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Keywords:

  • RELAXIN;
  • BMP;
  • BONE;
  • RXFP

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

Relaxin (Rln), a polypeptide hormone of the insulin superfamily, is an ovarian peptide hormone that is involved in a diverse range of physiological and pathological reactions. In this study, we investigated the effect of Rln on bone morphogenetic protein 2 (BMP-2)-induced osteoblast differentiation and bone formation. Expression of Rln receptors was examined in the primary mouse bone marrow stem cells (BMSCs) and mouse embryonic fibroblast cell line C3H/10T1/2 cells by RT-PCR and Western blot during BMP-2–induced osteoblast differentiation. The effect of Rln on osteoblast differentiation and mineralization was evaluated by measuring the alkaline phosphatase activity, osteocalcin production, and Alizarin red S staining. For the in vivo evaluation, BMP-2 and/or Rln were administered with type I collagen into the back of mice, and after 3 weeks, bone formation was analyzed by micro–computed tomography (µCT). Western blot was performed to determine the effect of Rln on osteoblast differentiation-related signaling pathway. Expression of Rxfp 1 in BMSCs and C3H/10T1/2 cells was significantly increased by BMP-2. In vitro, Rln augmented BMP-2–induced alkaline phosphatase expression, osteocalcin production, and matrix mineralization in BMSCs and C3H/10T1/2 cells. In addition, in vivo administration of Rln enhanced BMP-2–induced bone formation in a dose-dependent manner. Interestingly, Rln synergistically increased and sustained BMP-2–induced Smad, p38, and transforming growth factor-β activated kinase (TAK) 1 phosphorylation. BMP-2–induced Runx 2 expression and activity were also significantly augmented by Rln. These results show that Rln enhanced synergistically BMP-2–induced osteoblast differentiation and bone formation through its receptor, Rxfp 1, by augmenting and sustaining BMP-2–induced Smad and p38 phosphorylation, which upregulate Runx 2 expression and activity. These results suggest that Rln might be useful for therapeutic application in destructive bone diseases. © 2014 American Society for Bone and Mineral Research.


Introduction

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

Bone is a dynamic tissue that undergoes remodeling during vertebrate life to maintain the integrity of the skeleton and to regulate mineral homeostasis through the balanced between the activities of bone-forming osteoblasts and bone-degrading osteoclasts.[1, 2] An imbalance between these two activities results in systemic and local bone diseases such as osteoporosis and osteosclerosis.[3] Mesenchymal stem cells that reside in many tissues such as bone marrow, adipose tissue, periosteum, and hair follicle, are multipotent progenitor cells that can differentiate into osteogenic, chondrogenic, myogenic, and adipogenic lineages when stimulated under appropriate conditions.[4-11] Commitment of mesenchymal stem cells to the osteoblast lineage is controlled by growth factors, hormones, and cytokines.[12, 13]

Bone morphogenetic proteins (BMPs) play widely recognized roles in the regulation of morphogenesis and differentiation of various tissues.[14] Among the BMP family members, BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, and BMP-9, all have a strong osteogenic capacity both in vitro and in vivo.[15-19] BMPs function through type I and II receptors, and they induce phosphorylation of Smad 1/5/8 to promote nuclear transport, together with Smad 4 and/or other transcription factors for regulating target gene expression.[2, 20, 21] Numerous in vitro and in vivo studies have demonstrated that BMP receptor–Smad signaling regulates osteoblast differentiation of mesenchymal stem cells and osteoblast bone formation activity through upregulation of the expression of Runx 2 and Osterix, transcription factors that are essential for osteogenesis.[4, 22-26] In addition to the canonical BMP receptor–Smad pathway, BMPs can elicit the noncanonical MAPK pathway.[4, 20] BMPs have been shown to activate three kinds of MAPK signaling pathways: p38, ERK, and JNK MAPK pathways.[27, 28] A number of studies have demonstrated that the p38, ERK, and JNK MAPK signaling pathways are involved in osteoblast differentiation.[28-31] The p38 and JNK MAPK pathways that are activated by BMPs play a role in osteoblast differentiation in cooperation with the BMP receptor–Smad pathway through the activation of Runx 2 and Osterix.[26, 31-33] Furthermore, synergistic cooperation between Smad and MAPK pathways was established in the BMP pathway controlling limb development.[32, 34]

Relaxin (Rln) is a member of the insulin/Rln family of structurally related hormones and exerts autocrine, endocrine, and paracrine effects through membrane receptors known as Rxfps 1 to 4. Rln, which is known as a pregnancy hormone, has been shown to promote cervical softening and elongation of the interpubic ligaments, thus facilitating the rapid delivery of live young.[35] Rln is also involved in various mechanisms associated with collagen turnover, antifibrosis, angiogenesis, and tumor metastasis.[36, 37] Due to the wide range of activities of Rln, several experiments have been performed to test its clinical utility in the treatment of fibrosis and in facilitating orthodontic tooth movement.[38-40] Recently, Rln receptors have not only been detected in the reproductive tissues, but also in the bone tissues including osteoblasts, osteoclasts, and osteocytes.[41, 42] Previous studies have demonstrated that the Rln receptor, Rxfp 1, is expressed in human peripheral blood monocyte cells and Rln has been implicated not only in the differentiation of peripheral blood monocyte cells into mature osteoclasts, but also in the survival and activation of osteoclasts.[41] Furthermore, it has been reported that mutations in Rxfp 2 are associated with osteoporosis and the binding of insulin-like factor 3 to Rxfp 2 induces an increase in osteoblast proliferation and expression of characteristic osteoblastic genes.[42] Despite the important roles of Rln and its receptors in osteoclasts and osteoblasts, the effects of Rln on the differentiation of mesenchymal stem cells into osteoblasts and in vivo bone formation are still unknown.

In this study, we used the primary mouse bone marrow stem cells (BMSCs) and mouse embryonic fibroblast cell line C3H/10T1/2 that have the ability to differentiate into cells of connective tissue origins such as bone, cartilage, and fat to determine the synergistic effects of Rln on BMP-2–induced osteoblast differentiation. In addition, the effect of Rln on BMP 2-induced osteogenesis was determined in ectopic bone formation using an experimental mouse model. The effect of Rln on the canonical and noncanonical BMP-2 signaling pathways was also investigated.

Subjects and Methods

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

Animals

Specific pathogen-free C57BL/6 mice were obtained from the Orient Bio. Institute (Seongnam, Republic of Korea) and cared for in a controlled environment (25°C, 55% humidity). This study was conducted in accordance with the guidelines of the Chonnam National University Institutional Animal Care and Use Committee.

Cell culture and reagents

BMSCs were obtained from the bone marrow of 6-week-old C57BL/6 mice. The mice were killed by cervical dislocation, femurs and tibias were collected, and bone marrow was flushed using a syringe needle with Minimum Essential Medium alpha modification (GIBCO BRL, Grand Island, NY, USA) containing 10% fetal bovine serum (GIBCO BRL), 2 mM L-glutamine (GIBCO BRL), and 1% antibiotic-antimycotic (GIBCO BRL). Then, the cells were cultured at 37°C in a humidified 5% CO2 incubator. After 4 days, nonadherent cells were removed by replacing the medium. Subsequently, the culture medium was replaced every 3 days. Confluent cells were detached with trypsin-EDTA (GIBCO BRL) treatment and passaged. The three-passage and four-passage cells were used in this study.

For preparation of primary osteoblasts, the calvariae were isolated from 3-day-old neonatal mice and digested with 0.1% collagenase (Roche, Mannheim, Germany) at 37°C for 30 minutes. The calvariae were then digested four times. The last fractions were pooled and used as primary osteoblasts.

Peripheral blood mononuclear cells (PBMCs) were prepared from anticoagulated blood layered onto Histopaque 1077 (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 400g for 30 minutes to obtain the separation between blood components. The interface was washed with phosphate-buffered saline (PBS) twice and used as PBMCs.

Mouse embryonic fibroblast C3H/10T1/2 cells were obtained from ATCC (Manassas, VA) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 1% antibiotic-antimycotic.

Recombinant human BMP-2 and Rln were purchased from R&D Systems (Minneapolis, MN, USA), dissolved in PBS containing 0.1% BSA. Aliquots were stored at −80°C.

Induction of osteoblastic differentiation and Alizarin red S staining

For induction of osteogenic differentiation, cells were incubated in an osteogenic medium containing 50 µg/mL ascorbic acid (AA) and 5 mM β-glycerophosphate (β-GP) in the presence of 200 ng/mL BMP-2. The medium was changed every other day. Cells were treated with Rln for 2 days after the medium was changed to an osteogenic medium. The molecular ratio of BMP-2:Rln used in the in vitro study was 2.33:1, 23.3:1, or 233:1. On day 21, cells were rinsed with ice-cooled PBS and fixed with 70% ethanol. Cells were stained with 40 mM Alizarin red S solution (pH 4.2; Sigma-Aldrich) after washing three times with deionized water. The samples were observed under light microscope and the representatives were photographed. For quantitative analysis, the stains were extracted using 10% (wt/vol) cetylpyridinium chloride (CPC) in 10 mM sodium phosphate (pH 7.0) for 15 minutes and then measured at 540 nm using a multiplate reader (Bio-Tek Instruments, Winooski, VT, USA).

Cell proliferation assay

Cells were plated in 96-well plates and treated with 0.03, 0.3, and 3 nM Rln for 1 or 3 days. Cell viability was measured by Cell counting kit-8 (WST-8; Dojindo Laboratories, Tokyo, Japan). WST-8 reagent was added to each well to access dehydrogenase activity and incubated for 4 hours. Absorbance of supernatant was measured at 450 nm using a multiplate reader.

Transfection and luciferase reporter assay

For knockdown of Rxfp receptor expression, cells were transfected with specific siRNAs against Rln receptors or scrambled control RNA (Invitrogen, Carlsbad, CA, USA) using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. The expression level of Rln receptors was determined by Western blot analysis.

For the analysis of Runx 2-dependent transactivity of OG2-Luc promoter using a Luciferase assay kit (Promega, Madison, WI, USA), cell lysates were collected 24 hours after BMP-2 treatment. The results were repeated in at least three different experiments that were performed in triplicate. As an internal control, cytomegalovirus β-galactosidase plasmid was cotransfected in each transfection, and luciferase activity was normalized to β-galactosidase activity.

Western blot analysis

Cell extracts were prepared with PhosphoSafe Protein Extraction Reagent (Novagen, Madison, WI, USA). The extracts were electrophoresed on 10% SDS-polyacrylamide gel and transferred to a Protran nitrocellulose membrane (Whatman GmbH, Dassel, Germany). The membrane was blocked via 1 hour of incubation at room temperature in 10 mM Tris-buffered saline-0.1% Tween 20 containing 5% skim milk, followed by incubation with primary antibody overnight at 4°C with gentle shaking. Primary antibodies against Rxfp 1, Rxfp 3, and Runx 2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used. For the detection of total or phosphorylated Smads, MAPKs, and transforming growth factor-β activated kinase (TAK) 1, antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The purified mouse monoclonal primary antibody to β-actin (Sigma-Aldrich) was used as the reference. The blots were washed, and then incubated for 2 hours at room temperature with the horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG antibody (Cell Signaling). The blots were subsequently washed again and finally developed with the HRP Substrate Luminol Reagent (Millipore Corporation, Billerica, MA, USA) and photographed using LAS4000 loaded with ImageReader LAS-4000 software (Fujifilm; Minatoku, Tokyo, Japan). The relative phosphorylation level of each protein was quantified using the Scion Image software (Scion, Frederick, MD, USA) and shown as the ratio of phosphorylated to total protein level.

Real-time reverse transcription–PCR

The total RNA was extracted using Trizol Reagent (Gibco BRL). Reverse transcription (RT) was conducted with a RT system containing Moloney Murine Leukemia Virus reverse transcriptase (Promega) in accordance with the manufacturer's instructions. PCR was performed in a Palm-Cycler thermocycler (Corbett Life Science, Sydney, Australia) and the product was resolved in a 1.2% agarose gel. Real-time amplification of cDNA was conducted in a Rotor-Gene 3000 System (Corbett Research, Morklake, Australia) using the SYBR Green PCR Master Mix Reagent Kit (Qiagen, Valencia, CA, USA). The PCR conditions were as follows: incubation for 5 minutes at 95°C, followed by 30 cycles of denaturation for 15 seconds at 95°C, annealing for 15 seconds at 60°C, and extension for 15 seconds at 72°C. The primers used were as follows: mouse Rxfp 1: 5′-CCT CTT GGC AAG CAT CAT CC-3′ and 5′-CGG CTG TGC GTG CTT ATT GTA C-3′; mouse Rxfp 2: 5′-GTC TCC CCG TAG AGG CTT TG-3′ and 5′-CAC AGG TCC TAG AGC TGC CA-3′; mouse Rxfp 3: 5′-GCT CCT GAG TAG GGG ACT GC-3′ and 5′-GGC TGC ACT CAG CAT CAG TT-3′; mouse Rxfp 4: 5′-ACC CTC TTC TGG GTC AAT GG-3′ and 5′-AAA TTT CCC AGC AAG CCA AT-3′; mouse Runx 2: 5′-CCA GGC AGG TGC TTC AGA ACT G-3′ and 5′-ACA TGC CGA GGG ACA TGC CTG A-3′; mouse alkaline phosphatase (ALP): 5′-TAT GGT AAC GGG CCT GGC TAC-3′ and 5′-TGC TCA TGG ACG CCG TGA AGC A-3′; mouse osteocalcin (OC): 5′-TGA ACA GAC TCC GGC GCT AC-3′ and 5′-AGG GCA GCA CAG GTC CTA A-3′; and β-actin: 5′-GAT CTG GCA CCA CAC CTT CT-3′ and 5′-GGG GTG TTG AAG GTC TCA AA-3′. The relative levels of mRNA were calculated using the standard curve generated from the cDNA dilutions. The mean threshold cycle (Ct) values from quadruplicate measurements were employed in the calculation of gene expression, with normalization to β-actin employed as an internal control. Calculation of the relative level of gene expression was performed using Corbett Robotics Rotor-Gene software (Rotor-Gene 6 version 6.1, Build 90 software; Corbett Life Science).

ALP staining and activity assay

For ALP staining, media were removed and the cells were fixed with 3.7% formaldehyde for 15 minutes. After rinsing with deionized water three times, 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro-blue tetrazolium (NBT) substrate (Sigma-Aldrich) was added to each well. The reaction was stopped by addition of water.

The ALP assay was performed as described by Manolagas and colleagues.[43] Briefly, cell lysates were prepared by adding CytoBuster (Novagen). The ALP activity was measured in cell extracts using p-nitrophenyl phosphate substrate and normalized to the total amount of protein.

OC production assay

The level of OC secreted into the culture medium was determined using a mouse OC ELISA kit (Biomedical Technologies, Inc., Stoughton, MA, USA) according to the manufacturer's instructions.

In vivo experiments and X-ray and micro–CT scanning

For ectopic bone formation, mice (n = 3/group) were subcutaneously injected with 200 µL of type I collagen (BD Biosciences, Bedford, MA, USA) containing a mixture of BMP-2 and Rln, BMP-2 alone, or Rln alone. The molecular ratio of BMP-2:Rln used in the in vivo experiment was 23.3:1, 116:1, or 580:1. Ectopic bone formation was monitored by a radiographic apparatus (Hi-Tex, Osaka, Japan) at 35 kV and 400 µA (2D). The X-ray source was set at 50 kV and 200 A with a pixel size of 17.09 µm. Exposure time was 1.2 seconds. Four hundred and fifty projections were acquired over an angular range of 180 degrees (angular step of 0.4 degrees). The image slices were reconstructed using 3D CT analyzer software (CTAN; Skyscan). For static histomorphometry, the newly formed bone from each mouse was isolated and fixed in a 4% paraformaldehyde solution overnight at 4°C, followed by decalcification with 20% EDTA (pH 7.4). The samples were then dehydrated in a graded series of ethanol and embedded in paraffin. Four-micron-thick (4-µm-thick) sagittal sections were cut for hematoxylin and eosin staining. These specimens were visualized and photographed using an LSM confocal microscope (Carl Zeiss, Gottingen, Germany).

Statistical analysis

Statistical significance was assessed by the Student's t test. Statistical differences with a p value <0.05 were considered significant. All experiments were conducted three to five times independently. The results are shown as the mean ± SE of the mean of three different experiments that were performed in triplicate. Reproducible results were obtained, and representative data are shown in the figures.

Results

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

Rln enhances BMP-2–induced differentiation of mouse BMSCs into osteoblasts and mineralized nodule formation

First of all, we performed RT-PCR to evaluate the expression of the Rln receptors, Rxfp 1 to 4 in mouse BMSCs, primary osteoblasts, and PBMCs. BMSCs were cultured with or without BMP-2 in combination with AA and β-GP, which are classic inducers of osteoblast differentiation. In BMSCs, Rxfp 1 mRNA was endogenously expressed and increased by BMP-2, and the expression of Rxfps 2 to 4 was induced by BMP-2 as shown in Fig. 1A. Rxfp 1 expression was also detected in primary osteoblasts and PBMCs.

image

Figure 1. Effects of Rln on BMP-2–induced osteogenic differentiation and mineralized nodule formation in BMSCs. (A) BMSCs were cultured in an osteogenic medium containing AA and β-GP and treated with 200 ng/mL BMP-2 for 24 hours. Expression of Rln receptors, Rxfp 1 to 4 in BMSCs, primary osteoblasts, and PBMCs was analyzed by real-time RT-PCR. Data are shown as the mean ± SE. #p < 0.05, compared with the control group. BMSCs were treated with the indicated concentration of Rln in the presence of BMP-2 for 2 days after the first change to an osteogenic medium. After Rln treatment, the medium was changed to an osteogenic medium containing AA and β-GP in the presence or absence of BMP-2 and the medium was changed every other day. On day 10, ALP protein staining in cell layers (B) and OC production assay in culture medium (C) were performed. (D) On day 21, cells were fixed and stained with Alizarin red S stain solution. Alizarin red S concentration was measured as described in the Subjects and Methods section. Data were from three independent experiments performed in triplicate. *p < 0.05, compared with the indicated group.

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To investigate whether Rln plays a role in BMP-2–induced differentiation of BMSCs into osteoblasts, BMSCs were treated with BMP-2 in combination with AA and β-GP, and the expression levels of ALP and OC, which are typical osteogenic markers, were evaluated. Figure 1B, C shows an increase in ALP and OC protein production by BMP-2. Moreover, we found that Rln was able to significantly enhance BMP-2–induced ALP and OC expression. Next, we examined the ability of Rln to augment BMP-2–induced matrix mineralization. As shown in Fig. 1D, Rln in combination with BMP-2 dramatically augmented the amount of mineralization compared to that by BMP-2 alone. However, Rln alone under the normal conditions in the presence of AA and β-GP showed no effect on osteogenic differentiation and matrix mineralization. BMP-2–induced ALP and OC expression and matrix mineralization by Rln was enhanced in a dose-dependent manner and reached its maximum at a concentration of 0.3 nM (Fig. 1). Rln showed no effects on the proliferation and viability in BMSCs and C3H/10T1/2 cells (data not shown). Therefore, this effective dose of Rln was used in our subsequent experiments.

Rln enhances BMP-2–induced osteogenic differentiation and mineralization through binding with Rxfp 1 in mouse embryonic cell line C3H/10T1/2 cells

The expression of the Rln receptors, Rxfp 1 to 4 in C3H/10T1/2 cells was analyzed by RT-PCR and Western blot analysis. As shown in Fig. 2A, C3H/10T1/2 cells expressed Rxfp 1 and Rxfp 3 mRNAs and proteins, and the expression of these receptors was increased by BMP-2. However, Rxfp 2 and Rxfp 4 mRNAs could not be detected by RT-PCR analysis even after amplification for 40 cycles (Fig. 2A) and receptor proteins were also not detected by Western blot analysis (data not shown). To better investigate the Rxfp 1–mediated or Rxfp 3–mediated action of Rln on BMP-2–induced osteogenic activity, we silenced the Rxfp 1 and Rxfp 3 genes by treatment with specific siRNAs against Rxfp 1 and Rxfp 3, respectively. As shown in Fig. 2B, the receptors were silenced in C3H/10T1/2 cells that were cultured under normal conditions in the presence of AA and β-GP. BMP-2–upregulated Rxfp 1 and Rxfp 3 expressions were also successfully decreased by treatment with specific siRNAs. Silencing of Rxfp 1 and Rxfp 3 genes with specific siRNAs lasted 72 hours (data not shown).

image

Figure 2. Effects of Rln on BMP-2–induced osteoblast differentiation and mineralization in C3H/10T1/2 cells. Cells were cultured in an osteogenic medium containing AA and β-GP and treated with siRNAs against Rxfp 1 and Rxfp 3 for 6 hours. (A) Cells were treated with BMP-2 for 3 hours and mRNA levels of Rln receptors Rxfp1 to 4 were analyzed by real-time RT-PCR. (B) Protein levels of Rxfp 1 and Rxfp 3 were confirmed with Western blot analysis 24 hours after BMP-2 treatment. The relative expression of Rxfp 1 and Rxfp 3 was quantified by Scion Image software and normalized to β-actin expression. The result is a representative of three independent experiments. #p < 0.05, compared with the control group; †p < 0.05, compared with the BMP-treated group. After treatment of siRNAs, C3H/10T1/2 cells were cultured in an osteogenic medium containing AA and β-GP in the presence or absence of BMP-2, and the medium was changed every other day. Cells were treated with Rln for 2 days after the first change to an osteogenic medium. (C) On day 3, cells were prepared and mRNA of osteoblastic marker genes was analyzed by real-time RT-PCR and visualized on 1.2% agarose gel. (D) ALP protein in cell layers was stained on day 9 and ALP activity from the cell lysates was analyzed at the indicated times. (E) Culture medium was harvested for OC production assay. (F) On day 21, cells were fixed and stained with Alizarin red S stain solution. Alizarin red S concentration was measured. Data (mean ± SE) were from three independent experiments performed in triplicate. (*p < 0.05).

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To confirm the effect of Rln on BMP-2–induced osteoblast differentiation, C3H/10T1/2 cells were treated with BMP-2 in combination with AA and β-GP, and the expression levels of ALP and OC were evaluated. As shown in Fig. 2CE, Rln augmented not only the ALP and OC mRNA expressions but also the ALP activity and OC protein production induced by BMP-2. These effects of Rln on BMP-2–induced ALP and OC expressions were inhibited by treatment with siRNA against Rxfp 1, but not by treatment with control siRNA. The ability of Rln to augment in BMP-2–induced matrix mineralization was also confirmed. As shown in Fig. 2F, Rln in combination with BMP-2 dramatically augmented the amount of mineralization compared to that by BMP-2 alone. This augmentation of BMP-2–induced matrix mineralization by Rln was inhibited by treatment with siRNA against Rxfp 1, but not by treatment with Rxfp 3 and control siRNAs. Rln alone in the presence of AA and β-GP showed no effect on ALP activity, OC production and matrix mineralization (data not shown). siRNAs for Rln receptors did not show any effect on osteogenic differentiation and matrix mineralization induced by BMP-2 alone (data not shown). Taken together, the above results suggest that Rln can effectively enhance BMP-2–induced osteoblast differentiation and matrix mineralization through its receptor, Rxfp 1.

Rln increases BMP-2–induced ectopic bone formation in vivo in a dose-dependent manner

To further investigate the synergy between Rln and BMP-2, we conducted ectopic bone formation study using an experimental mouse model. A mixture of type I collagen and recombinant BMP-2 and Rln, a mixture of type I collagen and BMP-2 alone, or a mixture of type I collagen and Rln alone was injected subcutaneously into the back of mice and then analyzed 3 weeks later. Figure 3 shows the representative findings. Micro–CT (µCT) analysis showed that BMP-2 alone could induce the formation of measurable bone mass, and Rln effectively increased bone mass induced by BMP-2 in a dose-dependent manner (Fig. 3AC) at the injected site. However, treatment with type I collagen and Rln alone did not cause a discernible increase in bone mass (data not shown). In addition, histological analysis showed that the increase in the amount of newly formed mineralized matrix, which has an abundant amount of osteocytes and bone marrow, was greater in the BMP-2 plus Rln–treated group than in the BMP-2–treated group (Fig. 3C). These in vivo results strongly suggested that Rln may be acting synergistically with BMP-2 in bone formation, and a combination of BMP-2 and Rln may be an effective therapy for bone regeneration in destructive bone diseases.

image

Figure 3. Effect of Rln on BMP-2–induced ectopic bone formation in vivo. Type I collagen containing a mixture of BMP-2 (5 µg) and Rln, BMP-2 alone, or Rln alone was injected subcutaneously into the back of mice. (A, B) After 3 weeks, ectopic bone formation was analyzed by µCT. Data (mean ± SE) were from three independent experiments performed in triplicate. #p < 0.05, compared with the control group; †p < 0.05, compared with the BMP-treated group. (C) Each sample was formalin-fixed and paraffin-embedded, and then stained with hematoxylin and eosin: panel a, vehicle only; panel b, BMP; panel c, BMP + Rln 50 ng; panel d, magnified picture of the squared area in panel c.

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Rln augments BMP-2–induced Smad, p38, and TAK 1 phosphorylation

A number of in vitro and in vivo studies have shown that BMP-Smad signaling regulates osteoblastic differentiation of mesenchymal stem cells and osteoblastic bone formation activity.[23, 25] In addition, recent reports suggest that MAPKs activated by BMP-2 influence Runx 2 that acts downstream of BMP-2/Smad signaling, and Smad and MAPK pathways show synergistic interaction in the BMP pathway.[32, 33] Therefore, we examined the effect of Rln on canonical and noncanonical BMP-2 signaling pathway. As shown in Fig. 4A, Smad phosphorylation was progressively increased and reached its maximum at 30 minutes after BMP-2 treatment and declined thereafter. Surprisingly, Rln enhanced BMP-2–increased Smad phosphorylation and sustained it until 120 minutes. Moreover, BMP-2–induced p38 and ERK phosphorylation, but not JNK phosphorylation (data not shown). An increase of p38 phosphorylation caused by BMP-2 was enhanced and sustained by Rln, which was the same as that seen in Smad phosphorylation (Fig. 4C). Enhancement of BMP-2–induced Smad and p38 phosphorylation by Rln was inhibited by administration of Rxfp 1 siRNA, but not by treatment with control siRNA (Fig. 4B, D). siRNAs for Rln receptors did not show any effect on the phosphorylation levels of Smad and p38 induced by BMP-2 alone (data not shown). However, Rln did not show any effect on BMP-2–induced ERK phosphorylation (data not shown) and Rln alone induced a faint increase in Smad and p38 MAPK phosphorylation (Fig. 4A, B).

image

Figure 4. Effects of Rln on BMP-2–induced phosphorylation of Smad 1/5/8, p38 MAPK, and TAK 1. C3H/10T1/2 cells were treated with/without siRNAs against Rxfp 1 and Rxfp 3 for 6 hours. After overnight culture, the medium was changed to an osteogenic medium containing AA and β-GP in the presence or absence of 200 ng/mL BMP-2. At the indicated times, cells were harvested and cell lysates were prepared. Western blot was performed for the detection of Smad 1/5/8 (A, B), p38 MAPK (C, D), and TAK 1 phosphorylation (E, F). The phosphorylated protein levels were quantified by Scion Image software and normalized to total form of protein expression. The result is a representative of three independent experiments. Data are shown as the mean ± SE. (*p < 0.05). p-Smad = phosphorylated Smad; t-Smad = total Smad; p-p38 = phosphorylated p38 MAPK; t-p38 = total p38 MAPK; p-TAK 1 = phosphorylated TAK 1.

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TAK 1 is a mitogen-activated protein kinase kinase kinase activated by TGF-β and BMP and mediates the activation of p38 MAPK and Smad 1/5/8 phosphorylation in BMP signaling.[44] Therefore, we determined if Rln induced the activation of TAK 1, which regulated BMP-2–induced intracellular signaling, such as phosphorylation of Smad and p38. As shown in Fig. 4E, TAK 1 phosphorylation was induced and reached its maximum at 30 minutes after BMP-2 treatment and declined thereafter. In addition, Rln induced a weak but significant increase in TAK 1 phosphorylation and enhanced synergistically BMP-2–induced TAK 1 phosphorylation. Rln-induced TAK 1 phosphorylation and enhancement of BMP-2–induced TAK 1 phosphorylation by Rln was inhibited by administration of Rxfp 1 siRNA, but not by treatment with control siRNA (Fig. 4F). siRNAs did not show any effect on the phosphorylation level of TAK 1 induced by BMP-2 alone (data not shown).

Rln enhances BMP-2–increased Runx 2 expression and activity

Runx 2 is essential for the differentiation of MSCs into preosteoblasts and its transcriptional activity is influenced by MAPKs and Smad.[32] Therefore, we investigated the effect of Rln on BMP-2–increased Runx 2 expression and transcriptional activity. As expected, BMP-2 increased Runx 2 mRNA and protein expressions, and its effect was augmented significantly by Rln (Fig. 5A–C). In addition, BMP-2-induced Runx 2 transcriptional activity was also increased by Rln (Fig. 5D). Treatment with Rxfp 1 siRNA, but not treatment with control siRNA, induced downregulation of BMP-2–induced Runx 2 expression and transcriptional activity that were augmented by Rln (Fig. 5B–D)). Rxfp 1 siRNA did not show any effect on the expression level of Runx 2 induced by BMP-2 alone (data not shown). Collectively, these results suggest that Rln may exert a synergistic effect at least in part through upregulation of BMP-2–induced Smad and p38 phosphorylation, which have an influence on Runx 2 expression and activity.

image

Figure 5. Effects of Rln on BMP-2–induced expression and activity of Runx 2. C3H/10T1/2 cells were cultured in an osteogenic medium containing AA and β-GP and treated with 200 ng/mL BMP-2. (A) Expression of Runx 2 at the indicated times was analyzed by real-time RT-PCR. Data are shown as the mean ± SE. #p < 0.05, compared with the control group; †p < 0.05, compared with the BMP-treated group. C3H/10T1/2 cells were treated with/without siRNA against Rxfp 1 or pOG2-Luc for 6 hours. After overnight culture, the medium was changed to an osteogenic medium containing AA and β-GP in the presence or absence of BMP-2. (B) For RT-PCR analysis, cells were harvested 24 hours after BMP-2 treatment. (C) The protein level of Runx 2 was analyzed 3 days after BMP-2 treatment by Western blot. The relative protein expression of Runx 2 was quantified by Scion Image software and normalized to β-actin expression. The result is a representative of three independent experiments. (D) For promoter activity analysis, cells were harvested 1 day after BMP-2 treatment. Promoter activities were normalized to pCMV β-galactosidase activity. Data were from three independent experiments performed in triplicate. *p < 0.05, compared with the indicated group.

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Discussion

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

Bone remodeling is essential for skeletal renewal and calcium homeostasis, and this process is regulated strictly by a wide variety of systemic hormones and local acting factors such as PTH, BMPs, prostaglandins, and sex steroids.[1] Early studies showed that Rln, a peptide hormone known for its physiological role in the growth and differentiation of the reproductive tract during pregnancy, can play a role in bone physiology, diseases and metastasis.[35-37] We have previously demonstrated that a local physical force-induced orthodontic tooth movement, for which inevitably the remodeling process in the alveolar bone is needed, can affect the expression of Rln in the ovary.[45] Furthermore, it has been reported that Rln receptors are detected not only in osteoclasts, but also in osteoblasts.[41, 42] Based on the results of these studies, we have demonstrated the effect of Rln on osteoblast differentiation of MSCs and bone formation in vivo in this study. We showed that Rln receptor, Rxfp 1 was expressed in BMSCs and C3H/10T1/2 cells, and Rln effectively augmented BMP-2–induced production of osteogenic markers such as ALP and OC during osteoblast differentiation of BMSCs and C3H/10T1/2 cells. BMP-2–induced osteogenic differentiation and mineralization was also synergistically enhanced by Rln through the Rxfp 1 receptor in vitro. In our in vivo study, we found that Rln significantly augmented BMP-2–induced ectopic bone formation in a dose-dependent manner. These results suggested for the first time that Rln could be an effective molecule to augment BMP-2–induced osteoblasts differentiation and bone formation in vivo.

A number of studies have reported that Rln plays a role in a multitude of physiologic and pathologic processes such as collagen turnover, angiogenesis, renal function, cardiovascular disorder, and tumor metastasis.[36, 37] Together with its pleiotropic roles that are reported in various reproductive and non-reproductive tissues, recent findings demonstrate that Rln stimulates osteoclast differentiation from hematopoietic precursors and regulates the activity of mature osteoclasts.[41] Moreover, it has been demonstrated that mutations in the Rln receptor, Rxfp 2 are associated with osteoporosis, and insulin-like factor 3/Rxfp 2 ligand-receptor complex might play an important role in the osteogenic process by stimulating the transcription of genes that are related to osteoblast differentiation and activity.[42] In addition, we showed the synergistic effect of Rln on osteogenic activity of BMP-2 that induced osteoblast differentiation and bone formation in vivo. Therefore, the results of this study support the dual effect of Rln in bone metabolism, inducing bone formation and resorption.

Although Rln has a dual function in bone metabolism, Madan and colleagues[40] reported that clinical use of Rln had no effect on orthodontic tooth movement which necessarily accompanied bone remodeling. In our unpublished data, Rln augmented osteoblast differentiation of human mesenchymal stem cells in the medium containing dexamethasone, potassium phosphate, and ascorbic acid. This effect of Rln was better when Rln was administered in the early stage of osteogenic differentiation than in the later stages, and this effect was also better with the use of a single administration than with the use of repeated administration. These results suggested that the timing and frequency of treatment are very important for obtaining the effect of Rln on bone metabolism. Therefore, modulation of Rln and Rln receptor expression could be a useful strategy for regulating bone metabolism.

It has been well established that BMP-2 could activate MAPKs in a TAK 1–dependent manner in addition to the canonical Smad signaling pathway, which induces osteoblast differentiation and bone formation.[2, 46] Greenblatt and colleagues[44] have demonstrated that TAK 1 was required for the phosphorylation of p38 MAPK and Smad 1/5/8 in BMP signaling. Although BMP-induced phosphorylation of p38/ERK/JNK MAP kinases was reduced in TAK 1–deficient chondrocytes, the action of TAK 1 was attributed to activation of p38 in osteoblasts.[47] In our results, Rln induced TAK 1 phosphorylation and enhanced BMP-2 increased the phosphorylation of TAK 1. These suggest that Rln enhanced BMP-2–induced intracellular signaling, such as phosphorylation of Smad and p38 through increasing the phosphorylation of TAK 1 during osteoblastogenesis and provide an explanation of how Rln enhances the phosphorylation of Smad and p38 without affecting the phosphorylation of ERK or JNK.

Runx 2 has been widely accepted as the key osteogenic transcription factor and can physically interact with activated Smad 1 and Smad 5.[48] A number of studies have shown that activation of BMP-Smad signaling stimulates bone formation and osteoblastogenesis through the upregulation of Runx 2 expression and function.[2, 48, 49] Moreover, it has been demonstrated that p38 activated by BMP-2 plays a principal role in modulating Smad activation. In addition to many studies that identified the interaction between Smad and Runx 2, Lee and colleagues[32] reported that both the Smad and p38 MAPK pathways converge to regulate Runx 2 gene expression during mesenchymal precursor cell differentiation. These reports suggest that Runx 2 expression and function increased by Smad and p38 play an important role in BMP-2–induced osteoblast differentiation and bone formation. For studying molecular mechanisms of Rln activity, we investigated how Rln affects BMP-2 signaling pathway in osteogenesis. In our observations, BMP-2–induced Smad phosphorylation was strongly enhanced and sustained. Among MAPKs, p38 phosphorylation induced by BMP-2 was increased significantly and lasted for 120 minutes after BMP-2 treatment. Furthermore, the mechanical analysis showed that Runx 2 expression and activity increased by BMP-2 were augmented by treatment with Rln. Our results indicated that Rln augmented BMP-2–induced osteoblast differentiation and bone formation through an increase and sustenance of activated Smad, p38, TAK 1, and Runx 2. Nonetheless, the molecular mechanisms of Rln activity in the BMP-2 signaling pathway need to be investigated further.

In summary, we found that Rln effectively enhanced BMP-2–induced osteoblast differentiation and mineralization in vitro. BMP-2–induced bone formation was also significantly enhanced by Rln in vivo. Moreover, we found that Rln augmented BMP-2–induced Runx 2 expression and activity in addition to Smad and p38 phosphorylation. These results strongly demonstrated that Rln could not only effectively augment BMP-2–induced osteogenic differentiation of MSCs, but also ectopic bone formation in vivo through the upregulation of Runx 2 expression and activity by increasing and sustaining Smad and p38 phosphorylation. Furthermore, a combinational use of Rln and BMP-2 could be an effective therapeutics for bone regeneration.

Acknowledgments

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning 2011–0015019, and the Korea government (MSIP) 2011–0030121.

Authors' roles: Study design: JSM, SHK, and HMK. Study conduct: JSM, SHO, YWJ, JHK, HJS, BIP, and HMK. Data analysis: JSM, JTK, SHK, and HMK. Data interpretation: JCP, SB, MSK, SHK, and HMK. Drafting manuscript: JSM, JTK, SHK, and HMK. Approving final version of manuscript: all authors.

References

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References