CLONING AND IDENTIFICATION OF ANNEXIN-II AS AN AUTOCRINE PARACRINE FACTOR THAT INCREASES OSTEOCLAST FORMATION AND BONE-RESORPTION/

Autocrine products of osteoclasts such as interleukin-6 may play an im portant role in normal osteoclast formation and activity, To identify novel stimulatory factors for osteoclasts, we have prepared a mammalia n cDNA expression library generated from highly purified human osteocl ast-like multinucleated cells (MNC) formed in long term bone marrow cu ltures and screened this library for autocrine factors that enhance MN C formation. A candidate clone which stimulated MNC formation was isol ated. Sequence analysis showed that this cDNA encoded annexin II (AXII ). Purified recombinant AXII significantly increased MNC formation in human bone marrow cultures in the absence of 1,25-(OH)(2) vitamin D-3 and enhanced MNC formation in mouse bone marrow cultures treated with 10(-9) M 1,25-(OH)(2) vitamin D-3. The enhanced MNC formation in murin e marrow cultures resulted in increased bone resorption. Treatment of fetal rat long bones with AXII and 1,25 (OH)(2) vitamin D-3 significan tly increased bone resorption compared to 1,25-(OH)(2) vitamin D-3 alo ne. Reverse transcriptase polymerase chain reaction analysis demonstra ted that AXII mRNA was expressed at high levels in RNA isolated from h ighly purified giant cells from osteoclastomas, human osteoclast-like MNC, and pagetic bone. Western blot analysis of conditioned media coll ected from human marrow cultures showed that AXII was present in the m edia. Furthermore, approximately 50% of total AXII produced by cells t ransfected with AXII cDNA was present in the conditioned media. These data suggest that the AXII is an autocrine factor that enhances osteoc last formation and bone resorption and demonstrate a previous unknown function for AXII.