Annexin II increases osteoclast formation by stimulating the proliferationof osteoclast precursors in human marrow cultures

Annexin II (AXII), a calcium-dependent phospholipid-binding protein, has be en recently found to be an osteoclast (OCL) stimulatory factor that is also secreted by OCLs. In vitro studies showed that AXII induced OCL formation and bone resorption. However, the mechanism of action by which AXII acts as a soluble extracellular protein to induce OCL formation is unknown. In thi s paper, we demonstrate that AXII gene expression is upregulated by 1,25-di hydroxyvitamin D-3 [1,25-(OH)(2)D-3] and that addition of AXII significantl y increased OCL-like multinucleated cell formation. Time-course studies sug gested that AXII acted on the proliferative stage of OCL precursors and tha t AXII increased thymidine incorporation in OCL precursors. Moreover, AXII enhanced the growth of CFU-GM, the earliest identifiable OCL precursor, whe n bone marrow cultures were treated with low concentrations of GM-CSF. This capacity of AXII to induce OCL precursor proliferation was due to inductio n of GM-CSF expression, because the addition of neutralizing antibodies to GM-CSF blocked the stimulatory effect of AXII on OCL formation. RT-PCR anal ysis using RNA from highly purified subpopulations of marrow cells demonstr ated that T cells, especially CD4(+) T cells, produced GM-CSF in response t o AXII. Furthermore, FACS(R) analysis of T-cell subpopulations treated with fluorescein-labeled AXII suggested that the CD4+, but not CD8(+), subpopul ation of T cells express an AXII receptor. Taken together, these data sugge st that AXII stimulates OCL formation by activating T cells through a putat ive receptor to secrete GMCSF. GM-CSF then expands the OCL precursor pool t o enhance OCL formation.