5-azacytidine alters TGF-beta and BMP signaling and induces maturation in articular chondrocytes

Maintenance of the articular surface depends on the function of articular chondrocytes (ACs) which produce matrix and are constrained from undergoing the maturation program seen in growth plate chondrocytes. Only during pathologic conditions, such as in osteoarthritis, are maturational constraints lost causing recapitulation of the process that occurs during endochondral ossification. With the aim of establishing a model to identify regulatory mechanisms that suppress AC hypertrophy, we examined the capability of 5-azacytidine (Aza) to have an impact on the maturational program of these cells. Primary ACs do not spontaneously express markers of maturation and are refractory to treatment by factors that normally regulate chondrocyte maturation. However, following exposure to Aza, ACs (i) were induced to express type X collagen (colX), Indian hedgehog, and alkaline phosphatase and (ii) showed altered colX and AP expression in response to bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta (TGF-beta), and parathyroid hormone-related protein (PTHrP). Since Aza unmasked responsiveness of ACs to BMP-2 and TGF-beta, we examined the effect of Aza treatment on signaling via these pathways by assessing the expression of the TGF-beta Smads (2 and 3), the BMP-2 Smads (1 and 5), and the Smad2 and 3-degrading ubiquitin E3 ligase Smurf2. Aza-treated ACs displayed less Smad2 and 3 and increased Smad1, 5, and Smurf2 protein and showed a loss of TGF-beta signaling on the P3TP-luciferase reporter. Suggesting that Aza-induction of Smurf2 may be responsible for the loss of Smad2 and 3 protein via this pathway, immunoprecipitation and metabolic labeling experiments confirmed that Aza accelerated the ubiquitination and degradation of these targets. Overall, Aza-treated ACs represent a novel model for the study of mechanisms that regulate maturational potential of articular cartilage, with the data suggesting that maturation of these cells may be due to up-regulation of Smad1 and 5 coupled with a Smurf2-dependent degradation of Smad2 and 3 and loss of TGF-beta signaling.     

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.     

Human chondrocyte senescence and osteoarthritis

Although osteoarthritis (OA) is not an inevitable consequence of aging, a strong association exists between age and increasing incidence of OA. We hypothesized that this association is due to in vivo articular cartilage chondrocyte senescence which causes an age-related decline in the ability of the cells to maintain articular cartilage, that is, increasing age increases the risk of OA because chondrocytes lose their ability to replace their extracellular matrix. To test this hypothesis, we measured senescence markers in human articular cartilage chondrocytes from 27 donors ranging in age from one to 87 years. The markers included expression of the senescence-associated enzyme beta-galactosidase, mitotic activity measured by 3H-thymidine incorporation, and telomere length. beta-galactosidase expression increased with age (r=0.84, p=0.0001) while mitotic activity and mean telomere length declined (r=-0.774, p=0.001 and r=-0.71, p=0.0004, respectively). Decreasing telomere length was strongly correlated with increasing expression of beta-galactosidase and decreasing mitotic activity. These findings help explain the previously reported age related declines in chondrocyte synthetic activity and responsiveness to anabolic growth factors and indicate that in vivo articular cartilage chondrocyte senescence is responsible, at least in part, for the age related increased incidence of OA. The data also imply that people vary in their risk of developing OA because of differences in onset of chondrocyte senescence; and, the success of chondrocyte transplantation procedures performed to restore damaged articular surfaces in older patients could be limited by the inability of older chondrocytes to form new cartilage. New efforts to prevent the development or progression of OA might include strategies that delay the onset of chondrocyte senescence or replace senescent cells.     

In vitro morphogenesis of chick embryo hypertrophic cartilage

Dedifferentiated chick embryo chondrocytes (Castagnola, P., G. Moro, F. Descalzi-Cancedda, and R. Cancedda, 1986, J. Cell Biol., 102:2310-2317), when transferred to suspension culture on agarose-coated dishes in the presence of ascorbic acid, aggregate and remain clustered. With time in culture, clusters grow in size and adhere to each other, forming structures that may be several millimeters in dimension. These structures after 7 d of culture have the histologic appearance of mature hypertrophic cartilage partially surrounded by a layer of elongated cells resembling the perichondrium. Cells inside the aggregates have ultrastructural features of stage I (proliferating) or stage II (hypertrophic) chondrocytes depending on their location. Occurrence and distribution of type I, II, and X collagens in the in vitro-formed cartilage at different times of culture, show a temporal and spatial distribution of these antigens reminiscent of the maturation events occurring in the cartilage in vivo. A comparable histologic appearance is shown also by cell aggregates obtained starting with a population of cells derived from a single, cloned, dedifferentiated chondrocyte.     

Smurf2 induces degradation of GSK-3β and upregulates β-catenin in chondrocytes: A potential mechanism for Smurf2-induced degeneration of articular cartilage

We have previously demonstrated that Smurf2 is highly expressed in human osteoarthritis (OA) tissue, and overexpression of Smurf2 under the control of the type II collagen promoter (Col2a1) induces an OA-like phenotype in aged Col2a1-Smurf2 transgenic mice, suggesting that Smurf2 is located upstream of a signal cascade which initiates OA development. However, the factors downstream of Smurf2 in this signal cascade and how Smurf2-induced OA is initiated are largely unknown. In this study, we further characterized the phenotypic changes in Col2a1-Smurf2 transgenic and WT articular cartilage from the postnatal stage to adulthood. We found that the articular cartilage degeneration occurring at the cartilage surface in 6 month-old Col2a1-Smurf2 transgenic mice progressed from an expanded hypertrophic domain in the basal layer of the deep articular cartilage at 2.5 weeks of age, which may lead to an accelerated calcification and ectopic ossification of this region at 1 month of age, and aggregation and maturation of articular chondrocytes in the middle and deep zones at 2 months and 4.5 months of age, respectively. Furthermore, we discovered that ectopically expressed Smurf2 interacted with GSK-3β and induced its ubiquitination and subsequent proteasomal degradation, and hence upregulated β-catenin in Col2a1-Smurf2 transgenic chondrocytes ex vivo. It is therefore likely that Smurf2-mediated upregulation of β-catenin through induction of proteasomal degradation of GSK-β in chondrocytes may activate articular chondrocyte maturation and associated alteration of gene expression, the early events of OA.     

Dlx5 is a positive regulator of chondrocyte differentiation during endochondral ossification

The process of endochondral ossification in which the bones of the limb are formed after generation of cartilage models is dependent on a precisely regulated program of chondrocyte maturation. Here, we show that the homeobox-containing gene Dlx5 is expressed at the onset of chondrocyte maturation during the conversion of immature proliferating chondrocytes into postmitotic hypertrophying chondrocytes, a critical step in the maturation process. Moreover, retroviral misexpression of Dlx5 during differentiation of the skeletal elements of the chick limb in vivo results in the formation of severely shortened skeletal elements that contain excessive numbers of hypertrophying chondrocytes which extend into ectopic regions, including sites normally occupied by immature chondrocytes. The expansion in the extent of hypertrophic maturation detectable histologically is accompanied by expanded and upregulated domains of expression of molecular markers of chondrocyte maturation, particularly type X collagen and osteopontin, and by expansion of mineralized cartilage matrix, which is characteristic of terminal hypertrophic differentiation. Furthermore, Dlx5 misexpression markedly reduces chondrocyte proliferation concomitant with promoting hypertrophic maturation. Taken together, these results indicate that Dlx5 is a positive regulator of chondrocyte maturation and suggest that it regulates the process at least in part by promoting conversion of immature proliferating chondrocytes into hypertrophying chondrocytes. Retroviral misexpression of Dlx5 also enhances formation of periosteal bone, which is derived from the Dlx5-expressing perichondrium that surrounds the diaphyses of the cartilage models. This suggests that Dlx5 may be involved in regulating osteoblast differentiation, as well as chondrocyte maturation, during endochondral ossification.     

Transglutaminase-catalyzed matrix cross-linking in differentiating cartilage: identification of osteonectin as a major glutaminyl substrate

The expression of tissue transglutaminase in skeletal tissues is strictly regulated and correlates with chondrocyte differentiation and cartilage calcification in endochondral bone formation and in maturation of tracheal cartilage (Aeschlimann, D., A. Wetterwald, H. Fleisch, and M. Paulsson. 1993. J. Cell Biol. 120:1461-1470). We now demonstrate the transglutaminase reaction product, the gamma-glutamyl- epsilon-lysine cross-link, in the matrix of hypertrophic cartilage using a novel cross-link specific antibody. Incorporation of the synthetic transglutaminase substrate monodansylcadaverine (amine donor) in cultured tracheal explants reveals enzyme activity in the pericellular matrix of hypertrophic chondrocytes in the central, calcifying areas of the horseshoe-shaped cartilages. One predominant glutaminyl substrate (amine acceptor) in the chondrocyte matrix is osteonectin as revealed by incorporation of the dansyl label in culture. Indeed, nonreducible osteonectin-containing complexes of approximately 65, 90, and 175 kD can be extracted from mature tracheal cartilage. In vitro cross-linking of osteonectin by tissue transglutaminase gives similar products of approximately 90 and 175 kD, indicating that the complexes in cartilage represent osteonectin oligomers. The demonstration of extracellular transglutaminase activity in differentiating cartilage, i.e., cross-linking of osteonectin in situ, shows that tissue transglutaminase-catalyzed cross-linking is a physiological mechanism for cartilage matrix stabilization.     

SnoN Suppresses Maturation of Chondrocytes by Mediating Signal Cross-talk between Transforming Growth Factor-β and Bone Morphogenetic Protein Pathways

Hypertrophic maturation of chondrocytes is a crucial step in endochondral ossification, whereas abnormally accelerated differentiation of hypertrophic chondrocytes in articular cartilage is linked to pathogenesis of osteoarthritis. This cellular process is promoted or inhibited by bone morphogenetic protein (BMP) or transforming growth factor-β (TGF-β) signaling, respectively, suggesting that these signaling pathways cross-talk during chondrocyte maturation. Here, we demonstrated that expression of Tgfb1 was increased, followed by phosphorylation of Smad2, during BMP-2-induced hypertrophic maturation of ATDC5 chondrocytes. Application of a TGF-β type I receptor inhibitor compound, SB431542, increased the expression of Id1, without affecting the phosphorylation status of Smad1/5/8, indicating that the activated endogenous TGF-β pathway inhibited BMP signaling downstream of the Smad activation step. We searched for TGF-β-inducible effectors that are able to inhibit BMP signaling in ATDC5 cells and identified SnoN. Overexpression of SnoN suppressed the activity of a BMP-responsive luciferase reporter in COS-7 cells as well as expression of Id1 in ATDC5 cells and, subsequently, the expression of Col10a1, a hallmark of hypertrophic chondrocyte maturation. siRNA-mediated loss of SnoN showed opposite effects in BMP-treated ATDC5 cells. In adult mice, we found the highest level of SnoN expression in articular cartilage. Importantly, SnoN was expressed, in combination with phosphorylated Smad2/3, in prehypertrophic chondrocytes in the growth plate of mouse embryo bones and in chondrocytes around the ectopically existing hypertrophic chondrocytes of human osteoarthritis cartilage. Our results indicate that SnoN mediates a negative feedback mechanism evoked by TGF-β to inhibit BMP signaling and, subsequently, hypertrophic maturation of chondrocytes.     

IL-8/CXCL8 and growth-related oncogene alpha/CXCL1 induce chondrocyte hypertrophic differentiation

Foci of chondrocyte hypertrophy that commonly develop in osteoarthritic (OA) cartilage can promote dysregulated matrix repair and pathologic calcification in OA. The closely related chemokines IL-8/CXCL8 and growth-related oncogene alpha (GROalpha)/CXCL1 and their receptors are up-regulated in OA cartilage chondrocytes. Because these chemokines regulate leukocyte activation through p38 mitogen-activated protein kinase signaling, a pathway implicated in chondrocyte hypertrophic differentiation, we tested whether IL-8 and GROalpha promote chondrocyte hypertrophy. We observed that normal human and bovine primary articular chondrocytes expressed both IL-8Rs (CXCR1, CXCR2). IL-8 and the selective CXCR2 ligand GROalpha (10 ng/ml) induced tissue inhibitor of metalloproteinase-3 expression, markers of hypertrophy (type X collagen and MMP-13 expression, alkaline phosphatase activity), as well as matrix calcification. IL-8 and the selective CXCR2 ligand GROalpha also induced increased transamidation activity of chondrocyte transglutaminases (TGs), enzymes up-regulated in chondrocyte hypertrophy that have the potential to modulate differentiation and calcification. Under these conditions, p38 mitogen-activated protein kinase pathway signaling mediated induction of both type X collagen and TG activity. Studies using mouse knee chondrocytes lacking one of the two known articular chondrocyte-expressed TG isoenzymes (TG2) demonstrated that TG2 was essential for murine GROalpha homologue KC-induced TG activity and critically mediated induction by KC of type X collagen, matrix metalloproteinase-13, alkaline phosphatase, and calcification. In conclusion, IL-8 and GROalpha induce articular chondrocyte hypertrophy and calcification through p38 and TG2. Our results suggest a novel linkage between inflammation and altered differentiation of articular chondrocytes. Furthermore, CXCR2 and TG2 may be sites for intervention in the pathogenesis of OA.     

Potential involvement of oxidative stress in cartilage senescence and development of osteoarthritis: oxidative stress induces chondrocyte telomere instability and downregulation of chondrocyte function

Oxidative stress leads to increased risk for osteoarthritis (OA) but the precise mechanism remains unclear. We undertook this study to clarify the impact of oxidative stress on the progression of OA from the viewpoint of oxygen free radical induced genomic instability, including telomere instability and resulting replicative senescence and dysfunction in human chondrocytes. Human chondrocytes and articular cartilage explants were isolated from knee joints of patients undergoing arthroplastic knee surgery for OA. Oxidative damage and antioxidative capacity in OA cartilage were investigated in donor-matched pairs of intact and degenerated regions of tissue isolated from the same cartilage explants. The results were histologically confirmed by immunohistochemistry for nitrotyrosine, which is considered to be a maker of oxidative damage. Under treatment with reactive oxygen species (ROS; 0.1 μmol/l H₂O₂) or an antioxidative agent (ascorbic acid: 100.0 μmol/l), cellular replicative potential, telomere instability and production of glycosaminoglycan (GAG) were assessed in cultured chondrocytes. In tissue cultures of articular cartilage explants, the presence of oxidative damage, chondrocyte telomere length and loss of GAG to the medium were analyzed in the presence or absence of ROS or ascorbic acid. Lower antioxidative capacity and stronger staining of nitrotyrosine were observed in the degenerating regions of OA cartilages as compared with the intact regions from same explants. Immunostaining for nitrotyrosine correlated with the severity of histological changes to OA cartilage, suggesting a correlation between oxidative damage and articular cartilage degeneration. During continuous culture of chondrocytes, telomere length, replicative capacity and GAG production were decreased by treatment with ROS. In contrast, treatment with an antioxidative agent resulted in a tendency to elongate telomere length and replicative lifespan in cultured chondrocytes. In tissue cultures of cartilage explants, nitrotyrosine staining, chondrocyte telomere length and GAG remaining in the cartilage tissue were lower in ROS-treated cartilages than in control groups, whereas the antioxidative agent treated group exhibited a tendency to maintain the chondrocyte telomere length and proteoglycan remaining in the cartilage explants, suggesting that oxidative stress induces chondrocyte telomere instability and catabolic changes in cartilage matrix structure and composition. Our findings clearly show that the presence of oxidative stress induces telomere genomic instability, replicative senescence and dysfunction of chondrocytes in OA cartilage, suggesting that oxidative stress, leading to chondrocyte senescence and cartilage ageing, might be responsible for the development of OA. New efforts to prevent the development and progression of OA may include strategies and interventions aimed at reducing oxidative damage in articular cartilage.     

Changes in the chondrocyte and extracellular matrix proteome during post-natal mouse cartilage development

Skeletal growth by endochondral ossification involves tightly coordinated chondrocyte differentiation that creates reserve, proliferating, prehypertrophic, and hypertrophic cartilage zones in the growth plate. Many human skeletal disorders result from mutations in cartilage extracellular matrix (ECM) components that compromise both ECM architecture and chondrocyte function. Understanding normal cartilage development, composition, and structure is therefore vital to unravel these disease mechanisms. To study this intricate process in vivo by proteomics, we analyzed mouse femoral head cartilage at developmental stages enriched in either immature chondrocytes or maturing/hypertrophic chondrocytes (post-natal days 3 and 21, respectively). Using LTQ-Orbitrap tandem mass spectrometry, we identified 703 cartilage proteins. Differentially abundant proteins (q < 0.01) included prototypic markers for both early and late chondrocyte differentiation (epiphycan and collagen X, respectively) and novel ECM and cell adhesion proteins with no previously described roles in cartilage development (tenascin X, vitrin, Urb, emilin-1, and the sushi repeat-containing proteins SRPX and SRPX2). Meta-analysis of cartilage development in vivo and an in vitro chondrocyte culture model (Wilson, R., Diseberg, A. F., Gordon, L., Zivkovic, S., Tatarczuch, L., Mackie, E. J., Gorman, J. J., and Bateman, J. F. (2010) Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics. Mol. Cell. Proteomics 9, 1296-1313) identified components involved in both systems, such as Urb, and components with specific roles in vivo, including vitrin and CILP-2 (cartilage intermediate layer protein-2). Immunolocalization of Urb, vitrin, and CILP-2 indicated specific roles at different maturation stages. In addition to ECM-related changes, we provide the first biochemical evidence of changing endoplasmic reticulum function during cartilage development. Although the multifunctional chaperone BiP was not differentially expressed, enzymes and chaperones required specifically for collagen biosynthesis, such as the prolyl 3-hydroxylase 1, cartilage-associated protein, and peptidyl prolyl cis-trans isomerase B complex, were down-regulated during maturation. Conversely, the lumenal proteins calumenin, reticulocalbin-1, and reticulocalbin-2 were significantly increased, signifying a shift toward calcium binding functions. This first proteomic analysis of cartilage development in vivo reveals the breadth of protein expression changes during chondrocyte maturation and ECM remodeling in the mouse femoral head.     

Hypoxia-inducible factor-2α regulates Fas-mediated chondrocyte apoptosis during osteoarthritic cartilage destruction

Apoptosis of articular chondrocytes is associated with the pathogenesis of osteoarthritis (OA). Recently, we demonstrated that hypoxia-inducible factor (HIF)-2α, encoded by Epas1, causes OA cartilage destruction by regulating the expression of various matrix-degrading enzymes. Here, we investigated the involvement of HIF-2α in chondrocyte apoptosis and OA cartilage destruction. HIF-2α levels in human and mouse OA chondrocytes were markedly elevated in association with increased apoptosis of articular chondrocytes. Overexpression or knockdown of HIF-2α alone did not cause chondrocyte apoptosis. However, HIF-2α expression markedly increased chondrocyte apoptosis in the presence of an agonistic anti-Fas (CD95) antibody. HIF-2α enhanced Fas expression and potentiated downstream signaling pathways, increasing the activity of initiator and executioner caspases. Overexpression of HIF-2α in mouse cartilage tissue, either by intra-articular injection of Epas1 adenovirus (Ad-Epas1) or in the context of chondrocyte-specific Epas1 transgenic mice, increased chondrocyte apoptosis and cartilage destruction. In contrast, chondrocyte-specific knockout of Epas1 in mice suppressed DMM (destabilization of the medial meniscus)-induced chondrocyte apoptosis and inhibited OA cartilage destruction. Moreover, Fas-deficient mice exhibited diminished chondrocyte apoptosis and OA cartilage destruction in response to Ad-Epas1 injection or DMM surgery. Taken together, our results demonstrate that HIF-2α potentiates Fas-mediated chondrocyte apoptosis, which is associated with OA cartilage destruction.     

Transamidation by transglutaminase 2 transforms S100A11 calgranulin into a procatabolic cytokine for chondrocytes

In osteoarthritis (OA), low-grade joint inflammation promotes altered chondrocyte differentiation and cartilage catabolism. S100/calgranulins share conserved calcium-binding EF-hand domains, associate noncovalently as homodimers and heterodimers, and are secreted and bind receptor for advanced glycation end products (RAGE). Chondrocyte RAGE expression and S100A11 release are stimulated by IL-1beta in vitro and increase in OA cartilage in situ. Exogenous S100A11 stimulates chondrocyte hypertrophic differentiation. Moreover, S100A11 is covalently cross-linked by transamidation catalyzed by transglutaminase 2 (TG2), itself an inflammation-regulated and redox stress-inducible mediator of chondrocyte hypertrophic differentiation. In this study, we researched mouse femoral head articular cartilage explants and knee chondrocytes, and a soluble recombinant double point mutant (K3R/Q102N) of S100A11 TG2 transamidation substrate sites. Both TG2 and RAGE knockout cartilage explants retained IL-1beta responsiveness. The K3R/Q102N mutant of S100A11 retained the capacity to bind to RAGE and chondrocytes but lost the capacity to signal via the p38 MAPK pathway or induce chondrocyte hypertrophy and glycosaminoglycans release. S100A11 failed to induce hypertrophy, glycosaminoglycan release, and appearance of the aggrecanase neoepitope NITEGE in both RAGE and TG2 knockout cartilages. We conclude that transamidation by TG2 transforms S100A11 into a covalently bonded homodimer that acquires the capacity to signal through the p38 MAPK pathway, accelerate chondrocyte hypertrophy and matrix catabolism, and thereby couple inflammation with chondrocyte activation to potentially promote OA progression.     

Developmentally regulated synthesis of a low molecular weight protein (Ch 21) by differentiating chondrocytes

When transferred to suspension culture on agarose-coated dishes, dedifferentiated chick embryo chondrocytes resume the chondrocyte phenotype and continue their maturation to hypertrophic chondrocytes (Castagnola, P., G. Moro, F. Descalzi Cancedda, and R. Cancedda. 1986. J. Cell Biol. 102:2310-2317). In this paper we report the identification, purification, and characterization of a low molecular weight protein, named Ch 21, expressed and secreted by in vitro differentiating chondrocytes at a late stage of development. This protein is detectable in the cells after a short pulse labeling and is directly secreted in the culture medium. The Ch 21 protein has a peculiar resistance to limited pepsin digestion; nevertheless it is not collagenous in nature as revealed by its unaltered mobility when isolated from cells grown in the presence of alpha-alpha' dipyridyl, its resistance to bacterial collagenase, and its amino acid composition. By metabolic labeling of tissue slices and by immunohistochemistry, we show that in the chick embryo tibia the Ch 21 protein first appears at the boundary of the cone of hypertrophic cartilage and in the newly formed bone between the 6 and 10 d of embryo development and localizes in calcifying hypertrophic cartilage thereafter. The Ch 21 protein synthesized by the cultured chondrocytes is closely related and possibly identical to a 21K transformation- sensitive protein associated to the cell substratum of chick embryo fibroblasts.     

Cytokine-like 1 knock-out mice (Cytl1-/-) show normal cartilage and bone development but exhibit augmented osteoarthritic cartilage destruction

We have shown that cytokine-like 1 (Cytl1) is a novel autocrine regulatory factor that regulates chondrogenesis of mouse mesenchymal cells (Kim, J. S., Ryoo, Z. Y., and Chun, J. S. (2007) J. Biol. Chem. 282, 29359-29367). In this previous work, we found that Cytl1 expression was very low in mesenchymal cells, increased dramatically during chondrogenesis, and decreased during hypertrophic maturation, both in vivo and in vitro. Moreover, exogenous addition or ectopic expression of Cytl1 caused chondrogenic differentiation of mouse limb bud mesenchymal cells. In the current study, we generated a Cytl1 knock-out (Cytl1(-/-)) mouse to investigate the in vivo role of Cytl1. Deletion of the Cytl1 gene did not affect chondrogenesis or cartilage development. Cytl1(-/-) mice also showed normal endochondral ossification and long bone development. Additionally, ultrastructural features of articular cartilage, such as matrix organization and chondrocyte morphology, were similar in wild-type and Cytl1(-/-) mice. However, Cytl1(-/-) mice were more sensitive to osteoarthritic (OA) cartilage destruction. Compared with wild-type littermates, Cytl1(-/-) mice showed more severe OA cartilage destruction upon destabilization of the medial meniscus of mouse knee joints. In addition, expression levels of Cytl1 were markedly decreased in OA cartilage of humans and experimental mice. Taken together, our results suggest that, rather than regulating cartilage and bone development, Cytl1 is required for the maintenance of cartilage homeostasis, and loss of Cytl1 function is associated with experimental OA cartilage destruction in mice.     

Induction of proliferation or hypertrophy of chondrocytes in serum-free culture: the role of insulin-like growth factor-I, insulin, or thyroxine

In bone forming cartilage in vivo, cells undergo terminal differentiation, whereas most of the cells in normal articular cartilage do not. Chondrocyte hypertrophy can be induced also in vitro by diffusible signals. We have identified growth factors or hormones acting individually on 17-d chick embryo sternal chondrocytes cultured in agarose gels under strictly serum-free conditions. Insulin-like growth factor I or insulin triggered the first steps of chondrocyte maturation, i.e., cell proliferation and increased matrix deposition while the chondrocytic phenotype was maintained. However, cells did not progress to the hypertrophic stage. Proliferation and stimulated collagen production was preceded by a lag period, indicating that synthesis of other components was required before cells became responsive to insulin-like growth factor I or insulin. Very small amounts of FBS exerted effects similar to those of insulin-like growth factor I or insulin. However, FBS could act directly and elicited hypertrophy when constituting greater than 1% of the culture media. Basic FGF has been claimed to be the most potent chondrocyte mitogen, but had negligible effects under serum-free conditions. The same is true for PDGF, a major serum-mitogen. Under the direction of thyroxine, cells did not proliferate but became typical hypertrophic chondrocytes, extensively synthesizing collagen X and alkaline phosphatase.     

Reduction of basic fibroblasts growth factor receptor is coupled with terminal differentiation of chondrocytes.

Basic fibroblast growth factor (bFGF) stimulates proliferation of chondrocytes and their extracellular matrix synthesis but inhibits terminal differentiation to hypertrophic cells (Kato, Y., and Iwamoto, M., (1990) J. Biol. Chem. 265, 5903-5909). In the present study, we examined changes in bFGF binding during chondrocyte cytodifferentiation. In cultures of pelleted growth plate chondrocytes, binding of 125I-bFGF to 140-kDa receptors was observed during the mitotic and matrix-forming stages but decreased to a very low level as chondrocytes became hypertrophic. Scatchard plot analysis showed that the decrease in binding of bFGF was due to a decrease in the number not in the affinity of the receptor. The loss of bFGF receptor was associated with a decrease in biological responses to bFGF. On the other hand, the binding of transforming growth factor-beta and epidermal growth factor was constant throughout all stages of growth plate chondrocytes. A rapid decrease in bFGF binding was not observed with articular chondrocytes or bFGF-exposed growth plate chondrocytes, perhaps because they scarcely underwent terminal differentiation. A decrease in bFGF binding associated with terminal differentiation in situ was also demonstrated by examination of sequential slices of growth plates. These observations suggest that rapid reduction in bFGF receptor is a special event during terminal differentiation.