TGF-beta/Smad3 signals repress chondrocyte hypertrophic differentiation and are required for maintaining articular cartilage

Endochondral ossification begins from the condensation and differentiation of mesenchymal cells into cartilage. The cartilage then goes through a program of cell proliferation, hypertrophic differentiation, calcification, apoptosis, and eventually is replaced by bone. Unlike most cartilage, articular cartilage is arrested before terminal hypertrophic differentiation. In this study, we showed that TGF-beta/Smad3 signals inhibit terminal hypertrophic differentiation of chondrocyte and are essential for maintaining articular cartilage. Mutant mice homozygous for a targeted disruption of Smad3 exon 8 (Smad3(ex8/ex8)) developed degenerative joint disease resembling human osteoarthritis, as characterized by progressive loss of articular cartilage, formation of large osteophytes, decreased production of proteoglycans, and abnormally increased number of type X collagen-expressing chondrocytes in synovial joints. Enhanced terminal differentiation of epiphyseal growth plate chondrocytes was also observed in mutant mice shortly after weaning. In an in vitro embryonic metatarsal rudiment culture system, we found that TGF-beta1 significantly inhibits chondrocyte differentiation of wild-type metatarsal rudiments. However, this inhibition is diminished in metatarsal bones isolated from Smad3(ex8/ex8) mice. These data suggest that TGF-beta/Smad3 signals are essential for repressing articular chondrocyte differentiation. Without these inhibition signals, chondrocytes break quiescent state and undergo abnormal terminal differentiation, ultimately leading to osteoarthritis.     

The low permeability of healthy meniscus and labrum limit articular cartilage consolidation and maintain fluid load support in the knee and hip

The knee meniscus and hip labrum appear to be important for joint health, but the mechanisms by which these structures perform their functions are not fully understood. The fluid phase of articular cartilage provides compressive stiffness and aids in maintaining a low friction articulation. Healthy fibrocartilage, the tissue of meniscus and labrum, has a lower fluid permeability than articular cartilage. In this study we hypothesized that an important function of the knee meniscus and the hip labrum is to augment fluid retention in the articular cartilage of a mechanically loaded joint. Axisymmetric hyperporoelastic finite element models were analyzed for an idealized knee and an idealized hip. The results indicate that the meniscus maintained fluid pressure and inhibited fluid exudation in knee articular cartilage. Similar, but smaller, effects were seen with the labrum in the hip. Increasing the fibrocartilage permeability relative to that of articular cartilage gave a consolidation rate and loss of fluid load support comparable to that predicted by meniscectomy or labrectomy. The reduced articular cartilage fluid pressure that was calculated for the joint periphery is consistent with patterns of endochondral ossification and osteophyte formation in knee and hip osteoarthritis. High articular central strains and loss of fluid load support after meniscectomy could lead to fibrillation. An intact low-permeability fibrocartilage is important for limiting fluid exudation from articular cartilage in the hip and knee. This may be an important aspect of the role of fibrocartilage in protecting these joints from osteoarthritis.     

Effect of alendronate on post-traumatic osteoarthritis induced by anterior cruciate ligament rupture in mice

IntroductionPrevious studies in animal models of osteoarthritis suggest that alendronate (ALN) has antiresorptive and chondroprotective effects, and can reduce osteophyte formation. However, these studies used non-physiologic injury methods, and did not investigate early time points during which bone is rapidly remodeled prior to cartilage degeneration. The current study utilized a non-invasive model of knee injury in mice to investigate the effect of ALN treatment on subchondral bone changes, articular cartilage degeneration, and osteophyte formation following injury.MethodsNon-invasive knee injury via tibial compression overload or sham injury was performed on a total of 90 mice. Mice were treated with twice weekly subcutaneous injections of low-dose ALN (40 μg/kg/dose), high-dose ALN (1,000 μg/kg/dose), or vehicle, starting immediately after injury until sacrifice at 7, 14 or 56 days. Trabecular bone of the femoral epiphysis, subchondral cortical bone, and osteophyte volume were quantified using micro-computed tomography (μCT). Whole-joint histology was performed at all time points to analyze articular cartilage and joint degeneration. Blood was collected at sacrifice, and serum was analyzed for biomarkers of bone formation and resorption.ResultsμCT analysis revealed significant loss of trabecular bone from the femoral epiphysis 7 and 14 days post-injury, which was effectively prevented by high-dose ALN treatment. High-dose ALN treatment was also able to reduce subchondral bone thickening 56 days post-injury, and was able to partially preserve articular cartilage 14 days post-injury. However, ALN treatment was not able to reduce osteophyte formation at 56 days post-injury, nor was it able to prevent articular cartilage and joint degeneration at this time point. Analysis of serum biomarkers revealed an increase in bone resorption at 7 and 14 days post-injury, with no change in bone formation at any time points.ConclusionsHigh-dose ALN treatment was able to prevent early trabecular bone loss and cartilage degeneration following non-invasive knee injury, but was not able to mitigate long-term joint degeneration. These data contribute to understanding the effect of bisphosphonates on the development of osteoarthritis, and may support the use of anti-resorptive drugs to prevent joint degeneration following injury, although further investigation is warranted.     

Synovial perlecan is required for osteophyte formation in knee osteoarthritis

The osteophyte associated with osteoarthritis (OA) is a bony outgrowth formed at the margins of the affected joint through endochondral ossification-like processes. However, the mechanism of osteophyte formation and its pathogenesis are unclear. Perlecan (Hspg2), a heparan sulfate proteoglycan, is expressed in many extracellular tissues and plays critical roles in skeletal development and diseases. The aim of the present study is to identify the role of synovial perlecan in osteophyte formation using perinatal lethality rescued perlecan-knockout mice (Hspg2^?/?-Tg) wherein perlecan expression is lacking in the synovial and other tissues, except for cartilage. We analyzed the development of osteophytes in joints of Hspg2^?/?-Tg mice in two different animal models: the surgical OA model, in which the medial collateral ligament was transected and the medial meniscus was resected, and the TGF-β-induced osteophyte formation model. In the surgical OA model, the osteophyte size and maturation were significantly reduced in the OA joints of Hspg2^?/?-Tg mice compared with control mice, while OA developed on the medial side of the knee joints with no differences in the cartilage degradation score or synovitis score between control and Hspg2^?/?-Tg mice. The reduced osteophyte formation in Hspg2^?/?-Tg mice was associated with reduced cell proliferation and chondrogenesis. In the TGF-β model, the osteophyte size and maturation were also significantly reduced in Hspg2^?/?-Tg mice compared with control mice. Our findings suggest that synovial perlecan plays an important role in osteophyte development in OA, and they provide insights that may facilitate the development of OA therapy.     

Osteophyte formation and matrix mineralization in a TMJ osteoarthritis mouse model are associated with ectopic hedgehog signaling

The temporomandibular joint (TMJ) is a diarthrodial joint that relies on lubricants for frictionless movement and long-term function. It remains unclear what temporal and causal relationships may exist between compromised lubrication and onset and progression of TMJ disease. Here we report that Proteoglycan 4 (Prg4)-null TMJs exhibit irreversible osteoarthritis-like changes over time and are linked to formation of ectopic mineralized tissues and osteophytes in articular disc, mandibular condyle and glenoid fossa. In the presumptive layer of mutant glenoid fossa's articulating surface, numerous chondrogenic cells and/or chondrocytes emerged ectopically within the type I collagen-expressing cell population, underwent endochondral bone formation accompanied by enhanced Ihh expression, became entrapped into temporal bone mineralized matrix, and thereby elicited excessive chondroid bone formation. As the osteophytes grew, the roof of the glenoid fossa/eminence became significantly thicker and flatter, resulting in loss of its characteristic concave shape for accommodation of condyle and disc. Concurrently, the condyles became flatter and larger and exhibited ectopic bone along their neck, likely supporting the enlarged condylar heads. Articular discs lost their concave configuration, and ectopic cartilage developed and articulated with osteophytes. In glenoid fossa cells in culture, hedgehog signaling stimulated chondrocyte maturation and mineralization including alkaline phosphatase, while treatment with hedgehog inhibitor HhAntag prevented such maturation process. In sum, our data indicate that Prg4 is needed for TMJ integrity and long-term postnatal function. In its absence, progenitor cells near presumptive articular layer and disc undergo ectopic chondrogenesis and generate ectopic cartilage, possibly driven by aberrant activation of Hh signaling. The data suggest also that the Prg4-null mice represent a useful model to study TMJ osteoarthritis-like degeneration and clarify its pathogenesis.     

Increased physical activity severely induces osteoarthritic changes in knee joints with papain induced sulfate-glycosaminoglycan depleted cartilage

Introduction

Articular cartilage needs sulfated-glycosaminoglycans (sGAGs) to withstand high pressures while mechanically loaded. Chondrocyte sGAG synthesis is regulated by exposure to compressive forces. Moderate physical exercise is known to improve cartilage sGAG content and might protect against osteoarthritis (OA). This study investigated whether rat knee joints with sGAG depleted articular cartilage through papain injections might benefit from moderate exercise, or whether this increases the susceptibility for cartilage degeneration.

Methods

sGAGs were depleted from cartilage through intraarticular papain injections in the left knee joints of 40 Wistar rats; their contralateral joints served as healthy controls. Of the 40 rats included in the study, 20 rats remained sedentary, and the other 20 were subjected to a moderately intense running protocol. Animals were longitudinally monitored for 12 weeks with in vivo micro-computed tomography (μCT) to measure subchondral bone changes and single-photon emission computed tomography (SPECT)/CT to determine synovial macrophage activation. Articular cartilage was analyzed at 6 and 12 weeks with ex vivo contrast-enhanced μCT and histology to measure sGAG content and cartilage thickness.

Results

All outcome measures were unaffected by moderate exercise in healthy control joints of running animals compared with healthy control joints of sedentary animals. Papain injections in sedentary animals resulted in severe sGAG-depleted cartilage, slight loss of subchondral cortical bone, increased macrophage activation, and osteophyte formation. In running animals, papain-induced sGAG-depleted cartilage showed increased cartilage matrix degradation, sclerotic bone formation, increased macrophage activation, and more osteophyte formation.

Conclusions

Moderate exercise enhanced OA progression in papain-injected joints and did not protect against development of the disease. This was not restricted to more-extensive cartilage damage, but also resulted in pronounced subchondral sclerosis, synovial macrophage activation, and osteophyte formation.     

Changes in articular cartilage mechanics with meniscectomy: A novel image-based modeling approach and comparison to patterns of OA

Meniscectomy is a significant risk factor for osteoarthritis, involving altered cell synthesis, central fibrillation, and peripheral osteophyte formation. Though changes in articular cartilage contact pressure are known, changes in tissue-level mechanical parameters within articular cartilage are not well understood. Recent imaging research has revealed the effects of meniscectomy on the time-dependent deformation of physiologically loaded articular cartilage. To determine tissue-level cartilage mechanics that underlie observed deformation, a novel finite element modeling approach using imaging data and a contacting indenter boundary condition was developed. The indenter method reproduces observed articular surface deformation and avoids assumptions about tangential stretching. Comparison of results from an indenter model with a traditional femur-tibia model verified the method, giving errors in displacement, solid and fluid stress, and strain below 1% (RMS) and 7% (max.) of the absolute maximum of the parameters of interest. Indenter finite element models using real joint image data showed increased fluid pressure, fluid exudation, loss of fluid load support, and increased tensile strains centrally on the tibial condyle after meniscectomy-patterns corresponding to clinical observations of cartilage matrix damage and fibrillation. Peripherally there was decreased consolidation, which corresponds to reduced contact and fluid pressure in this analysis. Clinically, these areas have exhibited advance of the subchondral growth front, biological destruction of the cartilage matrix, cartilage thinning, and eventual replacement of the cartilage via endochondral ossification. Characterizing the changes in cartilage mechanics with meniscectomy and correspondence with observed tissue-level effects may help elucidate the etiology of joint-level degradation seen in osteoarthritis.     

Mechanisms for asporin function and regulation in articular cartilage

Osteoarthritis (OA), the most prevalent form of skeletal disease, represents a leading cause of disability following middle age. OA is characterized by the loss of articular cartilage; however, the details of its etiology and pathogenesis remain unclear. Recently, we demonstrated a genetic association between the cartilage extracellular matrix protein asporin and OA (Kizawa, H., Kou, I., Iida, A., Sudo, A., Miyamoto, Y., Fukuda, A., Mabuchi, A., Kotani, A., Kawakami, A., Yamamoto, S., Uchida, A., Nakamura, K., Notoya, K., Nakamura, Y., and Ikegawa, S. (2005) Nat. Genet. 37, 138-144). Furthermore, we showed that asporin binds to transforming growth factor-beta (TGF-beta), a key cytokine in OA pathogenesis, and inhibits TGF-beta-induced chondrogenesis. To date, functional data for asporin have come primarily from mouse cell culture models of developing cartilage rather than from human articular cartilage cells, in which OA occurs. Here, we describe mechanisms for asporin function and regulation in human articular cartilage. Asporin blocks chondrogenesis and inhibits TGF-beta1-induced expression of matrix genes and the resulting chondrocyte phenotypes. Small interfering RNA-mediated knockdown of asporin increases the expression of cartilage marker genes and TGF-beta1; in turn, TGF-beta1 stimulates asporin expression in articular cartilage cells, suggesting that asporin and TGF-beta1 form a regulatory feedback loop. Asporin inhibits TGF-beta/Smad signaling upstream of TGF-beta type I receptor activation in vivo by co-localizing with TGF-beta1 on the cell surface and blocking its interaction with the TGF-beta type II receptor. Our results provide a basis for elucidating the role of asporin in the molecular pathogenesis of OA.     

Celecoxib: considerations regarding its potential disease-modifying properties in osteoarthritis

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive loss of articular cartilage, subchondral bone sclerosis, osteophyte formation, and synovial inflammation, causing substantial physical disability, impaired quality of life, and significant health care utilization. Traditionally, non-steroidal anti-inflammatory drugs (NSAIDs), including selective cyclooxygenase (COX)-2 inhibitors, have been used to treat pain and inflammation in OA. Besides its anti-inflammatory properties, evidence is accumulating that celecoxib, one of the selective COX-2 inhibitors, has additional disease-modifying effects. Celecoxib was shown to affect all structures involved in OA pathogenesis: cartilage, bone, and synovium. As well as COX-2 inhibition, evidence indicates that celecoxib also modulates COX-2-independent signal transduction pathways. These findings raise the question of whether celecoxib, and potentially other coxibs, is more than just an anti-inflammatory and analgesic drug. Can celecoxib be considered a disease-modifying osteoarthritic drug? In this review, these direct effects of celecoxib on cartilage, bone, and synoviocytes in OA treatment are discussed.     

Degenerative osteoarthrosis in aged ICR mice

The tibia of 117 aged ICR mice was examined histologically to provide further information on degenerative osteoarthrosis. Incidence of the lesion became higher in the male older than 480 days of age and in the female older than 450 days. Main gross changes were roughening of the articular surface, narrowing or focal thickening of the articular cartilage, and osteophytes at the margins of the joint. Histology revealed loosening of the matrix, erosion, and marginal osteophytes in the articular cartilage, and sclerotic changes of the subchondral bone.     

Involvement of sensory nerves and immune cells in osteophyte formation in the ankle joint of adjuvant arthritic rats

To study the mechanism of osteophyte formation in the ankle joints of adjuvant arthritic (AA) rats, the localization of peripheral nerves and immune cells in the synovia were investigated in both axotomized AA rats, whose sciatic nerves were resected before adjuvant injection, and sham-operated ones, using immunohistochemistry for low-affinity nerve growth factor receptor (p75NGFR), growth-associated protein (GAP)-43, calcitonin gene-related peptide (CGRP), helper T cell (W3/25), monocyte/macrophage (ED1), and transforming growth factor (TGF)-beta1 and its receptor, TGF-betaRII. In sham-operated AA rats, dense plexuses of CGRP-positive fibers were observed in the inflamed synovia close to the osteophytes. Most of the CGRP-positive fibers were also positive for p75NGFR and GAP-43. These fibers appeared to be newly sprouted sensory nerves. In axotomized AA rats, the synovia were supplied with no CGRP-positive fibers and the sizes of the osteophytes were smaller than those in sham-operated animals. The ratio of the number of both W3/25- and ED1-positive cells in the inflamed synovia of sham-operated rats peaked at weeks 2-3 after adjuvant injection. The peak, however, lasted until week 4 in axotomized ones. In both animal groups, the macrophages and the osteoblasts were stained for TGF-beta1. The osteoblasts covering the osteophytes were also stained for TGF-betaRII. The present findings suggest that the sensory nerves and the macrophages may be involved in osteophyte formation in the ankle joints of AA rats.     

Do changes in the mechanical properties of articular cartilage promote catabolic destruction of cartilage and osteoarthritis?

Osteoarthritis (OA) is a joint disease characterized by cartilage degeneration, a thickening of subchondral bone, and formation of marginal osteophytes. Previous mechanical characterization of cartilage in our laboratory suggests that energy storage and dissipation is reduced in osteoarthritis as the extent of fibrillation and fissure formation increases. It is not clear whether the loss of energy storage and dissipation characteristics is a result of biochemical and/or biophysical changes that occur to hyaline cartilage in joints. The purpose of this study is to present data, on the strain rate dependence of the elastic and viscous behaviors of cartilage, in order to further characterize changes that occur in the mechanical properties that are associated with OA. We have previously hypothesized that the changes seen in the mechanical properties of cartilage may be due to altered mechanochemical transduction by chondrocytes. Results of incremental tensile stress-strain tests at strain rates between 100%/min and 10,000%/min conducted on OA cartilage indicate that the slope of the elastic stress-strain curve increases with increasing strain rate, unlike the reported behavior of skin and self-assembled collagen fibers. It is suggested that the strain-rate dependence of the elastic stress-strain curve is due to the presence of large quantities of proteoglycans (PGs), which protect articular cartilage by increasing the apparent stiffness. The increased apparent stiffness of articular cartilage at high strain rates may limit the stresses borne and prolong the onset of OA. It is further hypothesized that increased compressive loading of chondrocytes in the intermediate zone of articular cartilage occurs as a result of normal wear to the superficial zone or from excessive impact loading. Once the superficial zone of articular cartilage is worn away, the tension is decreased throughout all cartilage zones leading to increased chondrocyte compressive loading and up-regulation of mechanochemical transduction processes that elaborate catabolic enzymes.     

Transforming growth factor (TGF)-beta-activated kinase 1 mimics and mediates TGF-beta-induced stimulation of type II collagen synthesis in chondrocytes independent of Col2a1 transcription and Smad3 signaling

Transforming growth factor (TGF)-beta, bone morphogenetic protein (BMP), and interleukin-1beta activate TGF-beta-activated kinase 1 (TAK1), which lies upstream of the p38 MAPK, JNK, and NF-kappaB pathways. Our knowledge remains incomplete of TAK1 target genes, requirement for cooperative signaling, and capacity for shared or segregated ligand-dependent responses. We show that adenoviral overexpression of TAK1a in articular chondrocytes stimulated type II collagen protein synthesis 3-6-fold and mimicked the response to TGF-beta1 and BMP2. Both factors activated endogenous TAK1 and its activating protein, TAB1, and the collagen response was inhibited by dominant-negative TAK1a. Isoform-specific antibodies to TGF-beta blocked the response to endogenous and exogenous TGF-beta but not the response to TAK1a. Expression of Smad3 did not stimulate type II collagen synthesis or enhance that caused by TGF-beta1 or TAK1a, in contrast to its effects on its endogenous targets, CTGF and plasminogen-activated inhibitor-1. TAK1a, overexpressed alone and immunoprecipitated, phosphorylated MKK6 and stimulated the plasminogen-activated inhibitor-1 promoter following transient transfection; both effects were enhanced by TAB1 coexpression, but type II collagen synthesis was not. Stimulation by TAK1a or TGF-beta did not require increased Col2a1 mRNA, and TAK1 actually reduced Col2a1 mRNA in parallel with the cartilage markers, SRY-type HMG box 9 (Sox9) and aggrecan. Thus, TAK1 increased target gene expression (Col2a1) by translational or posttranslational mechanisms as a Smad3-independent response shared by TGF-beta1 and BMP2.     

Transforming growth factor beta regulates the metabolism of proteoglycans in bovine cartilage organ cultures

The effect of transforming growth factor beta (TGF-beta) has been studied in a bovine articular cartilage organ culture. The peptide stimulates synthesis of proteoglycans in a dose-dependent manner, reaching saturation at 10 ng/ml. This dose gave an approximate 7-fold increase in synthesis over basal controls. In addition, the peptide decreased the rates of catabolism of proteoglycans with an approximately 2-fold maximal effect seen at 5 ng/ml. At the latter concentration, TGF-beta prevented the 4-fold loss of proteoglycans which occurred in cultures maintained under basal conditions over the course of 3 weeks. There was no increase in cell (DNA) content of the cartilage explants under these conditions of TGF-beta treatment, and the net collagen content of the explants remained constant.     

Inhibition of transforming growth factor-beta type II receptor signaling accelerates tooth formation in mouse first branchial arch explants.

Members of the transforming growth factor-beta (TGF-beta) superfamily signal through their cognate receptors to determine cell phenotypes during embryogenesis. Our previous studies on the regulation of first branchial arch morphogenesis have identified critical components of a hierarchy of different TGF-beta isoforms and their possible functions in regulating tooth and cartilage formation during mandibular morphogenesis. Here we tested the hypothesis that TGF-beta type II receptor (TGF-beta IIR) is a critical component in the TGF-beta signaling pathway regulating tooth formation. To establish the precise location of TGF-beta ligand and its cognate receptor, we first performed detailed analyses of the localization of both TGF-beta2 and TGF-beta IIR during initiation and subsequent morphogenesis of developing embryonic mouse tooth organs. A possible autocrine functional role for TGF-beta and its cognate receptor (TGF-beta IIR) was inferred due to the temporal and spatial localization patterns during the early inductive stages of tooth morphogenesis. Second, loss of function of TGF-beta IIR in a mandibular explant culture model resulted in the acceleration of tooth formation to the cap stage while the mandibular explants in the control group only showed bud stage tooth formation. In addition, there was a significant increase in odontogenic epithelial cell proliferation following TGF-beta IIR abrogation. These results demonstrate, for the first time, that abrogation of the TGF-beta IIR stimulates embryonic tooth morphogenesis in culture and reverses the negative regulation of endogenous TGF-beta signaling upon enamel organ epithelial cell proliferation.     

Induction of angiogenesis by expression of soluble type II transforming growth factor-beta receptor in mouse hepatoma.

The biological effect of transforming growth factor-beta (TGF-beta) is cell type-specific and complex. The precise role of TGF-beta is not clear in vivo. To elucidate the regulation mechanism of endogenous TGF-beta on hepatoma progression, we modified the MH129F mouse hepatoma cell with a retroviral vector encoding the extracellular region of type II TGF-beta receptor (TRII). Soluble TRII (TRIIs) blocked TGF-beta binding to TRII on the membrane of hepatoma cells. Growth of MH129F cells was inhibited by TGF-beta1 treatment; however, soluble TRII-overexpressing cells (MH129F/TRIIs) did not show any change in proliferation after TGF-beta1 treatment. MH129F/TRIIs cells also increased vascular endothelial growth factor (VEGF) expression, endothelial cell migration, and tube formation. Implantation of MH129F/TRIIs cells into C3H/He mice showed the significantly enhanced tumor formation. According to Western blot and protein kinase C assay, the expression of VEGF, KDR/flk-1 receptor, and endothelial nitric-oxide synthase was enhanced, and the phosphorylation activity of protein kinase C was increased up to 3.7-fold in MH129F/TRIIs tumors. Finally, a PECAM-1-stained intratumoral vessel was shown to be 4.2-fold higher in the MH129F/TRIIs tumor. These results indicate that VEGF expression is up-regulated by a blockade of endogenous TGF-beta signaling in TGF-beta-sensitive hepatoma cells and then stimulates angiogenesis and tumorigenicity. Therefore, we suggest that endogenous TGF-beta is a major regulator of the VEGF/flk-1-mediated angiogenesis pathway in hepatoma progression.