Cocultures of MM cells inhibit RANKL-induced OC differentiation and bone resorption activity.

<p>(<b>A</b>) Quantitative analysis of multinuclear TRAP⁺ OCs per well of 24-well plate generated from cocultures of monocytes, either isolated from the PBMCs of five and other (data not shown) healthy donors or generated from cocultures of myelomonocytic cell line Raw264.7 (RAW), with ARP-1 cells in medium containing RANKL (50 ng/mL). (<b>B</b>) Real-time PCR showing relative expression levels of OC differentiation-associated proteins CTSK, TRAP, CALCA, and CALCR in monocytes cocultured with ARP-1 or MM.1S cells in medium without (Medium) or with RANKL (50 ng/mL). mRNA levels of CTSK, TRAP, CALCA, and CALCR in monocytes cocultured with ARP-1 or MM.1S cells were reduced compared with those in monocytes cultured alone in medium with RANKL. (<b>C</b>) Western blot showing downregulated levels of phosphorylated (p) NF-κB, JNK, ERK, and p38, but not Akt, in monocytes cocultured with ARP-1 or MM.1S cells, compared with those in monocytes cultured alone in medium with RANKL. (<b>D</b>) Relative expression levels of NFATc1, c-fos, and PU.1 measured by real-time PCR in monocytes cocultured with ARP-1 or MM.1S cells. Cells cultured in medium without RANKL served as controls. The TRAP 5b activity of mature OCs generated from monocytes cocultured with primary MM cells from 10 MM patients (Pt1 to Pt10) (E) or MM cell lines (F) in medium with RANKL was reduced compared with that of monocytes cultured alone. Cocultured with MM cells in medium without RANKL served as control. Representative results from four independent experiments are shown. *<i>P</i> ≤ 0.05; **<i>P</i> ≤ 0.01.