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C-reactive protein promotes bone destruction in human myeloma through the CD32–p38 MAPK–Twist axis

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Science Signaling  12 Dec 2017:
Vol. 10, Issue 509, eaan6282
DOI: 10.1126/scisignal.aan6282
  • Fig. 1 CRP enhances induction of bone lesions in myeloma-bearing mice.

    (A) Representative radiographic images of lytic lesions in the implanted human bones of primary myeloma-bearing severe combined immunodeficient (SCID)-hu mice before (0 w) or at week 4 (4 w) and week 8 (8 w) after injection of primary myeloma cells from one of five patients [Pt MM(multiple myeloma)] and treatment with phosphate-buffered saline (PBS), C-reactive protein (CRP) (20 μg per mouse), or serum amyloid P component (SAP) (20 μg per mouse). Tumor-free mice (no MM) served as controls. (B and C) Histomorphometric analysis of the osteolytic area (B) and bone volume density [(C); assessed as percentage of bone volume over total volume (BV/TV)] in the implanted human bones from the mice described in (A) at 8 weeks. (D) Representative radiographic images of lytic bone lesions in the distal femurs of SCID mice injected with one of two myeloma cell lines (ARP-1 or MM.1S) and treated with PBS or CRP (n = 10 mice per group). (E and F) Histomorphometric quantitative analysis of the femurs from the mice described in (D), assessing osteolytic area (E) and BV/TV (F). (G and H) Representative radiographic images of bone lesions (G) and histomorphometric quantitative analysis of osteolytic area (H) in the distal femurs of SCID mice (n = 10 per group) injected with CRP-expressing or vector-control myeloma cells. Arrows indicate osteolytic lesions in mouse femurs. (I) Micro–computed tomography scanning analysis of BV/TV in the femurs of SCID mice bearing CRP-expressing or vector-control myeloma cells, treated with neutralizing antibodies against CRP (αCRP) or control [immunoglobulin G (IgG)]. *P < 0.05, **P < 0.01 by Student’s t test. Images are representative and data are means ± SD of three independent experiments shown.

  • Fig. 2 CRP enhances osteoclastogenesis in myeloma-bearing mice.

    (A) Representative images of Tartrate-resistant acid phosphatase (TRAP)+ osteoclasts (OCs) in human trabecular bones from SCID-hu mice or 8 weeks after inoculation with patient myeloma cells (Pt 1) and treatment with CRP or PBS. Arrows indicate OCs in bone sections. No MM, tumor-free mice. Scale bars, 10 μm. (B and C) Histomorphometric quantitative analysis of (B) the number of OCs on the bone surface (Oc. S/BS) and (C) OC-mediated erosion of bone surface, expressed as a percentage of total bone surface (ES/BS), in the implanted human bones of SCID-hu mice that were either tumor-free (−) or bearing primary myeloma cells from one of five patients (Pt MM) and treated with CRP (+) or PBS (−). (D and E) Histomorphometric analysis of (D) Oc. S/BS and (E) ES/BS in the femurs of SCID mice (n = 10 per group) bearing myeloma cells and treated with CRP or PBS. (F) Oc. S/BS in the femurs of SCID mice bearing CRP-expressing (MM-CRP) or vector-control (MM-Vector) cells. (G) Oc. S/BS in the femurs of SCID mice bearing MM-CRP or MM-Vector cells treated with antibodies against CRP (αCRP) or control (IgG). Tumor-free mice (no MM) treated with agents or antibodies served as controls for baseline levels. Data from three independent experiments are shown. *P < 0.05, **P < 0.01 by Student’s t test.

  • Fig. 3 CRP enhances myeloma-induced OC formation and activity in vitro.

    (A and B) The number of multinuclear TRAP+ cells (A) and the amount of secreted TRAP5b (B) in cultures of OC precursor cells cultured alone or with either primary myeloma cells isolated from five myeloma patients (Pt MM) or with human myeloma cell lines (MM) in medium without receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) in the presence of CRP (5 μg/ml) or PBS. (C) Real-time polymerase chain reaction (PCR) analysis of the expression of CALCR, CTSK, and TRAP in mature OCs cocultured with OC precursors and ARP-1 or MM.1S cells with or without CRP (5 μg/ml). (D) TRAP staining for the number of multinuclear TRAP+ cells in cocultures of OC precursors with vector- or CRP-expressing ARP-1 or MM.1S cells (MM-Vector or MM-CRP) without RANKL. (E) As in (D), in the presence of antibodies against CRP (αCRP; 5 μg/ml) or control (IgG). *P < 0.05, **P < 0.01 by Student’s t test. Data from four independent experiments are shown.

  • Fig. 4 Knockdown of CD32 in myeloma cells reduces CRP-induced OC differentiation.

    (A) Western blot analysis for the abundance of CD32 in cultured wild-type (wt) ARP-1 or MM.1S cells and those transfected with nontargeted short hairpin RNA (shRNA) (Ctrl) or CD32 shRNA [CD32-knockdown (CD32-KD)]. β-Actin served as loading control. (B) The number of multinuclear TRAP+ cells in cocultures of OC precursors with control or CD32-deficient myeloma cells in the presence of CRP (5 μg/ml) or PBS. (C) Real-time PCR of gene expression in OCs cocultured with control or CD32-deficient ARP-1 myeloma cells and CRP (5 μg/ml) or PBS. (D to G) Histomorphometric analysis of (D) BV/TV, (E) Oc. S/BS, and immunohistochemical examination for (F) the percentage of CD138+ cells (myeloma marker) in the bone marrow and (G) staining for CD32 and CD138 by myeloma cells in bone sections of distal femurs from SCID mice (n = 10 per group) injected with Ctrl or CD32-KD ARP-1 cells. Mice receiving no CRP injection served as control in (D) and (E). Scale bar, 50 μm. ns, not significant. *P < 0.05, **P < 0.01 by Student’s t test. Data from three independent experiments are shown.

  • Fig. 5 Through myeloma cell CD32, CRP enhances the production of cytokines that mediate OC formation and activation.

    (A to D) Real-time PCR analysis of MCP-1, MIP-1α, and RANKL mRNA expression (A and B) and enzyme-linked immunosorbent assay (ELISA) analysis of secreted protein (C and D) in CD32-KD ARP-1 or MM.1S cells cultured with CRP (5 μg/ml) compared with those in nontarget control shRNA-transfected (Ctrl) cells. (E to H) Analysis of cytokine [monocyte chemoattractant protein–1 (MCP-1), macrophage inflammatory protein–1α (MIP-1α), and RANKL] abundance at the mRNA (E and F) and protein (G and H) levels in cultures of vector- or CD32-transfected (CD32) ARP-1 or MM.1S cells cultured with or without CRP (5 μg/ml). *P < 0.05, **P < 0.01 by Student’s t test. Data from three independent experiments are shown.

  • Fig. 6 Twist transcriptionally regulates CRP-induced cytokine expression in myeloma cells.

    (A) Heat map of the expression of transcriptional factors in myeloma cell lines treated with or without CRP (5 μg/ml) for 24 hours. (B) Chromatin immunoprecipitation (ChIP) assay for the interaction of Twist with the promoters of MCP-1 or RANKL using anti-Twist antibody or rabbit IgG. The input proteins served as controls. (C) Western blot for Twist abundance (top) and real-time PCR for MCP-1 and RANKL mRNAs in Twist-knockdown (Twist-KD) myeloma cells and nontargeted shRNA control (Ctrl) cells, cultured with or without CRP. (D and E) Luciferase activity in cultured ARP-1 (D) or MM.1S (E) cells transfected with a luciferase (Luc) reporter containing wild type, truncated mutants, or Twist binding site mutant RANKL or MCP-1 gene promoters. (F) Real-time PCR for gene expression in OC precursors cocultured with Twist-KD or control ARP-1 cells cultured with or without CRP. *P < 0.05, by Student’s t test. Data from four independent experiments are shown.

  • Fig. 7 Cross-linking CD32 by CRP activates p38 MAPK–Twist in myeloma cells.

    (A) Western blot for Twist abundance in ARP-1 or MM.1S cells treated with various doses of CRP for 24 hours. (B) Real-time PCR for TWIST mRNA levels in CD32-KD or control myeloma cells cultured with or without CRP. (C) Western blot analysis for dose-dependent effects of CRP on the phosphorylation (p) of p38 mitogen-activated protein kinase (MAPK), extracellular signal–regulated kinase (ERK), and NF-κB in myeloma cells. (D) Western blot analysis for effects of CRP on the phosphorylation of p38 MAPK in CD32-overexpressing (CD32) or knockdown (CD32-KD) ARP-1 cells compared with the respective controls. (E) Real-time PCR analysis of the expression of MCP-1, MIP-1α, and RANKL in ARP-1 cells cultured with or without CRP and the p38 MAPK inhibitor SB202190 (SB20). (F) Western blot analysis for the effect of SB20 on CRP-induced Twist abundance. *P < 0.05, **P < 0.01 by Student’s t test. Data from three independent experiments are shown.

  • Fig. 8 Relationship between serum CRP level and osteolytic bone lesion number in patients with newly diagnosed myeloma.

    (A) Correlation between serum CRP level and the number of lytic bone lesions (BLs) in 244 patients with newly diagnosed myeloma. CRP was measured by ELISA, and BLs were detected by radiography (x-ray or magnetic resonance imaging). (B) Representative images of immunohistochemical staining for CRP accumulation in the bone marrow of myeloma patients (three per group) with low (BL < 3) or high (BL ≥ 6) numbers of BLs. Scale bar, 50 μm. (C to E) Correlations between the level of circulating CRP and (C) MCP-1, (D) RANKL, and (E) CTx-1 in 28 randomly selected patients with newly diagnosed myeloma, calculated by Pearson’s correlation coefficient analysis.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/509/eaan6282/DC1

    Fig. S1. Injection of CRP does not affect tumor growth in vivo.

    Fig. S2. Generation of CRP-expressing myeloma cells.

    Fig. S3. CRP up-regulates myeloma cell production of cytokines important for OC activation.

    Fig. S4. Antibodies specific for OC-activating cytokines inhibit CRP-induced OC activation.

    Fig. S5. Knockdown or overexpression of CD32 does not affect myeloma cell proliferation or survival.

    Fig. S6. Overexpression of CD32 in myeloma cells enhances CRP-induced OC differentiation.

    Table S1. Primers used for real-time PCR.

  • Supplementary Materials for:

    C-reactive protein promotes bone destruction in human myeloma through the CD32–p38 MAPK–Twist axis

    Jing Yang,* Zhiqiang Liu, Huan Liu, Jin He, Jianling Yang, Pei Lin, Qiang Wang, Juan Du, Wencai Ma, Zheng Yin, Eric Davis, Robert Z. Orlowski, Jian Hou, Qing Yi*

    *Corresponding author. Email: yiq{at}ccf.org (Q.Y.); jiyang{at}mdanderson.org (J.Y.)

    This PDF file includes:

    • Fig. S1. Injection of CRP does not affect tumor growth in vivo.
    • Fig. S2. Generation of CRP-expressing myeloma cells.
    • Fig. S3. CRP up-regulates myeloma cell production of cytokines important for OC activation.
    • Fig. S4. Antibodies specific for OC-activating cytokines inhibit CRP-induced OC activation.
    • Fig. S5. Knockdown or overexpression of CD32 does not affect myeloma cell proliferation or survival.
    • Fig. S6. Overexpression of CD32 in myeloma cells enhances CRP-induced OC differentiation.
    • Table S1. Primers used for real-time PCR.

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    Citation: J. Yang, Z. Liu, H. Liu, J. He, J. Yang, P. Lin, Q. Wang, J. Du, W. Ma, Z. Yin, E. Davis, R. Z. Orlowski, J. Hou, Q. Yi, C-reactive protein promotes bone destruction in human myeloma through the CD32–p38 MAPK–Twist axis. Sci. Signal. 10, eaan6282 (2017).

    © 2017 American Association for the Advancement of Science

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