GDF5 coordinates bone and joint formation during digit development. (25/3372)

A functional skeletal system requires the coordinated development of many different tissue types, including cartilage, bones, joints, and tendons. Members of the Bone morphogenetic protein (BMP) family of secreted signaling molecules have been implicated as endogenous regulators of skeletal development. This is based on their expression during bone and joint formation, their ability to induce ectopic bone and cartilage, and the skeletal abnormalities present in animals with mutations in BMP family members. One member of this family, Growth/differentiation factor 5 (GDF5), is encoded by the mouse brachypodism locus. Mice with mutations in this gene show reductions in the length of bones in the limbs, altered formation of bones and joints in the sternum, and a reduction in the number of bones in the digits. The expression pattern of Gdf5 during normal development and the phenotypes seen in mice with single or double mutations in Gdf5 and Bmp5 suggested that Gdf5 has multiple functions in skeletogenesis, including roles in joint and cartilage development. To further understand the function of GDF5 in skeletal development, we assayed the response of developing chick and mouse limbs to recombinant GDF5 protein. The results from these assays, coupled with an analysis of the development of brachypodism digits, indicate that GDF5 is necessary and sufficient for both cartilage development and the restriction of joint formation to the appropriate location. Thus, GDF5 function in the digits demonstrates a link between cartilage development and joint development and is an important determinant of the pattern of bones and articulations in the digits.  (+info)

Systemic viral interleukin-10 gene delivery prevents cartilage invasion by human rheumatoid synovial tissue engrafted in SCID mice. (26/3372)

OBJECTIVE: To assess the effects of viral interleukin-10 (vIL-10) gene delivery on human rheumatoid synovial tissue. METHODS: SCID mice were engrafted subcutaneously with human rheumatoid synovial tissue and homologous cartilage before systemic injection of 10(9) plaque-forming units of type 5 E1a Elb-deficient non-replicative adenovirus vector containing the vIL-10 gene under control of the cytomegalovirus promoter (AdvIL-10; n = 10) or a control gene (AdvIL-10mut; n = 7). Three weeks later, the graft was removed for histologic analysis of cartilage invasion by synovial tissue. The number of CD3-positive mononuclear cells was assessed in the synovial tissue by immunohistology. Messenger RNA (mRNA) expression of matrix metalloproteinase 3 (MMP-3), tissue inhibitor of metalloproteinases 1 (TIMP-1), and proinflammatory cytokines was determined by polymerase chain reaction. RESULTS: Systemic vIL-10 gene transfer resulted in high sustained production of vIL-10 protein in SCID mouse sera (mean +/- SD 25 +/- 5 ng/ml on day 40 post vector injection). Moreover, vIL-10 mRNA expression was detected in the synovial tissue 3 weeks after intravenous injection of AdvIL-10, reflecting the gene transfer in the human graft. In animals treated with AdvIL-10, cartilage invasion by rheumatoid synovial tissue was significantly inhibited compared with the control vector (mean +/- SD histologic score 2.5 +/- 0.52 versus 0.75 +/-0.8; P < 0.0001). The number of T cells infiltrating the synovium and perichondral resorption in the animals treated with AdvIL-10 gene were not significantly modified relative to the control vector. In animals treated with AdvIL-10, the MMP-3-TIMP-1 balance was partially restored, independent of the effect on mRNA expression of tumor necrosis factor a, IL-1, IL-6, or IL-8. CONCLUSION: Systemic vIL-10 gene transfer prevented cartilage invasion by synovial tissue engrafted in SCID mice. This model offers the opportunity to study the biologic effects of gene transfer in vivo in rheumatoid synovium.  (+info)

Spatiotemporal pattern of the mouse chondromodulin-I gene expression and its regulatory role in vascular invasion into cartilage during endochondral bone formation. (27/3372)

During endochondral bone formation, vascular invasion into cartilage initiates the replacement of cartilage by bone. Chondromodulin-I, a 25 kDa glycoprotein purified from bovine epiphyseal cartilage, was recently identified as a novel endothelial cell growth inhibitor. Here we cloned the mouse chondromodulin-I cDNA from a mouse whole embryo cDNA library. Northern blot analysis revealed that the chondromodulin-I transcripts were expressed in association with the formation of cartilage expressing type II collagen from days 11 to 17 of gestation in mouse embryos, at which time cartilaginous bone rudiments were gradually replaced by bone. Chondromodulin-I mRNA was also detected in the thymus and eyes at a lower level. In situ hybridization revealed significant expression in all cartilaginous tissues in the embryos at days 13.5 and 16 of gestation. However, the expression was completely abolished in the hypertrophic cartilage zone prior to calcification. Upon chondrogenic differentiation of mouse ATDC5 cells in vitro, the expression of chondromodulin-I transcripts was induced concomitantly with the formation of type II collagen-expressing chondrocytes. The expression of the transcripts then declined as type X collagen-expressing hypertrophic chondrocytes appeared in the culture. Purified chondromodulin-I protein inhibited the vascular invasion into cartilage ectopically induced by demineralized bone matrix in nude mice, leading to the suppression of bone formation in vivo. These results suggest that chondromodulin-I is involved in the anti-angiogenic property of cartilage, and that the withdrawal of its expression allows the vascular invasion which triggers the replacement of cartilage by bone during endochondral bone development.  (+info)

Expression of N-cadherin, N-CAM, fibronectin and tenascin is stimulated by TGF-beta1, beta2, beta3 and beta5 during the formation of precartilage condensations. (28/3372)

Cell surface adhesion and extracellular matrix proteins are known to play a key role in the formation of cell condensations during skeletal development, and their formation is crucial for the expression of cartilage-specific genes. However, little is known about the relationship between adhesion molecules (N-cadherin and N-CAM), extracellular matrix proteins (fibronectin and tenascin) and TGF-beta1, TGF-beta2 and TGF-beta3 during in vitro precartilage condensations in mouse chondrogenesis. On these bases, we determined the participation of mammalian TGF-beta1, TGF-beta2 and TFG-beta3 and Xenopus TGF-beta5 on the expression of cell surface adhesion and extracellular matrix proteins during the formation of precartilage condensations. Also, we characterized the effects of TGF-betas on proteoglycan metabolism at different cellular densities in mouse embryonic limb bud mesenchymal cells. In TGF-beta1 and TGF-beta5-treated cultures, proteoglycan biosynthesis was higher than in controls, while there were no differences in proteoglycan catabolism, which caused the accumulation of cartilage extracellular matrix. When mesenchymal cells were seeded at three different cellular densities in the presence of TGF-betas, only high density cultures presented increased stimulation of proteoglycan biosynthesis, compared to low and intermediate densities. To determine whether the effect of TGF-betas on precartilage condensations is mediated through the expression of N-cadherin, N-CAM, fibronectin and tenascin, we evaluated their expression. Results showed that TGF-beta1, TGF-beta2, TGF-beta3, and TGF-beta5 differentially enhanced the expression of N-cadherin, N-CAM, fibronectin and tenascin in precartilage condensations, suggesting that TGF-beta isoforms play an important role in the establishment of cell-cell and cell-extracellular matrix interactions during precartilage condensations.  (+info)

Maturational disturbance of chondrocytes in Cbfa1-deficient mice. (29/3372)

Cbfa1, a transcription factor that belongs to the runt-domain gene family, plays an essential role in osteogenesis. Cbfa1-deficient mice completely lacked both intramembranous and endochondral ossification, owing to the maturational arrest of osteoblasts, indicating that Cbfa1 has a fundamental role in osteoblast differentiation. However, Cbfa1 was also expressed in chondrocytes, and its expression was increased according to the maturation of chondrocytes. Terminal hypertrophic chondrocytes expressed Cbfa1 extensively. The significant expression of Cbfa1 in hypertrophic chondrocytes was first detected at embryonic day 13.5 (E13.5), and its expression in hypertrophic chondrocytes was most prominent at E14.5-16.5. In Cbfa1-deficient mice, whose entire skeleton was composed of cartilage, the chondrocyte differentiation was disturbed. Calcification of cartilage occurred in the restricted parts of skeletons, including tibia, fibula, radius, and ulna. Type X collagen, BMP6, and Indian hedgehog were expressed in their hypertrophic chondrocytes. However, osteopontin, bone sialoprotein, and collagenase 3 were not expressed at all, indicating that they are directly regulated by Cbfa1 in the terminal hypertrophic chondrocytes. Chondrocyte differentiation was severely disturbed in the rest of the skeleton. The expression of PTH/PTHrP receptor, Indian hedgehog, type X collagen, and BMP6 was not detected in humerus and femur, indicating that chondrocyte differentiation was blocked before prehypertrophic chondrocytes. These findings demonstrate that Cbfa1 is an important factor for chondrocyte differentiation.  (+info)

Resistance of small leucine-rich repeat proteoglycans to proteolytic degradation during interleukin-1-stimulated cartilage catabolism. (30/3372)

A bovine nasal-cartilage culture system has been utilized to analyse the catabolic events occurring in response to interleukin-1beta over a 14-day period. An early event following the start of interleukin-1 treatment was the release of glycosaminoglycan into the culture medium. This release was accompanied by the appearance in the tissue, and shortly thereafter also in the culture media, of a globular domain (G1)-containing aggrecan degradation product generated by the action of aggrecanase. Link protein was also released from the cartilage with a similar timeframe to that of the G1 fragment, although there was no evidence of its proteolytic degradation. By comparison with aggrecan, the small leucine-rich repeat proteoglycans decorin, biglycan and lumican showed a resistance to both proteolytic cleavage and release throughout the culture period. In contrast, fibromodulin exhibited a marked decrease in size after day 4, presumably due to proteolytic modification, but the major degradation product was retained throughout the culture period. Also in contrast with the early changes in the components of the proteoglycan aggregate, type II collagen did not display signs of extensive degradation until much later in the culture period. Collagen degradation products compatible with collagenase action first appeared in the medium by day 10 and increased thereafter. These data demonstrate that the leucine-rich repeat proteoglycans are resistant to proteolytic action during interleukin-1-stimulated cartilage catabolism, compared with aggrecan. This resistance and continued interaction with the surface of the collagen fibrils may help to stabilize the collagen fibrillar network and protect it from extensive proteolytic attack during the early phases of cartilage degeneration.  (+info)

Effect of strontium on the epiphyseal cartilage plate of rat tibiae-histological and radiographic studies. (31/3372)

Following dietary administration of strontium carbonate, histological and radiographic changes in the epiphyseal cartilage plate of the rat tibiae were examined in the present study. The weight gain of the rat fed a strontium diet was less than that of the control rats. Longitudinal growth of tibiae and endochondral ossification were inhibited by strontium administration. The widths of both proximal and distal cartilage plates increased enormously as has also been shown by other investigators. Sizes of chondroblasts in columns of proximal cartilage plate in rats fed a strontium diet were smaller than those of the control rats and were not different between upper and lower parts. It is suggested that strontium inhibits bone growth through the inhibitory action on the maturation process of chondroblasts and the succeeding endochondral ossification.  (+info)

Cyclic mechanical stress induces extracellular matrix degradation in cultured chondrocytes via gene expression of matrix metalloproteinases and interleukin-1. (32/3372)

To clarify the mechanism of cartilage degradation induced by mechanical stress, we investigated the influence of cyclic tension force (CTF) on the metabolism of cultured chondrocytes. The chondrocytes were exposed to CTF using a Flexercell strain unit. Five or 15 kPa of high frequency CTF significantly inhibited the syntheses of DNA, proteoglycan, collagen, and protein. Fifteen kPa of high frequency CTF induced the expression of interleukin-1 (IL-1), matrix metalloproteinase (MMP)-2 and -9 mRNA, and increased the production of pro- and active-MMP-9. The degradation of proteoglycan was inhibited by and MMP inhibitor, indicating that MMPs are involved in the degradation of proteoglycans induced by high frequency CTF. Moreover, reducing the frequency of CTF from high to low decreased the inhibition of proteoglycan synthesis. These findings suggest that the CTF frequency is one of the key determinants of chondrocyte metabolism. Low magnitude CTF, whether high or low frequency, did not cause the gene expression of cartilage degradation factors, suggesting that this CTF magnitude causes only minor changes in the cartilage matrix. High magnitude and frequency CTF caused the gene expression of IL-1 and MMP-9, followed by increases in the production of MMP-2 and -9 proteins, suggesting that excessive and continuous cyclic mechanical stress induces the production of IL-1 and MMP-9, resulting in cartilage degradation.  (+info)