Glial Cell Line-Derived Neurotrophic Factor Influences Osteoblast Proliferation

Neurotrophic growth factors have been suggested to play a role in the development and regulation of dental tissues, but little is known about the role of neurotrophic factors in bone metabolism. Objectives: This study investigated the effects of the neurotrophic growth factor, glial cell line-derived neurotrophic factor (GDNF), on mouse MC3T3-E1 osteoblast-like cells. Methods: MC3T3-E1 cells were routinely cultured in DMEM/10%FBS and the expression of GDNF and its receptors, GFR1 and RET, was analysed by RT-PCR and immunocytochemistry. Cell proliferation was assessed by the WST-1 and BrdU incorporation assays after two days' of culture in the presence or absence of recombinant GDNF. Live/dead and lactate dehydrogenase assays were used to evaluate cell death in these cultures. Cellular alkaline phosphatase (AlP) activity was assayed biochemically by spectrophotometric analysis of p-nitrophenol production using p-nitrophenyl phosphate as substrate. Results: MC3T3-E1 cells expressed GDNF as well as its receptors, GFR1 and RET. Addition of recombinant GDNF to cultures in serum-containing medium modestly inhibited cell growth at high concentrations; however, under serum-free culture conditions GDNF dose-dependently increased cell proliferation. GDNF did not significantly affect the level of cell death or survival in these cultures. The effect of GDNF on cell growth was abolished in the presence of inhibitors to GFR1 and RET indicating that GDNF stimulates osteoblasts via its canonical receptors. GDNF inhibited relative AlP activity in serum-free cultures suggesting that GDNF stimulation of cell proliferation was associated with a concomitant decrease in early osteogenic differentiation. Conclusion: This study provides evidence for a direct, receptor-mediated effect of GDNF on osteoblasts highlighting a novel role for GDNF in bone physiology.