The present study explored whether bone morphogenetic proteins (BMPs) and Wnt/β-catenin signaling pathways were involved in the 1,25(OH)2D3-induced inhibition of osteogenic differentiation in bone marrow-derived mesenchymal stem cells (BMSCs). To evaluate the osteogenic differentiation of BMSCs, the expression levels of ossification markers, including BMP2, Runt-related transcription factor 2 (Runx2), Msh homeobox 2 (Msx2), osteopontin (OPN) and osteocalcin (OCN), and the activity of alkaline phosphatase (ALP), as well as the calcified area observed by Alizarin red-S staining, were investigated. Chromatin immunoprecipitation (ChIP) assay was used to detect the effect of 1,25(OH)2D3 on the DNA methylation and histone modification of BMP2, while an immunoprecipitation (IP) assay was performed to assess the crosstalk between Smad1 and disheveled-1 (Dvl-1) proteins. It was observed that 1,25(OH)2D3 significantly decreased the expression levels of BMP2, Runx2, Msx2, OPN and OCN, and reduced ALP activity and the calcified area in BMSCs, whereas these effects were rescued by BMP2 overexpression. ChIP assay revealed that BMSCs treated with 1,25(OH)2D3 exhibited a significant increase in H3K9me2 level and a decrease in the acetylation of histone H3 at the same BMP2 promoter region. In addition, 1,25(OH)2D3 treatment promoted the nuclear accumulation of β-catenin by downregulating BMP2. Furthermore, the β-catenin signaling inhibitor XAV-939 weakened the inhibitory effect of 1,25(OH)2D3 on osteogenic differentiation. Additionally, knockdown of β-catenin rescued the attenuation in Dvl-1 and Smad1 interaction caused by 1,25(OH)2D3. Overexpression of Smad1 also reversed the inhibitory effect of 1,25(OH)2D3 on osteogenic differentiation. Taken together, the current study demonstrated that 1,25(OH)2D3 inhibited the differentiation of BMSCs into osteoblast-like cells by inactivating BMP2 and activating Wnt/β-catenin signaling.
Osteoporosis is a common bone disease that mainly affects the elderly, and is characterized by low bone density and microarchitectural deterioration of bone tissue (
1,25(OH)2D3, the most active vitamin D metabolite, is a pleiotropic hormone. Through binding to its intra-nuclear receptor, vitamin D receptor (VDR), 1,25(OH)2D3 has numerous regulatory effects, including calcium homeostasis, cell proliferation and differentiation (
The canonical Wnt/β-catenin pathway has been identified to serve an important role in the regulation of the osteogenic differentiation of BMSCs (
In the present study, the aim was to explore the mechanism underlying the 1,25(OH)2D3-mediated suppression of osteogenic differentiation
In total, three Sprague-Dawley (SD) rats (140±10 g), aged 4-weeks-old, were purchased from Better Biotechnology Co., Ltd. (Nanjing, Jiangsu) and were maintained under specific pathogen-free conditions at 20–26°C with 55±5% humidity in 12 h light/dark cycle with
Subsequent to repeated flushing, the BMSCs were cultured in culture medium containing 89% Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.), and kept at 37°C with 5% CO2. The culture medium was refreshed twice per week. BMSCs at the 3rd passage were used in all the experiments.
To identify the BMSCs, cells at the 3rd passage were trypsinized, collected and subjected to flow cytometry analysis (CytoFLEX; Beckman Coulter Commercial Enterprise, Inc.) with antibodies against CD34 (1:50; cat. no. ab81289; Abcam), CD45 (1:10; cat. no. 554878; BD Bioscience, CD44 (1:10; cat. no. 550974; BD Bioscience) and CD90 (1:10; cat. no. 561973; BD Bioscience). Cells identified as BMSCs negatively expressed CD34 and CD45, and positively expressed CD44 and CD90 (
For osteogenic induction, BMSCs (1×106 cells/well) were seeded into 6-well plates and cultured in the complete culture medium supplemented with osteogenic induction medium (OIM) containing 10−7 M dexamethasone, 10 mM β-glycerophosphate and 50 µM ascorbate-2-phosphate (all purchased from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 14 days (
For 1,25(OH)2D3 treatment, BMSCs were incubated with 1, 5, 10, 20 or 50 nM of 1,25(OH)2D3 (Sigma-Aldrich; Merck KGaA) dissolved in ethanol for 48 h. Equivalent volume of 100% ethanol was used in the negative control group. Furthermore, XAV-939 (Selleck Chemicals, Shanghai, China) treatment was used to inhibit Wnt/β-catenin signaling in BMSCs at a concentration of 10 µM for 1 h, with equal volume of DMSO (Beyotime Institute of Biotechnology) as a negative control. For co-treatment of 1,25(OH)2D3 and XAV-939 (MedChemExpress, Inc.), the cells were first treated with 10 µM XAV-939 for 1 h at 37°C, followed by treatment with 10 nM 1,25(OH)2D3 for 48 h at 37°C.
In order to induce upregulation of BMP2 and Smad1 levels in BMSCs, overexpressing lentivirus vectors targeting the rat BMP2 (OE-BMP2) and Smad1 (OE-Smad1) genes were designed and synthesized by GenePharma Co., Ltd. Briefly, the BMSCs (5×105) were seeded in 6-well plates and cultured at 37°C overnight. Next, the cells were infected with OE-BMP2, OE-Smad1 or OE-NC (serving as the negative control vector) using 5 µg/ml polybrene (Hanbio Biotechnology Co., Ltd.), followed by incubation with 100 µg/ml G418 or 7 µg/ml puromycin for 14 days to select the stably infected cell lines, respectively.
Three siRNAs targeting the rat β-catenin gene (si-β-catenin) and a negative control vector (si-NC) were purchased from OriGene Technologies, Inc. (Rockville, MD, USA; cat. no. SR500644). The siRNAs (si-1, si-2 and si-3) were applied to knock down β-catenin expression in BMSCs, using Lipofectamine® 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Following 6 h of cell transfection, the medium was replaced with fresh DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS. The transfection efficiency was detected by using reverse transcription-quantitative PCR (RT-qPCR) and western blotting assays after 24 and 48 h of cell transfection. The siRNA sequences used were: si-1, 5′-CCAGCAAATCATGCGCCTT-3′; si-2, 5′-GCTGCATAATCTCCTGCTA-3′; and si-3, 5′-CCACTAATGTCCAGCGCTT-3′.
Total RNA was extracted from cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and the concentration was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). Next, the RNA was reversely transcribed into complementary DNA (cDNA) using a PrimeScript™ RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). Next, qPCR analysis was performed with SYBR GreenER™ qPCR SuperMix (Thermo Fisher Scientific, Inc.). The qPCR conditions involved pre-denaturation at 95°C for 5 min, 40 cycles of 95°C for 15 sec and 60°C for 1 min. The relative expressions of mRNAs were calculated by using the 2−ΔΔCq method (
Total protein was extracted from the BMSCs using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology), while the nuclear and cytoplasmic proteins were extracted from the cells using a CelLytic™ NuCLEAR™ Extraction kit (Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. Following the assessment of protein concentrations with a BCA kit (Thermo Fisher Scientific, Inc.), 25 µg protein from each sample was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by transfer onto PVDF membranes (Thermo Fisher Scientific, Inc.). The membranes were then blocked with 5% non-fat milk for 1 h at room temperature and probed overnight at 4°C with the following primary antibodies: GAPDH (1:10,000 dilution; ProteinTech), BMP2 (1:1,000 dilution; cat. no. ab14933; Abcam), Runx2 (1:1,000 dilution; cat. no. ab76956; Abcam), Msx2 (1:2,000 dilution; cat. no. HPA005652; Sigma-Aldrich), osteocalcin (OCN; 1:2,000 dilution; cat. no. ab93876; Abcam), osteopontin (OPN; 1:2,000 dilution; cat. no. ab8448; Abcam), β-catenin (1:2,500 dilution; cat. no. 9562; Cell Signaling Technology), disheveled-1 (Dvl-1; 1:2,000 dilution; cat. no. D3570; Sigma-Aldrich), Smad1 (1:1,000 dilution; cat. no. 9743; Cell Signaling Technology), tubulin (1:1,000 dilution; cat. no. 2148; Cell Signaling Technology) and histone (1:5,000 dilution; cat. no. ab1791; Abcam). Subsequently, incubation with a horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. SA00001-1/ SA00001-2; ProteinTech) was performed for 1 h at room temperature. GAPDH, histone and tubulin were used as the internal references for the total, nuclear and cytoplasmic protein expression, respectively. The band signals were enhanced by chemiluminescence (ECL reagent; EMD Millipore, Billerica, MA, USA) and visualized with a FluorChem Q system (SelectScience, Waltham, MA, USA). Protein expression quantification was performed using ImageJ software (version 1.48; National Institutes of Health, Bethesda, MD, USA).
To assess ALP activity, BMSCs at a density of 1×105 cells/well were seeded into 24-well plates and cultured for 14 days with OIM. Next, the ALP activity was measured with an ALP activity kit (Shanghai Suer Biological Technology Co., Ltd., Shanghai, China) according to the manufacturer's protocol. In brief, cells were lysed with RIPA lysis buffer on ice, and then the lysates were mixed with a working solution for 15 min at 37°C. The optical density values at 520 nm were subsequently measured, and the ALP activity was normalized to the total intracellular protein concentration.
In order to assess whether 1,25(OH)2D3 affected the osteogenic differentiation of BMSCs, after 12 days of incubation with OIM, BMSCs (5×105/well in 6-well plates) were cultured in OIM containing 1,25(OH)2D3 (10 nM) for a further 48 h and the ALP activity was determined. In addition, to assess the effects of BMP2 and Smad1 on osteogenic differentiation, the OE-BMP2/OE-Smad1 stable expressing cells were cultured in OIM for 14 days, followed by ALP detection.
Cells at a density of 5×106/well in 6-well plates treated with 1,25(OH)2D3 (10 nM) for 48 h or OE-BMP2/OE-Smad1 stable expression cell lines were washed with PBS and then fixed with 75% ethanol at 4°C for 30 min. Next, the cells were stained with Alizarin red-S (40 mM, pH 6.2) at room temperature for a total of 30 min. Any excess stain was removed by distilled water, and images of the plates were captured to visualize cell calcification. Quantification was performed using Image Pro Plus software (version 6.0; Media Cybernetics, Inc.).
A CCK-8 assay was used to detect cell proliferation. In brief, BMSCs were seeded in 96-well plates at a density of 3×103 cells/well and cultured at 37°C overnight. Subsequently, the cells were subjected to different treatments, including 1,25(OH)2D3 (10 nM)/ethanol (10 nM) and/or XAV-939 (10 µM)/DMSO (10 µM). Following incubation at 37°C for 48 h, the cell culture medium was replaced with 10 µl CCK-8 reagent (Beyotime Institute of Biotechnology) and 90 µl fresh medium, and incubated at 37°C for another 4 h. The absorbance at 450 nm was finally measured with a plate reader (model 680; Bio-Rad Laboratories, Inc.).
The fragment of rat BMP2 promoter region C that contains the VDR binding sites was amplified by PCR using the followed primers: Forward primer, 5′-ATTTGCCCTAAACTCGGGCATCTG-3′, and reverse primer, 5′-TTCGTCCCGAGCTGCCAAT-3′ (
ChIP assay was performed as previously described (
An IP assay was used to evaluate the interaction between Dvl-1 and Smad1 proteins. Briefly, cells were lysed in 5 ml lysis buffer (containing 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 0.5% Nonidet P40 and protease inhibitor cocktail) for 30 min at 4°C. After 1 h of incubation with 50 µl Dynabeads protein A (Invitrogen; Thermo Fisher Scientific, Inc.), the supernatants containing 200 µg proteins were incubated with antibody against Smad1 (2 mg; cat. no. 9743; Cell Signaling Technology) at 4°C overnight. Next, the Dynabeads were washed with lysis buffer for five times, followed by resuspension in SDS-PAGE loading buffer and assessment by western blotting using antibody against Dvl-1 (1:2,000; cat. no. D3570; Sigma-Aldrich; Merck KGaA).
Each experiment was repeated at least three times, and the data are represented as the mean ± standard deviation. Data analysis was performed with SPSS software, version 21.0 (IBM Corporation, Armonk, NY, USA), using the Student's t-test for comparisons between two groups, or one-way analysis of variance followed by Bonferroni post-hoc test for comparisons of more than two groups. P<0.05 was considered to denote a statistically significant difference.
The flow cytometry results demonstrated that the isolated cells positively expressed CD44 and CD90, while they negatively expressed CD34 and CD45 (
When cells were treated with 1,25(OH)2D3 (10 nM) for 48 h, the ALP activity and calcified area of BMSCs were significantly decreased, whereas BMP2 overexpression rescued these results (
Next, the current study explored the mechanism underlying 1,25(OH)2D3-induced BMP2 downregulation in BMSCs. Compared with the control (ethanol) group, 1,25(OH)2D3 treatment significantly decreased the activity of the pSV40-BMP2-Luciferase promoter (
Next, the study explored the role of β-catenin signaling in 1,25(OH)2D3-induced osteogenesis repression. The results revealed that 1,25(OH)2D3 treatment enhanced the nuclear expression level of β-catenin and decreased its cytoplasmic expression level, whereas BMP2 overexpression reversed the effects of 1,25(OH)2D3 (
As Wnt/β-catenin signaling closely interacts with BMP signaling pathway (
The imbalance in bone formation and bone resorption induced by the inactivity of osteoblasts and the hyperactivation of osteoclasts is considered as the main reason of osteoporosis (
It is well documented that Wnt/β-catenin and BMP signaling pathways are essential for BMSCs to differentiate into osteoblasts (
Previous evidence demonstrated that the 1,25(OH)2D3 role depends on its combination with its receptor VDR. Once 1,25(OH)2D3 binds to VDR, this receptor will heterodimerize with the retinoid X receptor and translocate into the nucleus to combine with vitamin D3 responsive elements (VDREs) in the promoter regions of the target genes of VDR, leading to upregulation or downregulation of gene transcription (
The present study further investigated the effects of the 1,25(OH)2D3/BMP2 axis on Wnt/β-catenin signaling activation. It was observed that 1,25(OH)2D3 treatment evidently enhanced the nuclear accumulation of β-catenin protein, while BMP2 overexpression reversed this effect, suggesting that 1,25(OH)2D3 activated β-catenin signaling through downregulating BMP2. However, the results of the present study were the opposite from those reported by Larriba
Increasing evidence has suggested that Wnt/β-catenin signaling closely interacts with the BMP signaling pathway. For instance, Haramis
In conclusion, the present study revealed that 1,25(OH)2D3 inhibited the differentiation of BMSCs into osteoclast-like cells through inactivating BMPs and activating Wnt/β-catenin signaling. The study provides a deeper understanding on the mechanisms of vitamin D in the inhibition of osteogenic differentiation, as well as reconsiders the role of vitamin D in osteoporosis treatment.
Not applicable.
No funding was received.
All data generated or analyzed during this study are included in this published article.
GC contributed to the design of the study and revised the manuscript. XH performed the experiments and data analysis, and wrote the manuscript. NZ and YW performed the experiments. All authors read and approved the final manuscript.
Experiments in this study involving animals were approved by the Review Committee for the Use of Human or Animal Subjects of Shanghai Jiao Tong University and were performed in accordance with the National Institutes of Health Guidelines on the Use of Laboratory Animals.
Not applicable.
The authors declare that they have no competing interests.
1,25(OH)2D3 repressed the osteogenic differentiation of BMSCs via BMP2 downregulation. (A) Flow cytometry was conducted to test the proportion of CD34+, CD44+, CD45+ and CD80+ in the isolated BMSCs. (B) mRNA and (C) protein levels of BMP2, determined by RT-qPCR and western blotting subsequent to stable infection of BMSCs with OE-BMP2 or OE-NC (n=3). (D) The effect of different concentrations of 1,25(OH)2D3 (1, 5, 10, 20 or 50 nM) on ALP activity in BMSCs, with ethanol serving as a negative control (n=3). (E) Cell viability determined by Cell Counting Kit-8 assay and (F) ALP activity in BMSCs treated with 1,25(OH)2D3 and/or OE-BMP2. (G) Alizarin red-S was used to assess the calcified nodules of BMSCs treated with 1,25(OH)2D3 and/or OE-BMP2. Magnification, ×200. (H) Western blotting of the protein levels of BMP2, Runx2, Msx2, OPN and OCN in BMSCs with different treatments. (I) RT-qPCR analysis of the mRNA levels of Runx2, BMP2 and Msx2 in differently treated BMSCs (n=3). *P<0.05 and **P<0.01, vs. corresponding control group (OE-NC, ethanol or ethanol + OE-NC group); #P<0.05 vs. 1,25(OH)2D3+OE-NC group. BMSCs, bone marrow-derived mesenchymal stem cells; BMP2, bone morphogenetic protein 2; ALP, alkaline phosphatase; Runx2, Runt-related transcription factor 2; Msx2, Msh homeobox 2; OPN, osteopontin; OCN, osteocalcin; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; OE, overexpressing vector; NC, negative control.
1,25(OH)2D3 decreased BMP2 expression through DNA methylation and histone modification. (A) The effect of 1,25(OH)2D3 (10 nM) on the transcriptional activity of BMP2 was assessed by luciferase report assay with pSV40-BMP2-Luciferase plasmid transfection. Chromatin immunoprecipitation assays were used to detect the crosstalk of BMP2 promoter with (B) histone H3-Ac or (C) H3K9me2 (n=3). **P<0.01 vs. control (ethanol) group. BMP2, bone morphogenetic protein 2; histone H3-Ac, acetyl-histone H3; H3K9me2, histone H3 dimethylated lysine 9.
Inhibition of β-catenin signaling rescued the repression of the osteogenic differentiation of BMSCs induced by 1,25(OH)2D3. (A) Western blotting of the expression of β-catenin in the nucleus and cytoplasm of BMSCs treated with ethanol plus OE-NC or OE-BMP2, or with 1,25(OH)2D3 (10 nM) plus OE-NC or OE-BMP2. *P<0.05 vs. ethanol + OE-NC group, #P<0.05 vs. 1,25(OH)2D3 + OE-NC group. (B) Cell viability determined by Cell Counting Kit-8 assay and (C) ALP activity in differently treated BMSCs (ethanol plus DMSO or XAV-939, and 1,25(OH)2D3 plus DMSO or XAV-939). (D) Alizarin red-S was used to assess the calcified nodules of BMSCs. Magnification, ×200. (E) Western blotting of the protein expression levels of BMP2, Runx2, Msx2, OPN and OCN after BMSCs were treated with ethanol plus DMSO or XAV-939, and with 1,25(OH)2D3 plus DMSO or XAV-939. (n=3). C-E, *P<0.05 vs. ethanol + DMSO group; #P<0.05 vs. 1,25(OH)2D3 + DMSO group. BMSCs, bone marrow-derived mesenchymal stem cells; BMP2, bone morphogenetic protein 2; ALP, alkaline phosphatase; Runx2, Runt-related transcription factor 2; Msx2, Msh homeobox 2; OPN, osteopontin; OCN, osteocalcin; OE, overexpressing vector; NC, negative control.
1,25(OH)2D3/β-catenin repressed the osteogenic differentiation of BMSCs through impairing the interaction between Dvl-1 and Smad1 proteins. (A) mRNA and (B) protein levels of β-catenin in BMSCs transfected with siRNAs were assessed by RT-qPCR and western blotting, respectively. **P<0.01 vs. si-NC group. (C) Immunoprecipitation assay, evaluating the crosstalk between Dvl-1 and Smad1 protein in BMSCs treated with 1,25(OH)2D3 (10 nM, 48 h) with or without si-β-catenin transfection. (D) mRNA and (E) protein expression levels of Smad1 in BSMCs stably infected with OE-Smad1 or OE-NC were determined by RT-qPCR and western blotting, respectively. *P<0.05 vs. OE-NC group. (F) BMSC viability determined by Cell Counting Kit-8 assay, and (G) ALP activity in BMSCs. *P<0.05 vs. ethanol + OE-NC group, #P<0.05 vs. 1,25(OH)2D3 + OE-NC group. (H) Alizarin red-S was used to assess the calcified nodules of BMSCs. Magnification, ×200. *P<0.05 vs. ethanol + OE-NC group, #P<0.05 vs. 1,25(OH)2D3 + OE-NC group. (n=3). BMSCs, bone marrow-derived mesenchymal stem cells; Dvl-1, disheveled-1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; si-/siRNA, small interfering RNA; ALP, alkaline phosphatase; OE, overexpressing vector; NC, negative control.