JBMR
ORIGINAL ARTICLE
Relaxin Augments BMP‐2–Induced Osteoblast Differentiation and Bone Formation Jung‐Sun Moon,1* Sun‐Hun Kim,1* Sin‐Hye Oh,1 Yong‐Wook Jeong,2 Jee‐Hae Kang,1 Jong‐Chun Park,2 Hye‐Ju Son,1 Suk Bae,3 Byung‐Il Park,1 Min‐Seok Kim,1 Jeong‐Tae Koh,1 and Hyun‐Mi Ko2 1
Dental Science Research Institute, Medical Research Center for Biomineralization Disorders, School of Dentistry, Chonnam National University, Gwangju, Korea 2 Department of Microbiology, College of Medicine, Seonam University, Namwon, Korea 3 Department of Biological Science, Natural Sciences, Chonnam National University, Gwangju, Korea
ABSTRACT 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 (mCT). 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‐b 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. KEY WORDS: RELAXIN; BMP; BONE; RXFP
Introduction
B
one 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
Received in original form May 27, 2013; revised form January 26, 2014; accepted February 6, 2014. Accepted manuscript online February 12, 2014. Address correspondence to: Hyun‐Mi Ko, PhD, Department of Microbiology, College of Medicine, Seonam University, Namwon, Chonbuk 590‐711, Republic of Korea. E‐mail: [email protected] These authors contributed equally to this work. Journal of Bone and Mineral Research, Vol. 29, No. 7, July 2014, pp 1586–1596 DOI: 10.1002/jbmr.2197 © 2014 American Society for Bone and Mineral Research
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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.
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.
Subjects and Methods
Cell proliferation assay
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.
Induction of osteoblastic differentiation and Alizarin red S staining For induction of osteogenic differentiation, cells were incubated in an osteogenic medium containing 50 mg/mL ascorbic acid (AA) and 5 mM b‐glycerophosphate (b‐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).
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 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%
Journal of Bone and Mineral Research
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.
RLN AUGMENTS BMP‐2–INDUCED OSTEOBLAST DIFFERENTIATION AND BONE FORMATION
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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 b‐galactosidase plasmid was cotransfected in each transfection, and luciferase activity was normalized to b‐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‐b activated kinase (TAK) 1, antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The purified mouse monoclonal primary antibody to b‐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: 50 ‐CCT CTT GGC AAG CAT CAT CC‐ 30 and 50 ‐CGG CTG TGC GTG CTT ATT GTA C‐30 ; mouse Rxfp 2: 50 ‐ GTC TCC CCG TAG AGG CTT TG‐30 and 50 ‐CAC AGG TCC TAG AGC TGC CA‐30 ; mouse Rxfp 3: 50 ‐GCT CCT GAG TAG GGG ACT GC‐30 and 50 ‐GGC TGC ACT CAG CAT CAG TT‐30 ; mouse Rxfp 4: 50 ‐ACC CTC TTC TGG GTC AAT GG‐30 and 50 ‐AAA TTT CCC AGC AAG CCA AT‐30 ; mouse Runx 2: 50 ‐CCA GGC AGG TGC TTC AGA ACT G‐30 and 50 ‐ACA TGC CGA GGG ACA TGC CTG A‐30 ; mouse alkaline phosphatase (ALP): 50 ‐TAT GGT AAC GGG CCT GGC TAC‐30 and 50 ‐ TGC TCA TGG ACG CCG TGA AGC A‐30 ; mouse osteocalcin (OC): 50 ‐TGA ACA GAC TCC GGC GCT AC‐30 and 50 ‐AGG GCA GCA CAG
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MOON ET AL.
GTC CTA A‐30 ; and b‐actin: 50 ‐GAT CTG GCA CCA CAC CTT CT‐30 and 50 ‐GGG GTG TTG AAG GTC TCA AA‐30 . 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 b‐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 ¼ 3/group) were subcutaneously injected with 200 mL 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 mA (2D). The X‐ray source was set at 50 kV and 200 A with a pixel size of 17.09 mm. 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‐mm‐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
ORIGINAL ARTICLE
Relaxin Augments BMP‐2–Induced Osteoblast Differentiation and Bone Formation Jung‐Sun Moon,1* Sun‐Hun Kim,1* Sin‐Hye Oh,1 Yong‐Wook Jeong,2 Jee‐Hae Kang,1 Jong‐Chun Park,2 Hye‐Ju Son,1 Suk Bae,3 Byung‐Il Park,1 Min‐Seok Kim,1 Jeong‐Tae Koh,1 and Hyun‐Mi Ko2 1
Dental Science Research Institute, Medical Research Center for Biomineralization Disorders, School of Dentistry, Chonnam National University, Gwangju, Korea 2 Department of Microbiology, College of Medicine, Seonam University, Namwon, Korea 3 Department of Biological Science, Natural Sciences, Chonnam National University, Gwangju, Korea
ABSTRACT 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 (mCT). 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‐b 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. KEY WORDS: RELAXIN; BMP; BONE; RXFP
Introduction
B
one 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
Received in original form May 27, 2013; revised form January 26, 2014; accepted February 6, 2014. Accepted manuscript online February 12, 2014. Address correspondence to: Hyun‐Mi Ko, PhD, Department of Microbiology, College of Medicine, Seonam University, Namwon, Chonbuk 590‐711, Republic of Korea. E‐mail: [email protected] These authors contributed equally to this work. Journal of Bone and Mineral Research, Vol. 29, No. 7, July 2014, pp 1586–1596 DOI: 10.1002/jbmr.2197 © 2014 American Society for Bone and Mineral Research
1586
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.
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.
Subjects and Methods
Cell proliferation assay
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.
Induction of osteoblastic differentiation and Alizarin red S staining For induction of osteogenic differentiation, cells were incubated in an osteogenic medium containing 50 mg/mL ascorbic acid (AA) and 5 mM b‐glycerophosphate (b‐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).
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 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%
Journal of Bone and Mineral Research
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.
RLN AUGMENTS BMP‐2–INDUCED OSTEOBLAST DIFFERENTIATION AND BONE FORMATION
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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 b‐galactosidase plasmid was cotransfected in each transfection, and luciferase activity was normalized to b‐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‐b activated kinase (TAK) 1, antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The purified mouse monoclonal primary antibody to b‐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: 50 ‐CCT CTT GGC AAG CAT CAT CC‐ 30 and 50 ‐CGG CTG TGC GTG CTT ATT GTA C‐30 ; mouse Rxfp 2: 50 ‐ GTC TCC CCG TAG AGG CTT TG‐30 and 50 ‐CAC AGG TCC TAG AGC TGC CA‐30 ; mouse Rxfp 3: 50 ‐GCT CCT GAG TAG GGG ACT GC‐30 and 50 ‐GGC TGC ACT CAG CAT CAG TT‐30 ; mouse Rxfp 4: 50 ‐ACC CTC TTC TGG GTC AAT GG‐30 and 50 ‐AAA TTT CCC AGC AAG CCA AT‐30 ; mouse Runx 2: 50 ‐CCA GGC AGG TGC TTC AGA ACT G‐30 and 50 ‐ACA TGC CGA GGG ACA TGC CTG A‐30 ; mouse alkaline phosphatase (ALP): 50 ‐TAT GGT AAC GGG CCT GGC TAC‐30 and 50 ‐ TGC TCA TGG ACG CCG TGA AGC A‐30 ; mouse osteocalcin (OC): 50 ‐TGA ACA GAC TCC GGC GCT AC‐30 and 50 ‐AGG GCA GCA CAG
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GTC CTA A‐30 ; and b‐actin: 50 ‐GAT CTG GCA CCA CAC CTT CT‐30 and 50 ‐GGG GTG TTG AAG GTC TCA AA‐30 . 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 b‐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 ¼ 3/group) were subcutaneously injected with 200 mL 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 mA (2D). The X‐ray source was set at 50 kV and 200 A with a pixel size of 17.09 mm. 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‐mm‐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
