Cloning and identification of annexin II as an autocrine/paracrine factor that increases osteoclast formation and bone resorption

Abstract

Autocrine products of osteoclasts such as interleukin-6 may play an important role in normal osteoclast formation and activity. To identify novel stimulatory factors for osteoclasts, we have prepared a mammalian cDNA expression library generated from highly purified human osteoclast-like multinucleated cells (MNC) formed in long term bone marrow cultures and screened this library for autocrine factors that enhance MNC formation. A candidate clone which stimulated MNC formation was isolated. 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 D3 and enhanced MNC formation in mouse bone marrow cultures treated with 10(-9) M 1,25-(OH)2 vitamin D3. The enhanced MNC formation in murine marrow cultures resulted in increased bone resorption. Treatment of fetal rat long bones with AXII and 1,25-(OH)2 vitamin D3 significantly increased bone resorption compared to 1,25-(OH)2 vitamin D3 alone. Reverse transcriptase polymerase chain reaction analysis demonstrated that AXII mRNA was expressed at high levels in RNA isolated from highly purified giant cells from osteoclastomas, human osteoclast-like MNC, and pagetic bone. Western blot analysis of conditioned media collected from human marrow cultures showed that AXII was present in the media. Furthermore, approximately 50% of total AXII produced by cells transfected with AXII cDNA was present in the conditioned media. These data suggest that the AXII is an autocrine factor that enhances osteoclast formation and bone resorption and demonstrate a previous unknown function for AXII.