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Introduction

Bone remodeling is a dynamic process orchestrated by bone-forming osteoblasts and bone-resorbing osteoclasts (1). The bone formation process involves the activation of different signaling pathways, which modulate multiple cellular and molecular events during osteoblast proliferation, differentiation and mineralization stages (1). Genetic studies in both humans and mice have revealed that the Wnt/β-catenin signaling pathway is an important mechanism for stimulating osteoblasts function (2).

The Wnt/β-catenin signaling pathway is activated by the binding of Wnt proteins with the Frizzled (Fzd)/low-density lipoprotein receptor-related protein (Lrp) (5/6) receptor complex. The formation of this receptor complex induces the cytoplasmic accumulation of β-catenin molecules and their nuclear translocation for the interaction with the T-cell specific transcription factor (Tcf)/lymphoid-enhancer binding factor (Lef) transcription factors, as well as the induction of expression of target genes, such as Lef1, Tcf7, NKD inhibitor of WNT signaling pathway 2 and anxin 2 (AXIN2) (2,3). In addition, the induction of AXIN2, a negative regulator of the signaling pathway, has been proposed to act at the transcriptional level and may create a negative feedback loop to silence the Wnt/β-catenin signaling pathway (4).

Under basal conditions, when the Wnt pathway is inactive, β-catenin is phosphorylated by glycogen synthase kinase 3 (GSK3), which forms part of a complex integrated by AXIN2 and the protein of the colon APC regulator of WNT signaling pathway (APC) gene, to be subsequently degraded in the proteasome (5). Therefore, the intracellular levels of β-catenin are kept relatively low. However, when the Wnt pathway is activated by the binding of the Wnt ligands to the Fxd/Lrp5/6 receptor complex, the decomposition of the intracellular AXIN2-APC-GSK3 complex is activated, which results in the inhibition of the phosphorylation of β-catenin (5). The hypophosphorylated β-catenin accumulates in the cytoplasm and translocates to the nucleus, where it regulates gene expression via the activation of various transcription factors, such as Tcf/Lef1(6). The Wnt/β-catenin signaling pathway is also regulated by several different extracellular antagonists, such as the family of secreted Fzd-related proteins 1-4 (SFRPs 1-4), the four members of the Dickkopf family (DKK 1-4) and the inhibitor Wnt inhibitory factor 1 (WIF1) (7). These molecules can act as decoys to compete with the Wnt ligands to bind with the receptors; for example, some SFRPs are secreted into the extracellular medium. Furthermore, members within the DKK protein family, in which type 1 (DKK-1) is particularly important in bone, can antagonize Wnt signals by binding with the Lrp5/6 co-receptors (4).

The roles of the Wnt signaling-related antagonists and their effect on bone metabolism and osteoblasts activities have not yet been fully elucidated. However, previous research suggests that they are involved in the regulation of osteoblast functions. For instance, studies in mice have reported that deletion of SFRP1 or elimination of one allele of DKK1, stimulates osteoblast proliferation and bone mass formation via the activation of the Wnt signaling pathway (8,9). Another study revealed that administration of recombinant SFRP2 or SFRP4 proteins enhanced the alkaline phosphatase (ALP) activity in mouse mesenchymal C3H10T1/2 cells, suggesting a role for SFRPs in osteoblastogenesis (9). Moreover, the overexpression of WIF1 in murine embryonic mesenchymal cells, inhibits osteoblast differentiation (10), and knockdown of DKK1 and DKK2 decreases matrix mineralization in KS483 mesenchymal stem cells (11). Similarly, clinical studies have observed an association between genetic variants in SFRP1 and SFRP4 and bone mass content in postmenopausal women (12,13). Taken together, these findings indicate that the Wnt signaling-related extracellular antagonists influence the osteoblast maturation process and bone formation, and that they can serve as potential targets to prevent the loss of bone mass. However, the expression levels of Wnt antagonists during the human osteoblast proliferation and differentiation stages remain unknown.

The aim of the present study was to investigate the dynamics of gene expression of extracellular antagonists, SFRP 1-4, DKK 1-4 and WIF1, during the proliferation and cell-differentiation stages of osteoblasts maturation. The hFOB 1.19 normal osteoblasts and the MG-63 and Saos-2 osteosarcoma cell lines were used as a model system. ALP activity and the expression levels of osterix (OSX) and RUNX family transcription factor 2 (RUNX2), which are markers of the early stage of osteoblast differentiation, were measured (14). In addition, AXIN2 expression was investigated to determine the activation status of the Wnt/β-catenin signaling pathway during the transition of osteoblasts, from proliferation to differentiation stage.

Materials and methods

Cell culture

The hFOB 1.19 (cat. no. CRL-11372), MG-63 (cat. no. CRL-1427) and Saos-2 (cat. no. HTB-85) cell lines were purchased from the American Type Culture Collection. hFOB 1.19 cells were maintained in DMEM/F-12 culture medium without phenol red (Sigma-Aldrich; Merck KGaA) and supplemented with 10% FBS (Biowest) and 0.3 mg/ml G418 (Sigma-Aldrich; Merck KGaA) at 37˚C and 5% CO2. MG-63 and Saos-2 cells were maintained in EMEM and McCoy's 5A (both Sigma-Aldrich; Merck KGaA), respectively, supplemented with 10% FBS (Biowest) and an antibiotic solution (penicillin and streptomycin, both 100 mg/ml; Gibco; Thermo Fisher Scientific, Inc.) at 37˚C and 5% CO2.

Cell viability and ALP activity assays

Cell viability and ALP activity were assessed in the hFOB 1.19, MG-63 and Saos-2 cell lines at 1, 3, 8, 15 and 21 days of cell culture. Cells were plated at a density of 70x104 cells/dish in their respective medium and cultured at 37˚C in a humidified incubator with 5% CO2 for the aforementioned time points. The culture media were removed and replaced with fresh medium every other day. The media were replaced with osteogenic media (100 µg/ml ascorbic acid and 5 mM β-glycerol phosphate) to maintain the osteoblast phenotype from day 8 onwards. Previous studies have reported that the decline in viability, which occurs after 8 days of cell culture, is essential to stimulate the osteoblast differentiation-related activities (15). Subsequently, the medium for hFOB 1.19 cells was replaced with culture medium supplemented with 0.01 µM menadione, 100 µg/ml ascorbic acid and 5 mM β-glycerol phosphate (16,17) (all Sigma-Aldrich; Merck KGaA). The medium for the MG-63 and Saos-2 cell lines was replaced by their respective medium supplemented with 100 µg/ml ascorbic acid and 5 mM β-glycerol phosphate (all Sigma-Aldrich; Merck KGaA).

In order to test the cell viability, the cells were harvested at the indicated times using a 0.05% trypsin/EDTA solution (GIBCO) for 2-3 min at 37˚C to detach the cells from the adherent substrate, the cell suspension was washed twice and resuspended in 1 ml of cold phosphate buffer saline (PBS) (4˚C). One part of the cell suspension was mixed with one part of 0.4% trypan blue, incubated for 3 min at room temperature and then visually examined to determine whether cells take up or exclude the dye, for direct identification and enumeration of live (unstained) and dead (blue) cells in a given population. and counted on a light inverted microscope using the 20x objective. The experiments were performed in triplicate and the cell viability was expressed as the cell number.

To determine the ALP activity, at the aforementioned time points, the cells were lysed with a solution containing 0.1 M Tris-HCl and 0.1% Tween-20 (pH 7.5) for 2 min on ice. Cell lysates were freeze-thawed (-70˚C/ice) twice and enzyme activity was determined using the Lowry method (18) using p-nitrophenyl phosphate (Sigma-Aldrich; Merck KGaA) as the substrate. Protein concentration was determined using the Bradford method, using the Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Inc.) with dilution from 0.2 to 0.9 mg/ml of BSA for the standard curve, following the manufacturer's protocol. All the experiments were performed three times in triplicate and results are expressed as enzymatic activity U/mg protein/min.

Gene expression studies

At the end of each incubation time point (1, 3, 8, 15 and 21 days of cell culture), the expression levels of RUNX2, OSX and AXIN2, as well as those of the extracellular antagonists were determined using reverse transcription-quantitative PCR (RT-qPCR). Total RNA was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to manufacturer's instructions. cDNA was transcribed from 1 µg total RNA using the TaqMan® Reverse Transcription Reagents kit (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions under the following conditions: 5 min at 65˚C, 2 min at 4˚C, 30 min at 37˚C, and 5 min, 95˚C. qPCR was performed using the following TaqMan gene expression assays (Applied Biosystems; Thermo Fisher Scientific, Inc.): RUNX2 (assay ID, Hs00231692_m1), OSX (assay ID, Hs01866874_s1) SFRP1 (assay ID, Hs00610060_m1), SFRP2 (assay ID, Hs00293258_m1), SFRP3 (assay ID, Hs0017350_m1), SFRP4 (assay ID, Hs00180066_m1), DKK1 (assay ID, Hs00183740_m1), DKK2 (assay ID, Hs00205294_m1), DKK3 (assay ID, Hs00183740_m1), DKK4 (assay ID, Hs00205290_m1), WIF1 (assay ID, Hs00183662_m1) and AXIN2 (assay ID, Hs00610344_m1) under universal cycling conditions (10 min at 95˚C; 15 sec at 95˚C, 1 min 60˚C, 40 cycles). PCR amplification was performed in triplicate using a QuantStudio™ 7 Flex Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Gene expression was normalized using GAPDH, and relative expression was calculated using the 2-ΔΔCq method (19).

Statistical analysis

Normal distribution was analyzed using the Shapiro-Wilk test and homogeneity of variance was determined using the Levene's test. Statistical differences were examined using a one-way ANOVA followed by Tukey's post hoc test. Correlations were evaluated using the Spearman correlation coefficient (rs). P<0.05 was considered to indicate a statistically significant difference. All analyses were performed using SPSS v20.0 software (IBM Corp). All assays were performed in triplicate.

Results

Cell viability and differentiation

To distinguish between the osteoblast proliferation and differentiation stages, the viability of the hFOB 1.19, MG-63 and Saos-2 osteoblastic cell lines during 21 days of cell culture was determined. The three cell lines exhibited distinct viability rates (Fig. 1A; Tables SI and SII). As expected, the Saos-2 cell line exhibited the highest viability rate (25.4-fold), followed by MG-63 (17.5-fold), while the hFOB 1.19 cell line had the lowest rate (13.5-fold). After day 8, the densities of the three osteoblastic cell lines increased significantly as compared with the respective day 1, in which the hFOB 1.19 and MG-63 cells reached saturation densities after 15 days of culture. It was observed that the cellular proliferation between day 15 and 21 was not significant.

Figure 1 Profiles of osteoblast markers of proliferation (evaluated as cell viability) and differentiation in normal hFOB 1.19 osteoblasts and MG-63 and Saos-2 osteosarcoma cell lines. (A) Cell number, (B) ALP activity and the expression levels of (C) RUNX2 and (D) OSX were determined at different time points during 21 days of cell culture. Data are presented as the mean ± SD of three experiments, in triplicate. *P<0.001 vs. respective day 1 of culture. RUNX2, RUNX family transcription factor 2; OSX, osterix; ALP, alkaline phosphatase.

To identify the osteoblast differentiation stage, ALP activity and the expression levels of the transcription factors RUNX2 and OSX, which are three well-known markers of osteoblast differentiation (20,21), were assessed during 21 days of cell culture. The ALP activity at the different time points is presented in Fig. 1B. It was found that the levels of ALP activity in the hFOB1.19, MG-63 and Saos-2 cell lines were correlated with their corresponding cells in terms of viability (Tables SI and SII). The highest level of ALP activity, expressed as units of enzyme activity, was found in the Saos-2 cell line since early days of culture and was maintained until day 21. Notably, the ALP activity level was similar in the hFOB 1.19 and MG-63 cell lines during 21 days of culture, in which the enzymatic activity increased in a time-dependent manner, reaching maximal levels at day 15 of cell culture compared to the respective day 1. However, the enzyme activity in Saos-2 cells was much higher compared with that other two cell lines, which had lower viability (Fig. 1B).

The expression levels of RUNX2 and OSX in the hFOB 1.19, MG-63 and Saos-2 cells at days 1, 3, 8, 15 and 21 of cell culture are illustrated in Fig. 1C and D, respectively. The expression profiles of RUNX2 in MG-63 and Saos-2 cells demonstrated a strong positive correlation between both cell lines, and the expression levels increased significantly in a time-dependent manner during the 21 days of culture, when compared with the day 1 of culture. By contrast, there was no significant increase in the expression levels of RUNX2 during the same days of culture in the hFOB 1.19 cell line (Fig. 1C). With respect to OSX, similar expression profiles were observed in the three cell lines (Table SIII).

High expression levels of OSX were observed at day 15 and reached maximum levels at day 21 in the three osteoblastic cell lines (Fig. 1D). These results indicated that the differentiation stage in osteoblast-like cell lines can be established from day 15 under the conditions used in the present study. The potential relationship existing between osteoblast markers is presented in Table SI.

Gene expression levels of Wnt-related antagonists

SFRPs genes

The expression levels of SFRP1, SFRP2, SFRP3 and SFRP4 in the hFOB 1.19, MG-63 and Saos-2 cell lines, during 21 days of cell culture are shown in Fig. 2. The expression levels of SFRP1 and SFRP3 were increased from day 3 until day 21 in the hFOB 1.19 cell line, compared with those on day 1. By contrast, the expression levels of SFRP2 and SFRP4 genes decreased from day 3 until day 21, compared with those on day 1 (Fig. 2A).

Figure 2 Expression profiles of SFRP1, SFRP2, SFRP3 and SFRP4 in the three osteoblast-like cell lines. (A) hFOB 1.19, (B) MG-63 and (C) Saos-2 cell lines were cultured for 21 days. Data are presented as the mean ± SD of three experiments, in triplicate. *P<0.05 vs. cells incubated at day 1. SFRP, secreted Frizzled-related protein.

In the MG-63 cell line, there was an increase in the expression of SFRP1 from day 3 until day 21, while high expression levels of SFRP2, SFRP3 and SFRP4 were detected after day 15 of cell culture, reaching maximum levels at day 21 (Fig. 2B). The Saos-2 cell line had higher expression levels of SFRP2, SFRP3 and SFRP4 compared with those in the hFOB 1.19 and MG-63 cell lines (Fig. 2C). These high levels were detected from day 15 and the maximum levels were detected at day 21. Notably, there were no significant differences in the expression levels of SFRP1 during the 21 days of culture of Saos-2 cells (Fig. 2C).

DKK genes

The expression levels of DKK1, DKK2, DKK3 and DKK4 in the hFOB 1.19, MG-63 and Saos-2 cell lines at days 1, 3, 8, 15 and 21 of cell culture are presented in Fig. 3. The hFOB 1.19 cell line demonstrated an increase in the expression of DKK1 after 3 days of culture, after which the expression decreased to levels below the basal line to day 21. The expression of DKK4 decreased at day 8 of cell culture compared with that on day 1. At day 15, there was a statistically significant increase in the expression of DKK4. With respect to DKK2, there was a decrease in its expression level during the 21 days of culture compared with that on day 1. There were no significant changes in DKK3 expression during the 21 days of cell culture (Fig. 3A).

Figure 3 Expression profiles of DKK1, DKK2, DKK3 and DKK4 in three osteoblastic cell lines. Expression profiles in (A) hFOB 1.19, (B) MG-63 and (C) Saos-2 cell lines. Data are presented as the mean ± SD of three experiments, in triplicate. *P<0.001 vs. respective day 1 of culture. DKK, Dickkopf.

In the MG-63 cell line, the expression levels of DKK2 and DKK3 increased in a time-dependent manner, reaching maximum levels at day 21 of cell culture. On the other hand, the expression levels of DKK1 and DKK4 were decreased below their basal levels during the 21 days of cell culture, as compared with day 1 (Fig. 3B). By contrast, there was high expression of DKK1 on day 3 in the Saos-2 cell line, which gradually decreased to levels below its basal level at day 21 of culture. Furthermore, the expression of DKK2 increased, reaching maximum levels on day 21, and there were no differences in the expression levels of DKK3 and DKK4 during 21 days of cell culture. The expression of DKK4 was not observed on day 21 (Fig. 3C).

WIF1 gene. WIF1

is an important negative regulator factor of the Wnt/β-catenin signaling pathway, and is structurally different from the SFRP and DKK families (22). WIF1 inhibits the activity of the Wnt signaling pathway by directly binding to Wnt proteins (22). Moreover, it is well-known that WIF1 can act as a tumor suppressor and its downregulation is associated with the development of various types of cancer (23). The expression of WIF1 in the hFOB 1.19, MG-63 and Saos-2 cell lines during the 21 days of culture is presented in Fig. 4. There was an increment on day 3, which remained constant until day 21 in the hFOB 1.19 cell line, while in Saos-2 cells, higher levels of WIF1 were observed from day 15 and reached maximum levels on day 21 when compared with day 1. By contrast, there were no changes in the expression of WIF1 in the MG-63 cell line.

Figure 4 Expression of WIF1 in hFOB 1.19, MG-63 and Saos-2 cell lines during 21 days of cell culture. Data are presented as the mean ± SD of three experiments, in triplicate. *P<0.05 vs. cells incubated at day 1. WIF1, Wnt inhibitory factor 1.

AXIN2 gene

AXIN2 is a negative intracellular regulator of the Wnt signaling pathway, which forms a complex with APC and GSK3, and results in the inhibition of the phosphorylation of β-catenin (5). The expression of AXIN2 in the hFOB 1.19, MG-63 and Saos-2 cell lines during days 1, 3, 8, 15 and 21 of cell culture is illustrated in Fig. 5. The three cell lines had a similar expression profile of AXIN2 (Table SIII). The results identified that AXIN2 was constantly expressed during the proliferative stage and there was a significant increase on day 15, which reached maximum levels on day 21 of culture in all three osteoblastic cell lines, as compared with the respective day 1. The relationship between AXIN2 and osteoblastic markers is presented in Tables SI and SII. A positive correlation was observed between the AXIN2 expression profile and cell markers of osteoblast proliferation (cell viability) and differentiation stages in hFOB 1.19, MG-63 and Saos-2 cell lines. On the other hand, a strong correlation between the mean of AXIN2 gene expression and differentially expressed extracellular Wnt antagonists during differentiation stage in the three cell lines was observed (Tables SIV-SVI).

Figure 5 Expression of AXIN2 in the hFOB 1.19, MG-63 and Saos-2 cell lines during 21 days of cell culture. Data are presented as the mean ± SD of three experiments in triplicate. *P<0.001 vs. cells incubated at day 1. AXIN2, axin 2.

Discussion

The results from the present study provide evidence of the differential expression of certain bone-related Wnt antagonists, during the proliferation (evaluated as cell viability) or differentiation stages in human osteoblast-like cells. The roles of these Wnt antagonists in the control of Wnt signaling have been only partially described, which is due to the complexity of their functions and the manner in which they have been investigated (4).

In the present study, to evaluate the dynamics of gene expression of the Wnt antagonists in the human osteoblasts, the transition from proliferation to differentiation stage was firstly determined in the hFOB 1.19, MG-63 and Saos-2 cells lines. The differentiation stage was defined by high confluency in cell culture, increased ALP activity and high expression levels of RUNX2 and OSX (20,21). The results demonstrated that arrest of cellular proliferation, judged by cell confluency at day 15 of cell culture, was associated with high levels of ALP activity and the expression of RUNX2 and OSX. The present results support the findings observed in previous studies by Stein et al (24) and Owen et al (25), in which high mRNA and protein expression levels of ALP, as well as no detectable expression of RUNX2 and OSX, were identified before day 12 of cell culture in rodent osteoblasts. Furthermore, in another study, high levels of ALP activity and osteocalcin, a differentiation marker, were observed at day 15 of cell culture in a neonatal rat calvarial osteoblasts model (26). Based on the present results, it was suggested that day 15 of cell culture was the beginning of the differentiation stage in the three osteoblast-like cell lines. To determine the expression levels of Wnt antagonists during the early differentiation stage, the present study was limited to days 15-21 of cell culture, when ALP reached maximum levels of activity. Previous studies have reported a significant decrease in enzyme activity when cell cultures progress into the mineralization stage (after day 25 of cell culture) (24,25). However, the association between the Wnt-pathway antagonists and the mineralization process was beyond the scope of the present research.

To characterize the expression profiles of the Wnt antagonists in the three osteoblast-like cell lines during the proliferation and differentiation stages, the cells were cultured for 21 days. Distinctive expression patterns were identified during both the proliferation and differentiation stages for each cell line. The differential expression patterns of Wnt antagonists suggested there was a possible balance between the temporal and spatial expression of Wnt-pathway antagonists during the progression of proliferative stage towards the differentiation of the osteoblasts (27,28). The present results demonstrated there was an overlap between SFPR2, SFRP3, SFRP4 and DKK2 gene expression levels in MG-63 and Saos-2 cell lines. The analysis of their expression patterns identified high levels on days 15 and 21 of cell culture, suggesting that these antagonists were upregulated during the differentiation stage in osteosarcoma cell lines. In addition, high expression of DKK3 in MG-63, but not in Saos-2 cells, was observed during the differentiation stage. On the other hand, in the hFOB1.19 cells, high expression of DKK4 was found during the differentiation stage (day 15). Several studies in murine osteoblasts cells have reported that the expression levels of SFRP2, SFRP4 (9,29) and SFRP3, activated by the β-catenin-independent pathway (30), can promote osteoblast differentiation by decreasing cell proliferation and inducing ALP activity. It has also been revealed that increased expression levels of DKK2 (31) or WIF1 (32) in murine osteoblasts and Saos-2 cells, promote in vitro mineralization. Moreover, DKK3, in Saos-2 and mesenchymal cells (33,34) and DKK4 in MC3T3-E1 cells (35), can increase cell proliferation and decrease or inhibit osteogenic differentiation.

WIF1 is a negative regulator, which acts upstream of the Wnt signaling pathway, and can inhibit the activation of the pathway by directly binding with the Wnt signaling proteins (22). WIF1 has also been found to act as a tumor suppressor protein (23). The current expression analysis of WIF1 identified that the MG-63 and Saos-2 cell lines exhibited low levels during the proliferative stage, and these were constantly low during the differentiation stage in the MG-63 cells, but not in Saos-2 cells. In the Saos-2 cell line, WIF1 was upregulated in a time-dependent manner from days 15 to 21. In normal hFOB1.19 cells, the expression of WIF1 was constant in both the proliferation and differentiation stages, but its levels were higher compared with those in MG-63 cells. These findings suggested that the activation of the Wnt/β-catenin signaling pathway may be associated with cell proliferation. A recent study reported an association between the decreased mRNA and protein expression levels of WIF1 and the increased levels of β-catenin and cyclin D1 expression in tumor tissues, compared with that in healthy tissues (36).

AXIN2 is a known intracellular negative regulator of the Wnt/β-catenin signaling pathway, which acts by preventing spontaneous signal transduction in the absence of a Wnt signal (5). At the same time, AXIN2 expression is repressed by the activation of the Wnt/β-catenin signaling pathway, which creates a negative feedback loop between the two (6). Several studies have revealed that high expression of AXIN2 is associated with the inhibition of the Wnt/β-catenin signaling pathway (6,37). The results from the present study demonstrated that high mRNA expression levels of AXIN2 during differentiation stage was associated with the overexpression of the Wnt antagonists. The expression levels of SFRP2, SFRP3, SFRP4 and DKK2 were upregulated in the Saos-2 and MG-63 cell lines, DKK3 was upregulated in the MG-63 cell line and WIF1 was upregulated in the Saos-2 cell line, while DKK4 was upregulated only in the hFOB 1.19 cell line. These results indicated that extracellular and intracellular antagonists could modulate the Wnt/β-catenin signaling pathway to decrease cell proliferation and promote osteoblast differentiation (37). However, additional studies are required to further assess this hypothesis.

The number of upregulated Wnt antagonists during cell proliferation is limited (17). The present study identified that DKK1 was the only gene that had a high expression in the proliferation stage in the hFOB 1.19 and Saos-2 cell lines. However, the underlying mechanism of this high expression is currently unknown. A previous study revealed that overexpression of DKK1 in the MG-63 and Saos-2 cell lines decreased the lag time prior to rapid exponential growth during cell proliferation (38).

The present results suggested that majority of the Wnt antagonists were downregulated during proliferation and/or differentiation stages; for instance, SFRP2, SFRP4, DKK1 and DKK2 in the hFOB 1.19 osteoblasts, DKK1 and DKK4 in the MG-63 cells and SFRP1 and DKK1 in the Saos-2 cells. The mechanism underlying the observed gene repression of the Wnt antagonist was not determined; however, recent studies have proposed a role for the small non-coding RNAs in the control of these genes (39,40). For example, overexpression of microRNA(miR)-29 modulates the intracellular mRNAs expression levels of DKK1 and SFRP2, thus promoting the differentiation of human osteoblasts (17). Furthermore, it has been shown that the high expression of miR-940 activates the Wnt/β-catenin signaling pathway by downregulating SFRP1 in human osteosarcoma tissue (41). Thus, the expression profile of these small non-coding RNAs in the progression of proliferation and differentiation in osteoblasts and osteosarcoma cell lines requires further research.

To the best of our knowledge, the current study provides for the first time, the distinctive and characteristic expression patterns of several Wnt antagonists during the proliferation and differentiation stages of human osteoblast cell lines (Fig. 6). However, the association between the gene expression levels and the protein levels, as well as their functional roles during both stages are still require further investigation. Moreover, other technologies, such as next-generation sequencing (seq), including RNA-seq and small-RNAseq, could be used to analyze additional aspects of RNA biology to identify the changes in the expression levels of the Wnt antagonists.

Figure 6 Schematic diagram of the effects of extracellular antagonists in the Wnt/β-cat signaling pathway during the proliferation and differentiation in human osteoblast. Activation of the Wnt signaling induces the expression of genes that promote cell proliferation. In the transition to differentiation, the expression of extracellular antagonists decreases Wnt signaling and promotes the expression of differentiation markers. During the differentiation, the upregulation of SFRP2, SFRP3, SFRP4 and DKK2 is observed in the Saos-2 and MG-63 cell lines. DKK3 and WIF1 are upregulated in MG-63 and Saos-2 respectively, while DKK4 is uniquely upregulated in the hFOB1.19 cell line. AXIN2, anxin 2; WIF1, Wnt inhibitory factor 1; DKK, Dickkopf; SFRP, secreted Frizzled-related protein; Fzd, Frizzled; LRP, Low-density lipoprotein receptor-related protein; GSK3, glycogen synthase kinase 3; CK1, casein kinase 1; β-cat, β-catenin; Tcf, T-cell specific transcription factor; Lef, lymphoid-enhancer binding factor; Dvl, Dishevelled.

In conclusion, the present results provide novel insights into the expression levels of Wnt antagonists during proliferation and differentiation stages in human osteoblast-like cell lines. In addition, the results offer a basis to evaluate novel potential targets for bone-related Wnt-signaling modulation and provide an additional area of research into Wnt-signaling in bone metabolism.

Supplementary Material

Table SI. Positive correlation between the expression levels of osteoblast markers in hFOB 1.19 cells.

Table SII. Positive correlation between the expression levels of osteoblast markers in the osteosarcoma cell lines MG-63 and Saos-2.

Table SIII. Correlation between the expression levels of osteoblast markers between hFOB 1.19, MG-63 and Saos-2 cell lines.

Table SIV. Correlation between the mean of AXIN2 gene expression and differentially expressed extracellular Wnt antagonists in hFOB 1.19 cells.

Table SV. Correlation between the mean of AXIN2 gene expression and differentially expressed extracellular Wnt antagonists in MG-63 cells.

Table SVI. Correlation between the mean of AXIN2 gene expression and differentially expressed extracellular Wnt antagonists in Saos-2 cells.

Acknowledgements

The authors would like to thank Mr. José Luis Cruz-Colín (National Institute of Genomic Medicine, INMEGEN) for his technical assistance with the culturing of cells.

Funding

The present study was partially supported by grants from Consejo Nacional de Ciencia y Tecnología (grant no. INFR-2016-01-270405) and the Instituto Nacional de Medicina Genómica (grant no. 266-17/2016/I).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

AYPT and RVC conceived and designed the study. JE, EGRS, RFJO and NP carried out the experiments, acquisition of data, analysis and interpretation of data. LMTE and MDJCL contributed to the statistical analysis and interpretation of data. AYPT, JE, EGRS, MDJCL and RVC drafted, reviewed and edited the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Authors' information

Miss Alma Y. Parra-Torres is a doctoral student from Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM) and received fellowship 421295 from CONACYT.

References