β‐catenin regulates parathyroid hormone/parathyroid hormone–related protein receptor signals and chondrocyte hypertrophy through binding to the intracellular C‐terminal region of the receptor

Objective

To investigate the underlying mechanisms of action and functional relevance of β‐catenin in chondrocytes, by examining the role of β‐catenin as a novel protein that interacts with the intracellular C‐terminal portion of the parathyroid hormone (PTH)/PTH‐related protein (PTHrP) receptor type 1 (PTHR‐1).

Methods

The β‐catenin–PTHR‐1 binding region was determined with deletion and mutagenesis analyses of the PTHR1 C‐terminus, using a mammalian two‐hybrid assay. Physical interactions between these 2 molecules were examined with an in situ proximity ligation assay and immunostaining. To assess the effects of gain‐ and loss‐of‐function of β‐catenin, transfection experiments were performed to induce overexpression of the constitutively active form of β‐catenin (ca‐β‐catenin) and to block β‐catenin activity with small interfering RNA, in cells cotransfected with either wild‐type PTHR1 or mutant forms (lacking binding to β‐catenin). Activation of the G protein α subunits G_αs and G_αq in the cells was determined by measurement of the intracellular cAMP accumulation and intracellular Ca²⁺ concentration, while activation of canonical Wnt pathways was assessed using a TOPflash reporter assay.

Results

In differentiated chondrocytes, β‐catenin physically interacted and colocalized with the cell membrane–specific region of PTHR‐1 (584–589). Binding of β‐catenin to PTHR‐1 caused suppression of the G_αs/cAMP pathway and enhancement of the G_αq/Ca²⁺ pathway, without affecting the canonical Wnt pathway. Inhibition of Col10a1 messenger RNA (mRNA) expression by PTH was restored by overexpression of ca‐β‐catenin, even after blockade of the canonical Wnt pathway, and Col10a1 mRNA expression was further decreased by knockout of β‐catenin (via the Cre recombinase) in chondrocytes from β‐catenin–floxed mice. Mutagenesis analyses to block the binding of β‐catenin to PTHR1 caused an inhibition of chondrocyte hypertrophy markers.

Conclusion

β‐catenin binds to the PTHR‐1 C‐tail and switches the downstream signaling pathway from G_αs/cAMP to G_αq/Ca²⁺, which is a possible mechanism by which chondrocyte hypertrophy may be regulated through the PTH/PTHrP signal independent of the canonical Wnt pathway.

     

Wnt/β‐catenin signaling is hyperactivated in systemic sclerosis and induces Smad‐dependent fibrotic responses in mesenchymal cells

Objective

Fibrosis in human diseases and animal models is associated with aberrant Wnt/β‐catenin pathway activation. The aim of this study was to characterize the regulation, activity, mechanism of action, and significance of Wnt/β‐catenin signaling in the context of systemic sclerosis (SSc).

Methods

The expression of Wnt signaling pathway components in SSc skin biopsy specimens was analyzed. The regulation of profibrotic responses by canonical Wnt/β‐catenin was examined in explanted human mesenchymal cells. Fibrotic responses were studied using proliferation, migration, and gel contraction assays. The cell fate specification of subcutaneous preadipocytes by canonical Wnt signaling was evaluated.

Results

Analysis of published genome‐wide expression data revealed elevated expression of the Wnt receptor FZD2 and the Wnt target LEF1 and decreased expression of Wnt antagonists DKK2 and WIF1 in skin biopsy specimens from subsets of patients with diffuse cutaneous SSc compared to the other distinct subsets. Immunohistochemical analysis showed increased nuclear β‐catenin expression in these biopsy specimens. In vitro, Wnt‐3a induced β‐catenin activation, stimulated fibroblast proliferation and migration, collagen gel contraction, and myofibroblast differentiation, and enhanced profibrotic gene expression. Genetic and pharmacologic approaches were used to demonstrate that these profibrotic responses involved autocrine transforming growth factor β signaling via Smads. In contrast, in explanted subcutaneous preadipocytes, Wnt‐3a repressed adipogenesis and promoted myofibroblast differentiation.

Conclusion

Canonical Wnt signaling was hyperactivated in SSc skin biopsy specimens. In explanted mesenchymal cells, Wnt‐3a stimulated fibrogenic responses while suppressing adipogenesis. Taken together, these results indicate that Wnts have potent profibrotic effects, and that canonical Wnt signaling plays an important role in the pathogenesis of fibrosis and lipoatrophy in SSc.

     

Smad3 controls β‐1,3‐glucuronosyltransferase 1 expression in rat nucleus pulposus cells: Implications of dysregulated expression in disc disease

Objective

To study the regulation of expression of β‐1,3‐glucuronosyltransferase 1 (GlcAT‐1), an important regulator of glycosaminoglycan (GAG) synthesis, by Smad3 in nucleus pulposus (NP) cells.

Methods

GlcAT‐1 expression was examined in rat NP and anulus fibrosus (AF) cells treated with transforming growth factor β (TGFβ). The effects of Smad signaling and Smad suppression on GlcAT‐1 were examined in rat NP cells. GlcAT‐1 expression was analyzed in the discs of Smad3‐null mice and in degenerated human NP tissue.

Results

TGFβ increased the expression of GlcAT‐1 in rat NP but not rat AF cells. Suppression of GlcAT‐1 promoter activity was evident with dominant‐negative ALK‐5 (DN‐ALK‐5). Cotransfection with Smad3 strongly induced promoter activity independent of TGFβ. Bioinformatics analysis indicated the presence of several Smad binding sites in the promoter; deletion analysis showed that the region between −274 and −123 bp was required for Smad3 response. DN‐Smad3, Smad 3 small interfering RNA, and Smad7 strongly suppressed basal as well as TGFβ‐induced promoter activity. Induction of promoter activity by Smad3 was significantly blocked by DN‐Smad3; Smad7 had a very small effect. Lentiviral transduction of NP cells with short hairpin RNA Smad3 resulted in a decrease in GlcAT‐1 expression and accumulation of GAG. Compared to wild‐type mice, significantly lower expression of GlcAT‐1 was seen in the discs of Smad3‐null mice. Analysis of degenerated human NP tissue specimens showed no positive correlation between GlcAT‐1 and TGFβ expression. Moreover, isolated cells from degenerated human tissue showed a lack of induction of GlcAT‐1 expression following TGFβ treatment, suggesting an altered response.

Conclusion

Our findings demonstrate that in healthy NP cells, the TGFβ–Smad3 axis serves as a regulator of GlcAT‐1 expression. However, an altered responsiveness to TGFβ during disc degeneration may compromise GAG synthesis.

     

In vitro and in vivo efficacy of a recombinant immunotoxin against folate receptor β on the activation and proliferation of rheumatoid arthritis synovial cells

Objective

To investigate the effects of the recombinant immunotoxin dsFv anti‐FRβ‐PE38, which consists of the disulfide‐stabilized Fv fragment (dsFv) of the anti–folate receptor β (anti‐FRβ) antibody and the 38‐kd portion of Pseudomonas exotoxin A (PE38), on the activation and proliferation of cells that function in inflammatory and degradative processes in rheumatoid arthritis (RA) synovial tissue.

Methods

The Ig V_H‐PE38 fusion protein and the Ig V_L protein were produced in Escherichia coli, and then joined with a disulfide bond by engineering cysteine residues in the framework regions of these proteins. The effects of dsFv anti‐FRβ‐PE38 on the activation and proliferation of cells in RA synovial tissue were investigated by immunohistochemistry; the numbers of cells expressing CD68, vascular cell adhesion molecule 1, angiopoietin 1, CD34, proliferating cell nuclear antigen, and interleukin‐6 and the numbers of apoptotic cells were counted in RA synovial tissue engrafted into SCID mice treated or not treated with dsFv anti‐FRβ‐PE38. The effects of dsFv anti‐FRβ‐PE38 on the generation of osteoclasts from RA adherent synovial mononuclear cells in vitro was investigated by counting the number of resorption pits on dentin slices treated or not treated with dsFv anti‐FRβ‐PE38.

Results

Administration of dsFv anti‐FRβ‐PE38 reduced the numbers of macrophages, activated fibroblast‐like cells, endothelial cells, and proliferating cells and increased the numbers of apoptotic cells in RA synovial tissue engrafted into SCID mice. In vitro, the generation of osteoclasts from RA adherent synovial mononuclear cells was largely suppressed by treatment with dsFv anti‐FRβ‐PE38.

Conclusion

Our findings show that dsFv anti‐FRβ‐PE38 immunotoxin would be a promising tool for the treatment of RA synovitis, especially when administered intraarticularly.

     

Interleukin‐6 plays an essential role in hypoxia‐inducible factor 2α–induced experimental osteoarthritic cartilage destruction in mice

Objective

Hypoxia‐inducible factor 2α (HIF‐2α) (encoded by Epas1) causes osteoarthritic (OA) cartilage destruction by regulating the expression of catabolic factor genes. We undertook this study to explore the role of interleukin‐6 (IL‐6) in HIF‐2α–mediated OA cartilage destruction in mice.

Methods

The expression of HIF‐2α, IL‐6, and catabolic factors was determined at the messenger RNA and protein levels in primary culture mouse chondrocytes, human OA cartilage, and mouse experimental OA cartilage. Experimental OA in wild‐type, HIF‐2α–knockdown (Epas1^+/−), and Il6^–/– mice was caused by intraarticular injection of Epas1 adenovirus or destabilization of the medial meniscus. The role of IL‐6 was determined by treating with recombinant IL‐6 protein or by injecting HIF‐2α adenovirus (AdEpas1) intraarticularly in mice with or without IL‐6–neutralizing antibody.

Results

We found that Il6 is a direct target gene of HIF‐2α in articular chondrocytes. Both Epas1 and Il6 were up‐regulated in human and mouse OA cartilage, whereas HIF‐2α knockdown in mice led to inhibition of both Il6 expression and cartilage destruction. Treatment with IL‐6 enhanced Mmp3 and Mmp13 expression; conversely, Il6 knockdown inhibited HIF‐2α–induced up‐regulation of Mmp3 and Mmp13. Injection of IL‐6 protein into mouse knee joints triggered OA cartilage destruction, whereas IL‐6 neutralization led to blocking of HIF‐2α–induced cartilage destruction with concomitant modulation of Mmp3 and Mmp13 expression. Moreover, Il6 knockout resulted in inhibition of AdEpas1‐induced and destabilization of the medial meniscus–induced cartilage destruction as well as inhibition of Mmp3 and Mmp13 expression.

Conclusion

Our findings indicate that IL‐6 acts as a crucial mediator of HIF‐2α–induced experimental OA cartilage destruction in mice via regulation of Mmp3 and Mmp13 levels.

     

Inhibition of interleukin‐1β‐stimulated production of matrix metalloproteinases by hyaluronan via CD44 in human articular cartilage

Objective

To investigate the mechanism of the inhibitory action of hyaluronan (HA) on interleukin‐1β (IL‐1β)‐stimulated production of matrix metalloproteinases (MMPs) in human articular cartilage.

Methods

IL‐1β was added to normal and osteoarthritic (OA) human articular cartilage in explant culture to stimulate MMP production. Articular cartilage was incubated or preincubated with a clinically used form of 800‐kd HA to assess its effect on IL‐1β‐induced MMPs. Levels of secreted MMPs 1, 3, and 13 in conditioned media were detected by immunoblotting; intracellular MMP synthesis in chondrocytes was evaluated by immunofluorescence microscopy. Penetration of HA into cartilage tissue and its binding to CD44 were analyzed by fluorescence microscopy using fluoresceinated HA. Blocking experiments with anti‐CD44 antibody were performed to investigate the mechanism of action of HA.

Results

Treatment and pretreatment with 800‐kd HA at 1 mg/ml resulted in significant suppression of IL‐1β‐stimulated production of MMPs 1, 3, and 13 in normal and OA cartilage explant culture. Fluorescence histocytochemistry revealed that HA penetrated cartilage tissue and localized in the pericellular matrix around chondrocytes. HA‐binding blocking experiments using anti‐CD44 antibody demonstrated that the association of HA with chondrocytes was mediated by CD44. Preincubation with anti‐CD44 antibody, which suppressed IL‐1β‐stimulated MMPs, reversed the inhibitory effect of HA on MMP production that was induced by IL‐1β in normal and OA cartilage.

Conclusion

This study demonstrates that HA effectively inhibits IL‐1β‐stimulated production of MMP‐1, MMP‐3, and MMP‐13, which supports the clinical use of HA in the treatment of OA. The action of HA on IL‐1β may involve direct interaction between HA and CD44 on chondrocytes.

     

Stimulation of superficial zone protein accumulation by hedgehog and Wnt signaling in surface zone bovine articular chondrocytes

Objective

To determine the roles of the hedgehog and Wnt signaling pathways in accumulation of superficial zone protein (SZP) in surface zone articular chondrocytes.

Methods

Explant cultures of disks of surface zone cartilage or isolated chondrocytes from the surface zone of articular cartilage of bovine stifle joints were cultured in serum‐free chemically defined medium. Accumulation of SZP in the culture medium, in response to hedgehog proteins (sonic hedgehog [SHH] and Indian hedgehog [IHH]), Wnt proteins (Wnt‐3a, Wnt‐5a, and Wnt‐11), agonists of the Wnt/β‐catenin pathway (glycogen synthase kinase 3β [GSK‐3β] inhibitors), and antagonists of the Wnt/β‐catenin pathway, was investigated. The interaction between transforming growth factor β1 (TGFβ1) and hedgehog proteins or antagonists of the Wnt/β‐catenin pathway was also investigated.

Results

Hedgehog proteins stimulated SZP accumulation. Activation of the Wnt/β‐catenin pathway by Wnt‐3a and GSK‐3β inhibitors led to inhibition of SZP accumulation; however, Wnt‐5a and Wnt‐11 had no influence on SZP accumulation. Conversely, antagonists of the Wnt/β‐catenin pathway stimulated SZP accumulation. In addition, there were combinatorial effects of TGFβ1 and hedgehog proteins or antagonists of the Wnt/β‐catenin pathway on SZP accumulation.

Conclusion

SHH and IHH signaling has a stimulatory effect on SZP accumulation in surface zone cartilage and isolated articular chondrocytes. These findings provide insight into the regulatory mechanisms of articular cartilage homeostasis and maintenance by morphogens.

     

Endogenous estrogen regulation of inflammatory arthritis and cytokine expression in male mice,predominantly via estrogen receptor α

Objective

A number of experimental observations have associated elevated estrogen levels with amelioration of inflammation. The involvement of estrogen and estrogen receptor (ER) isotypes in the regulation of inflammation in males is not well understood. In this study, we used specific ERα and ERβ agonists in male mice deficient in estrogen because of a deletion of aromatase (aromatase‐knockout [ArKO] mice) to investigate ER isotype utilization in estrogen regulation of inflammation.

Methods

Lipopolysaccharide (LPS)‐induced cytokine expression and antigen‐induced arthritis (AIA) were investigated in male ArKO and WT littermate mice, as well as in response to selective agonists of ERα (16α‐LE2) and ERβ (8β‐VE2). The therapeutic effect of selective ER agonists was also examined in mice with collagen‐induced arthritis (CIA).

Results

Estrogen deficiency in ArKO mice was associated with significant increases in LPS‐induced serum interleukin‐6 (IL‐6), tumor necrosis factor, monocyte chemotactic protein 1, and interferon‐γ levels, which were significantly abrogated by administration of 16α‐LE2, but not 8β‐VE2. In contrast, both 16α‐LE2 and 8β‐VE2 significantly increased LPS‐induced IL‐10 levels. Estrogen deficiency was also associated with significant exacerbation of AIA and antigen‐specific T cell proliferation, which was reversed by administration of either 16α‐LE2 or 8β‐VE2. ArKO mice showed increased antigen‐specific T cell proliferation in response to immunization with type II collagen (CII). Administration of 16α‐LE2, but not 8β‐VE2, significantly reduced the severity of CIA, which was associated with inhibition of anti‐CII–specific IgG.

Conclusion

These data indicate that endogenous estrogen plays an essential inhibitory role in inflammation in male mice and that ERα is the dominant receptor that mediates these effects.

     

Effectiveness of anti–folate receptor β antibody conjugated with truncated Pseudomonas exotoxin in the targeting of rheumatoid arthritis synovial macrophages

Objective

To define the distribution of folate receptor β (FRβ)–expressing cells in various tissues, including rheumatoid arthritis (RA) synovial tissues, and to verify the effects of an immunotoxin composed of an anti‐FRβ monoclonal antibody (mAb) and truncated Pseudomonas exotoxin A (PEA) on apoptosis and tumor necrosis factor α (TNFα) production by adherent synovial mononuclear cells from RA patients.

Methods

Anti‐FRβ mAb were produced by immunizing mice with FRβ‐transfected murine pre–B cells. The distribution of the FRβ antigen was examined by immunohistochemical analysis using anti‐FRβ mAb and macrophage‐specific anti‐CD163 mAb. Anti‐FRβ mAb was chemically crosslinked with truncated PEA. FRβ‐expressing macrophages were produced by the transfection of adenovirus vector containing the FRβ gene. Apoptotic cells were detected by staining with propidium iodide. TNFα was measured by enzyme‐linked immunosorbent assay.

Results

FRβ‐expressing cells were not present in peripheral blood leukocytes and their activated cells. In all of the tissues examined, most FRβ‐expressing cells were CD163+. The immunotoxin significantly induced the apoptosis of FRβ‐transfected macrophages and adherent RA synovial mononuclear cells and inhibited TNFα production by adherent RA synovial mononuclear cells.

Conclusion

We demonstrated the limited distribution of FRβ‐expressing cells in various tissues. The immunotoxin targeting FRβ‐expressing cells will provide a therapeutic tool for rheumatoid synovitis.

     

Resemblance of osteophytes in experimental osteoarthritis to transforming growth factor β–induced osteophytes: Limited role of bone morphogenetic protein in early osteoarthritic osteophyte formation

Objective

Osteoarthritis (OA) is characterized by cartilage damage, synovial fibrosis, and osteophyte formation. Both transforming growth factor β (TGFβ) and bone morphogenetic protein 2 (BMP‐2) can induce the formation of osteophytes during OA, but their specific role in this process is unclear. The purpose of this study was to investigate the respective contributions of TGFβ and BMP‐2 to OA.

Methods

Mouse knee joints injected with adenovirus (Ad‐TGFβ or Ad‐BMP‐2) were compared histologically with knee joints from murine models of OA (joints injected with collagenase and joints from STR/Ort mice with spontaneous OA). To further investigate the role of BMP during osteophyte formation, adenovirus Ad‐Gremlin was injected into knee joints that had previously been injected with Ad‐TGFβ or collagenase.

Results

BMP‐2 induced early osteophytes, which bulged from the growth plates on the femur and grew on top of the patella, whereas TGFβ induced early osteophyte formation on the bone shaft beneath the collateral ligament on the femur as well as on top of the patella. The pattern of osteophyte formation during experimental OA closely resembled that of TGFβ‐induced osteophyte formation, but differed from the pattern induced by BMP‐2. Ad‐Gremlin proved to be able to totally block BMP‐2–induced osteophyte formation. However, blocking BMP activity inhibited neither TGFβ‐induced nor experimental OA–associated osteophyte formation.

Conclusion

Our findings demonstrate that the role of BMP during the onset of TGFβ‐induced and experimental OA–induced osteophyte formation is limited. The latter finding does not rule out a role of BMP during osteophyte maturation.

     

Protein Kinase Cβ Is Required for Lupus Development in Sle Mice

Objective

To evaluate the requirement for protein kinase Cβ (PKCβ) in the development of lupus in mice, and to explore the potential of targeting PKCβ as a therapeutic strategy in lupus.

Methods

Congenic mice bearing the disease loci Sle1 or Sle1 and Sle3, which represent different stages of severity in the development of lupus, were crossed with PKCβ‐deficient mice. The effect of PKCβ deficiency in lupus development was analyzed. In addition, the effects of the PKCβ‐specific inhibitor enzastaurin on the survival of B cells from mice with lupus and human 9G4‐positive B cells as well as the in vivo effect of enzastaurin treatment on the development of lupus in Sle mice were investigated.

Results

In Sle mice, PKCβ deficiency abrogated lupus‐associated phenotypes, including high autoantibody levels, proteinuria, and histologic features of lupus nephritis. Significant decreases in spleen size and in the peritoneal B‐1 cell population, reduced numbers of activated CD4 T cells, and normalized CD4:CD8 ratios were observed. PKCβ deficiency induced an anergic B cell phenotype and preferentially inhibited autoreactive plasma cells and autoantibodies in mice with lupus. Inhibition of PKCβ enhanced apoptosis of both B cells from Sle mice and human autoreactive B cells (9G4 positive). Treatment of Sle mice with the PKCβ‐specific inhibitor enzastaurin prevented the development of lupus.

Conclusion

This study identifies PKCβ as a central mediator of lupus pathogenesis, suggesting that PKCβ represents a promising therapeutic target for the treatment of systemic lupus erythematosus. Moreover, the results indicate the feasibility of using a PKCβ inhibitor for the treatment of lupus.

     

Transforming growth factor β–transduced mesenchymal stem cells ameliorate experimental autoimmune arthritis through reciprocal regulation of Treg/Th17 cells and osteoclastogenesis

Objective

Bone marrow–derived mesenchymal stem cells (MSCs) can prevent various autoimmune diseases. We examined the therapeutic potential of transforming growth factor β (TGFβ)–transduced MSCs in experimental autoimmune arthritis, using an accepted animal model of collagen‐induced arthritis (CIA).

Methods

DBA/1J mice with CIA were treated with syngeneic TGFβ‐induced MSCs, whereas control mice received either vehicle or MSCs alone. Arthritis severity was assessed by clinical and histologic scoring. TGFβ‐transduced MSCs were tested for their immunosuppressive ability and differential regulation in mice with CIA. T cell responses to type II collagen were evaluated by determining proliferative capacity and cytokine levels. The effects of TGFβ‐transduced MSCs on osteoclast formation were analyzed in vitro and in vivo.

Results

Systemic infusion of syngeneic TGFβ‐transduced MSCs prevented arthritis development and reduced bone erosion and cartilage destruction. Treatment with TGFβ‐transduced MSCs potently suppressed type II collagen–specific T cell proliferation and down‐regulated proinflammatory cytokine production. These therapeutic effects were associated with an increase in type II collagen–specific CD4+FoxP3+ Treg cells and inhibition of Th17 cell formation in the peritoneal cavity and spleen. Furthermore, TGFβ‐transduced MSCs inhibited osteoclast differentiation.

Conclusion

TGFβ‐transduced MSCs suppressed the development of autoimmune arthritis and joint inflammation. These data suggest that enhancing the immunomodulatory activity of MSCs and modulating T cell–mediated immunity using gene‐modified MSCs may be a gateway for new therapeutic approaches to clinical rheumatoid arthritis.

     

Stromal cells and osteoclasts are responsible for exacerbated collagen‐induced arthritis in interferon‐β–deficient mice

Objective

Clinical trials using interferon‐β (IFNβ) in the treatment of rheumatoid arthritis have shown conflicting results. We undertook this study to understand the mechanisms of IFNβ in arthritis at a physiologic level.

Methods

Collagen‐induced arthritis (CIA) was induced in IFNβ‐deficient and control mice. The role of IFNβ was investigated in both the priming and effector phases of the disease. The effect of IFNβ deficiency on synovial cells, macrophages, and fibroblasts from preimmunized mice was analyzed by flow cytometry, immunohistochemistry, and enzyme‐linked immunosorbent assay. Differences in osteoclast maturation were determined in situ by histology of arthritic and naive paws and by in vitro maturation studies of naive bone marrow cells. The importance of IFNβ‐producing fibroblasts was determined by transfering fibroblasts into mice at the time of CIA immunization.

Results

Mice lacking IFNβ had a prolonged disease with a higher incidence compared with control mice. IFNβ deficiency was found to influence the effector phase, but not the priming phase, of arthritis. Compared with control mice, IFNβ‐deficient mice had greater infiltration of CD11b+ cells and greater production of tumor necrosis factor α in vivo, and their macrophages and fibroblasts were both more activated in vitro. Moreover, IFNβ‐deficient mice generated a greater number of osteoclasts in vitro, and mice immunized to induce arthritis, but not naive mice, had a greater number of osteoclasts in vivo compared with control mice. Importantly, IFNβ‐competent fibroblasts were able to ameliorate arthritis in IFNβ‐deficient recipients.

Conclusion

Our data indicate that IFNβ is involved in regulating the activation state of osteoclasts and stromal cells, including macrophages and fibroblasts, but that it has little effect on T cells.

     

Increased collagen and aggrecan degradation with age in the joints of Timp3−/− mice

Objective

To investigate the in vivo effect of an imbalance between metalloproteinases and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), in mouse articular cartilage.

Methods

Hind joints of Timp3^−/− and wild‐type mice were examined by routine staining and by immunohistochemical analysis using antibodies specific for type X collagen and for the neoepitopes produced on proteolytic cleavage of aggrecan (… VDIPEN and … NVTEGE) and type II collagen. The neoepitope generated on cleavage of type II collagen by collagenases was quantitated in sera by enzyme‐linked immunosorbent assay.

Results

Articular cartilage from Timp3‐knockout animals (ages ≥6 months) showed reduced Safranin O staining and an increase in …VDIPEN content compared with cartilage from heterozygous and wild‐type animals. There was also a slight increase in … NVTEGE content in articular cartilage and menisci of Timp3^−/− animals. Chondrocytes showed strong pericellular staining for type II collagen cleavage neoepitopes, particularly in the superficial layer, in knockout mice. Also, there was more type X collagen expression in the superficial zone of articular cartilage, especially around clusters of proliferating chondrocytes, in the knockout mice. More type II collagen cleavage product was found in the serum of Timp3^−/− mice compared with wild‐type animals. This increase was significant in 15‐month‐old animals.

Conclusion

These results indicate that TIMP‐3 deficiency results in mild cartilage degradation similar to changes seen in patients with osteoarthritis, suggesting that an imbalance between metalloproteinases and TIMP‐3 may play a pathophysiologic role in the development of this disease.