Notch- and TNFα-Activated Signaling Pathways Mediate Osteocyte Apoptosis Triggered By Multiple Myeloma Cells

Osteocytes comprise 95% of all bone cells and are central regulators of bone homeostasis and skeletal integrity. However the role of osteocytes in MM bone disease is unknown. We have previously shown that interactions with multiple myeloma cells have a profound effect on osteocytic gene expression, increasing Sost and RANKL transcripts and decreasing OPG. More recently, we and others have shown that myeloma cells increase the prevalence of osteocyte apoptosis, which might contribute to MM induced bone disease by increasing resorption in specific areas of bone. However, the mechanisms responsible for and the potential for targeting osteocyte apoptosis in myeloma are unknown. Osteocytes extensively communicate with each other and with cells on the bone surface and in the marrow, through cytoplasmic connections that run within canaliculi, which allows direct cell-to-cell contact and the distribution of secreted molecules among all bone and marrow cells including myeloma cells. Therefore, to explore the mechanism underlying osteocyte apoptosis induced by myeloma cells we used a co-culture system that allows both cell-to-cell contact and exchange of soluble factors between osteocytic MLO-A5 cells and the human JJN3 MM cell line or primary CD138+ cells isolated from MM patients. Osteocyte apoptosis was quantified by trypan blue uptake and chromatin condensation/nuclear fragmentation in the absence or presence of the caspase3 specific inhibitor DEVD. We found that osteocyte apoptosis was increased 2-3 fold when osteocytes were co-cultured with JJN3 cells compared to osteocytes cultured alone. Apoptosis occurred within 8h of co-culture and gradually increased for up to 48h. Osteocyte apoptosis was completely inhibited by DEVD. Importantly, co-culture with primary CD138+ MM cells from 5 different patients also increased osteocyte apoptosis, which was also blocked by DEVD. We next determined if direct cell-to-cell contact was required for MM cells to induce osteocyte apoptosis and whether Notch signaling, a signaling pathway with profound effects on the skeleton activated by such interactions, was involved. We found that osteocytes co-cultured with JJN3 cells exhibited 3-7 fold higher levels of expression of the Notch target genes Hes1 and Hey1, detected as early as 4h and maintained up to 48h of co-culture. The pharmacological specific Notch inhibitor GSIXX completely blocked osteocyte apoptosis induced by either JJN3 cells or primary CD138+ MM cells measured at 8h and 24h. Moreover, cultures of osteocytes grown on plates coated with the Notch ligand Delta 1 fused to IgG2 exhibited 2-5 times higher levels of apoptosis compared to osteocytes cultured on IgG2 control, and this effect was inhibited by GSIXX. In addition, overexpression of the Notch intracellular domains 1 or 2, known to activate Notch signaling, increased osteocyte apoptosis by 2 fold. These findings demonstrate that rapid activation Notch signaling in osteocytes triggered by direct cell-to-cell contact with myeloma cells induces osteocyte apoptosis. Interestingly, Notch inhibition by GSIXX only partially prevented osteocyte apoptosis induced by JJN3 cells measured at 48h, suggesting the involvement of other mechanisms. To further investigate this finding, we measured soluble levels of TNFα in JJN3 cultures since TNFα is a recognized inducer of osteocyte apoptosis. The levels of TNFα secreted by JJN3 cells increased 5 fold (from 2 to 9 pg/ml) during 4 to 48h of culture. Conditioned medium (CM) from JJN3 cells cultured alone for 48h increased osteocyte apoptosis, and this effect was blocked by DEVD and by a neutralizing anti-human TNFα antibody, but not by GSIXX. Moreover, combination of GSIXX and anti-TNFα antibody completely inhibited osteocyte apoptosis induced by co-culture with JJN3 cells, while each agent added separately only partially inhibited osteocyte apoptosis measured at 48h. These results demonstrate that direct interactions with MM cells induces caspase3-dependent osteocyte apoptosis, triggered by rapid activation of Notch signaling through cell-cell contact and is maintained by accumulation of MM-derived TNFα. Our findings suggest both Notch and TNFα signaling pathways are potential targets to reverse or prevent myeloma induced osteocyte apoptosis.