During skeletogenesis, cartilage develops to either permanent cartilage that persists through life or transient cartilage that is eventually replaced by bone. However, the mechanism by which cartilage phenotype is specified remains unclarified. Core binding factor α1 (Cbfa1) is an essential transcription factor for osteoblast differentiation and bone formation and has the ability to stimulate chondrocyte maturation in vitro. To understand the roles of Cbfa1 in chondrocytes during skeletal development, we generated transgenic mice that overexpress Cbfa1 or a dominant negative (DN)-Cbfa1 in chondrocytes under the control of a type II collagen promoter/enhancer. Both types of transgenic mice displayed dwarfism and skeletal malformations, which, however, resulted from opposite cellular phenotypes. Cbfa1 overexpression caused acceleration of endochondral ossification due to precocious chondrocyte maturation, whereas overexpression of DN-Cbfa1 suppressed maturation and delayed endochondral ossification. In addition, Cbfa1 transgenic mice failed to form most of their joints and permanent cartilage entered the endochondral pathway, whereas most chondrocytes in DN-Cbfa1 transgenic mice retained a marker for permanent cartilage. These data show that temporally and spatially regulated expression of Cbfa1 in chondrocytes is required for skeletogenesis, including formation of joints, permanent cartilages, and endochondral bones.

The Wnt antagonist Frzb-1 is expressed during limb skeletogenesis, but its roles in this complex multistep process are not fully understood. To address this issue, we determined Frzb-1 gene expression patterns during chick long bone development and carried out gain- and loss-of-function studies by misexpression of Frzb-1, Wnt-8 (a known Frzb-1 target), or different forms of the intracellular Wnt mediator LEF-1 in developing limbs and cultured chondrocytes. Frzb-1 expression was quite strong in mesenchymal prechondrogenic condensations and then characterized epiphyseal articular chondrocytes and prehypertrophic chondrocytes in growth plates. Virally driven Frzb-1 misexpression caused shortening of skeletal elements, joint fusion, and delayed chondrocyte maturation, with consequent inhibition of matrix mineralization, metalloprotease expression, and marrow/bone formation. In good agreement, misexpression of Frzb-1 or a dominant-negative form of LEF-1 in cultured chondrocytes maintained the cells at an immature stage. Instead, misexpression of Wnt-8 or a constitutively active LEF-1 strongly promoted chondrocyte maturation, hypertrophy, and calcification. Immunostaining revealed that the distribution of endogenous Wnt mediator beta-catenin changes dramatically in vivo and in vitro, from largely cytoplasmic in immature proliferating and prehypertrophic chondrocytes to nuclear in hypertrophic mineralizing chondrocytes. Misexpression of Frzb-1 prevented beta-catenin nuclear relocalization in chondrocytes in vivo or in vitro. The data demonstrate that Frzb-1 exerts a strong influence on limb skeletogenesis and is a powerful and direct modulator of chondrocyte maturation, phenotype, and function. Phases of skeletogenesis, such as terminal chondrocyte maturation and joint formation, appear to be particularly dependent on Wnt signaling and thus very sensitive to Frzb-1 antagonistic action.

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We cloned a novel mouse cDNA, CORS26 (collagenous repeat-containing sequence of 26-kDa protein), encoding a secretory protein by suppression subtractive hybridization between transforming growth factor-␤1-treated and untreated C3H10T1/2 cells. The deduced amino acid sequence of CORS26 consists of 246 amino acids with a secretory signal peptide and contains a collagenous region (Gly-X-Y repeats) at the NH 2 terminus and a complement factor C1q globular domain at the COOH terminus. CORS26 is structurally similar to C1q and to adipocyte-specific protein Acrp30. Transfection analysis suggested that CORS26 is a secretory protein.Northern blot analysis revealed that CORS26 mRNA was present at high levels in rib growth plate cartilage and at moderate levels in kidney of adult mice. In skeletal development in vertebrates, the formation of chondrocytes from undifferentiated mesenchymal cells is one of the important processes, but the molecular mechanisms are not well understood. Identifying the genes underlying the induction of chondrocyte differentiation will provide powerful tools for understanding skeletal development. The induction of chondrogenesis has been extensively studied in vitro using primary cells and clonal cell lines from a variety of sources (1-4). The mouse embryonic fibroblast cell line, C3H10T1/2, are multipotential cells and have been induced to undergo differentiation into myocytes, adipocytes, osteoblasts, and chondrocytes under specific culture conditions and treatments (5-8). The frequency of chondrogenic conversion in C3H10T1/2 cells was much lower and irregular compared with other types of conversion (5, 8), but it was recently reported that the induction of chondrogenesis and the formation of spheroids in C3H10T1/2 cells preferentially occurred when treated with transforming growth factor (TGF) 1 -␤1 (9), bone morphogenetic protein-2 (10), or a combination of osteoinductive bone proteins (11) in high-density micromass cultures. The formation of the spheroids resembled the condensation of mesenchymal cells seen in precartilage. Thus, C3H10T1/2 cells in high-density micromass cultures are suited for studying the molecular mechanisms involved in skeletal development.In the present study, to help clarify the mechanism for skeletal development, mRNAs expressed in TGF-␤1-treated C3H10T1/2 cells were subtracted with those in untreated C3H10T1/2 cells using the suppression subtractive hybridization (SSH) technique (12), and we isolated a novel gene, CORS26 (collagenous repeat-containing sequence of 26-kDa protein). Sequence analysis revealed that CORS26 possesses a collagenous structure at the NH 2 terminus and a complement factor C1q globular domain at the COOH terminus. Due to the structural similarity between CORS26 and subunits of complement factor C1q, this novel protein is thought to be a member of the C1q-related protein family. The presence of the signal peptide, plus the hydrophilic nature of CORS26, suggests that CORS26 is a secretory protein. Indeed, the CORS26 protein was secreted from COS-...

To examine the role of bone morphogenetic protein (BMP) signaling in chondrocytes during endochondral ossification, the dominant negative (DN) forms of BMP receptors were introduced into immature and mature chondrocytes isolated from lower and upper portions of chick embryo sternum, respectively. We found that control sternal chondrocyte populations expressed type IA, IB, and II BMP receptors as well as BMP-4 and -7. Expression of a DN-type II BMP receptor (termed DN-BMPR-II) in immature lower sternal (LS) chondrocytes led to a loss of differentiated functions; compared with control cells, the DN-BMPR- II–expressing LS chondrocytes proliferated more rapidly, acquired a fibroblastic morphology, showed little expression of type II collagen and aggrecan genes, and upregulated type I collagen gene expression. Expression of DN-BMPR-II in mature hypertrophic upper sternal (US) chondrocytes caused similar effects. In addition, the DN-BMPR-II–expressing US cells exhibited little alkaline phosphatase activity and type X collagen gene expression, while the control US cells produced both alkaline phosphatase and type X collagen. Both DN-BMPR-II–expressing US and LS chondrocytes failed to respond to treatment with BMP-2 . When we examined the effects of DN forms of types IA and IB BMP receptors, we found that DN-BMPR-IA had little effect, while DN-BMPR-IB had similar but weaker effects compared with those of DN-BMPR-II. We conclude that BMP signaling, particularly that mediated by the type II BMP receptor, is required for maintenance of the differentiated phenotype, control of cell proliferation, and expression of hypertrophic phenotype.