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The functions of Na(+) /H(+) exchangers (NHEs) during osteoclastic differentiation were investigated using the NHE inhibitor amiloride and a monoclonal antibody (MAb). Compared with sRANKL-stimulated control cells, amiloride decreased the number of large TRAP-positive osteoclast cells (OCs) with ≥10 nuclei and increased the number of small TRAP-positive OCs with ≤10 nuclei during sRANKL-dependent osteoclastic differentiation of RAW264.7 cells. NHE10 mRNA expression and OC differentiation markers were increased by sRANKL stimulation in dose- and time-dependent manners. NHEs 1-9 mRNA expression was not increased by sRANKL stimulation. Similar to amiloride, a rat anti-mouse NHE10 MAb (clone 6B11) decreased the number of large TRAP-positive OCs, but increased the number of small TRAP-positive OCs. These findings suggested that inhibition of NHEs by amiloride or an anti-NHE10 MAb prevented sRANKL-promoted cellular fusion. The anti-NHE10 MAb has the potential for use as an effective inhibitor of bone resorption for targeted bone disease therapy., (© 2015 International Federation for Cell Biology.)

The receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) system is a well-known key factor in osteoclast differentiation, and osteoblastic lineage cells are the major sources of RANKL and OPG in local bone tissue. Recently, a new molecule from the interleukin (IL)-1 family, IL-33, was identified. Here, we report the possible involvement of IL-33 in RANKL and OPG expression, and the signaling pathways that are required for maximal IL-33-induced RANKL expression in MC3T3-E1 osteoblastic cells. Stimulation with IL-33 increased the mRNA expression and secretion of RANKL in MC3T3-E1 cells. The IL-33-induced RANKL mRNA expression was inhibited by an anti-IL-33 monoclonal antibody. Furthermore, ERK and p38 MAPK inhibitors, but not a JNK inhibitor, suppressed IL-33-induced RANKL mRNA expression. On the other hand, IL-33 had no effect on OPG mRNA expression and protein secretion. These results taken together suggest that IL-33 stimulates RANKL expression through mechanisms dependent on the ERK and p38 MAPK pathways in MC3T3-E1 cells., (© 2014 International Federation for Cell Biology.)

Mechanical unloading conditions result in decreases in bone mineral density and quantity, which may be partly attributed to an imbalance in bone formation and resorption. To investigate the effect of mechanical unloading on osteoblast and osteoclast differentiation, and the expression of RANKL and OPG genes in osteoblasts, we used a three-dimensional (3D) clinostat system simulating microgravity to culture MC3T3-E1 and RAW264.7 cells. Long-term exposure (7 days) of MC3T3-E1 cells to microgravity in the 3D clinostat inhibited the expression of Runx2, Osterix, type I collagen alphaI chain, RANKL and OPG genes. Similarly, 3D clinostat exposure inhibited the enhancement of beta3-integrin gene expression, which normally induced by sRANKL stimulation in RAW264.7 cells. These results, taken together, demonstrate that long-term 3D clinostat exposure inhibits the differentiation of MC3T3-E1 cells together with suppression of RANKL and OPG gene expression, as well as the RANKL-dependent cellular fusion of RAW264.7 cells, suggesting that long-term mechanical unloading suppresses bone formation and resorption.