Osteoarthritis‐like changes and decreased mechanical function of articular cartilage in the joints of mice with the chondrodysplasia gene (cho)

Objective

To investigate whether heterozygosity for a loss‐of‐function mutation in the gene encoding the α1 chain of type XI collagen (Col11a1) in mice (chondrodysplasia, cho) causes osteoarthritis (OA), and to understand the biochemical and biomechanical effects of this mutation on articular cartilage in knee and temporomandibular (TM) joints.

Methods

Articular cartilage from the knee and TM joints of mice heterozygous for cho (cho/+) and their wild‐type littermates (+/+) was examined. The morphologic properties of cartilage were evaluated, and collagen fibrils were examined by transmission electron microscopy. Immunohistochemical staining was performed to examine the protein expression levels of matrix metalloproteinase 3 (MMP‐3) and MMP‐13 in knee joints. In 6‐month‐old animals, fixed‐charge density was determined using a semiquantitative histochemical method, and tensile stiffness was determined using an osmotic loading technique.

Results

The diameter of collagen fibrils in articular cartilage of knee joints from heterozygous cho/+ mice was increased relative to that in control cartilage, and histologic analysis showed OA‐like degenerative changes in knee and TM joints, starting at age 3 months. The changes became more severe with aging. At 3 months, protein expression for MMP‐3 was increased in knee joints from cho/+ mice. At 6 months, protein expression for MMP‐13 was higher in knee joints from cho/+ mice than in joints from their wild‐type littermates, and negative fixed‐charge density was significantly decreased. Moreover, tensile stiffness in articular cartilage of knee joints from cho/+ mice was moderately reduced and was inversely correlated with the increase in articular cartilage degeneration.

Conclusion

Heterozygosity for a loss‐of‐function mutation in Col11a1 results in the development of OA in the knee and TM joints of cho/+ mice. Morphologic and biochemical evidence of OA appears to precede significant mechanical changes, suggesting that the cho mutation leads to OA through a mechanism that does not initially involve mechanical factors.

     

Pathogenesis of osteoarthritis‐like changes in the joints of mice deficient in type IX collagen

Objective

To examine the pathogenetic mechanisms of osteoarthritis (OA)–like changes in Col9a1^−/− mice, which are deficient in type IX collagen.

Methods

Knee joints and temporomandibular joints (TMJs) from Col9a1^−/− mice and their wild‐type (Col9a1^+/+) littermates were examined by light microscopy. Immunohistochemical staining was performed to examine the expression of matrix metalloproteinase 3 (MMP‐3) and MMP‐13, degraded type II collagen, and the discoidin domain receptor 2 (DDR‐2) in knee joints. Cartilage mechanics were also evaluated for compressive properties by microindentation testing of the tibial plateau and for tensile properties by osmotic loading of the femoral condyle.

Results

Histologic analysis showed age‐dependent OA‐like changes in the knee and TMJs of Col9a1^−/− mice starting at the age of 3 months. At the age of 6 months, enhanced proteoglycan degradation was observed in the articular cartilage of the knee and TMJs of the mutant mice. The expression of MMP‐13 and DDR‐2 protein and the amount of degraded type II collagen were higher in the knee joints of Col9a1^−/− mice than in their wild‐type littermates at the age of 6 months. Changes in cartilage mechanics were observed in the femoral and tibial plateaus of Col9a1^−/− mice at 6 months, including a decrease in the compressive modulus and uniaxial modulus. At 3 and 6 months of age, tibial cartilage in Col9a1^−/− mice was found to be more permeable to fluid flow, with an associated compromise in the fluid pressurization mechanism of load support. All of these changes occurred only at medial sites.

Conclusion

Lack of type IX collagen in Col9a1^−/− mice results in age‐dependent OA‐like changes in the knee joints and TMJs.

     

Cyclic loading increases friction and changes cartilage surface integrity in lubricin‐mutant mouse knees

Objective

To investigate the effects of lubricin gene dosage and cyclic loading on whole joint coefficient of friction and articular cartilage surface integrity in mouse knee joints.

Methods

Joints from mice with 2 (Prg4^+/+), 1 (Prg4^+/−), or no (Prg4^−/−) functioning lubricin alleles were subjected to 26 hours of cyclic loading using a custom‐built pendulum. Coefficient of friction values were measured at multiple time points. Contralateral control joints were left unloaded. Following testing, joints were examined for histologic evidence of damage and cell viability.

Results

At baseline, the coefficient of friction values in Prg4^−/− mice were significantly higher than those in Prg4^+/+ and Prg4^+/− mice (P < 0.001). Cyclic loading continuously increased the coefficient of friction in Prg4^−/− mouse joints. In contrast, Prg4^+/− and Prg4^+/+ mouse joints had no coefficient of friction increases during the first 4 hours of loading. After 26 hours of loading, joints from all genotypes had increased coefficient of friction values compared to baseline and unloaded controls. Significantly greater increases occurred in Prg4^−/− and Prg4^+/− mouse joints compared to Prg4^+/+ mouse joints. The coefficient of friction values were not significantly associated with histologic evidence of damage or loss of cell viability.

Conclusion

Our findings indicate that mice lacking lubricin have increased baseline coefficient of friction values and are not protected against further increases caused by loading. Prg4^+/− mice are indistinguishable from Prg4^+/+ mice at baseline, but have significantly greater coefficient of friction values following 26 hours of loading. Lubricin dosage affects joint properties during loading, and may have clinical implications in patients for whom injury or illness alters lubricin abundance.

     

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.

     

Genetic inhibition of fibroblast growth factor receptor 1 in knee cartilage attenuates the degeneration of articular cartilage in adult mice

Objective

Fibroblast growth factor (FGF) family members are involved in the regulation of articular cartilage homeostasis. The aim of this study was to investigate the function of FGF receptor 1 (FGFR‐1) in the development of osteoarthritis (OA) and its underlying mechanisms.

Methods

FGFR‐1 was deleted from the articular chondrocytes of adult mice in a cartilage‐specific and tamoxifen‐inducible manner. Two OA models (aging‐associated spontaneous OA, and destabilization‐induced OA), as well as an antigen‐induced arthritis (AIA) model, were established and tested in Fgfr1‐deficient and wild‐type (WT) mice. Alterations in cartilage structure and the loss of proteoglycan were assessed in the knee joints of mice of either genotype, using these 3 arthritis models. Primary chondrocytes were isolated and the expression of key regulatory molecules was assessed quantitatively. In addition, the effect of an FGFR‐1 inhibitor on human articular chondrocytes was examined.

Results

The gross morphologic features of Fgfr1‐deficient mice were comparable with those of WT mice at both the postnatal and adult stages. The articular cartilage of 12‐month‐old Fgfr1‐deficient mice displayed greater aggrecan staining compared to 12‐month‐old WT mice. Fgfr1 deficiency conferred resistance to the proteoglycan loss induced by AIA and attenuated the development of cartilage destruction after surgically induced destabilization of the knee joint. The chondroprotective effect of FGFR‐1 inhibition was largely associated with decreased expression of matrix metalloproteinase 13 (MMP‐13) and up‐regulation of FGFR‐3 in mouse and human articular chondrocytes.

Conclusion

Disruption of FGFR‐1 in adult mouse articular chondrocytes inhibits the progression of cartilage degeneration. Down‐regulation of MMP‐13 expression and up‐regulation of FGFR‐3 levels may contribute to the phenotypic changes observed in Fgfr1‐deficient mice.

     

Developmental and osteoarthritic changes in Col6a1‐knockout mice: Biomechanics of type VI collagen in the cartilage pericellular matrix

Objective

Chondrocytes, the sole cell type in articular cartilage, maintain the extracellular matrix (ECM) through a homeostatic balance of anabolic and catabolic activities that are influenced by genetic factors, soluble mediators, and biophysical factors such as mechanical stress. Chondrocytes are encapsulated by a narrow tissue region termed the “pericellular matrix” (PCM), which in normal cartilage is defined by the exclusive presence of type VI collagen. Because the PCM completely surrounds each cell, it has been hypothesized that it serves as a filter or transducer for biochemical and/or biomechanical signals from the cartilage ECM. The present study was undertaken to investigate whether lack of type VI collagen may affect the development and biomechanical function of the PCM and alter the mechanical environment of chondrocytes during joint loading.

Methods

Col6a1^−/− mice, which lack type VI collagen in their organs, were generated for use in these studies. At ages 1, 3, 6, and 11 months, bone mineral density (BMD) was measured, and osteoarthritic (OA) and developmental changes in the femoral head were evaluated histomorphometrically. Mechanical properties of articular cartilage from the hip joints of 1‐month‐old Col6a1^−/−, Col6a1^+/−, and Col6a1^+/+ mice were assessed using an electromechanical test system, and mechanical properties of the PCM were measured using the micropipette aspiration technique.

Results

In Col6a1^−/− and Col6a1^+/− mice the PCM was structurally intact, but exhibited significantly reduced mechanical properties as compared with wild‐type controls. With age, Col6a1^−/− mice showed accelerated development of OA joint degeneration, as well as other musculoskeletal abnormalities such as delayed secondary ossification and reduced BMD.

Conclusion

These findings suggest that type VI collagen has an important role in regulating the physiology of the synovial joint and provide indirect evidence that alterations in the mechanical environment of chondrocytes, due to either loss of PCM properties or Col6a1^−/−‐derived joint laxity, can lead to progression of OA.

     

Chondroprotective role of the osmotically sensitive ion channel transient receptor potential vanilloid 4: Age‐ and sex‐dependent progression of osteoarthritis in Trpv4‐deficient mice

Objective

Mechanical loading significantly influences the physiology and pathology of articular cartilage, although the mechanisms of mechanical signal transduction are not fully understood. Transient receptor potential vanilloid 4 (TRPV4) is a Ca⁺⁺‐permeable ion channel that is highly expressed by articular chondrocytes and can be gated by osmotic and mechanical stimuli. The goal of this study was to determine the role of Trpv4 in the structure of the mouse knee joint and to determine whether Trpv4^–/– mice exhibit altered Ca⁺⁺ signaling in response to osmotic challenge.

Methods

Knee joints of Trpv4^–/– mice were examined histologically and by microfocal computed tomography for osteoarthritic changes and bone structure at ages 4, 6, 9, and 12 months. Fluorescence imaging was used to quantify chondrocytic Ca⁺⁺ signaling within intact femoral cartilage in response to osmotic stimuli.

Results

Deletion of Trpv4 resulted in severe osteoarthritic changes, including cartilage fibrillation, eburnation, and loss of proteoglycans, that were dependent on age and male sex. Subchondral bone volume and calcified meniscal volume were greatly increased, again in male mice. Chondrocytes from Trpv4^+/+ mice demonstrated significant Ca⁺⁺ responses to hypo‐osmotic stress but not to hyperosmotic stress. The response to hypo‐osmotic stress or to the TRPV4 agonist 4α‐phorbol 12,13‐didecanoate was eliminated in Trpv4^–/– mice.

Conclusion

Deletion of Trpv4 leads to a lack of osmotically induced Ca⁺⁺ signaling in articular chondrocytes, accompanied by progressive, sex‐dependent increases in bone density and osteoarthritic joint degeneration. These findings suggest a critical role for TRPV4‐mediated Ca⁺⁺ signaling in the maintenance of joint health and normal skeletal structure.

     

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.

     

Sclerostin is expressed in articular cartilage but loss or inhibition does not affect cartilage remodeling during aging or following mechanical injury

Objective

Sclerostin plays a major role in regulating skeletal bone mass, but its effects in articular cartilage are not known. The purpose of this study was to determine whether genetic loss or pharmacologic inhibition of sclerostin has an impact on knee joint articular cartilage.

Methods

Expression of sclerostin was determined in articular cartilage and bone tissue obtained from mice, rats, and human subjects, including patients with knee osteoarthritis (OA). Mice with genetic knockout (KO) of sclerostin and pharmacologic inhibition of sclerostin with a sclerostin‐neutralizing monoclonal antibody (Scl‐Ab) in aged male rats and ovariectomized (OVX) female rats were used to study the effects of sclerostin on pathologic processes in the knee joint. The rat medial meniscus tear (MMT) model of OA was used to investigate the pharmacologic efficacy of systemic Scl‐Ab or intraarticular (IA) delivery of a sclerostin antibody–Fab (Scl‐Fab) fragment.

Results

Sclerostin expression was detected in rodent and human articular chondrocytes. No difference was observed in the magnitude or distribution of sclerostin expression between normal and OA cartilage or bone. Sclerostin‐KO mice showed no difference in histopathologic features of the knee joint compared to age‐matched wild‐type mice. Pharmacologic treatment of intact aged male rats or OVX female rats with Scl‐Ab had no effect on morphologic characteristics of the articular cartilage. In the rat MMT model, pharmacologic treatment of animals with either systemic Scl‐Ab or IA injection of Scl‐Fab had no effect on lesion development or severity.

Conclusion

Genetic absence of sclerostin does not alter the normal development of age‐dependent OA in mice, and pharmacologic inhibition of sclerostin with Scl‐Ab has no impact on articular cartilage remodeling in rats with posttraumatic OA.

     

Loss of cartilage structure,stiffness, and frictional properties in mice lacking PRG4

Objective

To assess the role of the glycoprotein PRG4 in joint lubrication and chondroprotection by measuring friction, stiffness, surface topography, and subsurface histology of the hip joints of Prg4^−/− and wild‐type (WT) mice.

Methods

Friction and elastic modulus were measured in cartilage from the femoral heads of Prg4^−/− and WT mice ages 2, 4, 10, and 16 weeks using atomic force microscopy, and the surface microstructure was imaged. Histologic sections of each femoral head were stained and graded.

Results

Histologic analysis of the joints of Prg4^−/− mice showed an enlarged, fragmented surface layer of variable thickness with Safranin O–positive formations sometimes present, a roughened underlying articular cartilage surface, and a progressive loss of pericellular proteoglycans. Friction was significantly higher on cartilage of Prg4^−/− mice at age 16 weeks, but statistically significant differences in friction were not detected at younger ages. The elastic modulus of the cartilage was similar between cartilage surfaces of Prg4^−/− and WT mice at young ages, but cartilage of WT mice showed increasing stiffness with age, with significantly higher moduli than cartilage of Prg4^−/− mice at older ages.

Conclusion

Deletion of the gene Prg4 results in significant structural and biomechanical changes in the articular cartilage with age, some of which are consistent with osteoarthritic degeneration. These findings suggest that PRG4 plays a significant role in preserving normal joint structure and function.

     

Autophagy is a protective mechanism in normal cartilage,and its aging‐related loss is linked with cell death and osteoarthritis

Objective

Autophagy is a process for turnover of intracellular organelles and molecules that protects cells during stress responses. We undertook this study to evaluate the potential roles of Unc‐51–like kinase 1 (ULK1), an inducer of autophagy, Beclin1, a regulator of autophagy, and microtubule‐associated protein 1 light chain 3 (LC3), which executes autophagy, in the development of osteoarthritis (OA) and in cartilage cell death.

Methods

Expression of ULK1, Beclin1, and LC3 was analyzed in normal and OA human articular cartilage and in knee joints of mice with aging‐related and surgically induced OA, using immunohistochemistry and Western blotting. Poly(ADP‐ribose) polymerase (PARP) p85 expression was used to determine the correlation between cell death and autophagy.

Results

ULK1, Beclin1, and LC3 were constitutively expressed in normal human articular cartilage. ULK1, Beclin1, and LC3 protein expression was reduced in OA chondrocytes and cartilage, but these 3 proteins were strongly expressed in the OA cell clusters. In mouse knee joints, loss of glycosaminoglycans (GAGs) was observed at ages 9 months and 12 months and in the surgical OA model, 8 weeks after knee destabilization. Expression of ULK1, Beclin1, and LC3 decreased together with GAG loss, while PARP p85 expression was increased.

Conclusion

Autophagy may be a protective or homeostatic mechanism in normal cartilage. In contrast, human OA and aging‐related and surgically induced OA in mice are associated with a reduction and loss of ULK1, Beclin1, and LC3 expression and a related increase in apoptosis. These results suggest that compromised autophagy represents a novel mechanism in the development of OA.

     

Delayed gadolinium‐enhanced magnetic resonance imaging of the meniscus: An index of meniscal tissue degeneration?

Objective

Much attention has been focused recently on the need for a better understanding of the mechanisms and natural progression of knee osteoarthritis (OA), particularly in its early stages. One technique that has been used to investigate early OA is delayed gadolinium‐enhanced magnetic resonance imaging of cartilage (dGEMRIC), where T1(Gd) (T1 value after penetration of the MRI contrast agent gadopentate dimeglumine [Gd‐DTPA²⁻]) is used as an index of the molecular status of articular cartilage. The goal of this study was to explore T1(Gd) in the meniscus and its relationship with articular cartilage T1(Gd) in knee dGEMRIC image data sets.

Methods

T1(Gd) maps of the meniscus and articular cartilage were made from knee dGEMRIC images obtained from prior studies of dGEMRIC of the knee in 21 asymptomatic subjects and 9 patients with self‐reported OA.

Results

T1(Gd) of the meniscus covered a range of values (247–515 msec) and patterns (homogeneous and focal variations). In addition, T1(Gd) of the meniscus correlated with that of articular cartilage (R = 0.38, P = 0.037; R = 0.57, P = 0.001 for correlations of the medial posterior meniscus with the medial femoral and tibial cartilage, respectively; T1[Gd] of the anterior meniscus and lateral compartments also correlated, with R > 0.38 and P < 0.037), potentially demonstrating parallel degradative processes in the knee.

Conclusion

While the biophysical basis for the T1(Gd) results relative to meniscus molecular structure needs investigation, these findings introduce a potential means of examining the time course of meniscal tissue change in the development and progression of arthritis.

     

Water‐soluble C60 fullerene prevents degeneration of articular cartilage in osteoarthritis via down‐regulation of chondrocyte catabolic activity and inhibition of cartilage degeneration during disease development

Objective

Studies have shown the roles of oxidative stress in the pathogenesis of osteoarthritis (OA) and induction of chondrocyte senescence during OA progression. The aim of this study was to examine the potential of a strong free‐radical scavenger, water‐soluble fullerene (C60), as a protective agent against catabolic stress–induced degeneration of articular cartilage in OA, both in vitro and in vivo.

Methods

In the presence or absence of C60 (100 μM), human chondrocytes were incubated with interleukin‐1β (10 ng/ml) or H₂O₂ (100 μM), and chondrocyte activity was analyzed. An animal model of OA was produced in rabbits by resection of the medial meniscus and medial collateral ligament. Rabbits were divided into 5 subgroups: sham operation or treatment with C60 at 0.1 μM, 1 μM, 10 μM, or 40 μM. The left knee joint was injected intraarticularly with water‐soluble C60 (2 ml), while, as a control, the right knee joint received 50% polyethylene glycol (2 ml), once weekly for 4 weeks or 8 weeks. Knee bone and cartilage tissue were prepared for histologic analysis. In addition, in the OA rabbit model, the effect of C60 (10 μM) on degeneration of articular cartilage was compared with that of sodium hyaluronate (HA) (5 mg/ml).

Results

C60 (100 μM) inhibited the catabolic stress–induced production of matrix‐degrading enzymes (matrix metalloproteinases 1, 3, and 13), down‐regulation of matrix production, and apoptosis and premature senescence in human chondrocytes in vitro. In rabbits with OA, treatment with water‐soluble C60 significantly reduced articular cartilage degeneration, whereas control knee joints showed progression of cartilage degeneration with time. This inhibitory effect was dose dependent, and was superior to that of HA. Combined treatment with C60 and HA yielded a significant reduction in cartilage degeneration compared with either treatment alone.

Conclusion

The results indicate that C60 fullerene is a potential therapeutic agent for the protection of articular cartilage against progression of OA.

     

Articular cartilage and biomechanical properties of the long bones in Frzb‐knockout mice

Objective

Ligands and antagonists of the WNT pathway are linked to osteoporosis and osteoarthritis. In particular, polymorphisms in the FRZB gene, a secreted WNT antagonist, have been associated with osteoarthritis. The aim of this study was to examine cartilage and bone in Frzb^−/− mice.

Methods

The Frzb gene in mice was inactivated using a Cre/loxP strategy. Three models of osteoarthritis were used: collagenase, papain, and methylated bovine serum albumin induced. Bone biology was studied using density measurements and microfocal computed tomography. Bone stiffness and mechanical loading–induced bone adaptation were studied by compression of the ulnae.

Results

Targeted deletion of the Frzb gene in mice increased articular cartilage loss during arthritis triggered by instability, enzymatic injury, or inflammation. Cartilage damage in Frzb^−/− mice was associated with increased WNT signaling and matrix metalloproteinase 3 (MMP‐3) expression and activity. Frzb^−/− mice had increased cortical bone thickness and density, resulting in stiffer bones, as demonstrated by stress–strain relationship analyses. Moreover, Frzb^−/− mice had an increased periosteal anabolic response to mechanical loading as compared with wild‐type mice.

Conclusion

The genetic association between osteoarthritis and FRZB polymorphisms is corroborated by increased cartilage proteoglycan loss in 3 different models of arthritis in Frzb^−/− mice. Loss of Frzb may contribute to cartilage damage by increasing the expression and activity of MMPs, in a WNT‐dependent and WNT‐independent manner. FRZB deficiency also resulted in thicker cortical bone, with increased stiffness and higher cortical appositional bone formation after loading. This may contribute to the development of osteoarthritis by producing increased strain on the articular cartilage during normal locomotion but may protect against osteoporotic fractures.

     

Chondrocyte‐intrinsic Smad3 represses Runx2‐inducible matrix metalloproteinase 13 expression to maintain articular cartilage and prevent osteoarthritis

Objective

To identify mechanisms by which Smad3 maintains articular cartilage and prevents osteoarthritis.

Methods

A combination of in vivo and in vitro approaches was used to test the hypothesis that Smad3 represses Runx2‐inducible gene expression to prevent articular cartilage degeneration. Col2‐Cre;Smad3^fl/fl mice allowed study of the chondrocyte‐intrinsic role of Smad3 independently of its role in the perichondrium or other tissues. Primary articular cartilage chondrocytes from Smad3^fl/fl mice and ATDC5 chondroprogenitor cells were used to evaluate Smad3 and Runx2 regulation of matrix metalloproteinase 13 (MMP‐13) messenger RNA (mRNA) and protein expression.

Results

Chondrocyte‐specific reduction of Smad3 caused progressive articular cartilage degeneration due to imbalanced cartilage matrix synthesis and degradation. In addition to reduced type II collagen mRNA expression, articular cartilage from Col2‐Cre;Smad3^fl/fl mice was severely deficient in type II collagen and aggrecan protein due to excessive MMP‐13–mediated proteolysis of these key cartilage matrix constituents. Normally, transforming growth factor β (TGFβ) signals through Smad3 to confer a rapid and dynamic repression of Runx2‐inducible MMP‐13 expression. However, we found that in the absence of Smad3, TGFβ signals through p38 and Runx2 to induce MMP‐13 expression.

Conclusion

Our findings elucidate a mechanism by which Smad3 mutations in humans and mice cause cartilage degeneration and osteoarthritis. Specifically, Smad3 maintains the balance between cartilage matrix synthesis and degradation by inducing type II collagen expression and repressing Runx2‐inducible MMP‐13 expression. Selective activation of TGFβ signaling through Smad3, rather than p38, may help to restore the balance between matrix synthesis and proteolysis that is lost in osteoarthritis.

     

Akt1 in murine chondrocytes controls cartilage calcification during endochondral ossification under physiologic and pathologic conditions

Objective

To examine the role of the phosphoinositide‐dependent serine/threonine protein kinase Akt1 in chondrocytes during endochondral ossification.

Methods

Skeletal phenotypes of homozygous Akt1‐deficient (Akt1^−/−) mice and their wild‐type littermates were compared in radiologic and histologic analyses. An experimental osteoarthritis (OA) model was created by surgically inducing instability in the knee joints of mice. For functional analyses, we used primary costal and articular chondrocytes from neonatal mice and mouse chondrogenic ATDC5 cells with retroviral overexpression of constitutively active Akt1 or small interfering RNA (siRNA) for Akt1.

Results

Among the Akt isoforms (Akt1, Akt2, and Akt3), Akt1 was the most highly expressed in chondrocytes, and the total level of Akt protein was decreased in Akt1^−/− chondrocytes, indicating a dominant role of Akt1. Akt1^−/− mice exhibited dwarfism with normal proliferative and hypertrophic zones but suppressed cartilage calcification in the growth plate compared with their wild‐type littermates. In mice with surgically induced OA, calcified osteophyte formation, but not cartilage degradation, was prevented in the Akt1^−/− joints. Calcification was significantly suppressed in cultures of Akt1^−/− chondrocytes or ATDC5 cells overexpressing siRNA for Akt1 and was enhanced in ATDC5 cells overexpressing constitutively active Akt1. Neither proliferation nor hypertrophic differentiation was affected by the gain or loss of function of Akt1. The expression of ANK and nucleotide pyrophosphatase/phosphodiesterase 1, which accumulate pyrophosphate, a crucial calcification inhibitor, was enhanced by Akt1 deficiency or siRNA for Akt1 and was suppressed by constitutively active Akt1.

Conclusion

Our findings indicate that Akt1 in chondrocytes controls cartilage calcification by inhibiting pyrophosphate during endochondral ossification in skeletal growth and during osteophyte formation in OA.