Thyroid hormone-stimulated differentiation of primary rib chondrocytes in vitro requires thyroid hormone receptor beta.
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The active thyroid hormone, triiodothyronine (T(3)), binds to thyroid hormone receptors (TR) and plays an essential role in the control of chondrocyte proliferation and differentiation. Hypo- and hyperthyroidism alter the structure of growth plate cartilage and modify chondrocyte gene expression in vivo, whilst TR mutations or deletions in mice result in altered growth plate architecture. Nevertheless, the particular roles of individual TR isoforms in mediating T(3) action in chondrocytes have not been studied and are difficult to determine in vivo because of complex cellular and molecular interactions that regulate growth plate maturation. Therefore, we studied the effects of TRalpha and TRbeta on chondrocyte growth and differentiation in primary cultures of neonatal rib chondrocytes isolated from TRalpha- and TRbeta-deficient mice. T(3) decreased proliferation but accelerated differentiation of rib chondrocytes from wild-type mice. T(3) treatment resulted in similar effects in TRalpha-deficient chondrocytes, but in TRbeta-deficient chondrocytes, all T(3) responses were abrogated. Furthermore, T(3) increased TRbeta1 expression in wild-type and TRalpha-deficient chondrocytes. These data indicate that T(3)-stimulated differentiation of primary rib chondrocytes in vitro requires TRbeta and suggest that the TRbeta1 isoform mediates important T(3) actions in mouse rib chondrocytes. (+info)
Ceramide inhibition of chondrocyte proliferation and bone growth is IGF-I independent.
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Proinflammatory cytokines inhibit growth plate development. However, their underlying mechanisms of action are unclear. These effects may be mediated by ceramide, a sphingosine-based lipid second messenger, which is elevated in a number of chronic inflammatory diseases. To test this hypothesis, we determined the effects of C2-ceramide, a cell permeable ceramide analogue, on the growth of the ATDC5 chondrogenic cell line and on cultured fetal mice metatarsals. In ATDC5 cells, C2-ceramide significantly induced apoptosis at both 40 (82%; P < 0.05) and 25 microM (53%; P < 0.05). At 40 microM, C2-ceramide significantly reduced proliferation ([3H]-thymidine uptake/mg protein) (62%; P < 0.05). C2-ceramide did not markedly alter the differentiation state of the cells as judged by the expression of markers of chondrogenesis and differentiation (sox 9, collagen II and collagen X). The IGF-I signalling pathway is the major autocrine/paracrine regulator of bone growth. Both in the presence and absence of IGF-I, C2-ceramide (25 microM) induced an equivalent reduction in proliferation (60%; P < 0.001). Similarly, C2-ceramide (40 microM) induced a 31% reduction in fetal metatarsal growth both in the presence and absence of IGF-I (both P < 0.001). Furthermore, C2-ceramide reduced ADCT5 proliferation in the presence of AG1024, an IGF-I and insulin receptor blocker. Therefore, C2-ceramide-dependent inhibition appears to be independent of IGF-mediated stimulation of bone growth. Indeed, biochemical studies demonstrated that C2-ceramide (25 microM) pretreatment did not alter IGF-I-stimulated phosphorylation of insulin receptor substrate-1, Akt or P44/42 MAP kinase. In conclusion, C2-ceramide inhibits proliferation and induces apoptosis in growth plate chondrocytes through an IGF-I independent mechanism. (+info)
Regulation of chondrocyte differentiation by actin-severing protein adseverin.
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The importance of actin organization in controlling the chondrocyte phenotype is well established, but little is known about the cytoskeletal components regulating chondrocyte differentiation. Previously, we have observed up-regulation of an actin-binding gelsolin-like protein in hypertrophic chondrocytes. We have now identified it as adseverin (scinderin). Adseverin is drastically up-regulated during chondrocyte maturation, as shown by Northern blot analysis, in situ hybridization, and real-time RT-PCR. Its expression is positively regulated by PKC and MEK signaling as shown by inhibitory analyses. Over-expression of adseverin in non-hypertrophic chondrocytes causes rearrangement of the actin cytoskeleton, a change in cell morphology, a dramatic (3.5-fold) increase in cell volume, and up-regulation of Indian hedgehog (Ihh) and of collagen type X--all indicative of chondrocyte differentiation. These changes are mediated by ERK1/2 and p38 kinase pathways. Thus, adseverin-induced rearrangements of the actin cytoskeleton may mediate the PKC-dependent activation of p38 and Erk1/2 signaling pathways necessary for chondrocyte hypertrophy, as evidenced by changes in cell morphology, increase in cell size and expression of the chondrocyte maturation markers. These results demonstrate that interdependence of cytoskeletal organization and chondrogenic gene expression is regulated, at least in part, by actin-binding proteins such as adseverin. (+info)
Diminished bone formation during diabetic fracture healing is related to the premature resorption of cartilage associated with increased osteoclast activity.
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Histological and molecular analysis of fracture healing in normal and diabetic animals showed significantly enhanced removal of cartilage in diabetic animals. Increased cartilage turnover was associated with elevated osteoclast numbers, a higher expression of genes that promote osteoclastogenesis, and diminished primary bone formation. INTRODUCTION: Diminished bone formation, an increased incidence of nonunions, and delayed fracture healing have been observed in animal models and in patients with diabetes. Fracture healing is characterized by the formation of a stabilizing callus in which cartilage is formed and then resorbed and replaced by bone. To gain insight into how diabetes affects fracture healing, studies were carried out focusing on the impact of diabetes on the transition from cartilage to bone. MATERIALS AND METHODS: A low-dose treatment protocol of streptozotocin in CD-1 mice was used to induce a type 1 diabetic condition. After mice were hyperglycemic for 3 weeks, controlled closed simple transverse fractures of the tibia were induced and fixed by intramedullary pins. Histomorphometric analysis of the tibias obtained 12, 16, and 22 days after fracture was performed across the fracture callus at 0.5 mm proximal and distal increments using computer-assisted image analysis. Another group of 16-day samples were examined by microCT. RNA was isolated from a separate set of animals, and the expression of genes that reflect the formation and removal of cartilage and bone was measured by real-time PCR. RESULTS: Molecular analysis of collagen types II and X mRNA expression showed that cartilage formation was the same during the initial period of callus formation. Histomorphometric analysis of day 12 fracture calluses showed that callus size and cartilage area were also similar in normoglycemic and diabetic mice. In contrast, on day 16, callus size, cartilage tissue, and new bone area were 2.0-, 4.4-, and 1.5-fold larger, respectively, in the normoglycemic compared with the diabetic group (p < 0.05). Analysis of microCT images indicated that the bone volume in the normoglycemic animals was 38% larger than in diabetic animals. There were 78% more osteoclasts in the diabetic group compared with the normoglycemic group (p < 0.05) on day 16, consistent with the reduction in cartilage. Real-time PCR showed significantly elevated levels of mRNA expression for TNF-alpha, macrophage-colony stimulating factor, RANKL, and vascular endothelial growth factor-A in the diabetic group. Similarly, the mRNA encoding ADAMTS 4 and 5, major aggrecanases that degrade cartilage, was also elevated in diabetic animals. CONCLUSIONS: These results suggest that impaired fracture healing in diabetes is characterized by increased rates of cartilage resorption. This premature loss of cartilage leads to a reduction in callus size and contributes to decreased bone formation and mechanical strength frequently reported in diabetic fracture healing. (+info)
Increased collagen and aggrecan degradation with age in the joints of Timp3(-/-) mice.
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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 > or =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. (+info)
Development of the post-natal growth plate requires intraflagellar transport proteins.
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In the post-natal growth plate, chondrocytes are arranged in columns parallel to the long axis of the bone. Chondrocytes divide perpendicular to this axis and then move into position one on top of another in a process called "rotation" that maintains columnar organization. Primary cilia are non-motile microtubule base appendages extending from the surface of almost all vertebrate cells. Primary cilia were described on chondrocytes almost 40 years ago but the function of these structures in cartilage biology is not known. Intraflagellar transport (IFT) is the process by which primary cilia are generated and maintained. This study tested the hypothesis that IFT plays an important role in post-natal skeletal development. Kif3a, a subunit of the Kinesin II motor complex, that is required for intraflagellar transport and the formation of cilia, was deleted in mouse chondrocytes via Col2a-Cre-mediated recombination. Disruption of IFT resulted in subsequent depletion of cilia and post-natal dwarfism due to premature loss of the growth plate likely a result of reduced proliferation and accelerated hypertrophic differentiation of chondrocytes. Cell shape and columnar orientation in the growth plate were also disrupted suggesting a defect in the process of rotation. Alterations in chondrocyte rotation were accompanied by disruption of the actin cytoskeleton and alterations in the localization of activated FAK to focal adhesion-like structures on chondrocytes. This is the first report indicating a role for IFT and primary cilia in the development of the post-natal growth plate. The results suggest a model in which IFT/cilia act to maintain the columnar organization of the growth plate via the process of chondrocyte rotation. (+info)
COL10A1 nonsense and frame-shift mutations have a gain-of-function effect on the growth plate in human and mouse metaphyseal chondrodysplasia type Schmid.
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Missense, nonsense and frame-shift mutations in the collagen X gene (COL10A1) result in metaphyseal chondrodysplasia type Schmid (MCDS). Complete degradation of mutant COL10A1 mRNA by nonsense-mediated decay in human MCDS cartilage implicates haploinsufficiency in the pathogenesis for nonsense mutations in vivo. However, the mechanism is unclear in situations where the mutant mRNA persist. We show that nonsense/frame-shift mutations can elicit a gain-of-function effect, affecting chondrocyte differentiation in the growth plate. In an MCDS proband, heterozygous for a p.Y663X nonsense mutation, the growth plate cartilage contained 64% wild-type and 36% mutant mRNA and the hypertrophic zone was disorganized and expanded. The in vitro translated mutant collagen X chains, which are truncated, were misfolded, unable to assemble into trimers and interfered with the assembly of normal alpha1(X) chains into trimers. Unlike Col10a1 null mutants, transgenic mice (FCdel) bearing the mouse equivalent of a human MCDS p.P620fsX621 mutation, displayed typical characteristics of MCDS with disproportionate shortening of limbs and early onset coxa vara. In FCdel mice, the degree of expansion of the hypertrophic zones was transgene-dosage dependent, being most severe in mice homozygous for the transgene. Chondrocytes in the lower region of the expanded hypertrophic zone expressed markers uncharacteristic of hypertrophic chondrocytes, indicating that differentiation was disrupted. Misfolded FCdel alpha1(X) chains were retained within the endoplasmic reticulum of hypertrophic chondrocytes, activating the unfolded protein response. Our findings provide strong in vivo evidence for a gain-of-function effect that is linked to the activation of endoplasmic reticulum-stress response and altered chondrocyte differentiation, as a possible molecular pathogenesis for MCDS. (+info)
In vitro chondrogenesis of mesenchymal stem cells in recombinant silk-elastinlike hydrogels.
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PURPOSE: In this study the chondrocytic differentiation and cartilage matrix accumulation of human mesenchymal stem cells (hMSCs) were investigated after encapsulation in a genetically engineered silk-elastinlike protein polymer SELP-47 K as an injectable matrix for delivery of cell-based therapeutics. MATERIALS AND METHODS: hMSCs were encapsulated in SELP-47 K and cultured for 4 weeks in chondrogenic medium with or without transforming growth factor-beta3 (TGF). Chondrogenic differentiation was evaluated by histological, RNA and biochemical analyses for the expression of cartilage extracellular matrix components. RESULTS: Histological and immunohistochemical staining revealed that the cells acquired a rounded morphology and were embedded in significant amounts of chondrogenic extracellular matrix. Reverse transcriptase (RT)-PCR showed an up-regulation in aggrecan, type II and type X collagen and SOX9 in presence of TGF-beta3. By day 28, constructs cultured in the presence of TGF-beta3 exhibited significant increase in sulfated glycosaminoglycan and total collagen content up to 65 and 300%, respectively. CONCLUSIONS: This study demonstrates that SELP-47 K hydrogel can be used as a scaffold for encapsulation and chondrogenesis of hMSCs. The ability to use recombinant techniques to precisely control SELP structure enables the investigation of injectable protein polymer scaffolds for soft-tissue engineering with varied physicochemical properties. (+info)