Effects of Human Myeloma Cells on WNT Signaling System in Human Bone Marrow Osteoprogenitor Cells.

Abstract

The mechanisms by which multiple myeloma (MM) cells inhibit osteoblast formation are not completely clear. Recently we have demonstrated that human myeloma cells block the critical osteogenic transcription factor RUNX2 in osteoprogenitor cells inhibiting the osteoblastogenesis. However several data indicate that WNT signaling is also important, at least in murine system, in the regulation of osteoblast formation. Given that MM cells produce the WNT inhibitor DKK-1 that correlated with the presence of bone lesions, we have investigated the potential effects of human myeloma cells on WNT signaling in human bone marrow (BM) osteoprogenitor cells.First we checked human myeloma cell lines (HMCLs) (XG-1, XG-6, RPMI-8226, U266, OPM-2, JJN3) and purified CD138+ MM cells for the expression of the WNT inhibitors DKK-1 and secreted Frizzled-related proteins (sFRP)-1,-2,-3,-4 finding that 50% of HMCLs and 70% of MM patients were positive for DKK-1. A similar pattern of expression was observed for the sFRP-3 whereas MM cells were negative either for sFRP-2 with the exception of U266 or for sFRP-1 and sFRP-4. Further, we performed a co-culture system with BM osteoprogenitor cells (PreOB), obtained after two weeks of differentiation and MM cells, in the presence or absence of a transwell system for 12–72 hours. After the co-culture period in the cell-to-cell contact condition MM cells were depleted to avoid their contamination. WNT signaling pathway was analyzed in PreOB by microarray using a specific Oligo GEArray® kit and further evaluated by RT-PCR and western blot analysis on selected molecules. Any effect on the expression of DKK-1,-2,-3,-4 as well as sFRP1,-2,-3,-4 by PreOB was not observed. these results was also confirmed for DKK-1 and sFRPs by western blot. An inhibitory effect on beta-catenin and on DKK-1 receptor LRP5 as well as an up-regulation of the osteoblast regulators WISP-1 and WISP-2 and of WNT5a have been observed. The study of beta-catenin signaling was deepened in both HMCLs and PreOB using either specific antibodies for the active de-phosphorylated form and inactive phosphorilated one or ELISA assay to evaluate the total levels of beta-catenin in both cytosolic and nuclear extracts. We failed to observe an inhibitory effect on nuclear levels of active beta-catenin and total beta-catenin in PreOB after co-culture in both cell-to-cell contact and transwell conditions by westernblot and immunofluorescence. In addition any significant effect on the transcription factors LEF-1 and TCF was not observed in PreOB cells after the co-culture using the array and western blot. On the other hand we found that active beta-catenin levels were completely down-regulated in both cytosolic and nuclear extracts in the majority of HMCLs and MM cells tested suggesting that canonical WNT signaling was completely inhibited in myeloma cells but not in PreOB. The involvement of WNT signaling in the inhibition of osteoblastogenesis by was further evaluated testing the effect Wnt3a and DKK-1 on beta-catenin levels and osteoblast formation in vitro. Wnt3 stimulation did not restore the inhibitory effect induced by MM cells in co-culture on bone nodule formation whereas DKK-1 showed an inhibitory effect on beta-catenin and bone nodule formation only at very high concentrations. Our data indicate that MM cells may affect WNT signaling in human osteoprogenitor cells even if we failed to observe a block of this pathways suggesting that other mechanisms could be involved in DKK-1 mediated bone destruction in MM.