Interestingly, we also observed a higher population of adipocytes in the bone marrow of myeloma patients with the high levels of heparanase expression (Fig. 2). Primary calvarial osteoblast progenitor cells were cultured 1:1 in the

CM from HPSE-low or HPSE-high cells and osteogenic medium as indicated (Fig. 3A). Osteoblast differentiation (ALP staining) and mineralization (Von Kossa staining) were significantly suppressed by the CM of HPSE-high cells, compared to CM of HPSE-low cells. Conversely, significantly higher numbers of adipocytes were observed in cells cultured with HPSE-high CM versus HPSE-low CM (Fig. 3A), suggesting that the Tyrosine Kinase Inhibitor Library in vivo CM of HPSE-high cells supports the differentiation of mesenchymal progenitors toward the adipocyte lineage. To begin to understand the mechanism(s) involved in the suppression of osteoblastogenesis, we

next performed Western blot analysis. A significant decrease in Runx2 CT99021 expression (a marker of osteoblastogenesis) and increase in PPAR-γ expression (a marker of adipogenesis) were observed in HPSE-high CM treated cells, compared to HPSE-low CM treated cells (Fig. 3B). In a separate experiment, primary murine osteoblast progenitor cells were cultured in the CM of HPSE-low or HPSE-high cells with 1:1 adipocyte differentiation medium for 10 days. Oil Red O staining demonstrated a significant increase in adipocytes in the presence of HPSE-high CM, compared with HPSE-low CM (Fig. 3C). Taken together, these data suggest that HPSE-high myeloma cells secrete soluble factor(s) to inhibit osteoblastogenesis and promote adipogenesis, even if the cells are in a pro-osteoblastogenic environment in vitro. To identify what soluble factor(s) secreted by HPSE-high myeloma cells may be responsible for the decreased osteoblastogenesis and increased adipogenesis, we measured the levels of known regulators transforming growth factor beta (TGF-β), Bone Morphogenetic Protein 2 (BMP-2) and Dickkopf1 (DDK1) in the CM of HPSE-low and HPSE-high cells by ELISA. There was no significant

difference in the levels of TGF-β and BMP-2 in the CM of the two types of cell lines (data not shown). However, Tacrolimus (FK506) a significant increase in DKK1 was found in the CM of three different HPSE-high expressing cell lines, compared to the comparable HPSE-low cells (Fig. 4A). In addition, decreased β-catenin activation was observed in primary osteoblast progenitor cells cultured in the CM of HPSE-high cells (Fig. 4B), which was rescued by the addition of a potent and selective DKK1 inhibitor (Fig. 4B). These data demonstrate that the canonical Wnt signaling pathway was inhibited by increased DKK1 secretion from HPSE-high myeloma cells. We identified a high level of human heparanase uptake by murine C3H10T1/2 osteoblast precursor cells cultured in CM of human HPSE-high cells or in the presence of exogenous human rHPSE (Fig. 5A).