Opposing SMAD-6 and SMAD-7 Expression Profiles Correlate with the Sensitivity of Multiple Myeloma Cells to Bone Morphogenetic Protein (BMP)-2

Abstract

Objective: Multiple myeloma (MM) is a hematologic disease characterized by hyper-proliferation of antibody secreting B-cells. One of the most severe accompanying symptoms in MM is the destruction of bone tissue. Thus, the use of bone morphogenetic proteins (BMPs) for MM treatment seems to be a good option since these proteins are capable to induce formation of bone tissue in vivo and additionally can suppress cell proliferation of malignant Bcells. But, the different BMP family members vary in their biological activities also in terms of donor specific differences of addressed cells. In this study we analyzed signal transduction of two different TGFβ family members, BMP2 and GDF5, in different MM cell lines.Methods: Ten MM cell lines were stimulated either with BMP2 or GDF5 and cell proliferation was analyzed by WST-1 assays. Receptor expression levels were determined by qRT-PCR for relevant BMP receptors. SMAD-1/5/8 phosphorylation was analyzed by Western blot and correlated to the expression levels of inhibitory (i)-SMADs, SMAD-6 and SMAD-7 proteins, respectively.Results: Only three out of ten investigated cell lines were BMP2 responsive, one of which was additionally sensitive to GDF5. Depending on the expression of the required receptors the different cell lines could be divided into three subgroups. The first group expressed all receptor chains which are crucial for proper signal transduction and was ligand sensitive, the second also expressed the required receptors, but appeared ligand-resistant. The third subgroup instead missed at least one or more essential receptor rendering these cells also resistant to ligand exposure. Western blot analyses addressing phosphorylated SMAD-1/5/8 proteins revealed that the second group showed no or at least extremely reduced levels of SMAD-1/5/8 phosphorylation levels upon ligand exposure. Furthermore, Western blot analyses also showed that non-responsive cells expressed the inhibitory SMAD-7 protein at high levels prior to ligand stimulation. In contrast, the BMP2 responsive cells did not express SMAD-7 but instead expressed SMAD-6 at high levels prior to ligand stimulation.Conclusion: Aside of the expression of essential receptors, further factors are decisive for proper BMP signal transduction in MM cells such as the individual expression levels of SMAD-6 and SMAD-7 in unstimulated cells. The conspicuous opposing basal expression levels of either SMAD-6 or SMAD-7 might be used as prediction whether a particular MM cell line appears ligand-responsive or ligand-resistant.