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Acta Endocrinologica (Bucharest) logoLink to Acta Endocrinologica (Bucharest)
. 2020 Apr-Jun;16(2):156–164. doi: 10.4183/aeb.2020.156

CHANGES OF WNT/B-CATENIN SIGNALING AND DIFFERENTIATION POTENTIAL OF BONE MARROW MESENCHYMAL STEM CELLS IN PROCESS OF BONE LOSS IN OVARIECTOMIZED RATS

W Ren 1, D Gan 2, G Tan 2, H Xue 2, N Li 2, Z Xu 2,*
PMCID: PMC7535886  PMID: 33029231

Abstract

Background.

In vitro studies of the changes about osteoblastogenesis and adipogenesis potential of BMSCs were not clear. As it is the critical pathway for osteogenic differentiation and bone formation, whether or not Wnt/β-catenin signalling is involved in the changes of osteogenic and adipogenic potential of BMSCs and participates in bone content decrease of ovariectomized (OVX)osteoporosis rats has been rarely reported.

Material/Methods.

BMSCs from femurs of ovariectomzed rats were isolated and cultured in vitro. The proliferation potential of BMSCs was analysed by CCK-8 assays . Osteoblastic and adipogenic differentiation potential of the BMSCs was assessed by ALP activity assay, Alizarin red S staining, Oil red O staining and RT-PCR analysis.

Results.

The results demonstrated that BMSCs from bilateral ovariectomization rats were endowed with lower proliferation and osteoblastic differentiation potential but higher adipogenic potential than the control group in vitro. In addition, β-catenin was found to have been decreased in OVX BMSCs, indicating that Wnt/β-catenin signalling pathways were suppressed in OVX BMSCs .

Conclusions.

Results suggested that changes in the Wnt canonical signalling pathway may be related to imbalances of osteogenic and adipogenic potential of BMSCs, and this may be an important factor related to bone content decrease in ovariectomized osteoporosis rats.

Keywords: Postmenopausal osteoporosis, BMSCs, Wnt/β-catenin signalling, Osteoblastic differentiation, Adipogenic differentiation

INTRODUCTION

Postmenopausal osteoporosis is a common systemic skeletal disease, characterized by low density of bone mineral and microarchitecture deterioration. Estrogen deficiency is the most well-recognized cause of osteoporosis (1), thus, postmenopausal women are considered extensively vulnerable to osteoporosis. Decreases in bone mass and microarchitectural deterioration of bone tissues give rise to a high risk of fractures of the hip, vertebrae, pelvis and wrist, can cause disability, and even death (2). Research leading to a better understanding of the mechanisms of post-menopausal osteoporosis is very important and necessary for an increased and continued development of clinically based strategies for prevention, diagnosis, and therapeutic treatments for this affection.

Post-menopausal osteoporosis is mainly induced by the effects of the dynamics related to an imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Increasing research has reported that the balance between osteoblastogenesis and adipogenesis in bone marrow stromal cells (BMSCs) also plays an important role in the pathogenesis of postmenopausal osteoporosis (3, 4). BMSCs have the potential to differentiate into several different lineages, including osteoblasts, chondroblasts, adipocytes, and myoblasts; and of these lineages, osteogenic and adipogenic lineages are the most closely related (5-7). Several in vitro studies which used cultured rat and human BMSCs have suggested that there is a reciprocal relationship between the differentiation of osteogenic cells and adipocytes (8, 9). However, results about osteogenic and adipogenic differentiation potential of BMSCs have come to differing conclusions and the regulatory mechanism underlying these processes is extremely complicated and remains unclear. Thus, more experimentation is needed to elucidate such mechanisms.

In addition, it has been acknowledged that Wnts extensively participate in embryonic skeletal patterning, fetal skeletal development, and adult skeletal remodelling, and that the Wnt/β-catenin signalling pathway is critical for osteogenic differentiation and bone formation (10, 11). However, whether or not the Wnt/β-catenin signalling pathway is involved in the changes of osteogenic and adipogenic potential of BMSCs in ovariectomized osteoporosis rats has been rarely reported. Thus, we sought to examine and understand the mechanism of Post-menopausal Osteoporosis and its importance in the dynamics of imbalance of osteoblastic and adipogenic differentiation potential of BMSCs. We got BMSCs from ovariectomized (OVX) rats, and sought to evaluate whether or not the Wnt/β-catenin signalling pathway is involved in this process.

MATERIALS AND METHODS

Establishment of the OVX-induced osteoporosis model

Twelve female Sprague-Dawley (SD) rats (6 months of age and weighting 334.5±29.2 g) were raised separately and kept in an air-conditioned environment with relative steady temperature and humidity. Three days later, they were randomly divided into the sham operation group and bilateral ovariectomy group. In the ovariectomies group, bilateral ovariectomies were done to establish the osteoporosis model (10). In the sham operation group, a small amount of adipose tissue around the ovary was removed. At 3 months post-surgery, the bone mineral density (BMD) of the lumbar spine and femurs of all rats was determined by dual-energy X-ray to evaluate whether the osteoporosis model was successfully established. This study was approved by the Animal Ethics Committee of Shandong University of Traditional Chinese Medicine (SDUTCM2018091902).

Isolation and culture of BMSCs

Bone marrow stromal cells (BMSCs) were obtained from the bone marrow of the Sham and OVX rats by flushing the marrow cavity of the femur(11), as described previously (12). The cells were cultured in basic medium whose main component is dulbecco’s modified eagle medium (DMEM)with Low Glucose, supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA). Cells were maintained in a humidified incubator with 5% CO2 air atmosphere at 37 °C. After 3 days, nonadherent cells were removed by washing with phosphate-buffered saline (PBS), while the adherent cells were further cultured. At 80% cell confluence, the adherent cells were digested with trypsin-EDTA(Gibco, Grand Island, NY, USA), and passaged.

Cell proliferation assay

Cells (1×104 per well) were plated in a 96-well plate and cultured in basal medium. And cell proliferation was separately determined on days 1, 3, 5, 7 and 9 using the Cell Counting Kit-8 (CCK-8) (Boster, Wuhan, China) assay as instructed by the manufacturer. Absorbance was measured at 450nm using a microplate reader (Thermo Scientific, Beijing, China). Cell proliferation was expressed as the optical density (OD) value.

Osteogenic differentiation

After the 3 passage, the adherent cells were harvested by treatment with trypsin/EDTA. They were then counted and plated in culture plates (1 × 104 cells/cm2).When over 80% confluence was reached, the cells were cultured in osteogenic medium, which consisted of DMEM supplemented with 10% fetal bovine serum, 10 mM β-glycerophosphate, 50 mg/L ascorbic acid and 10-7 mol/L dexamethasone (all from Sigma, St Louis, MO, USA). Also the potential of Osteogenic differentiation was evaluated by measuring ALP activity, production of mineralized nodules by Alizarin red S staining, and gene expression of RUNX2,Osterix via RT-PCR.

Alkaline phosphatase (ALP) activity assay

ALP activity of osteoblastic differentiation BMSCs was determined as previously described(13). Briefly, Sham BMSCs and OVX BMSCs were subcultured in osteoblastic medium for 7, 10 and 14 days, then the medium was removed and rinsed with PBS, and digested by trypsin-EDTA. Cells were lysed with RIPA lysis buffer (Aidlab, Beijing, China). The ALP activity in the lysates was determined by the measurement of p-nitrophenyl phosphate (PNPP) using a commercial assay kit (Nanjing Jiancheng Bioengineering Institute, China).Absorbance of the reaction mixture was measured by a microplate reader at 405 nm. Total protein concentration of cell lysates was determined by BCA assay (Beyotime, China) at 562nm. The specific ALP activity was normalized by total protein content.

Alizarin red S staining and quantification

After 28 days of osteogenic induction, the cells were rinsed with PBS twice and fixed in 95% ethanol for 10 min. and then rinsed with deionized water twice. The cultures were then stained with 40mM Alizarin red S in deionized water (pH 4.2) for 10min at room temperature. After removing Alizarin red S solution, the cells were rinsed with fresh PBS and dried at room temperature. Red staining indicated the position and intensity of the calcium deposits. The quantification of Calcium deposit was evaluated by means of the cetyl pyridinium chloride (CPC) method. After rinsed with the deionized water, the cells were destained in 10% cetylpyridinium chloride monohydrate at room temperature for 30 min. The OD of the solution was measured at 540 nm(14).

Adipogenic differentiation

BMMSCs at passage 3 were plated in culture plates. The adipogenic medium was basic medium supplemented with 1μmol/L dexamethasone, 200 μmol/L indometacin, 0.5 mmol/L 3-isobutyl-1-methylxanthine (IBMX) and 10 μM insulin (all from Sigma, St Louis, MO, USA) (15). At 21 days cell differentiation was evaluated with Oil red O staining, gene expressions of PPARγ and LPL were detected via qRT-PCR.

Oil red O staining

Cells were cultured in an adipogenic induction medium for 21 days. Both groups were washed twice with PBS, fixed in 4% paraformaldehyde for 10 min, washed with 3% isopropanol, and stained with Oil red O staining solution for 10 min. After washing with PBS, BMSCs were destained in 100% isopropanol for 15 min, The optical density (OD) of the solution was measured (16).

Quantitative real-time RT-PCR

Sham BMSCs and OVX BMSCs were subcultured in osteoblastic and adipogenic medium for 14 days. Total RNA was extracted using TRIZOL reagent (Aidlab, Beijing, China). RNA concentrations were measured using a ND-1000 spectrophotometer (Kaiao Technology, Beijing, China), then reverse-transcribed to cDNA using Revert Aid First Strand cDNA Synthesis kit (Roche, Shanghai, China) according to the manufacturer’s instructions. Amplification reactions were conducted with the following thermal cycling parameters: an initial step at 95°C for 5 min, then 40 cycles of amplification (95 °C for 20s, 62 °C for 15 s and 72 °C for 15 s). The primer sequences are listed in Table 1. The osteoblastic marker genes used in this study included runt-related transcription factor 2 (RUNX2) and Osterix (OSX). While the adipogenic marker genes included peroxisome proliferators activated receptor gamma (PPARγ), and Lipoprotein lipase (LPL). Relative expression of the real time RT-PCR product was calculated using the comparative Ct method. The expression levels for each target gene were calculated using 2 (−ΔΔ C t) method and normalized to β-actin mRNA.

Table 1.

Gene Forward sequence (5’ to 3’) Reverse sequence (5’ to 3’)
RUNX2 5’-GAACCCACGGCCCTCCCTGAACTC-3’ 5’-AGCGGCGTGGTGGAATGGATGGAT-3’
Osterix 5’-CTGGGGGCAATTGGTTAGGTGGTG-3’ 5’-GGGGGCAAAGTCAGACGGGTAAGT-3’
PPARγ 5’-CGTCCCCGCCTTATTATT-3’ 5’-AACCGACAGTACTGACATTTATTT-3’
LPL 5’-AGCCCCTAGTCGCCTTTCTCCTG-3’ 5’-GCCTTGCTGGGGTTTTCTTCATTC-3’
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Western Blotting Assays

Cells were cultured in basic medium at 3 passage, the total protein was obtained using the RIPA lysis buffer (Aidlab, Beijing, China), Nucleoprotein was acquired by a Nuclear Extract Kit (Aidlab, Beijing, China), following the manufacturer’s instructions. The protein concentration of each group was measured by BCA Protein Assay Kit (Beyotime Shanghai, China). Immunoblotting was carried out as previously described (17). Samples were run on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (SDS-PAGE) and transferred onto a PVDF membrane. The membranes were incubated with β-catenin, β-actin, LaminB1 antibodies from rabbit for 2 h at room temperature. Goat anti-rabbit IgG-HRP was used as secondary antibody (all antibodies from Cell Signalling Technology, Beverly, MA, USA). β-actin was used as a standard for total protein while LaminB1 was used as a standard for Nucleoprotein. Protein bands were visualized via reaction with an ECL kit (ECL Plus, Amersham, Buckinghamshire, England).Images were analyzed with Quantity One software (BioRad, Hercules, CA) and band intensity was quantified and normalized against β-actin or LaminB1.

Statistical analysis

Data are presented as mean value ± standard deviation (SD). Differences between two groups were analyzed using the independent t-test or Mann-Whitney test (two-tailed), as appropriate. P<0.05 was considered to indicate a statistically significant difference. Unless otherwise noted, all experiments and samples were triplicated.

RESULTS

Assessment of the animal model

At 3 months post-surgery, all rats remained healthy, the group of ovariectomized rats had a significant increase in the body weight compared to the sham group (Fig. 1). Dual-energy X-ray demonstrated that bone mineral density of the spine and femur in the ovariectomized group was significantly lower than that in the sham operation group (P < 0.05). These indicate a significant bone content decrease in ovariectomized rats.

Figure 1.

Figure 1.

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a) Body weight of the two group of rats before surgery. b) Body weight of the two group of rats 3 months post-surgery; the group of ovariectomized rats had a more increase in the body weight than the sham group. Dual-energy X-ray of femur. c) and lumbar spine. d) At 3 months after Sham or OVX surgery, indicating that BMD of lumbar spine and femur in the OVX group was significantly lower than that in the sham operation group (*P < 0.05).

In vitro proliferation potential of BMSCs

The BMSCs cultured in basic medium were generally fusiform, asteroid, triangle or polygon cells with big, round or ovoid and prominent nucleolus, as well as an abundant amount of cytoplasm. There is no obvious difference in appearance of the two groups of BMSCs. The number of BMSCs increased with time over 9 days’ culture in basic medium. The proliferation potential of the two different sources of BMSCs was tested by using a CCK-8 kit. CCK-8 assays indicated that OVX BMSCs had a lower proliferation rate compared with control cells at days 5,7, and 9 and indicated a higher proliferation potential of BMSCs from Sham rats than from OVX rats (Fig. 2). On the tenth day of culture, control cell cultures are reaching confluency, and cell proliferation is inhibited by cell to cell contact. For this reason, it was difficult to compare proliferation rates after the tenth day.

Figure 2.

Figure 2.

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a), b) There is no obvious difference in appearance of the two group of BMSCs. c CCK-8 assays indicated a higher proliferation potential of BMSCs from Sham rats than that from OVX rats.

Osteoblastic differentiation potential of BMSCs

Alkaline phosphatase activity in cell lysates was examined as a functional indication of BMSCs differentiation to osteoblastic cells. The ALP activity of OVX BMSCs and Sham BMSCs cultured in osteoblastic medium were analyzed for 7, 10 and 14 days. The ALP activities in BMSCs from OVX rats always reached lower levels than in the control group (Fig. 3). After 28 days’ culture, as examined by ARS staining, OVX BMSCs demonstrated less calcium deposition in mineralized nodules (Fig. 4). And the OD value of ARS solution in Sham BMSCs was also significantly higher than in OVX BMSCs. Moreover, RT-PCR also showed that after 28 days’ osteoblastic induction, two osteogenic related genes, runt transcription factor (Runx2) and Osterix (Ost) were significantly greater in Sham BMSCs versus OVX BMSCs (Fig. 5) (p< 0.05 for all).

Figure 3.

Figure 3.

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ALP activity of OVX BMSCs and Sham BMSCs. The ALP activities in BMSCs from OVX rats always reached lower levels than in control group.

Figure 4.

Figure 4.

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a), a’), b), b’) OVX BMSCs demonstrated less calcium deposition in mineralized nodules in ARS staining. c) The OD value of ARS solution in Sham BMSCs was also significantly higher than in OVX BMSCs.

Figure 5.

Figure 5.

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a), b) RT-PCR analysis of the expression of osteoblastic specific genes (Runx2, Osterix). Runx2 and Osterix were significantly greater in Sham BMSCs versus OVX BMSCs (P < 0.05).

Adipogenic differentiation potential of BMSCs

After 21 days’ culture in adipogenic induction medium, the adipogenic factors successfully stimulated adipocytes formation. Lipid accumulation was measured using Oil red O staining. Oil red O-positive adipocytes were observed in both groups. However, the OD of the Oil red O destaining solution in Sham BMSCs was significantly lower than in OVX BMSCs (Fig. 6). Meanwhile, after 21 days’ adipogenic induction, the expression of three adipogenic differentiation genes, peroxisome proliferator-activate receptor γ(PPARγ) and Lipoprotein lipase (LPL) were investigated in this study. The levels of the two adipogenic marker genes in Sham group were lower than those of OVX group (Fig. 7).

Figure 6.

Figure 6.

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a) Oil red O staining of Sham BMSCs. b) Oil red O staining of OVX BMSCs subcultured in adipogenic medium. c) The OD of the Oil red O destaining solution in Sham BMSCs was significantly lower than in OVX BMSCs.

Figure 7.

Figure 7.

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a), b) RT-PCR analysis of the expression of adipose-specific genes (PPARγ, LPL). PPARγ and LPL in the OVX group were also lower than those from the OVX group (P < 0.05).

Western blot

β-catenin is a critical signalling molecule that plays an important role in Wnt/β-catenin signalling pathways. In our study, western blot was used to detect the levels of β-catenin. Our results showed that the total protein and nuclear protein levels of β-catenin were significantly decreased in OVX BMSCs compared with those in Sham rats (Fig. 8, P<0.05)

Figure 8.

Figure 8.

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a) The expression levels of β-catenin protein in OVX and Sham rats. b), c) The total protein and nuclear protein of β -catenin was significantly decreased compared with those in Sham rats (P<0.05).

DISCUSSION

Postmenopausal osteoporosis is a common disease characterized by low bone mineral density and microarchitecture deterioration and often is attributed to estrogen deficiency (18).

Yet, research on postmenopausal osteoporosis has mostly been limited to the link-coupled system between osteoblast and osteoclasts (19). However, an increasing amount of research has indicated that the balance between osteoblastogenesis and adipogenesis in BMSCs also plays an important role in the pathogenesis of postmenopausal osteoporosis (20, 21). In several in vitro studies of cultured rat and human BMSCs, there has been an observation of a reciprocal relationship between the differentiation of osteogenic cells and adipocytes (8, 9). However, the results of several additional in vitro studies that have examined changes of osteogenic and adipogenic differentiation potential have not been consistent with regard to osteogenic potential and cell proliferation in ovariectomized osteoporosis rats (22). These inconsistent results might be explained by aspects related to variability in study design, animal models variability and individual differences, according to Ying Gao (22). Thus, we sought to provide new experimental evidence in relation to questions addressing these aspects.

In the present study, we used ovariectomized osteoporosis rats (OVX rats) to act as experimental animal models for post-menopausal osteoporosis (23), and used the sham operation rats (Sham rats)as control animals. After three months of bilateral ovariectomization, all rats were examined by using dual-energy X-rays for the evaluation of BMD. The results indicated that all OVX rats had a significant reduction in their trabecular bone mass. It has also been reported that bone turnover increases during the first month and continues to three months post bilateral ovariectomization (24). Then, bone turnover is gradually reduced to the same levels as found in the control group (25). The imbalance in bone metabolism in OVX rats is similar to that in postmenopausal women with osteoporosis, but the rate of progression happens much more quickly than in humans (26).

The process of osteogenesis can be depicted in three major stages: proliferation, matrix maturation, and mineralization. One of the most frequently used markers of the osteoblast differentiation process are alkaline phosphatase (ALP) which is a significant enzyme signifying extracellular matrix maturation in the process of bone formation. ALP can enhance the mineralization of the bone matrix by transforming the phosphoric ester into inorganic phosphorus in order to increase phosphorus concentrations(27). In this study, ALP was used as an early marker of osteoblast differentiation. ALP activity of osteoblastic differentiation BMSCs was tested at 7, 10 and 14 day time steps. Results indicated that ALP activity of OVX BMSCs was always lower than BMSCs from the sham treatment group, which was consistent with the quantitative evaluation of mineralization nodules of BMSCs by ARS strain. 28 days post-induction into the osteoblastic medium, ARS staining was used to analyse the mineralization of nodules of the two treatment groups of BMSCs. For both treatment groups, calcium deposition was detected in mineralized nodules. However, the OD value of ARS solution in OVX BMSCs was significantly lower than in control group. Meanwhile, 14 days’ post-osteoblastic induction, the levels of expression for mRNA of Osterix and Runx2 were detected by RT-PCR, and these are considered as the master osteogenic transcription factors in the osteoblast maturation process as well as they play essential roles in the dynamics of gene expression of osteoblast markers. All results from osteoblastic marker tests were consistent and indicated that Sham BMSCs were superior to OVX BMSCs with regard to osteoblastic differentiation potential.

Adipogenic differentiation potential of two kinds of BMSCs was analyzed using Oil red O staining. After 21 days of culturing in adipogenic medium, lipid droplet formation was successfully stimulated and Oil red O-positive adipocytes were present in both groups. However, the OD of the destaining solution in Sham group was lower than that observed in the OVX BMSCs. Furthermore, according to results from RT-PCR analyses, the levels of PPARγ and LPL in the OVX group were also lower than those in the OVX group. Thus, our results indicated that OVX BMSCs had a higher adipogenic differentiation potential compared with Sham BMSCs.

Our results indicated that OVX BMSCs are endowed with a lower proliferation and osteoblastic differentiation potential but also a had higher adipogenic potential than Sham BMSCs in vitro. These findings may partly help to explain clinical observations of the excessive fat and decrease of trabecular bone volume (8, 9) in the marrow cavity of postmenopausal osteoporotic women.

Wnt/ß-catenin or canonical pathway is a critical pathway for osteogenic differentiation and bone formation (28). The canonical Wnt signalling pathway involves the formation of a complex including Wnt proteins, FZD, low density lipoprotein (LDL), receptor-related protein 5 (LRP5), LRP6 receptors, β-catenin and TCF/LEF (29). Previous research has indicated that the Wnt canonical pathway plays an important role in the differentiation of BMSCs towards osteoblasts and adipocytes. Expression of canonical pathway factors (including such as Wnts, TCF, LRP5, and β-catenin) can promote differentiation of BMSCs increasingly to osteoblasts by increasing expression of Runx2, collagen and osteocalcin mRNA (30). At the same time, activation of the Wnt canonical pathway can increase the expression of LRP5 which can induce inhibition of the adipogenic differentiation of BMSCs by down-regulating expression of PPAR-γ (31).

Given that the Wnt signalling pathway is essential for osteogenic differentiation of BMSCs (32), we investigated the possible involvement of the Wnt pathway in osteoblastic changes and in adipogenic differentiation potential of the ovariectomized rat BMSCs. β-catenin is a critical signalling molecule that plays an important role in Wnt/β-catenin signalling pathways. To some extent, the levels of expression of β-catenin represent the functional status of the Wnt/β-catenin signalling pathway (29). Thus, we examined whether or not β-catenin was changed in OVX BMSCs. According to the results from Western blotting, β-catenin was found to have been decreased in OVX BMSCs. These results indicated that Wnt/beta-catenin signalling pathways were suppressed in OVX BMSCs, and the result may also suggest that the Wnt canonical signalling pathway is involved in the regulation of the osteogenic and adipogenic processes of OVX BMSCs.

In conclusion, our experimental results indicated that the BMSCs from postmenopausal osteoporosis model rat had a stronger adipogenic differentiation potential and osteogenesis potential which had been weakened at the same time in vitro. However, results of the present study are not consistent with the increased bone turnover caused by a result of oestrogen-deficiency observed in vivo. This could relate to a feedback mechanism associated with rapid bone loss after estrogen withdrawal in vivo, while there was no such feedback mechanism in vitro. Meanwhile we preliminarily speculate that Wnt/β-catenin signalling pathways may be involved in these processes. Nonetheless, the specific mechanism of WNT canonical signalling pathways regulating osteogenic and adipocyte differentiation of BMSCs remains unclear and more experimentation is needed. In addition, reports have indicated that other signalling pathways are involved in the regulation of bone formation such as the MAPK pathway, BMP pathway, and Wnt noncanonical signalling pathways (33-35). Whether or not these signalling pathways also have an important influence or whether or not there is crosstalk among these signalling pathways in the imbalance of osteogenic and adipogenic differentiation is not entirely understood and further studies are needed.

Conflict of interest

The authors declare that they have no conflict of interest.

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