FGF-2 promotes osteocyte differentiation through increased E11/podoplanin expression

E11/ podoplaninis critical in the early stages of osteoblast-to- osteocytetransitions (osteocytogenesis), however, the upstream events which regulate E11 expression are unknown. The aim of this study was to examine the effects of FGF-2 on E11-mediated osteocytogenesis and to reveal the nature of the underlying signaling pathways regulating this process. Exposure of MC3T3 osteoblast-like cells and murine primary osteoblaststo FGF-2 (10 ng/ml) increased E11 mRNA and protein expression (p < 0.05) after 4, 6, and 24 hr. FGF-2 induced changes in E11 expression were also accompanied by significant (p < 0.01) increases in Phexand Dmp1( osteocytemarkers) expression and decreases in Col1a1, Postn, Bglap, and Alpl( osteoblastmarkers) expression. Immunofluorescent microscopy revealed that FGF-2 stimulated E11 expression, facilitated the translocation of E11 toward the cell membrane, and subsequently promoted the formation of osteocyte-like dendrites in MC3T3and primary osteoblasts. siRNA knock down of E11 expression achieved >70% reduction of basal E11 mRNA expression (p < 0.05) and effectively abrogated FGF-2-related changes in E11 expression and dendrite formation. FGF-2 strongly activated the ERK signaling pathway in osteoblast-like cells but inhibition of this pathway did not block the ability of FGF-2 to enhance E11 expression or to promote acquisition of the osteocytephenotype. The results of this study highlight a novel mechanism by which FGF-2 can regulate osteoblastdifferentiation and osteocyteformation. Specifically, the data suggests that FGF-2 promotes osteocytogenesis through increased E11 expression and further studies will identify if this regulatory pathway is essential for bone development and maintenance in health and disease.