Role of bone morphogenetic protein signals during skeletal development

Development of the skeletal components of limbs is initiated by mesenchymal cell condensation to form primordial cartilage, followed by endochondral ossification. Cartilage serves as the template for the formation of most bones. During development, proliferating chondrocytes differentiate into hypertrophic chondrocytes. In the final step in endochondral bone formation, the hypertrophic cartilage is invaded by blood vessels and osteoprogenitor cells, and the calcified cartilage is subsequently replaced by bone. Bone morphogenetic proteins (BMPs) were originally identified as secreted signaling molecules that could induce endochondral bone formation. Subsequent molecular cloning studies have revealed that the BMP family consists of various molecules, including members of the growth and differentiation factor (GDF) subfamily. BMPs have diverse biological activities during development of various organs and tissues, and the precise roles of BMP signals during mammalian skeletal development have yet to be determined. We previously isolated the promoter/enhancer sequence of the α 2(XI) collagen chain gene; this sequence is responsible for the chondrocyte-specific expression during mouse development. Using this sequence, we created transgenic mice that overexpress BMP4, GDF5 and Noggin (a BMP antagonist) in cartilage. Overproduction of BMP4 or GDF5 in cartilage increased cartilage production and enhanced chondrocyte differentiation. Noggin-expressing transgenic mice, in which BMP signals seem to be blocked in cartilage, lacked most of the cartilage and mature hypertrophic chondrocytes. These results indicate that BMP signals are essential for cartilage development.

BMP signals are mediated by Smad proteins intracellularly. We attempted to block Smad signaling in developing cartilage by overexpression of Smad6, an inhibitory Smad, in transgenic mice. In those mice, the cartilage grew almost normally until birth, but osteopenia developed after birth. Skeletal abnormalities were much less severe in the present Smad6 transgenic mice than in Noggin transgenic mice examined in a previous study; these two strains of transgenic mice had identical promoter/enhancer sequences. The relatively mild phenotype of Smad6 transgenic mice suggests a mechanism in which Smad6 alone cannot completely inhibit transduction of BMP signals in cartilage.

To analyze the roles of BMPs in the final stage of endochondral bone formation, we generated transgenic mice that expressed BMP4 or Noggin in osteoblasts. Expression of Noggin in osteoblasts inhibited bone formation, as expected. On the contrary, BMP4 overexpression in osteoblasts resulted in disruption of skeletal architecture. Histological examination revealed a reduced mineralized matrix in BMP4 transgenic mice. These results suggest that persistent expression of BMP4 dose not cause formation of solid bone.