Activation of Non-Canonical Wnt Pathway in Human Mesenchymal Cells Affects Osteogenic Differentiation: A Potential Target in Multiple Myeloma Microenvironment

Osteoblast suppression is the hallmark of Multiple Myeloma (MM) osteolytic bone lesions mainly due to the capacity of MM cells to inhibit the osteogenic differentiation of bone marrow (BM) mesenchymal cells (MSC). Many evidences suggest that Wnt signaling is critically involved in the regulation of osteoblast formation. Recently, in murine osteoprogenitor cells and in MM mouse models it has been shown that activation of canonical Wnt pathway stimulate osteoblast formation and blunts MM-induced bone destruction. In this study we have investigated whether modulation of both canonical and noncanonical Wnt signaling pathway may affect osteogenic differentiation of human MSC and counterbalance the suppressive effect of MM cells. First we checked the potential expression of Wnt activators and inhibitors by human MSC and osteoprogenitor cells (PreOB) by gene arrays. We found that both cells expressed the activator of non-canonical Wnt pathways Wnt5a but lack of express the main activators of canonical Wnt signaling as Wnt1, Wnt3a and Wnt8. The presence of the Wnt5a receptor FZD2 and FZD5 was also detected in both cells as well as that of FZD3, FZD6 and FDZ7 and the Wnt canonical co-receptors LRP5 and LRP6. On the other hand we found that both inhibitors of canonical and non-canonical Wnt pathways DKK-1 and sFRP-1 were expressed by MSC. Secondly, activation of either canonical or non-canonical Wnt signaling pathway by Wnt3a and Wnt5a treatment respectively was performed in human MSC to evaluate the effect on osteogenic differentiation and the expression of osteoblast related markers (Collagen I, Osteocalcin and Alkaline Phosphatase). We found that Wnt5a treatment but not Wnt3a significantly increased the early osteogenic differentiation and the expression of alkaline phosphatase in MSC. Consistently in a co-culture system with MM cells Wnt5a treatment blunted, at least in part, the inhibitory effect of MM cells on alkaline phosphatase expression by MSC and PreOB. To go further inside, we evaluated in both primary human BM MSC and the human MSC cell line HS-5 the effect of either the activation of non-canonical Wnt signaling by Wnt5a overexpression using a lentivirus vector or the Wnt5a suppression using siRNA. Wnt5a over-expression in MSC induced the activation of Wnt/Ca⁺⁺ non-canonical pathway as demonstrated by the increase of Wnt5a secretion and phospho-PKC expression detected by westernblot analysis. Consequently to non-canonical Wnt signal activation we found a significant increase of alkaline phosphatase expression by MSC cells as well as of their osteogenic differentiation. Interestingly, analyzing the gene expression profile by microarray, we found that Wnt5a overexpression in MSC also affects the expression of chemokines, inflammatory cytokines and pro-angiogenic molecules. In conclusion our data indicate that activation of non-canonical Wnt signal pathway may represent a potential target in MM microenvironment to counterbalance the inhibitory effect of MM cells on osteogenic differentiation of human MSC.