OBJECTIVETo study the effect of high glucose and mannitol (osmotic control) on receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis and Notch2 expression using bone marrow macrophages (BMMs) from mice. Furthermore, the effect of Notch2 activation on RANKL-induced osteoclastogenesis with high glucose concentration was explored.

METHODSPreosteoclasts were cultured and exposed to sustained high glucose (0, 5, 10, 20, 40 mmol x L(-1)) levels to mimic diabetic conditions. Osteoclast formation was analyzed using tartrate resistant acid phosphatase (TRAP) assay. Expression of Notch2 gene was analyzed using real-time polymerase chain reaction. Constitutively over-expressed active Notch2, via stable transfection of exogenous ICN2 (intracellular fragment of Notch2) in preosteoclasts and the effect of Notch2 over expression on osteoclastogenesis was analyzed using Western blotting and TRAP staining.

RESULTSThe osteoclast number with 20 mmol x L(-1) glucose (110.3 +/- 6.8) and 40 mmol x L(1) glucose (72.0 +/- 8.0) was significantly less than the group with 20mmol x L(-1) mannitol (152.7 +/- 7.0) and 40 mmol x L(-1) mannitol (157.0 +/- 12.5). The relative gene expression of Notch2 with 20 mmol x L(-1) glucose (1.65 +/- 0.23) and 40 mmol x L(-1) glucose (1.10 +/- 0.11) was significantly less than the group with 20 mmol x L(-1) mannitol (2.82 +/- 0.28) and 40 mmol x L(-1) mannitol (2.42 +/- 0.27) (P < 0.05). The osteoclast number after Notch2 activation (ICN2-OE) with 20 mmol x L(-1) glucose (206.7+/- 7.8) and 40 mmol x L(-1) glucose (178.3 +/- 11.5) was significantly more than the control group (EMPTY) (102.3 +/- 8.7 and 76.0 +/- 10.1 respectively) ( P < 0.05).

CONCLUSIONNotch2 signaling activation may promote osteoclastogenesis under high glucose concentration.