PURPOSE: Prostate cancer specifically metastasizes to bone where it leads to bone formation. We previously reported that coculturing MDA PCa 2b prostate cancer cells with primary mouse osteoblasts (PMOs) induced PMO proliferation and differentiation. An osteoblastic reaction was also observed in vivo after injection of MDA PCa 2b cells into the bones of severe combined immunodeficient disease mice. The aim of this study was to identify the sequence of events that leads to these osteoblastic lesions in vivo and, using this in vitro model, to define the contributions of various genes and cellular pathways in the pathophysiology of osteoblastic bone metastases of prostate cancer. EXPERIMENTAL DESIGN AND RESULTS: We show histological evidence of de novo bone formation as early as 2 weeks after injection of MDA PCa 2b cells in the bone of severe combined immunodeficient disease mice. In vitro, we show that PMOs induce MDA PCa 2b proliferation, suggesting a synergistic paracrine loop between these cells and PMOs. Endothelin (ET)-1, which is a mitogen for several cell types, is produced by all prostate cancer cell lines tested, and Atrasentan, an antagonist of ET-1 receptor A, partially reversed PMO proliferation induced by MDA PCa 2b cells. ET-1 is known to be comitogenic with a number of growth factors, including insulin-like growth factor (IGF)-I. In this study, we report that IGF-binding protein (IGFBP)-3 transcripts (that regulate levels of free IGF) are down-regulated in prostate cancer cells cocultured with PMO, whereas prostate-specific antigen (a protease known to cleave IGFBP-3) is detected in the 150-400 ng/ml range. Accordingly, IGFBP-3 has antiproliferative effects in PMOs, which were attenuated in our in vitro system. Taken together, our studies also implicate the IGF axis to play a role in this model of bone metastases. Secondly, the transcript level of mouse double minute 2 (a protein that regulate p53) was increased in prostate cancer cells grown with PMOs. The p53-dependent and -independent oncogenic activities of mouse double minute 2 suggest that osteoblasts induce a survival advantage in prostate cancer cells. Lastly, we show that expression of osteoprotegerin is decreased and of receptor activator of nuclear factor-kappaB ligand is increased in PMOs cultured in the presence of MDA PCa 2b cells, two events associated with osteoclast activation and bone resorption. CONCLUSIONS: Our results provide evidence that multiple and distinct molecular events affecting both bone formation and bone resorption concur to the increase bone mass in prostate cancer bone metastases. These data also provide a rationale for developing therapeutic strategies designed to target these molecular changes.