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Inhibition of osteoclast differentiation by overexpression of NDRG2 in monocytes
Biochemical and Biophysical Research Communications 468 (2015) 611e616
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Inhibition of osteoclast differentiation by overexpression of NDRG2 in monocytes Kyeongah Kang, Sorim Nam, Bomi Kim, Ji Hyun Lim, Young Yang, Myeong-Sok Lee, Jong-Seok Lim* Department of Biological Sciences and the Research Center for Women's Disease, Sookmyung Women's University, Seoul 140-742, Republic of Korea
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Article history: Received 16 October 2015 Accepted 31 October 2015 Available online 4 November 2015
N-Myc downstream-regulated gene 2 (NDRG2), a member of the NDRG family of differentiation-related genes, has been characterized as a regulator of dendritic cell differentiation from monocytes, CD34þ progenitor cells, and myelomonocytic leukemic cells. In this study, we show that NDRG2 overexpression inhibits the differentiation of U937 cells into osteoclasts in response to stimulation with a combination of macrophage colony-stimulating factor (M-CSF) and soluble receptor activator of NF-kB ligand (RANKL). U937 cells stably expressing NDRG2 are unable to differentiate into multinucleated osteoclast-like cells and display reduced tartrate-resistant acid phosphatase (TRAP) activity and resorption pit formation. Furthermore, NDRG2 expression significantly suppresses the expression of genes that are crucial for the proliferation, survival, differentiation, and function of osteoclasts, including c-Fos, Atp6v0d2, RANK, and OSCAR. The activation of ERK1/2 and p38 is also inhibited by NDRG2 expression during osteoclastogenesis, and the inhibition of osteoclastogenesis by NDRG2 correlates with the down-regulation of the expression of the transcription factor PU.1. Taken together, our results suggest that the expression of NDRG2 potentially inhibits osteoclast differentiation and plays a role in modulating the signal transduction pathway responsible for osteoclastogenesis. © 2015 Elsevier Inc. All rights reserved.
Keywords: NDRG2 Osteoclastic cytokines U937 cells Osteoclastogenesis
1. Introduction Bone is a dynamic tissue that undergoes continuous remodeling by bone-forming osteoblasts and bone-resorbing osteoclasts. The balance between the growth, differentiation, and activity of osteoblasts and osteoclasts is essential for maintaining a constant bone density and regulating the mineral homeostasis of the entire organism. However, this balance can be easily disrupted by inflammatory signals and systemic alterations, such as estrogen deficiency. Increased osteoclast activity leads to bone thinning and trabecular bone erosion, whereas increased osteoblast activity causes an increase in bone density [1,2]. Osteoclasts are multinucleated giant cells that are derived from hematopoietic cells of the monocyte/macrophage lineage. Osteoclast differentiation is a coordinated process involving the
* Corresponding author. Department of Biological Science and the Research Center for Women's Diseases, Sookmyung Women's University, Chungpa-Dong, Yongsan-Gu, Seoul, 140-742, Republic of Korea. Tel.: þ82 2 710 9560; fax: þ82 2 2077 7322. E-mail address: [email protected] (J.-S. Lim). http://dx.doi.org/10.1016/j.bbrc.2015.10.167 0006-291X/© 2015 Elsevier Inc. All rights reserved.
development of pre-osteoclasts, which express tartrate-resistant acid phosphatase (TRAP) and calcitonin receptor, followed by cellcell fusion and activation by various factors, such as the receptor activator of NF-kB ligand (RANKL), TNF-a, and LPS [2,3]. Osteoclast development is enhanced in destructive bone diseases, such as rheumatoid arthritis (RA), periodontitis, osteoporosis, and multiple myeloma [4]. Therefore, the specific inhibition of osteoclast differentiation and function has become an important strategy for the treatment of various metabolic bone diseases. Several transcription factors, including NF-kB, PU.1, microphthalmia transcription factor (MITF), c-Fos, and nuclear factor of activated T cells (NFAT) c1, participate in osteoclastogenesis [5]. Especially, PU.1, a member of the Ets family of transcription factors, is expressed in cells of multiple hematopoietic lineages, and enhanced PU.1 expression promotes macrophage and dendritic cell development [6,7]. In contrast, PU.1-null mice exhibit impaired myelopoiesis and inhibited osteoclastogenesis [8,9]. Moreover, PU.1 regulates the expression of colony stimulating factor 1 receptor (CSF-1R, or macrophage colony-stimulating factor receptor (MCSFR)), one of the most important genes in macrophage proliferation and function. In addition, PU.1- or CSF-1R-deficient mice exhibit osteopetrosis due to osteoclast defects [10].
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N-Myc downstream-regulated gene 2 (NDRG2) belongs to the NDRG family, a new family of differentiation-related genes comprising the proteins NDRG1-4. NDRG2 is highly expressed in the adult brain, salivary glands, and skeletal muscle and has been characterized as a regulator of dendritic cell differentiation from monocytes, CD34þ progenitor cells, and myelomonocytic leukemic cells [11,12]. NDRG2 has also been shown to regulate cell growth, apoptosis, and neurodegeneration [13e16] and has recently been suggested to be a novel intrinsic factor for the modulation of IL-10 production in myeloid cells [17]. Except for the role in the differentiation of dendritic cells, the precise role and function of NDRG2 in the process of myeloid cell differentiation and the regulatory mechanisms of differentiation-related signaling have not been extensively explored. In this study, we investigated the effects of NDRG2 expression on the osteoclast differentiation of U937 cells, a human myeloid leukemic cell line. Our findings demonstrate that NDRG2 overexpression in U937 cells and mouse primary cells inhibits TRAP activity, the expression of osteoclast-related genes, and resorption pit formation, suggesting the inhibition of differentiation into multinucleated osteoclast-like cells. 2. Materials and methods 2.1. Cell lines and reagents U937 (CRL-1593.2TM, American Type Culture Collection, Rockville, MD) were cultured in DMEM (Gibco/Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco). Phorbol 12-myristate 13-acetate (PMA) was purchased from SigmaeAldrich (St. Louis, MO). Recombinant human RANKL, murine RANKL, human M-CSF, and murine M-CSF were obtained from Peprotech, Inc. (Rocky Hill, NJ).
2.2. Experimental animals Male ICR mice were purchased from Samtako (Osan, Republic of Korea). The mice were maintained in a specific pathogen-free environment and used when they were 6 weeks old. All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee, approved by Institutional Ethical Committee of Sookmyung Women's University (Resolution No. SMU-IACUC-2011-0906-017). 2.3. Differentiation of U937 cells into osteoclast-like cells U937 cells were cultured in 0.1 mg/ml PMA for 2 days (day 2 to day 0) in a 24-well plate at a density of 5 104 cells/well. The cells were then stimulated with a combination of 50 ng/ml M-CSF and 100 ng/ml RANKL. The culture media and stimulators were replenished every 2e3 days. 2.4. Construction and transfection of U937 cells U937 cells expressing NDRG2 were previously generated in our laboratory [17]. The established clone #61 was used as a stable NDRG2-expressing U937 cell line. The MSCV and MSCV-PU.1 plasmids were kind gifts from Dr. H.-J. Lee (KRIBB, Chungbuk, Republic of Korea). Control and NDRG2 siRNAs were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). MSCV, MSCV-PU.1, and the control and NDRG2-targeting siRNAs were transfected into U937-NDRG2 cells using an Amaxa nucleofector (program V-001) (Cologne, Germany). 2.5. Differentiation of murine bone marrow-derived osteoclasts Bone marrow cells isolated from the femurs of ICR mice were harvested, and RBCs were depleted by treatment with RBC lysis buffer (SigmaeAldrich). The cells were pre-cultured in a-MEM (Gibco/Invitrogen) containing 10% FBS and 50 ng/ml M-CSF for 24 h and the non-adherent cells were collected and maintained for 3 days. Finally, the non-adherent cells were discarded, and the bone marrow-derived macrophages (BMM) were differentiated into osteoclasts by treatment with 50 ng/ml M-CSF and 100 ng/ml RANKL for 12 days. TRAP-positive multinuclear cells containing more than 3 nuclei were scored as osteoclasts. 2.6. Tartrate-resistant acid phosphatase (TRAP) staining and TRAP assay
Fig. 1. NDRG2 expression inhibits osteoclast differentiation. (A and B) NDRG2 expression levels were determined by real-time PCR and western blotting. (C and D) PMA-induced monocyte-like cells were treated with M-CSF and RANKL. (C) On day 7, the cells were stained for TRAP. TRAP-positive cells were viewed using a microscope. TRAP concentration was measured on day 4. The results represent the mean ± SD of duplicate experiments (**p < 0.01, Student's t-test). (D) On day 4, the cells were inoculated onto BD Biocoat™ Osteologic™ Discs and differentiated with each of the osteoclast differentiation factors for 10 days, and then the discs were stained by von Kossa staining. The resorption pits were quantified using ImageJ.
TRAP staining was performed using an acid phosphatase leukocyte kit from SigmaeAldrich. TRAP-positive multinuclear cells containing more than 3 nuclei were scored as osteoclasts and were examined using an Olympus microscope (100x) (Model IX71, Olympus Corp.) The TRAP concentrations were determined using the TRAP assay kit from Takara Bio. Inc. (Shiga, Japan). Briefly, 50 ml of cell extract and 50 ml of p-nitro-phenyl phosphate (pNPP) were mixed with a sodium tartrate solution and incubated at 37 C for 20e25 min. The reaction was stopped with 0.5 N NaOH, and the absorbance was measured at 405 nm. A solution containing acid phosphatase and pNPP, minus the sodium tartrate solution, was used as the control. 2.7. Resorption pit formation assay U937 cells were cultured with 0.1 mg/ml PMA for 2 days (day 2 to day 0) in a 24-well plate at a density of 5 104 cells/well. The cells were then stimulated with a combination of 50 ng/ml M-CSF and 100 ng/ml RANKL for 4 days. On day 4, the cells were harvested
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Fig. 2. NDRG2 inhibits the expression of osteoclast-related genes through PU.1 signaling. (AeD) PMA-induced monocyte-like cells were treated with M-CSF and RANKL for 3 days. The mRNA levels were measured by real-time PCR (A and B). PU.1 expression and MAPK kinase activation were measured by western blotting (C and D). (EeH) MSCV or MSCV-PU.1 was transfected into U937-NDRG2 cells using an Amaxa nucleofector (program V-001). PU.1 expression was measured by RT-PCR (E). TRAP concentration represents the mean ± SD of duplicate experiments (*p < 0.05, Student's t-test) (F). Expression levels of indicated genes were measured by real-time PCR (G and H).
and incubated with each stimulator in calcium phosphate-coated plates from BD Biosciences (BD BioCoat™ Osteologic™, San Jose, CA). The transfected primary cells were differentiated into osteoclasts in calcium phosphate-coated plates for 15 days. The resorption pits were observed by von Kossa staining according to the protocol supplied by BD Biosciences (Technical Bulletin #444). The cells were removed with 1 M NaOH, and the wells were stained with 5% silver nitrate for 30 min and then developed with 5% sodium carbonate in 4% formaldehyde for 1 min. After fixation with 5% sodium thiosulfate, the wells were examined under a microscope and quantified using ImageJ (Windows version of NIH ImageJ). 2.8. Reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR RNA was isolated using TRIzol (Gibco/Invitrogen) cDNA was synthesized from 1 mg of total RNA using the M-MLV reverse transcriptase (Promega, Madison, WI). The cDNAs were amplified by PCR. Quantitative real-time PCR was performed using a Power SYBR Green PCR Master Mix (Life Technology, Carlsbad, CA). Briefly, the cDNA was diluted with RNase-free water, and QuantiTect SYBR green PCR master mix and the indicated primers were added to the sample. The sample was amplified using the ABI StepOnePlus™ real-time PCR thermal cycler (Applied Biosystems, Foster City, CA). GAPDH was used as an endogenous control. 2.9. Western blot analysis Cells were washed with D-PBS and lysed in a protein extraction
solution (iNtRON Biotechnology, Seongnam, Republic of Korea). Proteins were separated on a 12% SDS-polyacrylamide gel and transferred to a PVDF membrane. The antibodies for p44/42 MAPK, phospho-p44/42 MAPK, phospho-p38 MAPK, SAPK/JNK, and phospho-SAPK/JNK (Thr183/Tyr185) were purchased from Cell Signaling Technology, Inc. (Beverly, MA), and the antibodies for p38 MAPK, PU.1, NDRG2, actin, and a-actinin were purchased from Santa Cruz Biotechnology. The antigeneantibody complexes were detected by enhanced chemiluminescence. 2.10. Statistical analysis Student's t-test and ANOVA followed by Bonferroni's modification of the Student's t-test were used for statistical analysis. Values are represented as the mean ± SD. A value of p < 0.05 was considered significant. 3. Results 3.1. NDRG2 expression inhibits osteoclast differentiation To investigate the effect of NDRG2 expression on osteoclast differentiation, we transfected U937 cells with a plasmid containing the NDRG2 gene and confirmed that the U937-NDRG2 cells, but not the U937-mock cells, expressed NDRG2 mRNA and protein (Fig. 1A and B). After 2 days of treatment with PMA, most of the U937-mock cells were adherent, suggesting that they had differentiated into monocyte-like cells. In contrast, more than 50% of the U937-NDRG2 cells remained in suspension (data not shown). The cells were treated with a combination of M-CSF and RANKL on day
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Fig. 3. NDRG2 down-regulation reverses the osteoclastogenesis. (AeF) Control or NDRG2 siRNA was transfected into U937-NDRG2 cells using an Amaxa nucleofector (program V001). NDRG2 levels were measured by RT-PCR and western blotting (A). TRAP concentration was measured in M-CSF- and RANKL-induced osteoclast-like cells harvested on day 6 (*p < 0.05, Student's t-test) (B). Expression levels of indicated genes were determined by real-time PCR (C and D). Western blotting was performed using specific MAPK antibodies (E). PU.1 mRNA levels were measured by RT-PCR (F).
0 to induce differentiation into osteoclast-like cells. To determine the osteoclast differentiation, we performed TRAP staining on cells after differentiation into osteoclast-like cells. The differentiated U937-mock cells were TRAP-positive and classified as multinucleated giant cells. In agreement with the TRAP staining data, the U937-mock cells had a higher TRAP concentration than the U937-NDRG2 cells (Fig. 1C). Moreover, although the differentiated U937-mock cells formed a higher number of resorption pits, the U937-NDRG2 cells showed a reduction in resorption pit area (Fig. 1D). Therefore, these data indicate that NDRG2 expression inhibits the differentiation into osteoclast-like cell.
3.2. NDRG2 inhibits the expression of osteoclast-related genes through PU.1 signaling It has been already known that many factors such as OSCAR, cFos, DC-STAMP and NFATc1 participate in osteoclastogenesis [18e21]. First, we investigated whether NDRG2 regulates the expression of osteoclast-related genes. The mRNA expression levels of OSCAR, RANK, c-Fos, DC-STAMP, NFATc1, Atp6V0d2, and MITF were reduced by NDRG2 expression (Fig. 2A). In addition, we confirmed the CSF-1R expression level, because CSF-1R is a receptor of M-CSF and its expression has been known to increase during osteoclastogenesis. The CSF-1R expression was increased in the U937-mock cells by PMA treatment. However, its expression was inhibited in U937-NDRG2 cells in spite of PMA treatment (Fig. 2B). It has been reported that RANKL induces the expression of
osteoclast-related genes through MAPK pathways [22]. Therefore, we investigated which signal is affected by NDRG2 expression. The activation of ERK1/2 and p38 increased during osteoclastogenesis in the U937-mock cells in the presence of M-CSF and RANKL, whereas the activation of these factors was attenuated or delayed by NDRG2 expression (Fig. 2C). Since PU.1 was known to be induced through ERK signaling [23], we investigated whether PU.1 is reduced by NDRG2 expression during M-CSF and RANKL-induced osteoclastogenesis. As expected, the U937-NDRG2 cells showed strongly reduced levels of PU.1 protein expression (Fig. 2D). We next determined whether increased PU.1 expression could induce osteoclast differentiation in U937-NDRG2 cells. U937-NDRG2 cells were transfected with PU.1, the expression of which was verified by RT-PCR (Fig. 2E). Regardless of whether the cells expressed NDRG2, the TRAP concentration was increased in the MSCV-PU.1transfected U937-NDRG2 cells (Fig. 2F). Moreover, the levels of osteoclast-regulating genes were increased in the cells differentiated from MSCV-PU.1-transfected U937-NDRG2 cells (Fig. 2G and H). Therefore, ectopic PU.1 expression was able to induce osteoclast differentiation in U937-NDRG2 cells. These data suggest that NDRG2 expression in U937 cells regulates osteoclastogenesis by down-regulating PU.1 through ERK1/2 and p38 signaling during osteoclastogenesis.
3.3. Osteoclastogenesis is recovered by NDRG2 down-regulation To confirm the inhibitory effect of NDRG2 expression on osteoclastogenesis, an NDRG2 siRNA was transfected into U937-
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Fig. 4. NDRG2 expression inhibits osteoclastogenesis in primary cells. (AeF) Murine bone marrow cells (BM) and macrophages (MØ) were transfected with pCMV/taq2B or pCMV/ taq2B-NDRG2. Diagram showing the scheme for transfecting primary bone marrow cells (A). After 8 h, NDRG2 expression was determined by RT-PCR (B). (C and D) The differentiated cells were stained for TRAP and examined using an Olympus microscope (100x) (Model IX71, Olympus Corp.) (C). The number of osteoclasts that contained more than 3 nuclei was determined for each well. The results represent the mean ± SD of cells from 10 wells (**p < 0.01, ***p < 0.001, Student's t-test) (D). (E and F) The transfected cells were differentiated into osteoclasts on BD Biocoat™ Osteologic™ Discs for 15 days. The discs were stained by von Kossa staining. The resorption pits were viewed using a microscope (100x) (E) and quantified using ImageJ (F).
NDRG2 cells (Fig. 3A). As expected, the TRAP concentration was significantly increased in the NDRG2 siRNA-transfected cells treated with M-CSF and RANKL (Fig. 3B). The expression levels of osteoclastic genes were also increased in the cells differentiated from NDRG2 siRNA-transfected cells (Fig. 3C and D). In addition, we confirmed again that ERK1/2 and p38 activation inhibited by NDRG2 expression was restored in the cells differentiated from NDRG2 siRNA-transfected cells (Fig. 3E). Finally, PU.1 expression decreased by NDRG2 was enhanced in the NDRG2 siRNAtransfected cells (Fig. 3F). Together, these findings demonstrate that NDRG2 expression inhibits osteoclastogenesis in U937 cells. 3.4. NDRG2 expression suppresses osteoclast differentiation in murine primary cells Lastly, we investigated whether the suppressive function of NDRG2 would also be observed in primary cells. Since the primary bone marrow cells and macrophages usually do not express NDRG2, both of primary bone marrow cells and M-CSF-induced macrophages were transfected with NDRG2, and the gene expression in two populations was confirmed by RT-PCR (Fig. 4A and B). In agreement with the results obtained using the U937 cell lines, there were far fewer pCMV/taq2B-NDRG2-transfected bone marrow cells and macrophages that had differentiated into TRAP-positive
osteoclasts and the number of osteoclasts that had differentiated from NDRG2 over-expressing cells was remarkably decreased compared to the control cells (Fig. 4C and D). Moreover, resorption pit formation was decreased by NDRG2 over-expression (Fig. 4E and F). Together, these results demonstrate that NDRG2 expression hinders osteoclast differentiation in the murine primary cells as well as in U937 cells treated with differentiation-inducing factors. 4. Discussion NDRG2 is regarded as a candidate tumor suppressor gene because its overexpression correlates with the induction of apoptosis, inhibition of MMP-9 activity, and decreased invasion potential [24,25]. NDRG2 is also involved in the differentiation of dendritic cells from myeloid cells and regulates IL-10 production in human myeloid cells [11,17]. In this study, we demonstrate, for the first time, the inhibitory effect of NDRG2 expression on osteoclast differentiation. Osteoclast differentiation, survival, and activation are also modulated by mitogen-activated protein kinase (MAPK), phosphoinositide-3-kinase (PI3K)/Akt, and NF-kB signaling. MAPK activation is triggered by RANK in osteoclasts and osteoclast precursors [18]. In the present study, the phosphorylation of SAPK/JNK was either unchanged or slightly elevated during the osteoclastogenesis
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of NDRG2-transfected U937 cells, whereas the activation of ERK1/2 and p38 in these cells was notably impaired. Most importantly, the phosphorylation of p38 MAPK in NDRG2-transfected U937 cells was almost undetectable, even after stimulation with differentiation-inducing factors, suggesting that the inhibitory effect of NDRG2 gene expression on osteoclastogenesis is induced via the suppression of p38 MAPK phosphorylation. In addition, when they were transfected with MSCV-PU.1 to investigate whether PU.1 overexpression is able to induce ERK1/2 activation during osteoclastogenesis, ERK activation was not restored in the cells differentiated from MSCV-PU.1-transfected U937-NDRG2 cells, suggesting that ERK may not be the direct target for PU.1 (data not shown). It has been reported that during osteoclast differentiation induced by M-CSF/RANKL, p38 MAPK was directly recruited to the target promoter and that the phosphorylated forms of MITF simultaneously appeared at these regions [26]. We have shown previously that NDRG2 overexpression suppresses MITF expression and its promoter activity in B16F10 melanoma cells [27]. Likewise, we also confirmed in this study that NDRG2 expression in U937 cells inhibited the expression of MITF during osteoclastogenesis. In earlier reports, MITF was implicated in the survival and differentiation of developmentally unrelated cell types, including melanocytes and osteoclasts [28,29], and it was suggested that the MITF may partly account for the ability of MITF to selectively regulate target genes during osteoclast differentiation [30]. Recently, it was reported that NDRG2-deficient mice exhibit vertebral defects in thoracic/lumbar and lumbar/sacral transitional regions and that the forced overexpression of NDRG2 in osteoblasts or chondrocytes also confers vertebral defects [31], indicating the critical function of NDRG2 at the osteochondrogenic differentiation stage. Previously, we reported that NDRG2 plays an essential role in determining the development of monocytic precursors into DCs [11]. Nonetheless, given that hematopoietic stem cells and myeloid precursors do not express NDRG2, it is highly unlikely that osteoclastogenesis in vivo is directly regulated by NDRG2 expression. Rather, we speculate that NDRG2 may affect the transient induction of gene expression in the presence of specific stimuli that are required for osteoclastogenesis. In fact, NDRG2 has been reported to be one of the important stress-inducible genes. Therefore, it remains to be determined in further study how and to what extent NDRG2 expression is involved in promoting or suppressing osteoclast formation and the development of specific cell lineages, such as DCs. Conflict of interest All authors declared no potential conflicts of interest. Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grants (2012R1A2A2A01046114) funded by Korean government (Ministry of Science, ICT and Future Planning) and partly by the Sookmyung Women's University Research Grant 2013. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.09.071. References [1] W.J. Boyle, W.S. Simonet, D.L. Lacey, Osteoclast differentiation and activation,
Nature 423 (2003) 337e342. [2] N.A. Sims, J.H. Gooi, Bone remodeling: multiple cellular interactions required for coupling of bone formation and resorption, Semin. Cell Dev. Biol. 19 (2008) 444e451. [3] A.P. Anandarajah, Role of RANKL in bone diseases, Trends Endocrinol. Metab. 20 (2009) 88e94. [4] G.D. Roodman, Treatment strategies for bone disease, Bone Marrow Transpl. 40 (2007) 1139e1146. [5] I.E. Adamopoulos, E.D. Mellins, Alternative pathways of osteoclastogenesis in inflammatory arthritis, Nat. Rev. Rheumatol. 11 (2015) 189e194. [6] K.S. Mak, A.P. Funnell, R.C. Pearson, M. Crossley, PU.1 and haematopoietic cell fate: dosage matters, Int. J. Cell Biol. 2011 (2011) 808524. [7] S. Carotta, A. Dakic, A. D'Amico, S.H. Pang, K.T. Greig, S.L. Nutt, et al., The transcription factor PU.1 controls dendritic cell development and Flt3 cytokine receptor expression in a dose-dependent manner, Immunity 32 (2010) 628e641. [8] S.R. McKercher, B.E. Torbett, K.L. Anderson, G.W. Henkel, D.J. Vestal, H. Baribault, et al., Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities, EMBO J. 15 (1996) 5647e5658. [9] E.W. Scott, M.C. Simon, J. Anastasi, H. Singh, Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages, Science 265 (1994) 1573e1577. [10] E.R. Stanley, V. Chitu, CSF-1 receptor signaling in myeloid cells, Cold Spring Harb. Perspect. Biol. 6 (2014). [11] S.C. Choi, K.D. Kim, J.T. Kim, J.W. Kim, D.Y. Yoon, Y.K. Choe, et al., Expression and regulation of NDRG2 (N-myc downstream regulated gene 2) during the differentiation of dendritic cells, FEBS Lett. 553 (2003) 413e418. [12] S.C. Choi, K.D. Kim, J.T. Kim, J.W. Kim, H.G. Lee, J.M. Kim, et al., Expression of human NDRG2 by myeloid dendritic cells inhibits down-regulation of activated leukocyte cell adhesion molecule (ALCAM) and contributes to maintenance of T cell stimulatory activity, J. Leukoc. Biol. 83 (2008) 89e98. [13] Y. Deng, L. Yao, L. Chau, S.S. Ng, Y. Peng, X. Liu, et al., N-Myc downstreamregulated gene 2 (NDRG2) inhibits glioblastoma cell proliferation, Int. J. Cancer 106 (2003) 342e347. [14] C. Mitchelmore, S. Buchmann-Moller, L. Rask, M.J. West, J.C. Troncoso, N.A. Jensen, NDRG2: a novel Alzheimer's disease associated protein, Neurobiol. Dis. 16 (2004) 48e58. [15] K. Takahashi, M. Yamada, H. Ohata, K. Honda, M. Yamada, Ndrg2 promotes neurite outgrowth of NGF-differentiated PC12 cells, Neurosci. Lett. 388 (2005) 157e162. [16] S.K. Shon, A. Kim, J.Y. Kim, K.I. Kim, Y. Yang, J.S. Lim, Bone morphogenetic protein-4 induced by NDRG2 expression inhibits MMP-9 activity in breast cancer cells, Biochem. Biophys. Res. Commun. 385 (2009) 198e203. [17] S.C. Choi, K.D. Kim, J.T. Kim, S.S. Oh, S.Y. Yoon, E.Y. Song, et al., NDRG2 is one of novel intrinsic factors for regulation of IL-10 production in human myeloid cell, Biochem. Biophys. Res. Commun. 396 (2010) 684e690. [18] N.S. Soysa, N. Alles, K. Aoki, K. Ohya, Osteoclast formation and differentiation: an overview, J. Med. Dent. Sci. 59 (2012) 65e74. [19] A. Arai, T. Mizoguchi, S. Harada, Y. Kobayashi, Y. Nakamichi, H. Yasuda, et al., Fos plays an essential role in the upregulation of RANK expression in osteoclast precursors within the bone microenvironment, J. Cell Sci. 125 (2012) 2910e2917. [20] M. Yagi, T. Miyamoto, Y. Sawatani, K. Iwamoto, N. Hosogane, N. Fujita, et al., DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells, J. Exp. Med. 202 (2005) 345e351. [21] H. Takayanagi, The role of NFAT in osteoclast formation, Ann. N. Y. Acad. Sci. 1116 (2007) 227e237. [22] X. Feng, RANKing intracellular signaling in osteoclasts, IUBMB Life 57 (2005) 389e395. [23] M.B. Miranda, D.E. Johnson, Signal transduction pathways that contribute to myeloid differentiation, Leukemia 21 (2007) 1363e1377. [24] A. Kim, M.J. Kim, Y. Yang, J.W. Kim, Y.I. Yeom, J.S. Lim, Suppression of NFkappaB activity by NDRG2 expression attenuates the invasive potential of highly malignant tumor cells, Carcinogenesis 30 (2009) 927e936. [25] H. Shi, N. Li, S. Li, C. Chen, W. Wang, C. Xu, et al., Expression of NDRG2 in esophageal squamous cell carcinoma, Cancer Sci. 101 (2010) 1292e1299. [26] S.M. Sharma, A. Bronisz, R. Hu, K. Patel, K.C. Mansky, S. Sif, et al., MITF and PU.1 recruit p38 MAPK and NFATc1 to target genes during osteoclast differentiation, J. Biol. Chem. 282 (2007) 15921e15929. [27] A. Kim, Y. Yang, M.S. Lee, Y.D. Yoo, H.G. Lee, J.S. Lim, NDRG2 gene expression in B16F10 melanoma cells restrains melanogenesis via inhibition of Mitf expression, Pigment Cell Melanoma Res. 21 (2008) 653e664. [28] J. Liu, M. Fukunaga-Kalabis, L. Li, M. Herlyn, Developmental pathways activated in melanocytes and melanoma, Arch. Biochem. Biophys. 563 (2014) 13e21. [29] J.H. Kim, N. Kim, Regulation of NFATc1 in osteoclast differentiation, J. Bone Metab. 21 (2014) 233e241. [30] R. Hu, S.M. Sharma, A. Bronisz, R. Srinivasan, U. Sankar, M.C. Ostrowski, Eos, MITF, and PU.1 recruit corepressors to osteoclast-specific genes in committed myeloid progenitors, Mol. Cell. Biol. 27 (2007) 4018e4027. [31] H. Zhu, J. Zhao, W. Zhou, H. Li, R. Zhou, L. Zhang, et al., Ndrg2 regulates vertebral specification in differentiating somites, Dev. Biol. 369 (2012) 308e318.
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Inhibition of osteoclast differentiation by overexpression of NDRG2 in monocytes Kyeongah Kang, Sorim Nam, Bomi Kim, Ji Hyun Lim, Young Yang, Myeong-Sok Lee, Jong-Seok Lim* Department of Biological Sciences and the Research Center for Women's Disease, Sookmyung Women's University, Seoul 140-742, Republic of Korea
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a b s t r a c t
Article history: Received 16 October 2015 Accepted 31 October 2015 Available online 4 November 2015
N-Myc downstream-regulated gene 2 (NDRG2), a member of the NDRG family of differentiation-related genes, has been characterized as a regulator of dendritic cell differentiation from monocytes, CD34þ progenitor cells, and myelomonocytic leukemic cells. In this study, we show that NDRG2 overexpression inhibits the differentiation of U937 cells into osteoclasts in response to stimulation with a combination of macrophage colony-stimulating factor (M-CSF) and soluble receptor activator of NF-kB ligand (RANKL). U937 cells stably expressing NDRG2 are unable to differentiate into multinucleated osteoclast-like cells and display reduced tartrate-resistant acid phosphatase (TRAP) activity and resorption pit formation. Furthermore, NDRG2 expression significantly suppresses the expression of genes that are crucial for the proliferation, survival, differentiation, and function of osteoclasts, including c-Fos, Atp6v0d2, RANK, and OSCAR. The activation of ERK1/2 and p38 is also inhibited by NDRG2 expression during osteoclastogenesis, and the inhibition of osteoclastogenesis by NDRG2 correlates with the down-regulation of the expression of the transcription factor PU.1. Taken together, our results suggest that the expression of NDRG2 potentially inhibits osteoclast differentiation and plays a role in modulating the signal transduction pathway responsible for osteoclastogenesis. © 2015 Elsevier Inc. All rights reserved.
Keywords: NDRG2 Osteoclastic cytokines U937 cells Osteoclastogenesis
1. Introduction Bone is a dynamic tissue that undergoes continuous remodeling by bone-forming osteoblasts and bone-resorbing osteoclasts. The balance between the growth, differentiation, and activity of osteoblasts and osteoclasts is essential for maintaining a constant bone density and regulating the mineral homeostasis of the entire organism. However, this balance can be easily disrupted by inflammatory signals and systemic alterations, such as estrogen deficiency. Increased osteoclast activity leads to bone thinning and trabecular bone erosion, whereas increased osteoblast activity causes an increase in bone density [1,2]. Osteoclasts are multinucleated giant cells that are derived from hematopoietic cells of the monocyte/macrophage lineage. Osteoclast differentiation is a coordinated process involving the
* Corresponding author. Department of Biological Science and the Research Center for Women's Diseases, Sookmyung Women's University, Chungpa-Dong, Yongsan-Gu, Seoul, 140-742, Republic of Korea. Tel.: þ82 2 710 9560; fax: þ82 2 2077 7322. E-mail address: [email protected] (J.-S. Lim). http://dx.doi.org/10.1016/j.bbrc.2015.10.167 0006-291X/© 2015 Elsevier Inc. All rights reserved.
development of pre-osteoclasts, which express tartrate-resistant acid phosphatase (TRAP) and calcitonin receptor, followed by cellcell fusion and activation by various factors, such as the receptor activator of NF-kB ligand (RANKL), TNF-a, and LPS [2,3]. Osteoclast development is enhanced in destructive bone diseases, such as rheumatoid arthritis (RA), periodontitis, osteoporosis, and multiple myeloma [4]. Therefore, the specific inhibition of osteoclast differentiation and function has become an important strategy for the treatment of various metabolic bone diseases. Several transcription factors, including NF-kB, PU.1, microphthalmia transcription factor (MITF), c-Fos, and nuclear factor of activated T cells (NFAT) c1, participate in osteoclastogenesis [5]. Especially, PU.1, a member of the Ets family of transcription factors, is expressed in cells of multiple hematopoietic lineages, and enhanced PU.1 expression promotes macrophage and dendritic cell development [6,7]. In contrast, PU.1-null mice exhibit impaired myelopoiesis and inhibited osteoclastogenesis [8,9]. Moreover, PU.1 regulates the expression of colony stimulating factor 1 receptor (CSF-1R, or macrophage colony-stimulating factor receptor (MCSFR)), one of the most important genes in macrophage proliferation and function. In addition, PU.1- or CSF-1R-deficient mice exhibit osteopetrosis due to osteoclast defects [10].
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N-Myc downstream-regulated gene 2 (NDRG2) belongs to the NDRG family, a new family of differentiation-related genes comprising the proteins NDRG1-4. NDRG2 is highly expressed in the adult brain, salivary glands, and skeletal muscle and has been characterized as a regulator of dendritic cell differentiation from monocytes, CD34þ progenitor cells, and myelomonocytic leukemic cells [11,12]. NDRG2 has also been shown to regulate cell growth, apoptosis, and neurodegeneration [13e16] and has recently been suggested to be a novel intrinsic factor for the modulation of IL-10 production in myeloid cells [17]. Except for the role in the differentiation of dendritic cells, the precise role and function of NDRG2 in the process of myeloid cell differentiation and the regulatory mechanisms of differentiation-related signaling have not been extensively explored. In this study, we investigated the effects of NDRG2 expression on the osteoclast differentiation of U937 cells, a human myeloid leukemic cell line. Our findings demonstrate that NDRG2 overexpression in U937 cells and mouse primary cells inhibits TRAP activity, the expression of osteoclast-related genes, and resorption pit formation, suggesting the inhibition of differentiation into multinucleated osteoclast-like cells. 2. Materials and methods 2.1. Cell lines and reagents U937 (CRL-1593.2TM, American Type Culture Collection, Rockville, MD) were cultured in DMEM (Gibco/Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco). Phorbol 12-myristate 13-acetate (PMA) was purchased from SigmaeAldrich (St. Louis, MO). Recombinant human RANKL, murine RANKL, human M-CSF, and murine M-CSF were obtained from Peprotech, Inc. (Rocky Hill, NJ).
2.2. Experimental animals Male ICR mice were purchased from Samtako (Osan, Republic of Korea). The mice were maintained in a specific pathogen-free environment and used when they were 6 weeks old. All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee, approved by Institutional Ethical Committee of Sookmyung Women's University (Resolution No. SMU-IACUC-2011-0906-017). 2.3. Differentiation of U937 cells into osteoclast-like cells U937 cells were cultured in 0.1 mg/ml PMA for 2 days (day 2 to day 0) in a 24-well plate at a density of 5 104 cells/well. The cells were then stimulated with a combination of 50 ng/ml M-CSF and 100 ng/ml RANKL. The culture media and stimulators were replenished every 2e3 days. 2.4. Construction and transfection of U937 cells U937 cells expressing NDRG2 were previously generated in our laboratory [17]. The established clone #61 was used as a stable NDRG2-expressing U937 cell line. The MSCV and MSCV-PU.1 plasmids were kind gifts from Dr. H.-J. Lee (KRIBB, Chungbuk, Republic of Korea). Control and NDRG2 siRNAs were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). MSCV, MSCV-PU.1, and the control and NDRG2-targeting siRNAs were transfected into U937-NDRG2 cells using an Amaxa nucleofector (program V-001) (Cologne, Germany). 2.5. Differentiation of murine bone marrow-derived osteoclasts Bone marrow cells isolated from the femurs of ICR mice were harvested, and RBCs were depleted by treatment with RBC lysis buffer (SigmaeAldrich). The cells were pre-cultured in a-MEM (Gibco/Invitrogen) containing 10% FBS and 50 ng/ml M-CSF for 24 h and the non-adherent cells were collected and maintained for 3 days. Finally, the non-adherent cells were discarded, and the bone marrow-derived macrophages (BMM) were differentiated into osteoclasts by treatment with 50 ng/ml M-CSF and 100 ng/ml RANKL for 12 days. TRAP-positive multinuclear cells containing more than 3 nuclei were scored as osteoclasts. 2.6. Tartrate-resistant acid phosphatase (TRAP) staining and TRAP assay
Fig. 1. NDRG2 expression inhibits osteoclast differentiation. (A and B) NDRG2 expression levels were determined by real-time PCR and western blotting. (C and D) PMA-induced monocyte-like cells were treated with M-CSF and RANKL. (C) On day 7, the cells were stained for TRAP. TRAP-positive cells were viewed using a microscope. TRAP concentration was measured on day 4. The results represent the mean ± SD of duplicate experiments (**p < 0.01, Student's t-test). (D) On day 4, the cells were inoculated onto BD Biocoat™ Osteologic™ Discs and differentiated with each of the osteoclast differentiation factors for 10 days, and then the discs were stained by von Kossa staining. The resorption pits were quantified using ImageJ.
TRAP staining was performed using an acid phosphatase leukocyte kit from SigmaeAldrich. TRAP-positive multinuclear cells containing more than 3 nuclei were scored as osteoclasts and were examined using an Olympus microscope (100x) (Model IX71, Olympus Corp.) The TRAP concentrations were determined using the TRAP assay kit from Takara Bio. Inc. (Shiga, Japan). Briefly, 50 ml of cell extract and 50 ml of p-nitro-phenyl phosphate (pNPP) were mixed with a sodium tartrate solution and incubated at 37 C for 20e25 min. The reaction was stopped with 0.5 N NaOH, and the absorbance was measured at 405 nm. A solution containing acid phosphatase and pNPP, minus the sodium tartrate solution, was used as the control. 2.7. Resorption pit formation assay U937 cells were cultured with 0.1 mg/ml PMA for 2 days (day 2 to day 0) in a 24-well plate at a density of 5 104 cells/well. The cells were then stimulated with a combination of 50 ng/ml M-CSF and 100 ng/ml RANKL for 4 days. On day 4, the cells were harvested
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Fig. 2. NDRG2 inhibits the expression of osteoclast-related genes through PU.1 signaling. (AeD) PMA-induced monocyte-like cells were treated with M-CSF and RANKL for 3 days. The mRNA levels were measured by real-time PCR (A and B). PU.1 expression and MAPK kinase activation were measured by western blotting (C and D). (EeH) MSCV or MSCV-PU.1 was transfected into U937-NDRG2 cells using an Amaxa nucleofector (program V-001). PU.1 expression was measured by RT-PCR (E). TRAP concentration represents the mean ± SD of duplicate experiments (*p < 0.05, Student's t-test) (F). Expression levels of indicated genes were measured by real-time PCR (G and H).
and incubated with each stimulator in calcium phosphate-coated plates from BD Biosciences (BD BioCoat™ Osteologic™, San Jose, CA). The transfected primary cells were differentiated into osteoclasts in calcium phosphate-coated plates for 15 days. The resorption pits were observed by von Kossa staining according to the protocol supplied by BD Biosciences (Technical Bulletin #444). The cells were removed with 1 M NaOH, and the wells were stained with 5% silver nitrate for 30 min and then developed with 5% sodium carbonate in 4% formaldehyde for 1 min. After fixation with 5% sodium thiosulfate, the wells were examined under a microscope and quantified using ImageJ (Windows version of NIH ImageJ). 2.8. Reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR RNA was isolated using TRIzol (Gibco/Invitrogen) cDNA was synthesized from 1 mg of total RNA using the M-MLV reverse transcriptase (Promega, Madison, WI). The cDNAs were amplified by PCR. Quantitative real-time PCR was performed using a Power SYBR Green PCR Master Mix (Life Technology, Carlsbad, CA). Briefly, the cDNA was diluted with RNase-free water, and QuantiTect SYBR green PCR master mix and the indicated primers were added to the sample. The sample was amplified using the ABI StepOnePlus™ real-time PCR thermal cycler (Applied Biosystems, Foster City, CA). GAPDH was used as an endogenous control. 2.9. Western blot analysis Cells were washed with D-PBS and lysed in a protein extraction
solution (iNtRON Biotechnology, Seongnam, Republic of Korea). Proteins were separated on a 12% SDS-polyacrylamide gel and transferred to a PVDF membrane. The antibodies for p44/42 MAPK, phospho-p44/42 MAPK, phospho-p38 MAPK, SAPK/JNK, and phospho-SAPK/JNK (Thr183/Tyr185) were purchased from Cell Signaling Technology, Inc. (Beverly, MA), and the antibodies for p38 MAPK, PU.1, NDRG2, actin, and a-actinin were purchased from Santa Cruz Biotechnology. The antigeneantibody complexes were detected by enhanced chemiluminescence. 2.10. Statistical analysis Student's t-test and ANOVA followed by Bonferroni's modification of the Student's t-test were used for statistical analysis. Values are represented as the mean ± SD. A value of p < 0.05 was considered significant. 3. Results 3.1. NDRG2 expression inhibits osteoclast differentiation To investigate the effect of NDRG2 expression on osteoclast differentiation, we transfected U937 cells with a plasmid containing the NDRG2 gene and confirmed that the U937-NDRG2 cells, but not the U937-mock cells, expressed NDRG2 mRNA and protein (Fig. 1A and B). After 2 days of treatment with PMA, most of the U937-mock cells were adherent, suggesting that they had differentiated into monocyte-like cells. In contrast, more than 50% of the U937-NDRG2 cells remained in suspension (data not shown). The cells were treated with a combination of M-CSF and RANKL on day
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Fig. 3. NDRG2 down-regulation reverses the osteoclastogenesis. (AeF) Control or NDRG2 siRNA was transfected into U937-NDRG2 cells using an Amaxa nucleofector (program V001). NDRG2 levels were measured by RT-PCR and western blotting (A). TRAP concentration was measured in M-CSF- and RANKL-induced osteoclast-like cells harvested on day 6 (*p < 0.05, Student's t-test) (B). Expression levels of indicated genes were determined by real-time PCR (C and D). Western blotting was performed using specific MAPK antibodies (E). PU.1 mRNA levels were measured by RT-PCR (F).
0 to induce differentiation into osteoclast-like cells. To determine the osteoclast differentiation, we performed TRAP staining on cells after differentiation into osteoclast-like cells. The differentiated U937-mock cells were TRAP-positive and classified as multinucleated giant cells. In agreement with the TRAP staining data, the U937-mock cells had a higher TRAP concentration than the U937-NDRG2 cells (Fig. 1C). Moreover, although the differentiated U937-mock cells formed a higher number of resorption pits, the U937-NDRG2 cells showed a reduction in resorption pit area (Fig. 1D). Therefore, these data indicate that NDRG2 expression inhibits the differentiation into osteoclast-like cell.
3.2. NDRG2 inhibits the expression of osteoclast-related genes through PU.1 signaling It has been already known that many factors such as OSCAR, cFos, DC-STAMP and NFATc1 participate in osteoclastogenesis [18e21]. First, we investigated whether NDRG2 regulates the expression of osteoclast-related genes. The mRNA expression levels of OSCAR, RANK, c-Fos, DC-STAMP, NFATc1, Atp6V0d2, and MITF were reduced by NDRG2 expression (Fig. 2A). In addition, we confirmed the CSF-1R expression level, because CSF-1R is a receptor of M-CSF and its expression has been known to increase during osteoclastogenesis. The CSF-1R expression was increased in the U937-mock cells by PMA treatment. However, its expression was inhibited in U937-NDRG2 cells in spite of PMA treatment (Fig. 2B). It has been reported that RANKL induces the expression of
osteoclast-related genes through MAPK pathways [22]. Therefore, we investigated which signal is affected by NDRG2 expression. The activation of ERK1/2 and p38 increased during osteoclastogenesis in the U937-mock cells in the presence of M-CSF and RANKL, whereas the activation of these factors was attenuated or delayed by NDRG2 expression (Fig. 2C). Since PU.1 was known to be induced through ERK signaling [23], we investigated whether PU.1 is reduced by NDRG2 expression during M-CSF and RANKL-induced osteoclastogenesis. As expected, the U937-NDRG2 cells showed strongly reduced levels of PU.1 protein expression (Fig. 2D). We next determined whether increased PU.1 expression could induce osteoclast differentiation in U937-NDRG2 cells. U937-NDRG2 cells were transfected with PU.1, the expression of which was verified by RT-PCR (Fig. 2E). Regardless of whether the cells expressed NDRG2, the TRAP concentration was increased in the MSCV-PU.1transfected U937-NDRG2 cells (Fig. 2F). Moreover, the levels of osteoclast-regulating genes were increased in the cells differentiated from MSCV-PU.1-transfected U937-NDRG2 cells (Fig. 2G and H). Therefore, ectopic PU.1 expression was able to induce osteoclast differentiation in U937-NDRG2 cells. These data suggest that NDRG2 expression in U937 cells regulates osteoclastogenesis by down-regulating PU.1 through ERK1/2 and p38 signaling during osteoclastogenesis.
3.3. Osteoclastogenesis is recovered by NDRG2 down-regulation To confirm the inhibitory effect of NDRG2 expression on osteoclastogenesis, an NDRG2 siRNA was transfected into U937-
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Fig. 4. NDRG2 expression inhibits osteoclastogenesis in primary cells. (AeF) Murine bone marrow cells (BM) and macrophages (MØ) were transfected with pCMV/taq2B or pCMV/ taq2B-NDRG2. Diagram showing the scheme for transfecting primary bone marrow cells (A). After 8 h, NDRG2 expression was determined by RT-PCR (B). (C and D) The differentiated cells were stained for TRAP and examined using an Olympus microscope (100x) (Model IX71, Olympus Corp.) (C). The number of osteoclasts that contained more than 3 nuclei was determined for each well. The results represent the mean ± SD of cells from 10 wells (**p < 0.01, ***p < 0.001, Student's t-test) (D). (E and F) The transfected cells were differentiated into osteoclasts on BD Biocoat™ Osteologic™ Discs for 15 days. The discs were stained by von Kossa staining. The resorption pits were viewed using a microscope (100x) (E) and quantified using ImageJ (F).
NDRG2 cells (Fig. 3A). As expected, the TRAP concentration was significantly increased in the NDRG2 siRNA-transfected cells treated with M-CSF and RANKL (Fig. 3B). The expression levels of osteoclastic genes were also increased in the cells differentiated from NDRG2 siRNA-transfected cells (Fig. 3C and D). In addition, we confirmed again that ERK1/2 and p38 activation inhibited by NDRG2 expression was restored in the cells differentiated from NDRG2 siRNA-transfected cells (Fig. 3E). Finally, PU.1 expression decreased by NDRG2 was enhanced in the NDRG2 siRNAtransfected cells (Fig. 3F). Together, these findings demonstrate that NDRG2 expression inhibits osteoclastogenesis in U937 cells. 3.4. NDRG2 expression suppresses osteoclast differentiation in murine primary cells Lastly, we investigated whether the suppressive function of NDRG2 would also be observed in primary cells. Since the primary bone marrow cells and macrophages usually do not express NDRG2, both of primary bone marrow cells and M-CSF-induced macrophages were transfected with NDRG2, and the gene expression in two populations was confirmed by RT-PCR (Fig. 4A and B). In agreement with the results obtained using the U937 cell lines, there were far fewer pCMV/taq2B-NDRG2-transfected bone marrow cells and macrophages that had differentiated into TRAP-positive
osteoclasts and the number of osteoclasts that had differentiated from NDRG2 over-expressing cells was remarkably decreased compared to the control cells (Fig. 4C and D). Moreover, resorption pit formation was decreased by NDRG2 over-expression (Fig. 4E and F). Together, these results demonstrate that NDRG2 expression hinders osteoclast differentiation in the murine primary cells as well as in U937 cells treated with differentiation-inducing factors. 4. Discussion NDRG2 is regarded as a candidate tumor suppressor gene because its overexpression correlates with the induction of apoptosis, inhibition of MMP-9 activity, and decreased invasion potential [24,25]. NDRG2 is also involved in the differentiation of dendritic cells from myeloid cells and regulates IL-10 production in human myeloid cells [11,17]. In this study, we demonstrate, for the first time, the inhibitory effect of NDRG2 expression on osteoclast differentiation. Osteoclast differentiation, survival, and activation are also modulated by mitogen-activated protein kinase (MAPK), phosphoinositide-3-kinase (PI3K)/Akt, and NF-kB signaling. MAPK activation is triggered by RANK in osteoclasts and osteoclast precursors [18]. In the present study, the phosphorylation of SAPK/JNK was either unchanged or slightly elevated during the osteoclastogenesis
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of NDRG2-transfected U937 cells, whereas the activation of ERK1/2 and p38 in these cells was notably impaired. Most importantly, the phosphorylation of p38 MAPK in NDRG2-transfected U937 cells was almost undetectable, even after stimulation with differentiation-inducing factors, suggesting that the inhibitory effect of NDRG2 gene expression on osteoclastogenesis is induced via the suppression of p38 MAPK phosphorylation. In addition, when they were transfected with MSCV-PU.1 to investigate whether PU.1 overexpression is able to induce ERK1/2 activation during osteoclastogenesis, ERK activation was not restored in the cells differentiated from MSCV-PU.1-transfected U937-NDRG2 cells, suggesting that ERK may not be the direct target for PU.1 (data not shown). It has been reported that during osteoclast differentiation induced by M-CSF/RANKL, p38 MAPK was directly recruited to the target promoter and that the phosphorylated forms of MITF simultaneously appeared at these regions [26]. We have shown previously that NDRG2 overexpression suppresses MITF expression and its promoter activity in B16F10 melanoma cells [27]. Likewise, we also confirmed in this study that NDRG2 expression in U937 cells inhibited the expression of MITF during osteoclastogenesis. In earlier reports, MITF was implicated in the survival and differentiation of developmentally unrelated cell types, including melanocytes and osteoclasts [28,29], and it was suggested that the MITF may partly account for the ability of MITF to selectively regulate target genes during osteoclast differentiation [30]. Recently, it was reported that NDRG2-deficient mice exhibit vertebral defects in thoracic/lumbar and lumbar/sacral transitional regions and that the forced overexpression of NDRG2 in osteoblasts or chondrocytes also confers vertebral defects [31], indicating the critical function of NDRG2 at the osteochondrogenic differentiation stage. Previously, we reported that NDRG2 plays an essential role in determining the development of monocytic precursors into DCs [11]. Nonetheless, given that hematopoietic stem cells and myeloid precursors do not express NDRG2, it is highly unlikely that osteoclastogenesis in vivo is directly regulated by NDRG2 expression. Rather, we speculate that NDRG2 may affect the transient induction of gene expression in the presence of specific stimuli that are required for osteoclastogenesis. In fact, NDRG2 has been reported to be one of the important stress-inducible genes. Therefore, it remains to be determined in further study how and to what extent NDRG2 expression is involved in promoting or suppressing osteoclast formation and the development of specific cell lineages, such as DCs. Conflict of interest All authors declared no potential conflicts of interest. Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grants (2012R1A2A2A01046114) funded by Korean government (Ministry of Science, ICT and Future Planning) and partly by the Sookmyung Women's University Research Grant 2013. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.09.071. References [1] W.J. Boyle, W.S. Simonet, D.L. Lacey, Osteoclast differentiation and activation,
Nature 423 (2003) 337e342. [2] N.A. Sims, J.H. Gooi, Bone remodeling: multiple cellular interactions required for coupling of bone formation and resorption, Semin. Cell Dev. Biol. 19 (2008) 444e451. [3] A.P. Anandarajah, Role of RANKL in bone diseases, Trends Endocrinol. Metab. 20 (2009) 88e94. [4] G.D. Roodman, Treatment strategies for bone disease, Bone Marrow Transpl. 40 (2007) 1139e1146. [5] I.E. Adamopoulos, E.D. Mellins, Alternative pathways of osteoclastogenesis in inflammatory arthritis, Nat. Rev. Rheumatol. 11 (2015) 189e194. [6] K.S. Mak, A.P. Funnell, R.C. Pearson, M. Crossley, PU.1 and haematopoietic cell fate: dosage matters, Int. J. Cell Biol. 2011 (2011) 808524. [7] S. Carotta, A. Dakic, A. D'Amico, S.H. Pang, K.T. Greig, S.L. Nutt, et al., The transcription factor PU.1 controls dendritic cell development and Flt3 cytokine receptor expression in a dose-dependent manner, Immunity 32 (2010) 628e641. [8] S.R. McKercher, B.E. Torbett, K.L. Anderson, G.W. Henkel, D.J. Vestal, H. Baribault, et al., Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities, EMBO J. 15 (1996) 5647e5658. [9] E.W. Scott, M.C. Simon, J. Anastasi, H. Singh, Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages, Science 265 (1994) 1573e1577. [10] E.R. Stanley, V. Chitu, CSF-1 receptor signaling in myeloid cells, Cold Spring Harb. Perspect. Biol. 6 (2014). [11] S.C. Choi, K.D. Kim, J.T. Kim, J.W. Kim, D.Y. Yoon, Y.K. Choe, et al., Expression and regulation of NDRG2 (N-myc downstream regulated gene 2) during the differentiation of dendritic cells, FEBS Lett. 553 (2003) 413e418. [12] S.C. Choi, K.D. Kim, J.T. Kim, J.W. Kim, H.G. Lee, J.M. Kim, et al., Expression of human NDRG2 by myeloid dendritic cells inhibits down-regulation of activated leukocyte cell adhesion molecule (ALCAM) and contributes to maintenance of T cell stimulatory activity, J. Leukoc. Biol. 83 (2008) 89e98. [13] Y. Deng, L. Yao, L. Chau, S.S. Ng, Y. Peng, X. Liu, et al., N-Myc downstreamregulated gene 2 (NDRG2) inhibits glioblastoma cell proliferation, Int. J. Cancer 106 (2003) 342e347. [14] C. Mitchelmore, S. Buchmann-Moller, L. Rask, M.J. West, J.C. Troncoso, N.A. Jensen, NDRG2: a novel Alzheimer's disease associated protein, Neurobiol. Dis. 16 (2004) 48e58. [15] K. Takahashi, M. Yamada, H. Ohata, K. Honda, M. Yamada, Ndrg2 promotes neurite outgrowth of NGF-differentiated PC12 cells, Neurosci. Lett. 388 (2005) 157e162. [16] S.K. Shon, A. Kim, J.Y. Kim, K.I. Kim, Y. Yang, J.S. Lim, Bone morphogenetic protein-4 induced by NDRG2 expression inhibits MMP-9 activity in breast cancer cells, Biochem. Biophys. Res. Commun. 385 (2009) 198e203. [17] S.C. Choi, K.D. Kim, J.T. Kim, S.S. Oh, S.Y. Yoon, E.Y. Song, et al., NDRG2 is one of novel intrinsic factors for regulation of IL-10 production in human myeloid cell, Biochem. Biophys. Res. Commun. 396 (2010) 684e690. [18] N.S. Soysa, N. Alles, K. Aoki, K. Ohya, Osteoclast formation and differentiation: an overview, J. Med. Dent. Sci. 59 (2012) 65e74. [19] A. Arai, T. Mizoguchi, S. Harada, Y. Kobayashi, Y. Nakamichi, H. Yasuda, et al., Fos plays an essential role in the upregulation of RANK expression in osteoclast precursors within the bone microenvironment, J. Cell Sci. 125 (2012) 2910e2917. [20] M. Yagi, T. Miyamoto, Y. Sawatani, K. Iwamoto, N. Hosogane, N. Fujita, et al., DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells, J. Exp. Med. 202 (2005) 345e351. [21] H. Takayanagi, The role of NFAT in osteoclast formation, Ann. N. Y. Acad. Sci. 1116 (2007) 227e237. [22] X. Feng, RANKing intracellular signaling in osteoclasts, IUBMB Life 57 (2005) 389e395. [23] M.B. Miranda, D.E. Johnson, Signal transduction pathways that contribute to myeloid differentiation, Leukemia 21 (2007) 1363e1377. [24] A. Kim, M.J. Kim, Y. Yang, J.W. Kim, Y.I. Yeom, J.S. Lim, Suppression of NFkappaB activity by NDRG2 expression attenuates the invasive potential of highly malignant tumor cells, Carcinogenesis 30 (2009) 927e936. [25] H. Shi, N. Li, S. Li, C. Chen, W. Wang, C. Xu, et al., Expression of NDRG2 in esophageal squamous cell carcinoma, Cancer Sci. 101 (2010) 1292e1299. [26] S.M. Sharma, A. Bronisz, R. Hu, K. Patel, K.C. Mansky, S. Sif, et al., MITF and PU.1 recruit p38 MAPK and NFATc1 to target genes during osteoclast differentiation, J. Biol. Chem. 282 (2007) 15921e15929. [27] A. Kim, Y. Yang, M.S. Lee, Y.D. Yoo, H.G. Lee, J.S. Lim, NDRG2 gene expression in B16F10 melanoma cells restrains melanogenesis via inhibition of Mitf expression, Pigment Cell Melanoma Res. 21 (2008) 653e664. [28] J. Liu, M. Fukunaga-Kalabis, L. Li, M. Herlyn, Developmental pathways activated in melanocytes and melanoma, Arch. Biochem. Biophys. 563 (2014) 13e21. [29] J.H. Kim, N. Kim, Regulation of NFATc1 in osteoclast differentiation, J. Bone Metab. 21 (2014) 233e241. [30] R. Hu, S.M. Sharma, A. Bronisz, R. Srinivasan, U. Sankar, M.C. Ostrowski, Eos, MITF, and PU.1 recruit corepressors to osteoclast-specific genes in committed myeloid progenitors, Mol. Cell. Biol. 27 (2007) 4018e4027. [31] H. Zhu, J. Zhao, W. Zhou, H. Li, R. Zhou, L. Zhang, et al., Ndrg2 regulates vertebral specification in differentiating somites, Dev. Biol. 369 (2012) 308e318.