Knockdown of macrophage inhibitory cytokine-1 in RPMI-8226 human multiple myeloma cells inhibits osteoclastic differentiation through inhibiting the RANKL-Erk1/2 signaling pathway

  • Authors:
    • Mingzhou Yuan
    • Junmin Chen
    • Zhiyong Zeng
  • View Affiliations

  • Published online on: October 24, 2016     https://doi.org/10.3892/mmr.2016.5879
  • Pages: 5199-5204
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Patients with multiple myeloma (MM) often develop myeloma bone disease (MBD). The development of MBD from MM is considered to be caused by an abnormal bone marrow microenvironment. Macrophage inhibitory cytokine-1 (MIC-1) is a member of the transforming growth factor‑β superfamily. In patients with MM, MIC‑1 is expressed at high levels, however, whether this increased expression of MIC‑1 is associated with the development of MBD from MM remains to be elucidated. The present study investigated whether MIC‑1 is essential for the osteoclastic differentiation of peripheral blood mononuclear cells (PBMNCs) by using a co‑culture system, in which the PBMNCs were co‑cultured with RPMI‑8226 cells. The expression of MIC‑1 in the RPMI‑8226 cells was knocked down using RNA interference. Osteoclastic differentiation was evaluated using tartrate‑resistant acid phosphatase staining and lacunar resorption on dentine slices. The expression of receptor activator of nuclear factor‑κB ligand (RANKL) and phosphorylation of extracellular signal‑regulated kinase (Erk)1/2 were measured using Western blotting. It was found that the reduced expression of MIC‑1 in the RPMI‑8226 cells inhibited the osteoclastic differentiation of PBMNCs and decreased the expression levels of RANKL and phosphorylated Erk1/2. It was concluded that MIC‑1 promoted the osteoclastic differentiation of PBMNCs via the RANKL‑Erk1/2 signaling pathway and, therefore, MIC‑1 may offer potential as a target in the design of strategies to treat MBD.

Introduction

Multiple myeloma (MM) is a malignant cancer of plasma cells, which represent ~13% of all hematological malignancies (1), with >80% of patients with MM developing myeloma bone disease (MBD). MBD is characterized by severe bone pain, vertebral compression fractures and pathological fractures, which are caused by osteoclastic bone resorption and impaired osteoblastic bone formation induced by the interaction between myeloma cells and the bone marrow microenvironment. However, the molecular mechanism by which MM leads to the development of MBD remains to be elucidated.

Genes that are expressed at high levels in the bone marrow of patients with MM have been identified (2), one of which is macrophage inhibitory cytokine-1 (MIC-1). MIC-1 belongs to the human transforming growth factor β superfamily. Also known as growth differentiation factor-15, placental bone morphogenetic protein, prostate-derived factor and NSAID-activated gene-1 (36), MIC-1 has multiple functions. In a previous in vitro study, MIC-1 was shown to inhibit the activation of macrophages (7). MIC-1 may be an important factor in the development of several types of tumor, including MM (8). In patients with MM, MIC-1 is expressed at high levels in the mesenchymal stem cells of the bone marrow, and high levels of MIC-1 in the patients' serum predicts a poor prognosis (2). MIC-1 may be involved in osteoclastogenesis; in patients with prostate cancer with bone metastases, MIC-1 has been found to induce the maturation of osteoclasts (9). However, whether MIC-1 is involved in the development of MBD in patients with MM remains to be elucidated. In present study, in order to evaluate the effect of target inhibition of MIC-1 in RPMI-8226 cells on osteoclastic differentiation of peripheral blood mononuclear cells (PBMNCs) and the mechanisms by which MIC-1 contributes to the maturation of osteoclasts, a lentiviral RNAi system directed toward the MIC-1 gene was designed and constructed in RPMI-8226 cells. A co-culture system was used in the present study to determine the role of MIC-1 on osteoclastic differentiation. The results from the present study offered a potential strategy for the treatment of MBD in patients with MM.

Materials and methods

Preparation of the lentiviral vector bearing MIC-1 short hairpin (sh)RNA

To understand the potential effect of MIC-1 in RPMI-8226 cells, an MIC-1 shRNA was designed to specifically knock down the gene expression of MIC-1. The MIC-1 shRNA was designed by Genechem (Shanghai, China), based on the MIC-1 cDNA sequence (GenBank accession no. AF019770.1; http://www.ncbi.nlm.nih.gov/nuccore/AF019770.1). A control shRNA encoding a nonspecific shRNA was used as a negative control. The MIC-1 shRNA and negative control shRNA were synthesized by Genechem (Shanghai, China). The synthesized oligonucleotides were designed, synthesized and inserted into a GV115 vector (GeneChem) to construct a lentiviral vector, according to the manufacturer's protocol, to generate lentiviral transfer plasmids: Lv-shRNA, bearing MIC-1 shRNA, and Lv-NC, bearing control shRNA as a negative control. The control shRNA had no significant homology to any human gene sequence. The lentiviral vectors were prepared by transfecting the Lv-shRNA or Lv-NC with packaging plasmids into 293T cells, as described previously (10).

Cell culture and lentiviral transduction

The RPMI-8226 cells (Wuhan University, Wuhan, China) were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere containing 5% CO2. After 3 days, the RPMI-8226 cells were transduced with the lentiviral vectors at a multiplicity of infection of 15 in the presence of 2 µg/ml polybrene (Sigma-Aldrich, St. Louis, MO, USA). Following incubation at 37°C for 8 h, the cells were washed twice with phosphate-buffered saline (PBS), and the mRNA and protein levels of MIC-1 in the cells were measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analyses, respectively, to evaluate the viral infection efficiency.

RT-qPCR

Total RNA was extracted from the cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The RNA (4 µg) was reverse transcribed into cDNA using Thermoscript RT-PCR System reagent (Gibco; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol, prior for use in qPCR. The qPCR was performed on an Applied Biosystems PRISM 7300 sequence detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using a SYBR Green PCR master mix (DRR041A; Takara Bio, Inc., Tokyo, Japan). A total volume of 20μl, containing 2μl cDNA, 10μl SYBR Premix Ex Taq, 0.4μl Forward Primer, 0.4μl Reverse Primer, 0.4μl ROX Reference Dye II and 6.8μl dH2O, was used in the q-PCR reaction. Each sample was analyzed in triplicate. The quantification cycle (Cq) value of each sample was normalized to an endogenous control, GAPDH. The 2−ΔΔCq method was used to relatively quantify the mRNA levels of MIC-1 (11). The primer (Genechem) sequences for human MIC-1 were: Sense 5′-GTTGCGGAAACGCTACGA−3′ and antisense 5′-AACAGAGCCCGGTGAAGG-3′. Primers for the control (GAPDH) were: Sense 5′-TGACTTCAACAGCGACACCCA-3′ and antisense 5′-CACCCTGTTGCTGTAGCCAAA-3′. The qPCR program was as follows: 45 cycles of 95°C for 15 sec, 60°C for 30 sec and 72°C for 45 sec, followed by 72°C for 10 min.

Isolation and culture of PBMNCs

The PBMNCs were isolated, as described previously (10) from the peripheral blood of healthy donors, who had provided written informed consent. The present study was approved by the Ethics Committee of Fujian Medical University. A total of 10 ml human blood was obtaubed from peripheral blood samples, and was mixed with 10 ml lymphocyte separation medium (Hao Yang Biological Manufacture Co. Ltd., Tianjin, China) in a centrifuge tube. The mixture was centrifuged at 2,000 × g at room temperature for 20 mins. Following washing with PBS once and α-minimal essential medium (α-MEM) twice, the nucleated cells were grown in α-MEM supplemented with 10% (v/v) fetal bovine serum (FBS), 50 ng/ml macrophage-colony-stimulating factor (M-CSF; PeproTech, Rocky Hill, NJ, USA) and 100 ng/ml receptor activator of nuclear factor-кB ligand (RANKL; Peprotech) at 37°C in 5% CO2 humidified air. The medium was replaced every 3 days.

Co-culture system

To investigate the potential effect of MIC-1 on the differentiation capacity of the PBMNCs, a Transwell insert system (Corning, Inc., New York, NY, USA) was used (Fig. 1) to co-culture the PBMNCs (1×106 cells/well) and the RPMI-8226 cells (2×103 cells/well) transduced with Lv-shRNA or Lv-NC. The PBMNCs were seeded into the lower compartment following placement of a cover glass and dentine slides on the bottom. The cells were cultured in α-MEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS, 50 ng/ml M-CSF and 100 ng/ml RANKL. After 12 h of culture at 37°C in 5% CO2, the non-adherent cells in the culture were removed by gently washing with fresh medium; the RPMI-8226 cells transduced with Lv-shRNA or Lv-NC were cultured in the upper compartment. RPMI-8226 cells without viral transduction were also used as a control.

Tartrate-resistant acid phosphatase (TRAP) staining and resorption pit formation assay

The PBMNCs (1×106 cells/well), which were cultured on cover glasses and dentine slides for 14 or 21 days, were stained using a leukocyte acid phosphatase kit (Sigma-Aldrich), and the cells were then counterstained with hematoxylin (Sigma-Aldrich), as previously described (10). A mature osteoclast was identified as a cell with at least three TRAP-positive nuclei. To determine the number of mature osteoclasts in each group, 10 randomly selected fields from the three cultures in each group were examined under a microscope (BX43; Olympus, Tokyo, Japan). For the resorption pit assay, the cells on dentine slices were stained with toluidine blue and then scanned under a microscope (BX43; Olympus). Osteoclastic bone resorption was measured, based on the area of resorption pits per field from three cultures in each group.

Western blot analysis

The PBMNCs were harvested after co-culture for 14 days for protein analysis. The protein extracts from these cells were separated by 6–15% SDS-PAGE gel electrophoresis and then transferred onto polyvinylidene fluoride (PVDF; Beyotime Institute of Biotechnology, Shanghai, China) membranes. Following blocking with Tris-buffered saline with 0.2% Tween (TBST) containing 5% non-fat milk at 4°C overnight, the membranes were incubated with the following antibodies diluted in TBST for 2 h at 4°C: Rabbit anti-RANKL (cat no. sc-9073; 1:2,000), rabbit anti–GAPDH (cat no. sc-25778; 1:2,000), rabbit anti-c-fos (cat no. sc-52, 1:2,000), rabbit anti-c-Jun N-terminal kinase (c-JNK; cat no. sc-572; 1:2,000), rabbit anti-p-c-Jun N-terminal kinase (p-c-JNK; cat no. sc-135642; 1:2,000), rabbit anti-P-38 (cat no. sc-535; 1:2,000), rabbit anti-p-P-38 (cat no. sc-101759; 1:2,000), rabbit anti-c-jun (cat no. sc-44; 1:2,000), rabbit anti-p-c-jun (cat no. sc-7980-R; 1:2,000), all purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), and rabbit anti-extracellular signal-regulated kinase 1/2 (Erk1/2; cat no. 4695; 1:2,000; Cell Signaling Technology, Inc., Danvers, MA, USA) or rabbit anti- p-Erk1/2 (cat no. 4376; 1:2,000; Cell Signaling Technology, Danvers, MA, USA). Following washing three times with TBST (pH 7.5), the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (cat no. bs-0295G; 1:1,000; Beijing Biosynthesis Biotechnology, Beijing, China) for 1 h at room temperature. The membranes were washed three times with TBST (pH 7.5). The resulting protein bands were visualized using an enhanced chemiluminescence reagent kit (Beyotime Institute of Biotechnology).

Statistical analysis

Statistical analysis was performed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). The data are expressed as the mean ± standard deviation. One-way analysis of variance and Student-Newman-Keuls analysis were used to evaluate the statistical significance of differences among the groups. P<0.05 was considered to indicate a statistically significant difference.

Results

Expression of MIC-1 in RPMI-8226 cells

To determine whether MIC-1 shRNA successfully knocked down the gene expression of MIC-1, the present study prepared lentiviral vectors bearing either MIC-1 shRNA (Lv-shRNA) or non-specific sequence (Lv-NC). Following transduction with Lv-shRNA or Lv-NC for 14 days, the RPMI-8226 cells were harvested and the expression levels of MIC-1 in these cells were analyzed using RT-qPCR and Western blot analyses. As shown in Fig. 2, the mRNA and protein levels of MIC-1 were significantly lower in the cells transduced with Lv-shRNA, compared with those in the cells transduced with Lv-NC or those without viral tranduction.

MIC-1 is essential for the osteoclastic differentiation of PBMNCs

To investigate the role of MIC-1 in the osteoclastic differentiation of PBMNCs, the PBMNCs were stained for TRAP following co-culture with RPMI-8226 cells for 14 days. The number of TRAP(+) cells was significantly lower when the PBMNCs were co-cultured with the RPMI-8226 cells transduced with Lv-shRNA, compared with when they were co-cultured with RPMI-8226 cells transduced with Lv-NC or without viral transduction (Fig. 3A and B). To investigate the role of MIC-1 in bone resorption by differentiated PBMNCs, the PBMNCs were stained with toluidine blue following co-culture with RPMI-8226 cells for 14 days. The percentage of pit area within a randomly selected area was significantly lower when the PBMNCs were co-cultured with the RPMI-8226 cells transduced with Lv-shRNA, compared with those co-cultured with the RPMI-8226 cells transduced with Lv-NC or without viral transduction (Fig. 3C and D). Taken together, these results suggested that MIC-1 was required for the osteoclastic differentiation and bone resorption activities of PBMNCs.

To confirm the role of MIC-1 in the osteoclastic differentiation of PBMNCs, the present study examined whether the expression of the osteoclast-specific gene, RANKL, was altered by the decreased expression of MIC-1. As shown in Fig. 4A and B, the protein level of RANKL in the PBMNCs was significantly lower when the cells were co-cultured with the RPMI-8226 cells transduced with Lv-shRNA, compared with those co-cultured with the RPMI-8226 cells transduced with Lv-NC or without viral transduction.

Subsequently, the present study investigated which signaling pathway was activated by RANKL by analyzing the phosphorylation status of the ERK1/2, c-JNK and p38 mitogen-activated protein kinase (MAPK) proteins using Western blot analysis. It was found that the protein levels of phosphorylated Erk1/2, but not those of c-JNK or p38, were significantly lower in the PBMNCs co-cultured with the RPMI-8226 cells transduced with Lv-shRNA, compared with those co-cultured with RPMI-8226 cells transduced with Lv-NC or without viral transduction (Fig. 4C and D), indicating that the MCI-1-induced increase in RANKL may activate the Erk1/2 pathway.

The present study then investigated whether c-fos and c-jun, important downstream molecules of ERK1/2, were involved in the differentiation of osteoclasts by analyzing the phosphorylation status of c-fos and c-jun using Western blot analysis. It was found that the protein levels of total c-fos were markedly reduced in the PBMNCs co-cultured with the RPMI-8226 cells transduced with Lv-shRNA, compared with those co-cultured with the RPMI-8226 cells transduced with Lv-NC or without viral transduction. However, no changes were observed in the protein levels of total c-jun or phosphorylated c-jun in the PBMNCs co-cultured with the RPMI-8226 cells transduced with either Lv-shRNA or Lv-NC (Fig. 4C and D). Taken together, these results suggested that MIC-1 regulated the osteoclastic differentiation and bone resorption activities of the PBMNCs via the Erk1/2 signaling pathway.

Discussion

In the present study, the role of MIC-1 in the osteoclastic differentiation of PBMNCs was examined. A co-culture system was used, in which PBMNCs co-cultured with RPMI-8226 cells were induced to differentiate into osteoclasts and to resorb bone. To knock down the expression of MIC-1 in the RPMI-8226 cells, a lentiviral vector was used to deliver the MIC-1 shRNA to the RPMI-8226 cells. It was found that MIC-1 shRNA efficiently decreased the expression of MIC-1 in the RPMI-8226 cells (Fig. 2). The present study then demonstrated that reduced expression of MIC-1 inhibited the osteoclastic differentiation of PBMNCs (Fig. 3). Finally, it was shown that the expression of the osteoclast-specific gene, RANKL, and the levels of dephosphorylated Erk1/2 were decreased in those PBMNCs, which were co-cultured with the RPMI-8226 cells transduced with Lv-shRNA (Fig. 4A and C). These results led to the conclusion that MIC-1 promoted osteoclastic differentiation of PBMNCs through inhibition of the RANKL-Erk1/2 signaling pathway.

Patients with MM often develop MBD with osteoclastic differentiation, induced by cytokines and other soluble factors in the bone marrow (12). In the bone marrow of patients with MM, MIC-1 is expressed at high levels (2). It has been suggested that MIC-1 may be involved in the regulation of osteoclastogenesis (13,14). The results of the study demonstrated that MIC-1 was required in order for RPMI-8226 to induce osteoclastic differentiation of the PBMNCs (Figs. 3 and 4), supporting the role of MCI-1 in the development of MBD in patients with MM.

The osteoclast-specific marker gene, RANKL, is involved in MCI-1 stimulated osteoclastic differentiation. Binding to its receptor RANK, RANKL stimulates the osteoclastic differentiation of monocyte macrophages and the maturation of osteoclasts (15). In RANKL-deficient mice, severe osteopetrosis and a defect in tooth eruption have been observed (16). In patients with MM, RANKL has been shown to be involved osteoclastic differentiation (1719). In the present study, it was shown that the expression of RANKL in the PBMNCs was reduced when the expression of MIC-1 in the co-cultured RPMI-8226 cells was knocked down (Fig. 4). RANKL binds to RANK and activates several MAPK signaling pathways (20), including the Erk1/2, JNK and P-38 pathways (21). The present study demonstrated that reduced expression of MIC-1 in the RPMI-8226 cells appeared to decrease the phosphorylation of Erk1/2, but not JNK or P-38, indicating the involvement of the Erk1/2 pathway in MIC-1-induced osteoclastic differentiation of PBMNCs (22,23). Consistent with this observation, the expression levels of c-fos and c-jun, downstream molecules of the Erk1/2 signaling pathway in PBMNCs, were also decreased when the expression of MCI-1 in the co-cultured RPMI-8226 cells was reduced.

In conclusion, the present study found that MIC-1 was involved in the development of MBD in patients with MM by promoting the osteoclastic differentiation of PBMNCs by activating the RANKL-Erk1/2 signaling pathway. Thus, MIC-1 may offer potential as a target gene in the development of strategies to treat MM.

References

1 

Kyle RA and Rajkumar SV: Criteria for diagnosis, staging, risk stratification and response assessment of multiple myeloma. Leukemia. 23:3–9. 2009. View Article : Google Scholar : PubMed/NCBI

2 

Corre J, Mahtouk K, Attal M, Gadelorge M, Huynh A, Fleury-Cappellesso S, Danho C, Laharrague P, Klein B, Rème T and Bourin P: Bone marrow mesenchymal stem cells are abnormal in multiple myeloma. Leukemia. 21:1079–1088. 2007.PubMed/NCBI

3 

Hromas R, Hufford M, Sutton J, Xu D, Li Y and Lu L: PLAB, a novel placental bone morphogenetic protein. Biochim Biophys Acta. 1354:40–44. 1997. View Article : Google Scholar : PubMed/NCBI

4 

Paralkar VM, Vail AL, Grasser WA, Brown TA, Xu H, Vukicevic S, Ke HZ, Qi H, Owen TA and Thompson DD: Cloning and characterization of a novel member of the transforming growth factor-beta/bone morphogenetic protein family. J Biol Chem. 273:13760–13767. 1998. View Article : Google Scholar : PubMed/NCBI

5 

Böttner M, Suter-Crazzolara C, Schober A and Unsicker K: Expression of a novel member of the TGF-beta superfamily, growth/differentiation factor-15/macrophage-inhibiting cytokine-1 (GDF-15/MIC-1) in adult rat tissues. Cell Tissue Res. 297:103–110. 1999. View Article : Google Scholar : PubMed/NCBI

6 

Baek SJ, Horowitz JM and Eling TE: Molecular cloning and characterization of human nonsteroidal anti-inflammatory drug-activated gene promoter. Basal transcription is mediated by Sp1 and Sp3. J Biol Chem. 276:33384–33392. 2001. View Article : Google Scholar : PubMed/NCBI

7 

Bootcov MR, Bauskin AR, Valenzuela SM, Moore AG, Bansal M, He XY, Zhang HP, Donnellan M, Mahler S, Pryor K, et al: Mic-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-beta superfamily. Proc Natl Acad Sci USA. 94:11514–11519. 1997. View Article : Google Scholar : PubMed/NCBI

8 

Tarkun P, Atesoglu E Birtas, Mehtap O, Musul MM and Hacihanefioglu A: Serum growth differentiation factor 15 levels in newly diagnosed multiple myeloma patients. Acta Haematol. 131:173–178. 2014. View Article : Google Scholar : PubMed/NCBI

9 

Wakchoure S, Swain TM, Hentunen TA, Bauskin AR, Brown DA, Breit SN, Vuopala KS, Harris KW and Selander KS: Expression of macrophage inhibitory cytokine-1 in prostate cancer bone metastases induces osteoclast activation and weight loss. Prostate. 69:652–661. 2009. View Article : Google Scholar : PubMed/NCBI

10 

Zeng Z, Zhang C and Chen J: Lentivirus-mediated RNA interference of DC-STAMP expression inhibits the fusion and resorptive activity of human osteoclasts. J Bone Miner Metab. 31:409–416. 2013. View Article : Google Scholar : PubMed/NCBI

11 

Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI

12 

Roodman GD: Pathogenesis of myeloma bone disease. Leukemia. 23:435–441. 2009. View Article : Google Scholar : PubMed/NCBI

13 

Khosla S: Minireview: The OPG/RANKL/RANK system. Endocrinology. 142:5050–5055. 2001. View Article : Google Scholar : PubMed/NCBI

14 

Sung B, Prasad S, Yadav VR, Gupta SC, Reuter S, Yamamoto N, Murakami A and Aggarwal BB: RANKL signaling and osteoclastogenesis is negatively regulated by cardamonin. PLoS One. 8:e641182013. View Article : Google Scholar : PubMed/NCBI

15 

Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, et al: Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell. 93:165–176. 1998. View Article : Google Scholar : PubMed/NCBI

16 

Kong YY, Yoshida H, Sarosi I, Tan HL, Timms E, Capparelli C, Morony S, Oliveira-dos-Santos AJ, Van G, Itie A, et al: OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature. 397:315–323. 1999. View Article : Google Scholar : PubMed/NCBI

17 

Schramek D, Leibbrandt A, Sigl V, Kenner L, Pospisilik JA, Lee HJ, Hanada R, Joshi PA, Aliprantis A, Glimcher L, et al: Osteoclast differentiation factor RANKL controls development of progestin-driven mammary cancer. Nature. 468:98–102. 2010. View Article : Google Scholar : PubMed/NCBI

18 

Sung B, Oyajobi B and Aggarwal BB: Plumbagin inhibits osteoclastogenesis and reduces human breast cancer-induced osteolytic bone metastasis in mice through suppression of RANKL signaling. Mol Cancer Ther. 11:350–359. 2012. View Article : Google Scholar : PubMed/NCBI

19 

Ai LS, Sun CY, Zhang L, Zhou SC, Chu ZB, Qin Y, Wang YD, Zeng W, Yan H, Guo T, et al: Inhibition of BDNF in multiple myeloma blocks osteoclastogenesis via down-regulated stroma-derived RANKL expression both in vitro and in vivo. PLoS One. 7:e462872012. View Article : Google Scholar : PubMed/NCBI

20 

Wada T, Nakashima T, Hiroshi N and Penninger JM: RANKL-RANK signaling in osteoclastogenesis and bone disease. Trends Mol Med. 12:17–25. 2006. View Article : Google Scholar : PubMed/NCBI

21 

Huang P, Han J and Hui L: MAPK signaling in inflammation-associated cancer development. Protein Cell. 1:218–226. 2010. View Article : Google Scholar : PubMed/NCBI

22 

Zhang W and Liu HT: MAPK signal pathways in the regulation of cell proliferation in mammalian cells. Cell Res. 12:9–18. 2002. View Article : Google Scholar : PubMed/NCBI

23 

Feng X: RANKing intracellular signaling in osteoclasts. IUBMB Life. 57:389–395. 2005. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

December-2016
Volume 14 Issue 6

Print ISSN: 1791-2997
Online ISSN:1791-3004

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Yuan M, Chen J and Zeng Z: Knockdown of macrophage inhibitory cytokine-1 in RPMI-8226 human multiple myeloma cells inhibits osteoclastic differentiation through inhibiting the RANKL-Erk1/2 signaling pathway. Mol Med Rep 14: 5199-5204, 2016
APA
Yuan, M., Chen, J., & Zeng, Z. (2016). Knockdown of macrophage inhibitory cytokine-1 in RPMI-8226 human multiple myeloma cells inhibits osteoclastic differentiation through inhibiting the RANKL-Erk1/2 signaling pathway. Molecular Medicine Reports, 14, 5199-5204. https://doi.org/10.3892/mmr.2016.5879
MLA
Yuan, M., Chen, J., Zeng, Z."Knockdown of macrophage inhibitory cytokine-1 in RPMI-8226 human multiple myeloma cells inhibits osteoclastic differentiation through inhibiting the RANKL-Erk1/2 signaling pathway". Molecular Medicine Reports 14.6 (2016): 5199-5204.
Chicago
Yuan, M., Chen, J., Zeng, Z."Knockdown of macrophage inhibitory cytokine-1 in RPMI-8226 human multiple myeloma cells inhibits osteoclastic differentiation through inhibiting the RANKL-Erk1/2 signaling pathway". Molecular Medicine Reports 14, no. 6 (2016): 5199-5204. https://doi.org/10.3892/mmr.2016.5879