Cartilage provides mechanical strength to resist compression in joints. In development, cartilage serves as the template for the growth of most bones. When cartilage formation is impaired, malformation of the limbs, craniofacial bones, and appendicular skeleton occurs. Cartilage formation is initiated by mesenchymal cell condensation to form primordial cartilage followed by chondrocyte differentiation, which includes resting, proliferative, prehypertrophic, and hypertrophic chondrocytes. As a final step in endochondral bone formation, hypertrophic cartilage is invaded by blood vessels and osteoprogenitor cells, and the calcified cartilage is subsequently replaced by bone. Thus, spatial and temporal regulation of chondrocyte differentiation is essential in determining the length and width of skeletal components.? ? Transforming growth factor-beta (TGF-beta), one of the most abundant cytokines in the bone matrix, regulates bone formation and homeostasis. The roles of ALK-5 in development of the skeletal system have not been fully elucidated. We have created mesenchymal tissue-specific conditional knockout mice for ALK-5 by mating floxed-ALK-5 mice with Dermo1-Cre knockin mice, which express Cre recombinase specific to mesenchymal progenitors under the control of the Dermo1 promoter. These conditional ALK-5 knockout mice died perinatally and had severe defects in the axial and appendicular skeletal system. Both endochondral and membranous ossifications were inhibited in mutant mice, and multiple cartilaginous protuberances were observed in the growth plate. The perichondrium and periosteum layers were thin, and bone mineralization was poor because of the reduced proliferation of the periosteum. Analyses of cell proliferation and formation of mineralized bone nodules by using primary calvarial osteoblast cultures showed that homozygous genetic ablation of ALK-5 inhibited proliferation and promoted differentiation of osteoblasts. These findings suggest that ALK-5 functions in promoting proliferation of the periosteum, a process that provides a sufficient osteoblast cell population for the proper bone size.? Perlecan (Perl), a heparan sulfate proteoglycan, is expressed in cartilage. We previously showed that perlecan deficiency (Perl-/-) in mice and humans causes perinatal lethal chondrodysplasia, indicating that perlecan is essential for cartilage development. However, the functions of perlecan in cartilage development are unknown. The Perl-/- growth plate is short with severely impaired endochondral bone formation and vascularization. Chondrocyte proliferation is reduced, and the hypertrophic matrix is disorganized. Although chondrocytes are differentiated, the expression of FGF downstream genes is dysregulated. We found that both FGFR3 and FGFR1 were activated in the Perl-/- growth plate. Expression of VEGF, a FGF/FGFR target gene, was upregulated by Perl-/- hypertrophic chondrocytes, suggesting that the lack of vascularization into the hypertrophic zone is not due to the reduced VEGF expression. We found that expression of the perlecan transgene specifically in cartilage of Perl-/- mice rescued the perinatal lethality of Perl-/- mice, and vascularization into the growth plate was restored, indicating that perlecan in the growth plate is critical in this process. These results suggest that perlecan plays a critical role in endochondral bone formation via modulating FGF/VEGF signaling and promoting angiogenesis.
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