Distinct roles of Smad pathways and p38 pathways in cartilage-specific gene expression in synovial fibroblasts.
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The role of TGF-beta/bone morphogenetic protein signaling in the chondrogenic differentiation of human synovial fibroblasts (SFs) was examined with the adenovirus vector-mediated gene transduction system. Expression of constitutively active activin receptor-like kinase 3 (ALK3CA) induced chondrocyte-specific gene expression in SFs cultured in pellets or in SF pellets transplanted into nude mice, in which both the Smad and p38 pathways are essential. To analyze downstream cascades of ALK3 signaling, we utilized adenovirus vectors carrying either Smad1 to stimulate Smad pathways or constitutively active MKK6 (MKK6CA) to activate p38 pathways. Smad1 expression had a synergistic effect on ALK3CA, while activation of p38 MAP kinase pathways alone by transduction of MKK6CA accelerated terminal chondrocytic differentiation, leading to type X collagen expression and enhanced mineralization. Overexpression of Smad1 prevented MKK6CA-induced type X collagen expression and maintained type II collagen expression. In a mouse model of osteoarthritis, activated p38 expression as well as type X collagen staining was detected in osteochondrophytes and marginal synovial cells. These results suggest that SFs can be differentiated into chondrocytes via ALK3 activation and that stimulating Smad pathways and controlling p38 activation at the proper level can be a good therapeutic strategy for maintaining the healthy joint homeostasis and treating degenerative joint disorders. (+info)
Indian hedgehog and syndecans-3 coregulate chondrocyte proliferation and function during chick limb skeletogenesis.
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Hedgehog proteins exert critical roles in embryogenesis and require heparan sulfate proteoglycans (HS-PGs) for action. Indian hedgehog (Ihh) is produced by prehypertrophic chondrocytes in developing long bones and regulates chondrocyte proliferation and other events, but it is not known whether it requires HS-PGs for function. Because the HS-PG syndecan-3 is preferentially expressed by proliferating chondrocytes, we tested whether it mediates Ihh action. Primary chick chondrocyte cultures were treated with recombinant Ihh (rIhh-N) in absence or presence of heparinase I or syndecan-3 neutralizing antibodies. While rIhh-N stimulated proliferation in control cultures, it failed to do so in heparinase- or antibody-treated cultures. In reciprocal gain-of-function studies, chondrocytes were made to overexpress syndecan-3 by an RCAS viral vector. Cells became more responsive to rIhh-N, but even this response was counteracted by heparinase or antibody treatment. To complement the in vitro data, RCAS viral particles were microinjected in day 4-5 chick wing buds and effects of syndecan-3 misexpression were monitored over time. Syndecan-3 misexpression led to widespread chondrocyte proliferation and, interestingly, broader expression and distribution of Ihh. In addition, the syndecan-3 misexpressing skeletal elements were short, remained cartilaginous, lacked osteogenesis, and exhibited a markedly reduced expression of collagen X and osteopontin, products characteristic of hypertrophic chondrocytes and bone cells. The data are the first to indicate that Ihh action in chondrocyte proliferation involves syndecan-3 and to identify a specific member of the syndecan family as mediator of hedgehog function. (+info)
The cyclin-dependent kinase inhibitor p57(Kip2) mediates proliferative actions of PTHrP in chondrocytes.
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Parathyroid hormone-related peptide (PTHrP) is a positive regulator of chondrocyte proliferation during bone development. In embryonic mice lacking PTHrP, chondrocytes stop proliferating prematurely, with accelerated differentiation. Because the bone phenotype of mice lacking the cyclin-dependent kinase inhibitor p57(Kip2) is the opposite of the PTHrP-null phenotype, we hypothesized that PTHrP's proliferative actions in chondrocytes might be mediated by opposing p57. We generated p57/PTHrP-null embryos, which showed partial rescue of the PTHrP-null phenotype. There was reversal of the loss of proliferative chondrocytes in most bones, with reversal of the accelerated differentiation that occurs in the PTHrP-null phenotype. p57 mRNA and protein were upregulated in proliferative chondrocytes in the absence of PTHrP. Metatarsal culture studies confirmed the action of PTHrP to decrease p57 mRNA and protein levels in a model in which parathyroid hormone (PTH), used as an analog of PTHrP, increased chondrocyte proliferation rate and the length of the proliferative domain. PTH treatment of p57-null metatarsals had no effect on proliferation rate in round proliferative chondrocytes but still stimulated proliferation in columnar chondrocytes. These studies suggest that the effects of PTHrP on both the rate and extent of chondrocyte proliferation are mediated, at least in part, through suppression of p57 expression. (+info)
The aromatase inhibitor letrozole increases epiphyseal growth plate height and tibial length in peripubertal male mice.
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Sex hormones may influence longitudinal growth, either indirectly, by affecting the growth-hormone-insulin-like growth factor I (IGF-I) axis, or directly, by affecting changes within the epiphyseal growth plate (EGP). The aim of the present study was to investigate the effects of letrozole, an aromatase inhibitor, on longitudinal growth and changes in the EGP in vivo. Eighteen peripubertal male mice were divided into three groups. The first group was killed at baseline, the second was injected with letrozole (Femara) s.c., 2 mg/kg body weight/day, for 10 days, and the third was injected with the vehicle alone. Serum testosterone levels were found to be significantly higher in the treated group than in the controls. Letrozole induced a significant increase in body weight, tail length and serum growth hormone level, but had no significant effect on the level of serum IGF-I. On histomorphometric study, there was a significant increase (12%) in EGP height in the treated animals compared with controls. Immunohistochemistry showed a 3.4-fold letrozole-induced increase in the proliferation of the EGP chondrocytes, as estimated by the number of proliferation cell nuclear antigen-stained cells, and a decrease in the differentiation of the EGP chondrocytes, as estimated by type X collagen staining. Letrozole did not interfere with type II collagen levels. The study group also showed a twofold increase in the number of IGF-I receptor-positive cells compared with controls. In conclusion, the aromatase inhibitor, letrozole, appears to increase the linear growth potential of the EGP in mice. (+info)
Forward mandibular positioning enhances condylar adaptation in adult rats.
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The aim of this investigation was to assess quantitatively the adaptive changes in the condyles of adult rats to forward mandibular positioning. The level of types II and X collagen expressed in the condyles of adult rats was compared with that formed in response to forward mandibular positioning and the levels of expression were correlated to the amount of bone formed in response to mandibular advancement. Seventy-eight 120-day-old female Sprague-Dawley rats were included in this study. The rats were randomly allocated to six groups. Each group consisted of nine rats with bite-jumping devices and four untreated controls. The animals in each group were sacrificed on days 3, 7, 14, 21, 30, and 60. Immunostaining was used for the detection of types II and X collagen, while Alcian blue-PAS was used to observe the extracellular matrix and new bone formation. The results showed that new cartilage was formed in the posterior condyle. The highest level of expression of types II and X collagen were present on day 21, the amount of increase was 247.99 and 540.08 per cent, respectively. The highest level of new bone formation was measured at day 30 of advancement when the amount of increase in new bone formation was 318.91 per cent. These findings indicate that forward mandibular positioning causes changes in the biophysical environment of the temporomandibular joint (TMJ) of adult rats that leads to condylar adaptation. (+info)
Hyaline cartilage engineered by chondrocytes in pellet culture: histological, immunohistochemical and ultrastructural analysis in comparison with cartilage explants.
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Cartilage engineering is a strategic experimental goal for the treatment of multiple joint diseases. Based on the process of embryonic chondrogenesis, we hypothesized that cartilage could be engineered by condensing chondrocytes in pellet culture and, in the present study, examined the quality of regenerated cartilage in direct comparison with native cartilage. Chondrocytes isolated from the sterna of chick embryos were cultured in pellets (4 x 10(6) cells per pellet) for 2 weeks. Cartilage explants from the same source were cultured as controls. After 2 weeks, the regenerated cartilage from pellet culture had a disc shape and was on average 9 mm at the longest diameter. The chondrocyte phenotype was stabilized in pellet culture as shown by the synthesis of type II collagen and aggrecan, which was the same intensity as in the explant after 7 days in culture. During culture, chondrocytes also continuously synthesized type IX collagen. Type X collagen was negatively stained in both pellets and explants. Except for fibril orientation, collagen fibril diameter and density in the engineered cartilage were comparable with the native cartilage. In conclusion, hyaline cartilage engineered by chondrocytes in pellet culture, without the transformation of cell phenotypes and scaffold materials, shares similarities with native cartilage in cellular distribution, matrix composition and density, and ultrastructure. (+info)
Chondrocyte terminal differentiation, apoptosis, and type X collagen expression are downregulated by parathyroid hormone.
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Parathyroid hormone (PTH) regulates calcium and phosphate homeostasis through the endocrine system. Parathyroid hormone-related peptide (PTHrP) is a heterogeneous polypeptide with sequence homology to PTH in its first 13 amino acid residues. Both bind and activate a common receptor, the type 1 PTH/PTHrP receptor (PTH1R). Activation of this G-protein-coupled receptor by PTHrP has been shown to regulate chondrogenesis in a manner that attenuates chondrocyte hypertrophy. Here, we report the dose-response (10(-7) to 10(-15) M) effects of PTH on chondrogenesis using an avian sternal organ culture model. PTH increased cartilaginous tissue length and downregulated the deposition of type X collagen and its mRNA expression. In addition, PTH increased chondrocyte cell diameter in prehypertrophic and proliferative regions while decreasing chondrocyte apoptosis in the hypertrophic zone. In conclusion, these experiments demonstrate that PTH regulates cartilage growth, chondrocytic apoptosis, deposition of type X collagen protein, and expression of type X collagen mRNA. Type X collagen mRNA expression was downregulated by PTH in this organ culture model, but cell size, another marker for terminal differentiation, increased. (+info)
External GTP-bound transglutaminase 2 is a molecular switch for chondrocyte hypertrophic differentiation and calcification.
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Chondrocyte maturation to hypertrophy, associated with up-regulated transglutaminase 2 (TG2) expression, mediates not only physiologic growth plate mineralization but also pathologic matrix calcification and dys-regulated matrix repair in osteoarthritic articular cartilage. TG2-/- mouse chondrocytes demonstrate markedly inhibited progression to hypertrophic differentiation in response to both retinoic acid and the chemokine CXCL1. Here, our objectives were to test if up-regulated TG2 alone is sufficient to promote chondrocyte hypertrophic differentiation and to identify TG2 molecular determinants and potential downstream signals involved. TG2 activities, regulated by nucleotides and calcium, include cross-linking of cartilage matrix proteins, binding of fibronectin, and hydrolysis of GTP and ATP. Following transfection of TG2 site-directed mutants into chondrocytic cells, we observed that wild type TG2, and TG catalytic site and fibronectin-binding mutants promoted type X collagen expression and matrix calcification consistent with chondrocyte hypertrophic differentiation. In contrast, transfected mutants of TG2 GTP binding (K173L) and externalization (Y274A) sites did not stimulate chondrocyte hypertrophy. Recombinant TG2 treatment of bovine cartilage explants demonstrated that extracellular TG2 induced hypertrophy more robustly in the GTP-bound state, confirming an essential role of TG2 GTP binding. Finally, TG2 treatment induced type X collagen in a beta1 integrin-mediated manner, associated with rapid phosphorylation of both Rac1 and p38 kinases that were inhibited by mutation of the TG2 GTP binding site. In conclusion, externalized GTP-bound TG2 serves as a molecular switch for differentiation of chondrocytes to a hypertrophic, calcifying phenotype in a manner that does not require either TG2 transamidation activity or fibronectin binding. (+info)