Bone marrow stromal cells derived MCP-1 reverses the inhibitory effects of multiple myeloma cells on osteoclastogenesis by upregulating the RANK expression

Abstract

Multiple myeloma (MM) cells are responsible for aberrant osteoclast (OC) activation. However, when cocultured monocytes, but not OC precursors, with MM cells, we made a novel observation that MM cells inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced increase of OC differentiation, OC gene expression, signaling pathways and bone resorption activity. Our results showed that MM cells produced multiple inhibitory cytokines of osteoclastogenesis, such as IL-10, which activated STAT3 signaling and induce OC inhibition. However, cocultures of bone marrow stromal cells (BMSCs) reversed MM-induced OC inhibition. We found that MM cells increased production of MCP-1 from BMSCs and BMSC-derived MCP-1 enhanced OC formation. Mechanistic studies showed that IL-10 downregulated RANK expression in monocytes and thus, inhibited RANKL-induced OC formation. In contrast, MCP-1 upregulated RANK expression and thus, enhanced OC formation. Overall, our studies for the first time demonstrated that MM cell have inhibitory effects on osteoclastogenesis by producing inhibitory cytokines. Our results further indicate that activation of osteoclastogenesis in bone marrow requests the crosstalk of MM cells, BMSCs and their produced cytokines. Thus, our studies provide evidences that targeting bone marrow microenvironmental cells and/or cytokines may be a new approach to treating MM bone destruction.

Figure 1. MM cells inhibit mature OC formation from monocytes.

(A and B) Representative images of TRAP staining for mature OCs, defined as multinuclear TRAP+ cells, in cocultures of preOCs or monocytes without (Medium) or with primary MM cells isolated from a MM patient (Pt1) or ARP-1 cells in medium without or with RANKL (50 ng/mL). (C and D) Quantitative analysis of mature OCs generated from cocultures of monocytes with primary MM cells isolated from 10 MM patients (Pt1 to Pt10) or six MM cell lines in medium without or with RANKL. Numbers of multinuclear TRAP+ OCs per well of 24-well plate were counted. Representative results from five independent experiments are shown. *P ≤ 0.05; **P ≤ 0.01.

Figure 2. Cocultures of MM cells inhibit RANKL-induced OC differentiation and bone resorption activity.

(A) 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) 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. (C) 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. (D) 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. *P ≤ 0.05; **P ≤ 0.01.

Figure 3. Identification of cytokines that regulate osteoclastogenesis.

(A) Quantitative analysis of multinuclear TRAP⁺ OCs generated from monocytes cocultured with ARP-1 or MM.1S cells with cell-cell contact (cell-cell) or separated by transwell inserts (Trans) or cultures of monocytes in medium with the addition of conditioned medium (CM) and RANKL (50 ng/mL). Monocyte cultures in medium without RANKL served as controls.(B and C) Representative images (B) and densitometric Data (C) of a cytokine array showing the profile of cytokine expression in medium with no cell culture (Medium) and conditioned medium collected from cultures of ARP-1 cells. Increased levels of cytokines in ARP-1-conditioned medium (indicated by boxes) compared with those in medium with no cell culture are shown. (D) Representative images of a cytokine array showing the profile of cytokine expression in conditioned medium collected from cultures of BMSCs alone or cocultures of BMSCs and ARP-1 cells. Increased levels of cytokines in the conditioned medium from cocultures of BMSCs and ARP-1 cells (ARP-1+BMSCs) compared with those in conditioned medium from cultures of BMSCs alone are shown. In panel B and D, IL-10 was marked in red, while other cytokines were marked in black. (E) Densitometric data of the array showing the profile of cytokine expression in conditioned medium with no cell culture, cultures of ARP-1 cells alone, cultures of BMSCs alone, or cocultures of ARP-1 and BMSCs (MSC). Cocultured with MM cells in medium without RANKL served as control. Representative results from three independent experiments are shown.*P ≤ 0.05; **P ≤ 0.01.

Figure 4. MM-derived cytokines inhibit osteoclastogenesis.

(A) Reverse transcription PCR showing mRNA expression of IL-8, IL-10, MCP-1, VEGF, IFN-γ, and thrombopoietin (TPO) in three normal B cells isolated from healthy donors, primary MM cells isolated from six MM patients (Pt1-Pt6) or six MM cell lines (ARP-1, MM.1S, RPMI8266, U266, ARK and CAG). (B) Secreted IL-10 and IL-8 levels in B cells from healthy donors, primary MM cells from six MM patients (PtMM) and six MM cell lines (MM lines), determined by ELISA. (C) ARP-1 cells produced higher levels of secreted IL-10 than those produced by U266 cells; the levels of secreted IL-8 produced by the two cell lines were similar. (D) The numbers of multinuclear TRAP⁺ OCs generated from cocultures of monocytes with ARP-1 cells were lower than those generated from cocultures of monocytes with U266 cells (with the addition of RANKL for both), as determined by TRAP staining. (E) The numbers of multinuclear TRAP⁺ OCs generated from cultures of monocytes with the addition of RANKL and recombinant IL-10, but not IL-8, were significantly lower than those generated from cultures with the addition of RANKL only, as determined by TRAP staining. The numbers of multinuclear TRAP⁺ OCs generated from cultures of monocytes in medium containing RANKL, ARP-1-conditioned medium (CM), and neutralizing antibodies against IL-10 (αIL-10) but not IL-8 (αIL-8), were higher than those generated from the control cultures containing control IgG (Ctrl IgG). Representative results from five independent experiments are shown. *P ≤ 0.05; **P ≤ 0.01.

Figure 5. IL-10 inhibits osteoclastogenesis via STAT3 activation.

(A) Western blot showing upregulated levels of phosphorylated STAT3 (pSTAT3) and (B) downregulated expression of phosphorylated NF-κB (pNF-κB) in monocytes cultured with the addition of ARP-1 conditioned medium (CM) or recombinant IL-10 and reduced levels of pSTAT3 in monocytes cultured with the addition of ARP-1 CM and neutralizing antibodies against IL-10 (αIL-10), compared with those in monocytes cultured with the addition of ARP-1 CM and control IgG (Ctrl IgG). β-actin served as a protein loading control. (C) Real-time PCR showing enhanced expression of OC differentiation-associated proteins CTSK, TRAP, CALCA, and CALCR in monocytes cultured with the addition of RANKL, ARP-1 CM and STAT3 inhibitors (STAT3 inh), compared with those in monocytes cultured with RANKL and ARP-1 CM only. (D) TRAP staining showing enhanced numbers of multinuclear TRAP⁺ OCs, generated from cultures of monocytes in medium without or with RANKL, ARP-1 CM, and/or IL-10, in the presence or absence of STAT3 inhibitors (STAT3 inh), compared with those in cultures without STAT3 inhibitors. Representative results from four independent experiments are shown. *P ≤ 0.05; **P ≤ 0.01.

Figure 6. BMSC-derived MCP-1 enhances osteoclastogenesis.

TRAP staining showing increased numbers of multinuclear TRAP⁺ OCs generated from cocultures of monocytes with BMSCs and either primary MM cells (PtMM) isolated from six MM patients (A) or ARP-1 cells (B) in medium with RANKL, compared with those in cultures with MM cells only or with BMSCs only. BMSCs were generated from bone marrow aspirates of MM patients or generated from human healthy fetal bones. Similar results were obtained for cocultures with MM.1S and other MM cell lines. (C) The levels of secreted MCP-1 in conditioned medium from cocultures of ARP-1 cells and BMSCs were higher than those from cultures of ARP-1 cells alone or cultures of BMSCs alone. Similar results were obtained for conditioned medium from cocultures of MM.1S cells and BMSCs. The numbers of multinuclear TRAP⁺ OCs per well of 24-well plate as determined by TRAP staining (D) and the expression levels of OC-associated proteins CTSK and TRAP, determined by real-time PCR, in OCs generated from cocultures of monocytes with ARP-1 cells and BMSCs in medium containing RANKL (E) were higher than those in cocultures of monocytes with ARP-1 cells only or with BMSCs only. The numbers of OCs (D) and the expression levels of CTSK and TRAP in OCs generated from cocultures of ARP-1 and BMSCs with the addition of RANKL and neutralizing antibodies against MCP-1 (αMCP-1) (E) were lower than with those from cocultures containing control IgG (Ctrl IgG). Similar results were obtained for cocultures of monocytes with BMSCs and MM.1S cells. Representative results from four independent experiments are shown. **P ≤ 0.01.

Figure 7. Regulation of RANK expression by IL-10 and MCP-1 affects osteoclastogenesis.

Real-time PCR results for RANK mRNA levels (A, D) and flow cytometry results for RANK surface protein levels (B, E) in monocytes cultured in medium containing recombinant IL-10 (A, B) or MCP-1 (D, E). The levels of surface RANK on monocytes were reduced in cultures with the addition of conditioned medium (CM) of ARP-1 or MM.1S cells (C) and increased in cultures with the addition of CM from cocultures of BMSCs and either ARP-1 cells or MM.1S cells (F) compared with those in cultures without MM CM or in cultures without CM of BMSCs and MM cells, as determined by flow cytometry. In addition, the levels of surface RANK on monocytes were increased in cultures with the addition of CM of ARP-1 or MM.1S cells and neutralizing antibodies against IL-10 (αIL-10) (C) and reduced in cultures with the addition of CM of BMSC and ARP-1 or MM.1S cells and neutralizing antibodies against MCP-1 (αMCP-1) (F) compared with those in cultures containing control IgG (Ctrl IgG). Representative results from three independent experiments are shown. **P ≤ 0.01.