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Keywords:

  • MAFB;
  • MICRORNA;
  • OSTEOCLASTOGENESIS;
  • OSTEOPOROSIS;
  • PBMCS

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

MicroRNAs (miRNAs) play crucial roles in bone metabolism. In the present study, we found that miR-148a is dramatically upregulated during osteoclastic differentiation of circulating CD14+ peripheral blood mononuclear cells (PBMCs) induced by macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). Overexpression of miR-148a in CD14+ PBMCs promoted osteoclastogenesis, whereas inhibition of miR-148a attenuated osteoclastogenesis. V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) is a transcription factor negatively regulating RANKL-induced osteoclastogenesis. miR-148a directly targeted MAFB mRNA by binding to the 3′ untranslated region (3′UTR) and repressed MAFB protein expression. In vivo, our study showed that silencing of miR-148a using a specific antagomir-inhibited bone resorption and increased bone mass in mice receiving ovariectomy (OVX) and in sham-operated control mice. Furthermore, our results showed that miR-148a levels significantly increased in CD14+ PBMCs from lupus patients and resulted in enhanced osteoclastogenesis, which contributed to the lower bone mineral density (BMD) in lupus patients compared with normal controls. Thus, our study provides a new insight into the roles of miRNAs in osteoclastogenesis, and contributes to a new therapeutic pathway for osteoporosis. © 2013 American Society for Bone and Mineral Research.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Bone resorption is controlled in part by microRNAs (miRNAs), which are small (∼22 nucleotides), single-stranded noncoding RNAs that may control over 30% of all human genes.1, 2 miRNAs are required for normal osteoclastogenesis in vivo,3, 4 and several miRNAs have been characterized that control osteoclast differentiation and function.5–9 miR-21 regulates osteoclastogenesis of bone marrow–derived monocyte/macrophage precursors (BMMs) via targeting programmed cell death protein 4 (PDCD4).5 miR-223 expresses in the RAW264.7 osteoclast precursor cell line and controls osteoclast differentiation by regulating nuclear factor I-A (NFI-A) and the macrophage colony-stimulating factor receptor (M-CSFR) levels.6 In contrast, Mann and colleagues7 found that miR-155 was downregulated in osteoclasts and upregulated in macrophages. Nakasa and colleagues8 found that miR-146a expression inhibits osteoclastogenesis. However, the functions of miRNAs in osteoclast differentiation still needs further investigation.

The osteoclast is the primary bone-resorbing cell and is formed from fusion of precursors. CD14+ peripheral blood mononuclear cells (PBMCs) serve as early progenitors of osteoclasts.10 A recent research revealed osteoclast formation in the presence of both macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) observed in culture of the circulating CD14+ PBMCs.11

The present study was undertaken to investigate the role of target miRNA in osteoclast differentiation. First, we performed miRNA profiling and identified the most significantly regulated miRNA during osteoclastogenesis using CD14+ PBMCs culture exposure to M-CSF + RANKL. Then the function of miRNA was investigated.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

CD14+ PBMC cultures

Whole blood was obtained from healthy donors under a protocol approved by the Ethnic Committee of the Second Xiangya Hospital of Central South University. PBMCs were isolated from buffy coats (Duo-Add blood bags; Baxter, Deerfield, IL, USA) using Ficoll-Paque (Amersham Pharmacia Biotech, Roosendaal, Netherlands) as described.11 Before further purification, granulocyte contamination was reduced to <1% by a second Ficoll-Paque separation step. Cell purification was achieved using CD14 antibody-coated magnetic cell sorting (MACS) MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Purity was assessed using flow cytometry (FACSCalibur; Becton Dickinson, Franklin Lakes, NJ, USA). The CD14+ PBMCs (2.5 × 105/well in 48-well cluster plate) were cultured in α modified essential medium (α-MEM) containing 10% fetal bovine serum (FBS) as osteoclasts precursors in osteoclast formation experiments.

Osteoclastic differentiation was induced by changing to media containing 10% FBS supplemented with 25 ng/mL recombinant human M-CSF and 30 ng/mL RANKL (R&D Systems Inc, Minneapolis, MN, USA). The growth and function of osteoclasts was assessed by tartrate-resistant acid phosphatase (TRAP) staining, TRAP activity detection in culture medium, and mRNA detection of osteoclast differentiation marker genes including TRAP and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1).

miRNA microarray assay

The RNA of CD14 + PBMCs cultured with or without M-CSF + RANKL for 14 days were extracted. Small RNA was isolated and labeled either with Cy3 or Cy5. miRNA microarray assay was completed by LC Sciences. The probe content was accordant with Version 14.0 of the Sanger miRBase database. After subtracting the background, the fluorescence value was detected. The ratio of the two subgroups (log2 transformed) and p values of the t test were calculated. The miRNAs with p value <0.01 and fluorescence value >500 were selected.

Bone mineral density measurement

The left femur of each mouse was fixed onto the scanning table along the longitudinal axis, and the whole femur was scanned by dual-energy X-ray absorptiometry (DXA) using a PIXImus densitometer (GE Lunar, Fairfield, CT, USA) to determine the bone mineral density (BMD).12

Human BMD was measured using a DXA fan-beam bone densitometer (Hologic QDR 4500A; Hologic, Bedford, MA, USA) at the lumbar spine (L1–L4) and the left femoral neck as described.13, 14 All BMD results were expressed in g/cm2. The reference ranges for the Z-scores were derived from our own databases.13 The control spine phantom scan performed each day had a long-term (more than 10 years) coefficient of variation of less than 0.43%.

Study population

The clinical study was approved by the Ethics Committee of the Second Xiangya Hospital of Central South University, and written informed consents were obtained from all participants. The study population was comprised of 31 Chinese premenopausal women including 16 lupus patients and 15 normal controls. The lupus patients met at least four American College of Rheumatology (ACR) criteria for systemic lupus erythematosus (SLE) and had clinical definition of SLE without glucocorticoids therapy history. All subjects were screened using a detailed questionnaire, by disease history, and by physical examination.15 Information on dietary calcium intake through a food or drug frequency questionnaire, as well as habitual physical activity and smoking habits was investigated. Total calcium intake was calculated by adding dietary calcium intake and calcium from supplements. Subjects were excluded from the study if they had oligomenorrhea or amenorrhea before 40 years of age. Subjects were excluded from the study if they had conditions affecting bone metabolism, including diseases of the kidney, liver, parathyroid, thyroid, diabetes mellitus, hyperprolactinemia, oophorectomy, rheumatoid arthritis, ankylosing spondylitis, malabsorption syndromes, malignant tumors, hematologic diseases, previous pathological fractures, or traumatic fractures within 1 year. Subjects were also excluded if they had hypertension, chronic liver disease, coronary artery disease, angiopathy, myocardial infarction, cerebral infarction, or infectious disease. If the subjects had received treatment with glucocorticoids, estrogens, thyroid hormone, parathyroid hormone, fluoride, bisphosphonate, calcitonin, thiazide diuretics, barbiturates, anti-seizure medication, vitamin D, or calcium-containing drugs, they were also excluded. Body height and weight were measured using a stadiometer and standardized balance-beam scale, respectively. All women gave their informed consent to participate. All subjects were selected on the basis of having similar levels of serum estradiol, vitamin D, and parathyroid hormone. Table 1 shows the characteristics, bone, and biochemical parameters of 31 subjects.

Table 1. Subject Characteristics, and Bone and Biochemical Parameters of 31 Chinese Women
 Premenopausal normal womenSLE patients
  1. Values are shown as mean ± SD.

  2. SLE = systemic lupus erythematosus; BMI = body mass index; BMD = bone mineral density; PTH = parathyroid hormone.

  3. *p < 0.05.

n1516
Age (years)30.53 ± 5.9930.19 ± 6.36
Height (cm)158.53 ± 4.11159.66 ± 2.73
Body weight (kg)54.81 ± 3.4055.32 ± 3.23
BMI (kg/m2)21.83 ± 1.4021.70 ± 1.12
Calcium intake (mg/d)655.47 ± 18.88656.13 ± 19.48
Physical activity (hours/week)6.54 ± 0.476.50 ± 0.43
Smoking (%)00
Lumbar spine BMD (g/cm2)0.75 ± 0.040.62 ± 0.04
Lumbar spine Z-score–0.33 ± 0.38*–1.76 ± 0.38
Femoral neck BMD (g/cm2)0.77 ± 0.030.63 ± 0.04
Femoral neck Z-score–0.20 ± 0.36*–1.68 ± 0.32
25-Hydroxyvitamin D (ng/mL)32.90 ± 10.0933.24 ± 12.62
PTH (pg/mL)52.51 ± 24.5852.25 ± 18.15
Estradiol (pM)51.85 ± 19.0254.64 ± 20.62

Statistics

Data are presented as mean ± SD. Comparisons were made using one-way ANOVA. All experiments were repeated at least three times, and representative experiments are shown. Differences were considered significant at p < 0.05.

Supplemental Materials and Methods

Technical details for performing qRT-PCR analysis, Western blot, Northern blot, plasmid constructs and transfections, luciferase reporter assay, mice, micro–computed tomography (µCT) analysis, histomorphometric analysis, and biochemical analysis are documented in the Supplemental Materials (Supplemental Tables S1–S3 and Figs. S1–S4).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

miR-148a dramatically upregulated during osteoclast differentiation

To investigate the miRNA expression profile during osteoclastogenesis, microarray assays were performed. CD14+ PBMCs from young healthy volunteers were induced to osteoclastogenesis by treatment with M-CSF + RANKL. CD14+ PBMCs from the same subject were cultured without M-CSF + RANKL as controls. Our results showed that 27 miRNAs were differently expressed during the differentiation of PBMCs into osteoclasts (Fig. 1A, Supplemental Table S1). Also, hsa-miR-148a, hsa-miR-483, hsa-miR-223, hsa-miR-21, and hsa-miR-214 were upregulated. Conversely, hsa-miR-155, hsa-miR-125a, hsa-miR-27b, hsa-miR-145 were downregulated. The results of real-time PCR were consistent with that of miRNA microarray (Fig. 1B).

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Figure 1. miR-148a dramatically upregulated during osteoclast differentiation. (A) Microarray assays were performed in the process of osteoclast differentiation. Red and green indicate high expression levels and low expression levels, respectively. Shown are the parts of the miRNAs that changed more than twofold. (B) The results of qRT-PCR were consistent with miRNA microarray. (C) Northern blot and qRT-PCR show miR-148a increased progressively with time during osteoclast differentiation. The level of miR-148a was normalized to U6 (n = 5).

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As the most dramatically upregulated miRNA, we chose to study miR-148a and investigated its expression pattern during osteoclast differentiation. Northern blot showed miR-148a started to be expressed on 5 days in CD14+ PBMCs after treatment with M-CSF + RANKL and increased progressively. On day 15 after induction, the expression of miR-148a reached the maximum (Fig. 1C). Theses results suggested that miR-148a plays important roles in osteoclast differentiation.

miR-148a regulated M-CSF + RANKL–induced osteoclastogenesis

To investigate the role of miR-148 during osteoclast differentiation, PBMCs were transfected with the miRNA precursor (pre-miR-148a) and the level of mature miR-148a was assessed by Northern blotting (Fig. 2A). After treatment with M-CSF + RANKL for 6 days, osteoclast formation was investigated. Nuclear factor of activated T-cells cytoplasmic (NFATc1) and TRAP were used as reliable markers of osteoclasts.16, 17 Our results showed that after pre-miR-148a transfection, TRAP+ multinucleated giant cells (Fig. 2B), TRAP activity and expression, and NFATc1 expression (Fig. 2C, D) increased significantly. Conversely, 2′-O-methyl antisense inhibitory oligoribonucleotides (anti-miR-148a), a proven effective blocker of miR-148a, inhibited osteoclastogenesis significantly (Fig. 2BD). Furthermore, we transfected the CD14+ PBMCs with pre-miR-148a adenoviral vector. Our results showed that after treatment with M-CSF + RANKL for 15 days, overexpression of miR-148a significantly increased the bone resorption area on dentin slices (Fig. 2E), indicating that miR-148a also regulates osteoclast differentiation at this late stage.

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Figure 2. miR-148a regulated M-CSF + RANKL–induced osteoclastogenesis of CD14+ PBMCs. (A) CD14+ PBMCs from young healthy volunteers (n = 5) were transfected with pre-miR-148a or antagomiR-148a. miR-148a levels were detected using Northern blot. (B) After osteoclast differentiation was induced with M-CSF + RANKL for 6 days, representative microscopic views of TRAP+ osteoclasts formation and TRAP+ multinucleated giant cell numbers are shown. (C) TRAP+ multinucleated giant cell numbers and TRAP activity are shown. (D) TRAP and NFATc1 mRNA levels were determined using qRT-PCR and are shown as fold induction relative to β-actin. (E) Bone resorption area on dentin slices was measured by image analysis. Data are shown as means ± SD. *p < 0.05 versus control.

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We also investigated the role of miR-148a in osteoclastogenesis of bone marrow cells from mice. After treatment with M-CSF + RANKL for 7 days, our results showed overexpression of miR-148a enhanced osteoclast differentiation whereas inhibition of miR-148a decreased osteoclast differentiation (Supplemental Fig. S1).

These results showed that miR-148a played a positive regulatory role in osteoclast differentiation.

miR-148a directly targeted V-maf musculoaponeurotic fibrosarcoma oncogene homolog B

It has been demonstrated that miRNAs executed regulatory function on the expression of mRNAs by binding the 3′ untranslated region (3′UTR) of the target gene.18, 19 To predict the possible targets of miR-148a, TargetScan release 5.0 was used (Bioinformatics and Research Computing, Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA, USA; http://www.targetscan.org/vert_50/).20 In the TargetScan database, only conserved miRNAs that target conserved gene transcripts were considered. We predicted the target gene of hsa-miR-148a and got a total of 536 possible target genes. We focused on the predicted genes that had been reported in the regulatory relationship with osteoclastogenesis. V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB), a reported negative regulator of osteoclastogenesis, was one of the proper genes we chose.21 TargetScan provided us one conserved miR-148a target in the 3′UTR of MAFB (Fig. 3A). The results suggested the potential regulatory effect of miR-148a on the MAFB gene.

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Figure 3. miR-148a directly targeted MAFB. (A) TargetScan was used to predict the miR-148a putative target site in human MAFB mRNA. A schematic of miR-148a with WT and MUT 3′UTR regions of MAFB are shown in complementary pairing. The three mutated nucleotides are underlined. (B) CD14+ PBMCs from postmenopausal normal women (n = 5) were cotransfected with the luciferase reporter carrying WT-pGL3-MAFB-3′UTR or MUT-pGL3-MAFB-3′UTR, phRL-null, and miR-C or pre-miR-148a. Effects of miR-148a and miR-C on the reporter constructs were determined 48 hours after transfection. Firefly luciferase values normalized for Renilla luciferase are presented. (C) CD14+ PBMCs were transfected with pre-miR-148a or antagomiR-148a, then induced with M-CSF + RANKL for 6 days. MAFB mRNA levels determined by qRT-PCR are shown as fold induction relative to β-actin. MAFB protein was determined by Western blot and expressed as densitometry of RANK/β-actin. Data are shown as means ± SD. *p < 0.05 versus control.

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To validate whether miR-148a can directly regulate MAFB, the luciferase reporter construct containing 3′UTR of MAFB was generated and three mutations were introduced into the predicted miRNA-binding site. We introduced the luciferase expression vector containing the 3′UTR of MAFB (WT-pGL3-MAFB) with pre-miR-148a into PBMCs and measured the effects of miR-148a on luciferase translation by the level of luciferase enzyme activity. Overexpression of miR-148a could suppress the luciferase activity of the MAFB 3′UTR reporter gene. Mutation of the three nucleotides within the sequences of the putative target site in the 3′UTR of MAFB (MUT-pGL3-MAFB) abolished this repression, confirming the specificity of the action (Fig. 3B).

To directly verify whether this conserved site was the actual binding region or not, we transfected PBMCs with pre-miR-148a or anti-miR-148a and measured the mRNA and protein levels of MAFB by qRT-PCR and Western blotting. Relative to the control, overexpression of miR-148a decreased MAFB protein expression and inhibition of miR-148a increased MAFB protein expression, whereas no changes in MAFB mRNA levels were noted (Fig. 3C). Furthermore, we cotransfected the CD14+ PBMCs with anti-miR-148a and short hairpin RNA (shRNA) against MafB. MafB protein decreased significantly after shRNA transfection (Supplemental Fig. S2A). After treatment with M-CSF + RANKL for 6 days, our results showed that the inhibited osteoclastogenesis was rescued by shRNA against MafB (Supplemental Fig. S2B), indicating that MafB was the main target of miR-148a in osteoclasts.

Because it has been reported that miR-148a regulates microphthalmia-associated transcription factor (Mitf) expression in melanoma cells, we investigated the effect of miR-148a on Mitf gene expression in osteoclasts. Our results showed that after overexpression or inhibition of miR-148a, no significant changes of Mitf isoforms A and E protein levels were noted, indicating that Mitf was not the target of miR-148a in osteoclasts (Supplemental Fig. S3).

These results showed that MAFB was the target of miR-148a in osteoclast differentiation.

miR-148a regulated bone metabolism in vivo

To investigate the function of miR-148a in vivo, mice that had undergone sham operation (SHAM group) or ovariectomy (OVX group) were given antagomiR-148a via a single tail vein injection. AntagomiR-148a is a chemically modified oligonucleotide that can specifically enhance or inhibit the expression of mir-148a in vivo. Mut-antagomiR-148a and PBS were used as controls. Northern blotting showed that after mice were injected with antagomir, the inhibition of miR-148a in bone could last for 3 weeks, whereas mutant antagomir and PBS had no effect on miR-148a expression (Fig. 4A). To maintain the effect of miR-148a on mice, the mice received another injection of antagomir-148a on days 1 to 3 in the fourth week after the first injection. All mice were euthanized for analysis by the end of the sixth week after the first injection.

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Figure 4. AntagomiR-148a treated mice showed increased bone mass and decreased bone resorption. (A) Mice were injected intravenously with PBS, mut-antagomiR-148a, or antagomir-148a. miR-148a expression was analyzed by Northern blot at 4 days, 3 weeks, and 6 weeks after the first injection in bones. (B) Six weeks after the first injection, BMD of femurs was measured using a PIXImus densitometer. (C) µCT section of proximal tibia diaphyses are shown. (D) Structure parameter BV/TV was measured by µCT. (E) Bone resorption parameter N.Oc/B.Pm of tibia was measured by histomorphometric analysis. (F) Bone formation parameter BFR/BS was measured by histomorphometric analysis. (G) qRT-PCR analysis of TRAP mRNA in bone. (H) Serum activity of TRAP5b. (I) MAFB mRNA levels in bone were determined by qRT-PCR and are shown as fold induction relative to β-actin. MAFB protein levels in bone were determined by Western blot and expressed as densitometry of RANK/β-actin. Data are shown as means ± SD of 6 mice per group. *p < 0.05 versus control.

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The results showed that in both SHAM and OVX groups, antagomiR-148a–treated mice exhibited a significant increase in femur BMD by PIXImus in comparison with mice treated with mut-antagomiR-148a or PBS (Fig. 4B). Quantification of the tibia bone volume/tissue volume ratio (BV/TV) and trabecular thickness (Tb.Th) was conducted using µCT (Fig. 4C). The assay revealed that in SHAM mice, the absence of miR-148a increased BV/TV and Tb.Th (Fig. 4D, Supplemental Fig. S4A). OVX mice treated with mutant antagomir-148a or PBS exhibited a significant decrease in BV/TV and Tb.Th. However, antagomir-148a improved these indices (Fig. 4D, Supplemental Fig. S4A). These results revealed that miR-148a was involved in the regulation of bone metabolism.

We next measured several parameters of bone formation and resorption by histomorphometric analysis. We assessed bone resorption parameters; ie, osteoclast surface per bone surface (Oc.S/BS) and the number of osteoclasts per bone perimeter (N.Oc/B.Pm). OVX augmented the levels of Oc.S/BS and N.Oc/B.Pm, whereas antagomir-148a treatment decreased the levels of bone resorption parameters (Fig. 4E, Supplemental Fig. S4B). We also assessed bone formation parameters. Results showed that in SHAM groups, antagomiR-148a–treated mice exhibited decreased bone formation rate per bone surface (BFR/BS), mineral appositional rate, osteoblast surface per bone surface (Ob.S/BS), and number of osteoblasts per bone perimeter (N.Ob/B.Pm). These bone formation parameters were increased in OVX mice treated with mutant antagomiR-148a or PBS. However, antagomiR-148a decreased the levels of these bone formation parameters (Fig. 4F, Supplemental Fig. S4C).

Furthermore, antagomiR-148a treatment decreased the levels of osteoclast activity markers such as TRAP and NFATc1 mRNA in bone tissue (Fig. 4G, Supplemental Fig. S4D). Levels of osteoblast activity marker alkaline phosphatase (ALP) mRNA in bone tissue changed similarly (Supplemental Fig. S4E). Serum TRAP activity and ALP activity were also significantly decreased in antagomiR-148a treatment mice (Fig. 4H, Supplemental Fig. S4F).

By Western blot, we noticed that in SHAM and OVX groups, antagomiR-148a–treated mice exhibited a significant increase in MAFB protein levels. However, no MAFB mRNA change was detected (Fig. 4I). Furthermore, we checked MafB expression in bone marrow cells of mice using immunohistochemical analysis. Our results showed that after antagomiR-148a injection in vivo, the MafB level decreased significantly in osteoclast precursors (Supplemental Fig. S4G).

These results collectively showed that inhibition of miR-148a elevated bone mass by decreasing bone resorption.

miR-148a levels significantly increased in CD14+ PBMCs of SLE patients

Several investigators have reported a relationship between SLE and lower BMD in patients never receiving corticosteroids,22–24 suggesting that SLE per se may be a risk factor for bone loss. Pan and colleagues25 have found that miR-148a was overexpressed in T cells from lupus patients. To investigate whether miR-148a also increases in CD14+ PBMCs of lupus patients and contributes to their lower BMD, we selected 16 female lupus patients who had never received corticosteroid therapy. Fifteen age-matched normal women served as controls. Compared with controls, lupus patients had lower BMD (Table 1). CD14+ PBMCs were isolated from both subgroups and miR-148a levels were detected using qRT-PCR. Our results showed that miR-148a levels were significantly increased in CD14+ PBMCs from lupus patients, who had lower BMD compared with normal controls (Fig. 5A).

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Figure 5. CD14+ PBMCs of SLE patients had increased miR-148a levels and enhanced osteoclastogenesis. (A) MiR-148a levels of CD14+ PBMCs from SLE patients (n = 16) and normal controls (n = 15) were determined by qRT-PCR and are shown as fold induction relative to U6. (B) qRT-PCR showed that miR-148a increased progressively with time during osteoclast differentiation in SLE patients. The level of miR-148a was normalized to U6 (n = 5). (C) After osteoclast differentiation was induced with M-CSF + RANKL for 6 days, TRAP+ multinucleated giant cell numbers are shown. (D) TRAP activity was determined using an ELISA kit. (E) NFATc1 and TRAP mRNA levels were determined using qRT-PCR and are shown as fold induction relative to β-actin. Data are shown as means ± SD. *p < 0.05 versus control.

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miR-148a contributed to the increased osteoclastogenesis of CD14+ PBMCs in lupus patients

CD14+ PBMCs from lupus patients and normal controls described in the previous section were induced to osteoclastogenesis by M-CSF + RANKL. The miR-148a expression pattern of CD14+ PBMCs from lupus patients was investigated during 15 days of induction. Our results showed that miR-148a increased progressively with time during osteoclast differentiation (Fig. 5B), which is similar to the expression pattern of miR-148a from normal controls. Furthermore, osteoclast formation, and NFATc1 and TRAP expression were investigated after 6 days of induction. Our results showed that TRAP+ multinucleated giant cells (Fig. 5C), TRAP activity (Fig. 5D) and expression, and NFATc1 expression (Fig. 5E) increased more obviously in cultured CD14+ PBMCs from lupus patients, indicating the enhanced ability to osteoclastogenesis. However, after inhibition of miR-148a with anti-miR-148a, the increased osteoclastogenesis of CD14+ PBMCs from lupus patients was restrained (Fig. 6A, B). These results suggested miR-148a contributed to the enhanced osteoclastogenesis of CD14+ PBMCs in lupus patients.

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Figure 6. Inhibition of miR-148a restrained the increased osteoclastogenesis of CD14+ PBMCs from lupus patients. (A) After inhibition of miR-148a with anti-miR-148a, CD14+ PBMCs from SLE patients (n = 16) were induced to osteoclast differentiation with M-CSF + RANKL for 6 days. TRAP+ multinucleated giant cell numbers and TRAP activity are shown. (B) TRAP and NFATc1 mRNA levels were determined using qRT-PCR and are shown as fold induction relative to β-actin. Data are shown as means ± SD. *p < 0.05 versus control.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

In the present study, we found that miR-148a dramatically upregulated during M-CSF + RANKL-induced osteoclastogenesis of CD14+ PBMCs. Overexpression of miR-148a promoted osteoclastogenesis, whereas inhibition of miR-148a attenuated osteoclastogenesis in vitro and in vivo. miR-148a regulated osteoclastogenesis by targeting the MAFB. Furthermore, miR-148a contributed to the increased osteoclastogenesis of CD14+ PBMCs and lower BMD in lupus patients.

Bone metabolism is balanced by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Osteoporosis occurred when osteoclastogenesis exceeding osteoblastogenesis.26 RANKL-RANK-osteoprotegerin (OPG) is the most important system regulating osteoclastogenesis.27 As early progenitors of osteoclasts, circulating CD14+ PBMCs express RANK that recognizes RANKL through direct cell-to-cell interaction with osteoblast/stromal cells and differentiate into mature osteoclasts. OPG competes with RANK, which results in the inhibition of osteoclastogenesis and bone resorption.28 However, the mechanism regulating osteoclastogenesis still needs further investigation.

miRNAs are a kind of small, functional, highly conserved, noncoding RNAs of 19 to 23 nucleotides, inducing posttranscriptional regulation by either translational repression or cleaving the target mRNAs. miRNAs play a key role in various biological processes including osteoblast differentiation and mineralization.29–31 Osteoclastogenesis is also regulated by miRNAs. In the present study, we performed microarray assays and found miR-148a dramatically upregulated during osteoclast differentiation. Furthermore, we investigated the expression pattern of miR-148a during osteoclast differentiation and found that miR-148a increased progressively after M-CSF + RANKL induction of CD14+ PBMCs. These results indicated that miR-148a may participate in osteoclastogenesis.

To determine whether miR-148a is directly coupled to osteoclastogenesis, we investigated the action of miR-148a in the process of M-CSF + RANKL–induced osteoclastogenesis. CD14+ PBMCs were transfected with pre-miR-148a or anti-miR-148a, and then induced to osteoclast differentiation by M-CSF + RANKL. miR-148a overexpression promoted osteoclastogenesis; ie, increased the levels of NFATc1 and TRAP, the number of TRAP+ multinucleated giant cells, and the bone resorption area on dentin slices. In contrast, inhibition of miR-148a expression attenuated osteoclastogenesis in CD14+ PBMCs. These results showed that miR-148a was involved in osteoclast differentiation, and then the mechanism was investigated.

miRNAs have been shown to inhibit mRNA translation or to decrease mRNA stability by binding sequences in the 3′UTR of the target mRNA. To further investigate the role of miR-148a in osteoclast differentiation, the bioinformatic algorithm TargetScan was used to predict the target genes. TargetScan predicts biological targets of miRNAs by searching for the presence of conserved 8mer and 7mer sites that match the seed region of each miRNA. We found that MAFB, a reported negative regulator of osteoclastogenesis, was one of the predicted genes by TargetScan. TargetScan provided us one conserved miR-148a target in the 3′UTR of MAFB (Fig. 3A). The results suggested the potential regulatory effect of miR-148a on the MAFB gene.

MAFB is a bZIP transcription factor, important for podocyte differentiation,32 rhombomere specification in the early hindbrain, and for respiratory control.33 Moreover, in the hematopoietic system, MAFB regulates myeloid differentiation and promotes macrophage differentiation.34 Recently, it has been reported that MAFB negatively regulates RANKL-induced osteoclastogenesis by attenuating DNA binding of the key regulators NFATc1, c-Fos, and Mitf. This suggested that miR-148a induces osteoclast differentiation by repressing MAFB.

Several lines of evidence indicate that MAFB is the target of miR-148a. First, the seed region of miR-148a binds to the 3′UTR of MAFB mRNA with complementarity, suggesting miR-148a directly regulates MAFB expression. Second, we constructed luciferase reporter vectors and found that overexpression of miR-148a inhibited the activity of WT-pGL3-MAFB whereas MUT-pGL3-MAFB abolished this repression. Third, overexpression of miR-148a decreased MAFB protein whereas inhibition of miR-148a increased MAFB protein without changing mRNA levels. The last, the inhibited osteoclastogenesis by anti-miR-148a, was rescued by shRNA against MafB. All these results showed that miR-148a directly targeted MAFB and repressed MAFB protein expression by inhibiting mRNA translation, not by targeting mRNA for degradation.

Because miR-148a facilitates osteoclast differentiation in vitro, we built mice models to investigate its role in bone metabolism in vivo. Antagomir-148a was injected into a single tail vein to generate mice lacking miR-148a. Our results showed antagomir-148a treatment led a significant increase of tibia BMD in both SHAM and OVX mice. Bone parameters BV/TV and Tb.Th also increased. Furthermore, antagomir-148a treatment decreased osteoclast numbers and the levels of osteoclast activity markers such as TRAP and NFATc1 in bone tissue, accompanied by decreased osteoblast activity. Because miR-148a does not express in osteoblasts,29 the decrease of osteoblast function after antagomir-148a treatment may be a result of the decreased osteoclast activity that led to impaired coupling of resorption and formation. Many studies have showed bone resorption was coupled with bone formation.35–37 Tang and colleagues35 found that active transforming growth factor-β1 (TGF-β1) released from bone matrix during bone resorption directs the migration of bone marrow mesenchymal stem cells to form new bones. Dai and colleagues38 found that osteoclast deficiency results in disorganized matrix, reduced mineralization, and abnormal osteoblast behavior in developing bone. Thus, the decreased bone formation in mice may be attributed to the decreased bone resorption after antagomir-148a treatment. Conversely, with the decreased bone resorption in antagomir-148a treated mice, MAFB protein expression was increased. These data suggested that miR-148a influenced bone mass by regulating bone resorption in vivo, predominantly through its effect on MAFB.

SLE is a complex autoimmune disease and osteoporosis is an important and potential comorbidity among SLE patients. Although corticosteroid therapy may contribute to the lower BMD, several studies have showed a relationship between SLE and lower BMD in patients never receiving corticosteroids, suggesting that SLE per se may be a risk factor for bone loss. Because miR-148a plays an important role in osteoclastogenesis, and Pan and colleagues25 found that miR-148a is overexpressed in T cells from lupus patients, we were very interested in whether miR-148a also increases in CD14+ PBMCs of lupus patients and contributes to their lower BMD. Our results showed that miR-148a was significantly increased in CD14+ PBMCs from lupus patients compared with normal controls. Compared with controls, lupus patients also had lower BMD. Furthermore, the osteoclastogenesis ability of CD14+ PBMCs from lupus patients increased. After inhibition of miR-148a, the enhanced osteoclastogenesis of CD14+ PBMCs was restrained. These results suggested that miR-148a contributed to the increased osteoclastogenesis of CD14+ PBMCs and lower BMD in lupus patients.

In conclusion, the present study provides evidence that miR-148a in CD14+ PBMCs regulates osteoclast differentiation by targeting MAFB protein. Furthermore, increased miR-148a levels in CD14+ PBMCs resulted in the enhanced osteoclastogenesis and contributed to the lower BMD in lupus patients. Our findings provide a new insight into the roles of miRNAs in osteoclastogenesis, and contribute to a new therapeutic pathway for osteoporosis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

This work was supported by China National Funds for Distinguished Young Scientists grant 81125006, National Natural Science Foundation grant 30870925 and 81000122 from China, and Specialized Research Fund for the Doctoral program of High Education grant 20110162110038 from China.

Authors' roles: Study design: XHL. Study conduct: PC, CC, and RH. Data collection: PC, RH, HBH, and WZ. Bioinformatics analysis: HX and CC. Data analysis: CC, HDZ, and XPW. Data interpretation: RCD and EYL. Drafting manuscript: PC and CC. Revising manuscript: XHL and PC. Approving final version of manuscript: All authors. XHL and PC take responsibility for the integrity of the data analysis.

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  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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