Smad proteins and the regulation of endochondral bone formation.

Bone morphogenetic proteins (BMPs) are required for endochondral bone formation and control chondrocyte proliferation, survival, and differentiation. It is believed that BMPs mediate these events predominantly through canonical Smad pathways, although p38 MAPK pathways may be involved. Receptor Smads 1, 5, and 8 are activated by BMP stimulation, while receptor Smads 2 and 3 are activated by TGFbeta stimulation. Inhibitory Smads 6 and 7 negatively regulate BMP and TGFbeta signaling. Smad proteins share structural homology, yet the extent to which they are functionally redundant, alternatively regulate distinct targets, and control endochondral bone formation in vivo is unknown. In this study, Smad1/5CKO;Smad8 -/- mice were generated by combining cartilage-specific deletions of Smads1 and 5 with a global deletion of Smad8. Mutant mice exhibit severe chondrodysplasia, demonstrating that BMP signaling through canonical Smad proteins is required for chondrogenesis and that the majority of BMP effects are mediated through Smad pathways rather than MAPK pathways. Mice deficient in a single R-Smad in cartilage develop normally, however the Smad1/5CKO mutant phenocopies the Smad1/5CKO;Smad8-/- mutant, indicating Smads 1 and 5 are functionally redundant and essential for endochondral bone formation. The lethal Smad1/5CKO; Smad8-/- mutation exhibits little to no mineralization of the axial skeleton. The shortened rudimentary limbs of these mice lack an organized growth plate and display excessive growth of the bone collar due to a block in chondrocyte differentiation and an imbalance between BMP, FGF, and Ihh/PTHrP pathways. This study also demonstrates that elevated canonical signaling through loss of inhibitory Smads 6 and 7 results in altered chondrocyte differentiation and patterning defects of the axial skeleton phenotypically distinct from those seen with loss of receptor Smads. Furthermore, severe chondrodyspasia is not seen in viable Smad2CKO;Smad3-/- mutants that exhibit a cartilage-specific disruption of TGFbeta canonical signaling. Smad2CKO;Smad3-/- mice are progressively runted as adults and display joint defects, indicating TGFbeta canonical signaling is required for postnatal skeletal growth and homeostasis. Collectively, these results demonstrate that canonical Smad signaling pathways orchestrate multiple aspects of skeletogenesis and demonstrate that receptor Smads 1 and 5 are required for endochondral bone formation.