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Tissue Engineering Part A Estrogen modulates BMP-induced sclerostin expression via the Wnt signaling pathway (doi: 10.1089/ten.TEA.2014.0585) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
1
Estrogen modulates BMP-induced sclerostin expression via the Wnt signaling pathway
Ri Youn Kima†, Hoon Joo Yangb†, Yun Mi Songc, In Sook Kimc*, Soon Jung Hwanga,b,c*
a
Department of Maxillofacial Cell and Developmental Biology, School of Dentistry, Seoul
National University, BK21 plus, Seoul, Republic of Korea b
Department of Oral and Maxillofacial Surgery, School of Dentistry, Seoul National
University, BK21 Plus, Seoul, Republic of Korea c
Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic
of Korea
†
These authors contributed equally.
*
Corresponding author at: Department of Oral and Maxillofacial Surgery, School of
Dentistry, Seoul National University, Seoul 110-749, Republic of Korea. Tel: +82-2-20723061, Fax: +82-2-766-4948, E-mail address: [email protected] (Hwang, SJ DDS, MD)
or
Dental Research Institute, Seoul National University, Seoul 110-749 Republic of Korea. Tel:+82-2-740-8736, Fax: +82-2-766-4948, E-mail address: [email protected] (Kim, IS, PhD)
1
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ABSTRACT Clinical data show that estrogen levels are inversely associated with the production of sclerostin, a Wnt antagonist that recently attracted great attention over the use of its antibody in the anabolic treatment of osteoporotic conditions. However, the molecular link between sclerostin expression and estrogen signaling is not yet known. We investigated the mechanisms by which estrogen modulates sclerostin (SOST) gene expression at the cellular level in human osteoblast cells in association with bone morphogenetic protein (BMP)2 signaling, given that BMP2 is a potential inducer of SOST in human mesenchymal stromal cells (hMSCs). 17-estradiol (E2) alone had no effect on SOST expression, which was significantly induced by treatment with BMP2 in hMSCs and osteoblasts derived from the mandibles of female donors. However, E2 suppressed the induction of SOST and other BMP2 target genes by BMP2 in hMSCs. E2 signaling was independent of the Smad pathway, which plays a critical role in SOST induction mediated by BMP2. Instead, E2 increased the transcriptional expression of -catenin and levels of its activated form. Silencing of the gene encoding estrogen receptor (ER) decreased E2 activity in -catenin activation and the suppression of SOST induction by BMP2 but had no influence on BMP2-mediated SOST induction in the same conditions. Similar results were obtained after treatment with ER antagonist as a Wnt inhibitor. In human osteoblasts, the effect of E2 on SOST expression was either suppressive or absent, depending on the cell donor. Interestingly, the SOST expression pattern after treatment with BMP2 or BMP2/E2 in human osteoblasts showing a pattern of E2-suppression on SOST induction by BMP2 correlated with the ratio of RANKL to OPG expression. These results demonstrate that estrogen signaling in osteoblasts negatively regulates SOST expression in an indirect manner through interaction with BMP2 signaling and that this regulation involves the Wnt/ER and -catenin pathway. This study highlights 2
Tissue Engineering Part A Estrogen modulates BMP-induced sclerostin expression via the Wnt signaling pathway (doi: 10.1089/ten.TEA.2014.0585) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 3 of 50
3
several interactions between estrogen and BMP cascades in osteoblasts that may provide a
basis for therapeutic intervention for the modification of bone mass density.
Key words: estrogen, sclerostin/SOST, BMP2, human mesenchymal stromal cells, human
osteoblasts, Wnt pathway, estrogen receptor , RANKL/OPG
3
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4
Introduction
Estrogen clearly leads to a reduction in bone resorption in women.1 The anti-resorptive activity of estrogen is mainly observed in osteoclasts, in which estrogen inhibits development and activity, and increases apoptosis.2,3 Recent clinical studies demonstrated a close relationship between estrogen and the bone resorption marker sclerostin.4,5 Postmenopausal women have higher serum sclerostin levels than premenopausal women, and sclerostin levels are inversely associated with the circulating free estradiol index.4,6 Moreover, chronic estrogen treatment of postmenopausal women leads to a reduction in sclerostin levels.5,7 Although these findings suggest that estrogen may regulate sclerostin production, the molecular mechanism underlying the inverse relationship between these factors remains unclear. Sclerostin, the protein product of the SOST gene, is expressed in osteogenic cells, including hypertrophic chondrocytes, osteoblasts, and osteocytes.8 There is abundant evidence that SOST functions as a critical negative regulator of bone formation by antagonizing the Wnt signaling pathway.9-11 It has been suggested that sclerostin signaling in bone is induced as part of the mechanical adaptive response of osteocytes.8,12 Mechanical loading induces downregulation of SOST expression in osteocytes,13 leading to either systemic or local regulation of bone formation in a paracrine fashion, which could in turn antagonize canonical Wnt signaling in osteoblasts.14 Furthermore, SOST knockout mice have a high bone mass phenotype and have been shown to resist bone loss in a hind limb unloading model.14,15 Recent reports demonstrated that SOST is induced by bone morphogenetic protein (BMP) in cells of the osteoblast lineage, where it acts as a BMP antagonist.16 In contrast with the known role of BMPs as potent osteoinducers,17,18 a few studies have reported that mice with 4
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5
conditional knockout (cKO) of BMP receptor type 1A (Bmpr1A) exhibit a unique phenotype with increased bone mass resulting from severely suppressed bone resorption, despite little change in the rate of bone formation.19 This research group demonstrated that SOST plays a critical role in the phenotype of Bmpr1A cKO mice. SOST is upregulated by BMP and inhibits Wnt/-catenin signaling by binding to the co-receptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6).
9,20
Sclerostin eventually enhances
resorption by triggering RANKL-OPG pathway-induced osteoclastogenesis, leading to a decrease in bone mass.9,10,20 These observations led to the idea of therapeutic intervention using sclerostin inhibitors to increase bone mineral density. In preclinical studies, a sclerostin neutralizing monoclonal antibody (Scl-Ab)II increased bone mass and bone strength in aged ovariectomized rats and aged male rats to levels greater than those found in control rats.21 In addition, an investigation using humanized Scl-AbIV in normal female primates showed clear anabolic effects, with marked dose-dependent increases in bone formation.22 Clinical trials using an anti-sclerostin antibody series have been of in performed with encouraging results for the efficacy and safety.23,24 Taken together, these findings indicate that estrogen and BMP signaling converge on SOST expression and Wnt pathways in bone cells.4,10,25,26 However, studies performed to date very rarely provide any molecular evidence for a direct effect of estrogen on SOST expression or whether the effect of estrogen on SOST expression involves BMP signaling. Therefore, we hypothesized that estrogen modulation of SOST expression might be indirectly mediated by BMP signaling at the level of the bone cell, given the potent induction of SOST by BMP2. We expect that this study will be helpful in understanding the molecular mechanism underlying estrogen regulation of SOST expression, which will also provide clues to the potential mechanism of the bone catabolic effect of BMP2. The major aims of this study were threefold: (a) to characterize the effect of estrogen on the induction of SOST by BMP2 and 5
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the expression of other BMP-responsive genes using undifferentiated primary human mesenchymal stromal cells (hMSCs) from female donors; (b) to investigate the molecular mechanism of estrogen action involved in the modulation of SOST expression in association with BMP2 signaling by examining the involvement of Wnt/-catenin/estrogen receptor (ER) and the Smad pathway in hMSCs; (c) to investigate the estrogen effect on BMP2mediated gene expression, including SOST, and determine markers that correlate with SOST expression in osteoblasts derived from the mandibles of female donors.
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Materials and Methods
Chemicals
17‚-estradiol (E2) was purchased from Sigma-Aldrich (St. Louis, MO, USA). TCF/LEF reporter for Wnt and Smad signaling was obtained from Qiagen (Hilden, Germany). The Dual-Luciferase Reporter Assay system was purchased from Promega (Fitchburg, WI, USA). ER and Smad4 siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Transfections for the reporter assay and siRNA were performed using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). Antibodies for total/active catenin, ER, and Smad4 were obtained from Cell Signaling Technology (Danvers, MA, USA), Abcam (Burlingame, CA, USA), and Santa Cruz Biotechnology, respectively. ELISA reagents for sclerostin, Opg, and IGF1 were obtained from R&D Systems (Minneapolis, MN, USA). The Smad inhibitor dorsomorphin was obtained from Calbiochem (Gibbstown, NJ, USA) and the Wnt inhibitor ICI 182,780 was purchased from R&D Systems. Recombinant human BMP2 (rhBMP2), prepared from E. coli, was obtained from Novosis®-Dent, BioAlpha Inc. (Gyeonggi-do, South Korea); we demonstrated its osteogenic activity in a previous study.27
Isolation and culture of hMSCs and human osteoblasts
Bone marrow was obtained from the iliac crest of three non-osteoporotic healthy female donors (19-25 years old) who provided informed consent. Procedures were approved by the local ethics committee (IRB) of Seoul National University Dental Hospital (SNUDH), 7
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8
according to the legal regulations for human tissue and organs in Korea (CRI05008). hMSCs from bone marrow were cultured as described previously.28,
29
Nucleated cells that
concentrated at the interface after Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) treatment were collected and washed with phosphate-buffered saline (PBS). Collected cells were plated at a density of 2×106 cells/100 mm and cultured in expansion medium comprised of low-glucose Dulbecco’s modified Eagle’s medium (DMEM; Welgene, Inc., Daegu, South Korea), 100 units/mL penicillin, 100 mg/mL streptomycin, and 10% heat-inactivated fetal bovine serum (HIFBS) under a humidified atmosphere of 5% CO2 at 37°C, with medium changes every 3 or 4 days. Cells were passaged when they reached 70% confluence, and reseeded in a new culture plate at a density of 3×105 cells/cm2. Cells from the second to fifth passages were used for all experiments. The ability of hMSCs to differentiate into osteoblasts, chondrocytes, or adipocytes was confirmed according to previously published protocols.29 Primary human osteoblasts were derived from mandible biopsy specimens of four healthy female human donors (19-25 years old) who provided informed consent. To obtain bone-derived cells, bone samples were minced and washed twice with PBS. The samples were incubated for 40 minutes with gentle shaking in a solution containing 0.1% type I collagenase (Sigma-Aldrich) and 0.2% dispase (Roche, Indianapolis, IN, USA) in serum-free DMEM at 37°C. This process was performed twice, and the cells that were obtained were combined for each case and cultured separately. To investigate the effect of estrogen on SOST expression in association with BMP signaling and osteoblast differentiation, hMSCs were treated with E2 or a combination of E2 and BMP2 in osteogenic differentiation medium with DMEM supplemented with ascorbic acid (50 µg/mL) and -glycerophosphate (10 mM) without dexamethasone (DEX), added at 1 or 2 days post-plating when the cells were confluent. 8
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Transient transfection and luciferase reporter assays
Transient transfections for luciferase reporter assays were performed using Lipofectamine 2000 reagent (Invitrogen). hMSCs were transfected with 1 ng/l of TOPFlash luciferase reporter plasmids or Smad reporter plasmids in serum-free DMEM without antibiotics. Positive and negative reporters, corresponding to each reporter assay, were also included according to the manufacturer’s instruction. After 5 hours of transfection, the cells were stabilized by culturing overnight in DMEM with 10% HI-FBS and then plated in 96well plates (5,000 cells/well). The next day, the cells were treated with E2 (100 nM), BMP2 (200 ng/mL), E2/BMP2, or no treatment for 24 hrs. The cells were washed once with PBS and lysed with 1× passive lysis buffer. Luciferase activity was measured using a Turner 20/20 luminometer with a Dual-Luciferase Reporter Assay kit (Promega) according to the manufacturer’s instructions. Luciferase activity was calculated as the ratio of firefly luciferase activity to that of Renilla luciferase.
Gene silencing using siRNA targeting ER or SMAD
Endogenous ER or SMAD4 expression was knocked down by siRNA transfection using human ER siRNA (sc-29305) or Smad4 siRNA (sc-29484), which refers to a pool of four target-specific, 19- to 25-oligonucleotide siRNAs. siRNA-A, a non-targeting 20- to 25oligonucleotide (sc-37007), was used as the control. hMSCs were transfected with human sequence-specific ER/Smad4 siRNA or control siRNA-A at a final concentration of 30 pM using Lipofectamine 2000 reagent in serum free-DMEM without antibiotics for 5-6 hours, according to the manufacturer’s instructions (Invitrogen). After transfection, the cells were 9
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10
stabilized by culturing overnight in DMEM with 10% HI-FBS and then the medium was replaced with DMEM with 1% HI-FBS. After incubation for 24 hrs, the cells were treated with E2 (100 nM), BMP2 (200 ng/mL), or E2/BMP2 or left untreated.
Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR)
Total RNAs were extracted by the addition of 0.5 mL of TRIzol reagent (Life Technologies) to cells on a plate as described in the manufacturer’s instructions. One microgram of RNA was subjected to cDNA synthesis with SuperScriptTM Reverse Transcriptase II and oligo12–18 primers (Invitrogen). We used SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) to detect the accumulation of PCR product during cycling with the ABI Prism 7700 Sequence Detection System (Applied Biosystems). The thermocycling conditions were as follows: predenaturation at 95C for 10 min; 40 cycles of denaturation at 95C for 15 s and annealing and extension at 60C for 1 min; followed by a final dissociation cycle at 95C for 15 s, 60C for 1 min, and 95C for 15 s. Real-time RTPCR was carried out in triplicate in at least three independent experiments (n>3). Oligonucleotide primers were designed using real-time RT-PCR system sequence detection software v1.3 (Applied Biosystems) and their sequences are provided in Table 1. Fold differences in the expression level of each gene were calculated for each treatment group using CT values normalized to transcript levels of the housekeeping gene 18S rRNA, according to the manufacturer’s instructions.
Alkaline phosphatase (ALP) assay
10
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11 We assayed ALP activity by measuring the amount of -nitrophenol produced using nitrophenol phosphate substrate. Cell lysates were mixed with alkaline buffer solution and gently shaken for 10 min. ALP substrate was added at room temperature for 30 min. After the reaction was stopped with 0.05 N NaOH, the absorbance at 405 nm was read and compared with a standard curve prepared with -nitrophenol standard solution. Enzyme activity was normalized to the protein concentration of the cell layer determined using a protein assay (Bio-Rad, Hercules, CA, USA).
Enzyme-linked immunosorbent assay (ELISA)
Sclerostin or Opg levels in the culture supernatants were determined using an ELISA kit (R&D Systems). After centrifugation, cell culture supernatants (n=4-5) were added to 96-well ELISA plates. Standards for cytokines (0–1,000 pg/mL) were run in each series. After incubation, aspiration, and washing, a human conjugate of each protein (100 µL/well) was added according to the manufacturer’s instructions. The OD of each well was determined within 30 min using a microplate reader set to a 450-nm wavelength with correction for optical imperfections in the plate.
Western blotting
Total cell lysates of cultured hMSCs were prepared by lysing cells
in RIPA buffer
containing 50 mM Tris buffer (pH 7.5), 50 mM NaCl, 1% Triton X-100, 1 mM EGTA, 10 mM Na4P2O9, 5 mM Na3VO4, 50 mMNaF, and protease inhibitors (10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 μg/ml pepstatin A, 0.1 mM PMSF, and 1 mM DTT). Nuclear and cytoplasmic fractionation of cultured hMSCs was performed using NE-PERTM Nuclear and 11
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cytoplasmic extraction reagents (Thermo SCIENTIFIC, Rokford, IL, USA) according to the manufacturer’s instructions. Total cell lysates (20 µg/lane) or nuclear/cytoplasmic fraction (15 µg/lane) were subjected to electrophoresis through a 10% SDS-polyacrylamide gel and transferred to PolyScreen PVDF membranes (PerkinElmer Life Sciences, Hopkinton, MA, USA). Membranes were blocked with 5% nonfat dry milk in TBST buffer (0.1 M Trisbuffered saline [pH 7.5]/0.1% Tween) and probed with antibodies. The following antibodies were used in this study: polyclonal rabbit anti-total -catenin (Abnova, Taipei, Taiwan); polyclonal rabbit anti–non-phospho (active) -catenin antibody, diluted 1:2000; polyclonal rabbit anti-ER antibody, diluted 1:200; monoclonal mouse anti-Smad4 antibody, diluted 1:200; and monoclonal mouse anti--tubulin antibody, diluted 1:200. Primary antibodies were detected with HRP-conjugated anti-rabbit IgG (Santa Cruz Biotechnology) for total/active -catenin and ER, or with HRP-conjugated anti-mouse IgG (Dako) for Smad4 and -tubulin. Finally, blots were stained with an enhanced chemiluminescence detection system (ECL, Amersham Biosciences) according to the manufacturer’s instructions.
Immunofluorescent staining
Sclerostin or -catenin expression in hMSCs was detected at an average cell density of 3,000 cells per coverslip. Cells were washed three times with PBS, fixed in 4% paraformaldehyde for 30 min, and blocked in blocking solution for 30 min. Cells were incubated with antibody against sclerostin (anti-rabbit, 1:100; Abnova) or active -catenin (anti-rabbit, 1:400; Cell Signaling Technology, Inc) overnight at 4C, washed twice with PBS, and then incubated with secondary antibody labeled with FITC (anti-rabbit IgG, 1:500; Biomeda) for 1 hr. Primary antibody controls were processed in parallel using only the 12
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secondary antibody. Slides were washed in PBS for 10 min and incubated with PBS-buffered 4΄,6-diamino-2-phenylindole (DAPI) solution (1 mg/mL; Santa Cruz Biotechnology) using a dilution ratio of 1:1000 for nuclear staining. Section images of the stained cells were captured using an Olympus Fluoview FV300 confocal laser scanning microscope and Fluoview software (Olympus Optical Co. Ltd.).
Statistical analysis
All data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using SPSS 20 (IBM Co., Armonk, NY, USA). Differences between two groups were evaluated using a two-tailed Student’s t-test, and the comparison of data for more than two groups was performed using with two-way analysis of variance post hoc via the Bonferroni method. Differences with p
Tissue Engineering Part A Estrogen modulates BMP-induced sclerostin expression via the Wnt signaling pathway (doi: 10.1089/ten.TEA.2014.0585) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
1
Estrogen modulates BMP-induced sclerostin expression via the Wnt signaling pathway
Ri Youn Kima†, Hoon Joo Yangb†, Yun Mi Songc, In Sook Kimc*, Soon Jung Hwanga,b,c*
a
Department of Maxillofacial Cell and Developmental Biology, School of Dentistry, Seoul
National University, BK21 plus, Seoul, Republic of Korea b
Department of Oral and Maxillofacial Surgery, School of Dentistry, Seoul National
University, BK21 Plus, Seoul, Republic of Korea c
Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic
of Korea
†
These authors contributed equally.
*
Corresponding author at: Department of Oral and Maxillofacial Surgery, School of
Dentistry, Seoul National University, Seoul 110-749, Republic of Korea. Tel: +82-2-20723061, Fax: +82-2-766-4948, E-mail address: [email protected] (Hwang, SJ DDS, MD)
or
Dental Research Institute, Seoul National University, Seoul 110-749 Republic of Korea. Tel:+82-2-740-8736, Fax: +82-2-766-4948, E-mail address: [email protected] (Kim, IS, PhD)
1
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ABSTRACT Clinical data show that estrogen levels are inversely associated with the production of sclerostin, a Wnt antagonist that recently attracted great attention over the use of its antibody in the anabolic treatment of osteoporotic conditions. However, the molecular link between sclerostin expression and estrogen signaling is not yet known. We investigated the mechanisms by which estrogen modulates sclerostin (SOST) gene expression at the cellular level in human osteoblast cells in association with bone morphogenetic protein (BMP)2 signaling, given that BMP2 is a potential inducer of SOST in human mesenchymal stromal cells (hMSCs). 17-estradiol (E2) alone had no effect on SOST expression, which was significantly induced by treatment with BMP2 in hMSCs and osteoblasts derived from the mandibles of female donors. However, E2 suppressed the induction of SOST and other BMP2 target genes by BMP2 in hMSCs. E2 signaling was independent of the Smad pathway, which plays a critical role in SOST induction mediated by BMP2. Instead, E2 increased the transcriptional expression of -catenin and levels of its activated form. Silencing of the gene encoding estrogen receptor (ER) decreased E2 activity in -catenin activation and the suppression of SOST induction by BMP2 but had no influence on BMP2-mediated SOST induction in the same conditions. Similar results were obtained after treatment with ER antagonist as a Wnt inhibitor. In human osteoblasts, the effect of E2 on SOST expression was either suppressive or absent, depending on the cell donor. Interestingly, the SOST expression pattern after treatment with BMP2 or BMP2/E2 in human osteoblasts showing a pattern of E2-suppression on SOST induction by BMP2 correlated with the ratio of RANKL to OPG expression. These results demonstrate that estrogen signaling in osteoblasts negatively regulates SOST expression in an indirect manner through interaction with BMP2 signaling and that this regulation involves the Wnt/ER and -catenin pathway. This study highlights 2
Tissue Engineering Part A Estrogen modulates BMP-induced sclerostin expression via the Wnt signaling pathway (doi: 10.1089/ten.TEA.2014.0585) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 3 of 50
3
several interactions between estrogen and BMP cascades in osteoblasts that may provide a
basis for therapeutic intervention for the modification of bone mass density.
Key words: estrogen, sclerostin/SOST, BMP2, human mesenchymal stromal cells, human
osteoblasts, Wnt pathway, estrogen receptor , RANKL/OPG
3
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4
Introduction
Estrogen clearly leads to a reduction in bone resorption in women.1 The anti-resorptive activity of estrogen is mainly observed in osteoclasts, in which estrogen inhibits development and activity, and increases apoptosis.2,3 Recent clinical studies demonstrated a close relationship between estrogen and the bone resorption marker sclerostin.4,5 Postmenopausal women have higher serum sclerostin levels than premenopausal women, and sclerostin levels are inversely associated with the circulating free estradiol index.4,6 Moreover, chronic estrogen treatment of postmenopausal women leads to a reduction in sclerostin levels.5,7 Although these findings suggest that estrogen may regulate sclerostin production, the molecular mechanism underlying the inverse relationship between these factors remains unclear. Sclerostin, the protein product of the SOST gene, is expressed in osteogenic cells, including hypertrophic chondrocytes, osteoblasts, and osteocytes.8 There is abundant evidence that SOST functions as a critical negative regulator of bone formation by antagonizing the Wnt signaling pathway.9-11 It has been suggested that sclerostin signaling in bone is induced as part of the mechanical adaptive response of osteocytes.8,12 Mechanical loading induces downregulation of SOST expression in osteocytes,13 leading to either systemic or local regulation of bone formation in a paracrine fashion, which could in turn antagonize canonical Wnt signaling in osteoblasts.14 Furthermore, SOST knockout mice have a high bone mass phenotype and have been shown to resist bone loss in a hind limb unloading model.14,15 Recent reports demonstrated that SOST is induced by bone morphogenetic protein (BMP) in cells of the osteoblast lineage, where it acts as a BMP antagonist.16 In contrast with the known role of BMPs as potent osteoinducers,17,18 a few studies have reported that mice with 4
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5
conditional knockout (cKO) of BMP receptor type 1A (Bmpr1A) exhibit a unique phenotype with increased bone mass resulting from severely suppressed bone resorption, despite little change in the rate of bone formation.19 This research group demonstrated that SOST plays a critical role in the phenotype of Bmpr1A cKO mice. SOST is upregulated by BMP and inhibits Wnt/-catenin signaling by binding to the co-receptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6).
9,20
Sclerostin eventually enhances
resorption by triggering RANKL-OPG pathway-induced osteoclastogenesis, leading to a decrease in bone mass.9,10,20 These observations led to the idea of therapeutic intervention using sclerostin inhibitors to increase bone mineral density. In preclinical studies, a sclerostin neutralizing monoclonal antibody (Scl-Ab)II increased bone mass and bone strength in aged ovariectomized rats and aged male rats to levels greater than those found in control rats.21 In addition, an investigation using humanized Scl-AbIV in normal female primates showed clear anabolic effects, with marked dose-dependent increases in bone formation.22 Clinical trials using an anti-sclerostin antibody series have been of in performed with encouraging results for the efficacy and safety.23,24 Taken together, these findings indicate that estrogen and BMP signaling converge on SOST expression and Wnt pathways in bone cells.4,10,25,26 However, studies performed to date very rarely provide any molecular evidence for a direct effect of estrogen on SOST expression or whether the effect of estrogen on SOST expression involves BMP signaling. Therefore, we hypothesized that estrogen modulation of SOST expression might be indirectly mediated by BMP signaling at the level of the bone cell, given the potent induction of SOST by BMP2. We expect that this study will be helpful in understanding the molecular mechanism underlying estrogen regulation of SOST expression, which will also provide clues to the potential mechanism of the bone catabolic effect of BMP2. The major aims of this study were threefold: (a) to characterize the effect of estrogen on the induction of SOST by BMP2 and 5
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6
the expression of other BMP-responsive genes using undifferentiated primary human mesenchymal stromal cells (hMSCs) from female donors; (b) to investigate the molecular mechanism of estrogen action involved in the modulation of SOST expression in association with BMP2 signaling by examining the involvement of Wnt/-catenin/estrogen receptor (ER) and the Smad pathway in hMSCs; (c) to investigate the estrogen effect on BMP2mediated gene expression, including SOST, and determine markers that correlate with SOST expression in osteoblasts derived from the mandibles of female donors.
6
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Tissue Engineering Part A Estrogen modulates BMP-induced sclerostin expression via the Wnt signaling pathway (doi: 10.1089/ten.TEA.2014.0585) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
7
Materials and Methods
Chemicals
17‚-estradiol (E2) was purchased from Sigma-Aldrich (St. Louis, MO, USA). TCF/LEF reporter for Wnt and Smad signaling was obtained from Qiagen (Hilden, Germany). The Dual-Luciferase Reporter Assay system was purchased from Promega (Fitchburg, WI, USA). ER and Smad4 siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Transfections for the reporter assay and siRNA were performed using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). Antibodies for total/active catenin, ER, and Smad4 were obtained from Cell Signaling Technology (Danvers, MA, USA), Abcam (Burlingame, CA, USA), and Santa Cruz Biotechnology, respectively. ELISA reagents for sclerostin, Opg, and IGF1 were obtained from R&D Systems (Minneapolis, MN, USA). The Smad inhibitor dorsomorphin was obtained from Calbiochem (Gibbstown, NJ, USA) and the Wnt inhibitor ICI 182,780 was purchased from R&D Systems. Recombinant human BMP2 (rhBMP2), prepared from E. coli, was obtained from Novosis®-Dent, BioAlpha Inc. (Gyeonggi-do, South Korea); we demonstrated its osteogenic activity in a previous study.27
Isolation and culture of hMSCs and human osteoblasts
Bone marrow was obtained from the iliac crest of three non-osteoporotic healthy female donors (19-25 years old) who provided informed consent. Procedures were approved by the local ethics committee (IRB) of Seoul National University Dental Hospital (SNUDH), 7
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according to the legal regulations for human tissue and organs in Korea (CRI05008). hMSCs from bone marrow were cultured as described previously.28,
29
Nucleated cells that
concentrated at the interface after Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) treatment were collected and washed with phosphate-buffered saline (PBS). Collected cells were plated at a density of 2×106 cells/100 mm and cultured in expansion medium comprised of low-glucose Dulbecco’s modified Eagle’s medium (DMEM; Welgene, Inc., Daegu, South Korea), 100 units/mL penicillin, 100 mg/mL streptomycin, and 10% heat-inactivated fetal bovine serum (HIFBS) under a humidified atmosphere of 5% CO2 at 37°C, with medium changes every 3 or 4 days. Cells were passaged when they reached 70% confluence, and reseeded in a new culture plate at a density of 3×105 cells/cm2. Cells from the second to fifth passages were used for all experiments. The ability of hMSCs to differentiate into osteoblasts, chondrocytes, or adipocytes was confirmed according to previously published protocols.29 Primary human osteoblasts were derived from mandible biopsy specimens of four healthy female human donors (19-25 years old) who provided informed consent. To obtain bone-derived cells, bone samples were minced and washed twice with PBS. The samples were incubated for 40 minutes with gentle shaking in a solution containing 0.1% type I collagenase (Sigma-Aldrich) and 0.2% dispase (Roche, Indianapolis, IN, USA) in serum-free DMEM at 37°C. This process was performed twice, and the cells that were obtained were combined for each case and cultured separately. To investigate the effect of estrogen on SOST expression in association with BMP signaling and osteoblast differentiation, hMSCs were treated with E2 or a combination of E2 and BMP2 in osteogenic differentiation medium with DMEM supplemented with ascorbic acid (50 µg/mL) and -glycerophosphate (10 mM) without dexamethasone (DEX), added at 1 or 2 days post-plating when the cells were confluent. 8
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Transient transfection and luciferase reporter assays
Transient transfections for luciferase reporter assays were performed using Lipofectamine 2000 reagent (Invitrogen). hMSCs were transfected with 1 ng/l of TOPFlash luciferase reporter plasmids or Smad reporter plasmids in serum-free DMEM without antibiotics. Positive and negative reporters, corresponding to each reporter assay, were also included according to the manufacturer’s instruction. After 5 hours of transfection, the cells were stabilized by culturing overnight in DMEM with 10% HI-FBS and then plated in 96well plates (5,000 cells/well). The next day, the cells were treated with E2 (100 nM), BMP2 (200 ng/mL), E2/BMP2, or no treatment for 24 hrs. The cells were washed once with PBS and lysed with 1× passive lysis buffer. Luciferase activity was measured using a Turner 20/20 luminometer with a Dual-Luciferase Reporter Assay kit (Promega) according to the manufacturer’s instructions. Luciferase activity was calculated as the ratio of firefly luciferase activity to that of Renilla luciferase.
Gene silencing using siRNA targeting ER or SMAD
Endogenous ER or SMAD4 expression was knocked down by siRNA transfection using human ER siRNA (sc-29305) or Smad4 siRNA (sc-29484), which refers to a pool of four target-specific, 19- to 25-oligonucleotide siRNAs. siRNA-A, a non-targeting 20- to 25oligonucleotide (sc-37007), was used as the control. hMSCs were transfected with human sequence-specific ER/Smad4 siRNA or control siRNA-A at a final concentration of 30 pM using Lipofectamine 2000 reagent in serum free-DMEM without antibiotics for 5-6 hours, according to the manufacturer’s instructions (Invitrogen). After transfection, the cells were 9
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stabilized by culturing overnight in DMEM with 10% HI-FBS and then the medium was replaced with DMEM with 1% HI-FBS. After incubation for 24 hrs, the cells were treated with E2 (100 nM), BMP2 (200 ng/mL), or E2/BMP2 or left untreated.
Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR)
Total RNAs were extracted by the addition of 0.5 mL of TRIzol reagent (Life Technologies) to cells on a plate as described in the manufacturer’s instructions. One microgram of RNA was subjected to cDNA synthesis with SuperScriptTM Reverse Transcriptase II and oligo12–18 primers (Invitrogen). We used SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) to detect the accumulation of PCR product during cycling with the ABI Prism 7700 Sequence Detection System (Applied Biosystems). The thermocycling conditions were as follows: predenaturation at 95C for 10 min; 40 cycles of denaturation at 95C for 15 s and annealing and extension at 60C for 1 min; followed by a final dissociation cycle at 95C for 15 s, 60C for 1 min, and 95C for 15 s. Real-time RTPCR was carried out in triplicate in at least three independent experiments (n>3). Oligonucleotide primers were designed using real-time RT-PCR system sequence detection software v1.3 (Applied Biosystems) and their sequences are provided in Table 1. Fold differences in the expression level of each gene were calculated for each treatment group using CT values normalized to transcript levels of the housekeeping gene 18S rRNA, according to the manufacturer’s instructions.
Alkaline phosphatase (ALP) assay
10
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11 We assayed ALP activity by measuring the amount of -nitrophenol produced using nitrophenol phosphate substrate. Cell lysates were mixed with alkaline buffer solution and gently shaken for 10 min. ALP substrate was added at room temperature for 30 min. After the reaction was stopped with 0.05 N NaOH, the absorbance at 405 nm was read and compared with a standard curve prepared with -nitrophenol standard solution. Enzyme activity was normalized to the protein concentration of the cell layer determined using a protein assay (Bio-Rad, Hercules, CA, USA).
Enzyme-linked immunosorbent assay (ELISA)
Sclerostin or Opg levels in the culture supernatants were determined using an ELISA kit (R&D Systems). After centrifugation, cell culture supernatants (n=4-5) were added to 96-well ELISA plates. Standards for cytokines (0–1,000 pg/mL) were run in each series. After incubation, aspiration, and washing, a human conjugate of each protein (100 µL/well) was added according to the manufacturer’s instructions. The OD of each well was determined within 30 min using a microplate reader set to a 450-nm wavelength with correction for optical imperfections in the plate.
Western blotting
Total cell lysates of cultured hMSCs were prepared by lysing cells
in RIPA buffer
containing 50 mM Tris buffer (pH 7.5), 50 mM NaCl, 1% Triton X-100, 1 mM EGTA, 10 mM Na4P2O9, 5 mM Na3VO4, 50 mMNaF, and protease inhibitors (10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 μg/ml pepstatin A, 0.1 mM PMSF, and 1 mM DTT). Nuclear and cytoplasmic fractionation of cultured hMSCs was performed using NE-PERTM Nuclear and 11
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cytoplasmic extraction reagents (Thermo SCIENTIFIC, Rokford, IL, USA) according to the manufacturer’s instructions. Total cell lysates (20 µg/lane) or nuclear/cytoplasmic fraction (15 µg/lane) were subjected to electrophoresis through a 10% SDS-polyacrylamide gel and transferred to PolyScreen PVDF membranes (PerkinElmer Life Sciences, Hopkinton, MA, USA). Membranes were blocked with 5% nonfat dry milk in TBST buffer (0.1 M Trisbuffered saline [pH 7.5]/0.1% Tween) and probed with antibodies. The following antibodies were used in this study: polyclonal rabbit anti-total -catenin (Abnova, Taipei, Taiwan); polyclonal rabbit anti–non-phospho (active) -catenin antibody, diluted 1:2000; polyclonal rabbit anti-ER antibody, diluted 1:200; monoclonal mouse anti-Smad4 antibody, diluted 1:200; and monoclonal mouse anti--tubulin antibody, diluted 1:200. Primary antibodies were detected with HRP-conjugated anti-rabbit IgG (Santa Cruz Biotechnology) for total/active -catenin and ER, or with HRP-conjugated anti-mouse IgG (Dako) for Smad4 and -tubulin. Finally, blots were stained with an enhanced chemiluminescence detection system (ECL, Amersham Biosciences) according to the manufacturer’s instructions.
Immunofluorescent staining
Sclerostin or -catenin expression in hMSCs was detected at an average cell density of 3,000 cells per coverslip. Cells were washed three times with PBS, fixed in 4% paraformaldehyde for 30 min, and blocked in blocking solution for 30 min. Cells were incubated with antibody against sclerostin (anti-rabbit, 1:100; Abnova) or active -catenin (anti-rabbit, 1:400; Cell Signaling Technology, Inc) overnight at 4C, washed twice with PBS, and then incubated with secondary antibody labeled with FITC (anti-rabbit IgG, 1:500; Biomeda) for 1 hr. Primary antibody controls were processed in parallel using only the 12
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secondary antibody. Slides were washed in PBS for 10 min and incubated with PBS-buffered 4΄,6-diamino-2-phenylindole (DAPI) solution (1 mg/mL; Santa Cruz Biotechnology) using a dilution ratio of 1:1000 for nuclear staining. Section images of the stained cells were captured using an Olympus Fluoview FV300 confocal laser scanning microscope and Fluoview software (Olympus Optical Co. Ltd.).
Statistical analysis
All data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using SPSS 20 (IBM Co., Armonk, NY, USA). Differences between two groups were evaluated using a two-tailed Student’s t-test, and the comparison of data for more than two groups was performed using with two-way analysis of variance post hoc via the Bonferroni method. Differences with p
