c-Raf promotes angiogenesis during normal growth plate maturation

Extracellular phosphate plays a key role in growth plate maturation by inducing Erk1/2 (Mapk3/1) phosphorylation, leading to hypertrophic chondrocyte apoptosis. The Raf kinases induce Mek1/2 (Map2k1/2) and Erk1/2 phosphorylation; however, a role for Raf kinases in endochondral bone formation has not been identified. Ablation of both A-Raf (Araf) and B-Raf (Braf) in chondrocytes does not alter growth plate maturation. Because c-Raf (Raf1) phosphorylation is increased by extracellular phosphate and c-Raf is the predominant isoform expressed in hypertrophic chondrocytes, chondrocyte-specific c-Raf knockout mice (c-Raf(f/f); ColII-Cre(+)) were generated to define a role for c-Raf in growth plate maturation. In vivo studies demonstrated that loss of c-Raf in chondrocytes leads to expansion of the hypertrophic layer of the growth plate, with decreased phospho-Erk1/2 immunoreactivity and impaired hypertrophic chondrocyte apoptosis. However, cultured hypertrophic chondrocytes from these mice did not exhibit impairment of phosphate-induced Erk1/2 phosphorylation. Studies performed to reconcile the discrepancy between the in vitro and in vivo hypertrophic chondrocyte phenotypes revealed normal chondrocyte differentiation in c-Raf(f/f); ColII-Cre(+) mice and lack of compensatory increase in the expression of A-Raf and B-Raf. However, VEGF (Vegfa) immunoreactivity in the hypertrophic chondrocytes of c-Raf(f/f); ColII-Cre(+) mice was significantly reduced, associated with increased ubiquitylation of VEGF protein. Thus, c-Raf plays an important role in growth plate maturation by regulating vascular invasion, which is crucial for replacement of terminally differentiated hypertrophic chondrocytes by bone.