Hypophosphatemia causes rickets by impairing hypertrophic chondrocyte apoptosis. Phosphate induction of Mek1/2;Erk1/2 phosphorylation in hypertrophic chondrocytes is required for phosphate-mediated apoptosis and growth plate maturation. Mek1/2 can be activated by numerous molecules including Raf isoforms. A- and B-Raf ablation in chondrocytes does not alter skeletal development while ablation of C-Raf decreases hypertrophic chondrocyte apoptosis and impairs vascularization of the growth plate. However, ablation of C-Raf does not impair phosphate-induced Erk1/2 phosphorylation in vitro, but leads to rickets by decreasing VEGF protein stability. To determine if Raf isoforms are required for phosphate-induced hypertrophic chondrocyte apoptosis, mice lacking all three Raf isoforms in chondrocytes were generated. Raf deletion caused neonatal death and a significant expansion of the hypertrophic chondrocyte layer of the growth plate, accompanied by decreased cleaved caspase-9. This was associated with decreased pErk1/2 immunoreactivity in the hypertrophic chondrocyte layer and impaired vascular invasion. These data further demonstrated that Raf Kinases are required for phosphate-induced Erk1/2 phosphorylation in cultured hypertrophic chondrocytes and perform essential, but partially redundant roles in growth plate maturation.
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