely used to study the development and progression of these diseases using genetically engineered mouse strains along with surgical or biochemical interventions. However, due to the small size and thickness of murine cartilage, the relationship between mechanical properties, molecular structure and cartilage composition has not been well studied. We adapted a recently developed AFM-based nano-rheology system to probe the dynamic nanomechanical properties of murine cartilage over a wide frequency range of 1 Hz to 10 kHz, and studied the role of glycosaminoglycan (GAG) on the dynamic modulus and poroelastic properties of murine femoral cartilage. We showed that poroelastic properties, highlighting fluid-solid interactions, are more sensitive indicators of loss of mechanical function compared to equilibrium properties in which fluid flow is negligible. These fluid-flow-dependent properties include the hydraulic permeability (an indicator of the resistance of matrix to fluid flow) and the high frequency modulus, obtained at high rates of loading relevant to jumping and impact injury in vivo. Utilizing a fibril-reinforced finite element model, we estimated the poroelastic properties of mouse cartilage over a wide range of loading rates for the first time, and show that the hydraulic permeability increased by a factor ~16 from knormal=7.80×10(-16)±1.3×10(-16) m(4)/N s to kGAG-depleted=1.26×10(-14)±6.73×10(-15) m(4)/N s after GAG depletion. The high-frequency modulus, which is related to fluid pressurization and the fibrillar network, decreased significantly after GAG depletion. In contrast, the equilibrium modulus, which is fluid-flow independent, did not show a statistically significant alteration following GAG depletion.

Key role of suppressor of cytokine signaling 3 in regulating gp130 cytokine-induced signaling and limiting chondrocyte responses during murine inflammatory arthritis.

OBJECTIVE: To examine the impact of the gp130 cytokine family on murine articular cartilage and to explore a potential regulatory role of suppressor of cytokine signaling 3 (SOCS-3) in murine chondrocytes. METHODS: In wild-type (WT) mouse chondrocytes, baseline receptor expression levels and gp130 cytokine-induced JAK/STAT signaling were determined by flow cytometry, and expression of SOCS-3 was assessed by quantitative polymerase chain reaction. The role of endogenous SOCS-3 was examined in cartilage explants and chondrocytes from mice with conditional deletion of Socs3 driven by the Col2a1 promoter in vitro (Socs3(Δ/Δcol2) ) and from mice during CD4+ T cell-dependent inflammatory monarthritis. Bone erosions in the murine joints were analyzed by micro-computed tomography. RESULTS: On chondrocytes from WT mice, gp130 and the oncostatin M (OSM) receptor were strongly expressed, whereas the transmembrane interleukin-6 (IL-6) receptor was expressed at much lower levels. Compared to other gp130 cytokines, OSM was the most potent activator of the JAK/STAT pathway and of SOCS-3 induction. Treatment of Socs3(Δ/Δcol2) mouse cartilage explants and chondrocytes with gp130 cytokines prolonged JAK/STAT signaling, enhanced cartilage degradation, increased the expression of Adamts4, Adamts5, and RANKL, and elevated the production of IL-6, granulocyte colony-stimulating factor, CXCL1, and CCL2. Socs3(Δ/Δcol2) mice developed exacerbated inflammation and joint damage in response to gp130 cytokine injections, and these histopathologic features were also observed in mice with inflammatory monarthritis. CONCLUSION: The results of this study highlight a key role for SOCS-3 in regulating chondrocyte responses during inflammatory arthritis. Within the gp130 cytokine family, OSM is a potent stimulus of chondrocyte responses, while IL-6 probably signals via trans-signaling. The gp130 cytokine-driven production of RANKL in chondrocytes may link chondrocyte activation and bone remodeling during inflammatory arthritis. Thus, these findings suggest that the inhibition of OSM might reduce the development and severity of structural joint damage during inflammatory arthritis.