Alpha_VBeta₃ (α_vβ₃) Integrin Drives the Osteoclastogenesis through a Osteoclast-Like Functional Differentiation of Myeloma Cells.

Accelerated osteoclastogenesis is the major event promoting the skeletal impairment in multiple myeloma (MM). Osteoclasts (OC) are directly activated by myeloma cells (MC), although these cells themselves may apparently undergo to OC-like morphologic transformation and produce bone erosion in vitro. Since OCs exert their function and promote osteoclastogenesis through activation of several adhesion molecules, including a_vb₃, we investigated the role of this integrin expressed by MCs in their OC-like activity in vitro. Bone marrow MCs were purified from eight patients with severe skeletal disease (group A) and from two patients without bone lesions (group B). U266 and RPMI-8226 MC lines were the controls. Semi-nested PCR assessed the CDR3 immunoglobulin (Ig) gene rearrangement, whereas OC markers including TRAcP, cathepsin-k, calcitonin-receptor, carbonic anhydrase and vATPase were evaluated by RT-PCR. The cytoskeletal rearrangement of F-actin was analyzed by immunofluorescence. a_v and b₃ expression on MCs was evaluated by flow-cytometry, whereas bone erosion on calcium phosphate discs and number of pits was measured by dedicated software. The effect of a_vb₃ stimulation on the activation of osteoclastogenic function was investigated by exploring the phosphorilation of transcriptional kinases and downstream molecules, as ERK1/2 and cFos, respectively. The primary role of a_vb₃ in OC-like functional transdifferentiation was explored in MCs by siRNA silencing for both chains. Ontogenetic derivation from the B-cell lineage was confirmed by the monoclonal CDR3 rearrangement, CD138/CD38 and Pax-5 expression. Cells from patients of group A expressed OC markers, in contrast with those of group B or U266 and RPMI-8226. Formation of the F-actin ring confirmed the differentiation of MCs toward the OC-like phenotype. Cells from group A expressed a_v and b₃ (80±7% and 75±9%) similarly to U266 and RPMI-8226 (>90% in both instances), whereas a minimal expression was demonstrated in group B (a_v:6±2%; b₃:8±3%). a_vb₃⁺ cells produced a high number of erosive pits, at variance from a_vb₃⁻ cells (35±8 vs. 4±1 pits/cm² ). The highest phosphorilation of ERK1/2 and expression of cFos was revealed in patients of group A as compared to B (840±110 OD and 905±210 OD vs. 270±35 OD and 315±80 OD, p<0.0001 in both instances). Finally, the silencing of a_v and/or b₃ chain inhibited the erosion by a_vb₃⁺ cells, resulting in a reduced number of pits (7±2 pits/cm²) similar to the pattern obtained with a_vb₃⁻ cells. Similarly, an inhibition of both ERK1/2 and cFos was demonstrated in silenced cells from group A with values similar to those of group B. Since a_vb₃ drives both adhesion of Ocs to extracellular matrix and activation of osteoclastogenesis, it is conceivable that a_vb₃⁺ MCs may functionally transdifferentiate to OCs in response to integrin activation induced by the contact with stromal cells within the marrow environment, thus resulting in a typical OC-like behaviour. In addition, our data suggest that MCs induce this effect by a_vb₃, since its silencing reduces the OC-like activity in vitro.