Differential localization of myosin II isoforms in resting and activated osteoclasts.

Osteoclasts resorb bone through a cyclical process of attachment to matrix, polarization, retraction, and migration. Although this process requires major alterations in the organization of actin structures, little is known about roles that myosins play in osteoclast cytoskeletal dynamics. We performed immunolocalization of myosin II using antibodies against heavy chain isoforms IIA and IIB and found that osteoclasts expressed the isoforms in distinct subcellular locations. Myosin IIA was enriched in dynamic cytoskeletal compartments, including the sealing zones of polarized and unpolarized osteoclasts. In contrast, myosin IIB was generally absent from these regions and maintained a comparatively static distribution during different phases of the osteoclast activation cycle. Inhibition of myosin II in osteoclasts by treatment with 2,3-butanedione monoxime caused detachment of unpolarized, but not polarized, cells from the bone matrix. These results suggest that myosin IIA is critical to development of an activated osteoclast phenotype.

Vacuolar H+-ATPase activity and expression in mouse bone marrow cultures.

We examined vacuolar H+-ATPase (V-ATPase) structure, enzymatic properties, and protein and mRNA expression from mouse marrow cultured in the presence or absence of 1,25-dihydroxyvitamin D3 (1, 25(OH)2D3), which stimulates formation of bone-resorptive osteoclasts. V-ATPases from osteoclast-containing cultures were similar in ion and inhibitor sensitivities to the enzyme from kidney-derived sources. Immunopurified V-ATPase from 1,25(OH)2D3-stimulated cultures exhibited 20-fold greater ATPase activity than the enzyme from unstimulated cultures, which do not contain osteoclasts. In contrast, 1,25(OH)2D3-treated cultures contained only 2-fold more assembled V-ATPase, as determined by immunoprecipitation. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunoblot analysis similarly showed approximately 2-fold increases of V-ATPase mRNA and protein levels in 1,25(OH)2D3-treated cultures. The bulk of the relative difference in V-ATPase activity between the two cultures was due to a 10-fold difference in enzyme specific activity. Quantitative RT-PCR also revealed that expression levels of V-ATPase mRNAs reflected the stoichiometry of enzyme subunits in the assembled complex. These data indicate that in mouse bone marrow cultures, V-ATPase expression is controlled at the level of mRNA, and that increases in subunit expression and assembly cannot account for the 20-fold difference in enzyme activity in osteoclast-containing cultures. Therefore, osteoclast V-ATPase activity may be regulated by subtle alterations in enzyme structure or associated factors.

A novel transcription factor regulates expression of the vacuolar H+-ATPase B2 subunit through AP-2 sites during monocytic differentiation.

During monocyte-to-macrophage differentiation, the cellular content of vacuolar H+-ATPase (V-ATPase) increases more than 4-fold. We have shown previously that amplified expression of the B2 subunit of the V-ATPase occurs solely by increased transcription, and that the 5'-untranslated region of the B2 gene, containing multiple consensus binding sites for the transcription factors AP-2 and Sp1, is required for this expression. The present study demonstrates that AP-2 binding sequences are essential for increased transcription from the B2 promoter during monocyte-macrophage differentiation and that AP-2, expressed exogenously in THP-1 and other cells, activates transcription from the B2 promoter. In mobility shift assays, a nuclear factor from THP-1 and U-937 cells was identified that binds to several AP-2 response elements within the B2 promoter, but does not react with AP-2 antibodies, and has a DNA sequence binding affinity profile that differs from AP-2. These findings suggest that a novel AP-2-like transcription factor is responsible for V-ATPase B subunit amplification during monocyte differentiation.

Osteoclasts express the B2 isoform of vacuolar H(+)-ATPase intracellularly and on their plasma membranes.

Osteoclasts express high levels of vacuolar H(+)-ATPase (V-ATPase) in their ruffled membranes, driving the secretion of H+ required for normal bone resorption. Previous reports have suggested that the B subunit of the osteoclast V-ATPase differs from those expressed in kidney and other tissues. In this study, B subunit isoform-specific antibodies and cDNA probes were used to examine which B subunit isoform is expressed in osteoclasts and osteoclast-like cells. Immunoblotting and RNA hybridization analysis were used to demonstrate that cells from an osteoclast-rich mouse bone marrow culture model express the B2 but not the B1 subunit isoform. Immunocytochemical staining of murine osteoclasts generated in vitro and of native rat osteoclasts in bone sections showed that the B2 but not the B1 isoform was expressed at high levels and was polarized to the ruffled membrane. Human marrow cultures and monocyte-derived macrophages, used as models for osteoclasts, also expressed the B2 but not the B1 subunit isoform. These results indicate that V-ATPases containing the B2 subunit isoform mediate osteoclast bone resorption.