Effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast differentiation via the WNT1/β-catenin signaling pathway

Background WNT1 c.110 T>C and c.505G>T missense mutations have been identified in patients with osteogenesis imperfecta (OI). Whether these mutations affect osteoblast differentiation remains to be determined. This study aimed to investigate the effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast function, gene expression, and pathways involved in OI. Methods Empty vector (negative control), wild-type WNT1, WNT1 c.110 T>C, WNT1 c.505G>T, and WNT1 c.884C>A (positive control) mutant plasmids were constructed and transfected into preosteoblast (MC3T3-E1) cells to investigate their effect on osteoblast differentiation. The expressions of osteoblast markers, including BMP2, RANKL, osteocalcin, and alkaline phosphatase (ALP), were determined using quantitative real-time polymerase chain reaction (RT-qPCR), western blotting (WB), enzyme-linked immunosorbent assay, and ALP staining assay, respectively. The mRNA and protein expression levels of WNT1 or the expression levels of the relevant proteins involved in the WNT1/β-catenin signaling pathway were also determined using RT-qPCR, WB, and immunofluorescence (IF) assays after the different plasmids were transfected into MC3T3-E1 cells. Results Compared with those in the wild-type group, in the mutation groups, the mRNA and protein expression levels of BMP2 were suppressed, the expressions of osteocalcin and ALP were inhibited, and the mRNA and protein expression levels of RANKL were enhanced in MC3T3-E1 cells. WB and IF assays revealed that the protein expression levels of WNT1 in MC3T3-E1 cells were downregulated in the mutation groups compared with those in the wild-type WNT1 group. Furthermore, the expression levels of nonphosphorylated β-catenin (non-p-β-catenin) and phosphorylated GSK-3β (p-GSK-3β) were downregulated in the mutation groups compared with those in the wild-type group. However, no significant changes in the expression level of non-p-β-catenin or p-GSK-3β were observed in the mutation groups. Conclusions WNT1 c.110 T>C and c.505G>T mutations may alter the proliferation and osteogenic phenotype of MC3T3-E1 linked to the progression of OI via the inhibition of the WNT1/β-catenin signaling pathway. This is the first study to confirm the effect of WNT1 c.110 T>C and c.505G>T missense mutations on osteoblast differentiation and propose a new molecular mechanism for OI development.

WNT1, a member of the WNT protein family, plays a vital role in regulating bone mass and maintains the homeostasis of bone metabolism. In vitro experiments have shown that WNT1 defects can result in significantly decreased bone formation, whereas WNT1 overexpression can promote bone formation [9]. In OI, studies have shown that biallelic mutations in WNT1 result in recessive OI, whereas heterozygous mutations in WNT1 are associated with early-onset osteoporosis in dominant hereditary families [10,11]. More than 27 diseasecausing WNT1 mutations have been discovered thus far [12]. However, the biological functions in most of the WNT1 mutations have not yet been elucidated.
The canonical WNT1 pathway initiates a signaling cascade by binding to the Frizzled and LRP5 receptors on the cell surface, resulting in the accumulation of nonphosphorylated β-catenin (non-p-β-catenin) in cells, and functions as a transcription factor to stimulate the transcription of the downstream target genes of the WNT signaling pathway [13]. A study has shown that WNT1 mutations can affect the activation of the canonical WNT pathway and the mineralization of osteoblasts [11]. Another study has also shown that a missense mutation in exon 3 (c.505G>T) of WNT1 resulted in the substitution of glycine by cysteine at position 169 (p.G169C); besides, the missense mutation in exon 2 (c.110G>T) of WNT1 resulted in the conversion of isoleucine to threonine at position 37 (p.I37T) in patients with OI [6]. However, whether the mechanism of the WNT1 c.110 T>C and c.505G>T mutations affect osteoblast differentiation remains to be determined.
For a better evaluation of the potential molecular mechanisms of WNT1 mutations, a nonsense mutation (c.884C>A) was set as a positive control. This mutation causes the truncation of the last 76 amino acids of WNT1, which has been confirmed by western blotting (WB). Hence, c.884C>A expression can be detected at a position below the molecular weight of the wild-type WNT1 [11]. Therefore, in this study, WNT1 c.110 T>C, WNT1 c.505G>T, and WNT1 c.884C>A (positive control) mutant plasmids were constructed and transfected into osteoblasts to investigate the effects of the mutations on cell viability, expression levels of osteoblast markers, and activation of the WNT1/β-catenin pathway in MC3T3-E1 cells. This study showed the effect of WNT1 c.110 T>C and c.505G>T mutations on osteoblast differentiation for the first time and proposed a new molecular mechanism for the development of OI.

Plasmid construction and transfection
Wild-type WNT1, WNT1 c.110 T>C, WNT1 c.505G>T, and WNT1 c.884C>A mutant plasmids were synthesized by General Biosystems, Inc (Anhui, China). Empty plasmids were used as the negative control, and c.884C>A was used as a positive control. The MC3T3-E1 cells were grown for 24 h in 6-well plates with an initial cell density of 5 × 10 5 cells/mL. Cells in each well were transfected with Vector, WNT1, c.110 T>C, c.505G>T, and c.884C>A using Lipofectamine 3000 (Invitrogen, CA, USA) according to the manufacturer's instructions for subsequent quantitative real-time polymerase chain reaction (RT-qPCR) and WB experiments. MC3T3-E1 cell suspensions were transfected with different plasmids using an osteogenic differentiation medium (#MUXMT-90021, Cyagen Biosciences, Guangzhou, China). Cells were cultured in the osteogenic differentiation medium and seeded in coverslips of 6-well plates at a density of 2 × 10 4 cells/mL, and then the plasmids were transfected into the MC3T3-E1 cells every 72 h for a total of 14 days. Samples were then collected for the enzyme-linked immunosorbent assay (ELISA) and alkaline phosphatase (ALP) staining assay. Three independent assays were performed.

ELISA and ALP activity assay
During the cultivation of MC3T3-E1 cells in the osteogenic differentiation medium for 14 days, the supernatants were collected for the ELISA experiments. The Mouse OT/BGP ELISA Kit (#CSB-E06917m) provided by Cusabio (Wuhan, China) was used. Briefly, cell culture media were added to 96-well plates, incubated with 100 μL of biotinylated antibodies for 60 min at room temperature, washed five times, incubated with 100 μL of HRP-conjugated streptavidin for 20 min at room temperature in the dark, incubated with 3,3′,5,5′-tetramethylbenzidine solution for 20 min, and incubated with the termination solution. The relative expression levels were then determined by measuring the absorbance at 450 nm. Three independent assays were performed.
Similarly, during the cultivation of cells in the osteogenic differentiation medium for 14 days, the cells were also collected and examined. Cells were stained with ALP according to the instructions of the BCIP/NBT Alkaline Phosphatase Color Development Kit (#C3206, Beyotime Biotechnology, Jiangsu, China). The coverslips were removed, washed twice with phosphate buffer saline, and fixed with 95% ethanol for 8 min. Subsequently, the cells were air-dried and incubated with a substrate solution at 37°C for 30 min in the dark. After the reaction, the samples were washed with double-distilled water, counterstained with methyl green for 2 min, washed three times with double-distilled water, and airdried. Five sections per sample were randomly selected for analysis under a microscope (OPTEC, TP510, Chongqing, China). Three independent assays were performed.

Statistical analysis
GraphPad Prism 7 was used for visualization. One-way analysis of variance followed by Tukey's post hoc test for multiple comparisons was performed to compare differences between groups. P < 0.05 was considered to indicate a statistically significant difference.

WNT1 c.110 T>C and c.505G>T mutations affect MC3T3-E1 cell proliferation
We transfected empty vector, wild-type WNT1, WNT1 c.110 T>C, WNT1 c.505G>T, and WNT1 c.884C>A plasmids into MC3T3-E1 cells for 24 h individually, and then detected the expression levels of WNT1. The RT-qPCR results showed that the mRNA expression levels of WNT1 were upregulated in the mutation and wildtype groups compared with those in the empty vector (Vector) group and that the mRNA levels of WNT1 were downregulated in the WNT1 c.110 T>C and c.505G>T mutation groups compared with those in the wild-type WNT1 group, indicating that these plasmids were successfully transfected into the MC3T3-E1 cells and the WNT1 mutations in MC3T3-E1 cells were successfully established (Fig. 1A). To investigate the effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast proliferation, empty vector, wild-type WNT1, WNT1 c.110 T>C, WNT1 c.505G>T, and WNT1 c.884C>A plasmids were transfected into the MC3T3-E1 cells for 24 and 48 h, respectively, and the cell viability in each group was measured using the MTS assay. Cell viability was found to be increased in the wild-type WNT1 group compared with that in the Vector group. However, cell viability in the mutation groups was significantly lower than that in the wild-type WNT1 group at 24 and 48 h, suggesting that WNT1 mutations inhibited the viability of MC3T3-E1 cells (Fig. 1B).

WNT1 C.110 T>C and C.505G>T mutations affect the expression of osteoblast markers
For further investigation of the effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast characteristics, the expression levels of the osteoblast differentiation marker BMP2 and osteoclast differentiation marker RANKL were analyzed using RT-qPCR and WB in MC3T3-E1 cells transfected with empty vector, wildtype WNT1, WNT1 c.110 T>C, WNT1 c.505G>T, and WNT1 c.884C>A plasmids. Compared with the Vector group, the cells transfected with wild-type WNT1 plasmid had upregulated mRNA and protein expression levels of BMP2 and downregulated mRNA and protein expression levels of RANKL ( Fig. 2A-E). In addition, compared with the cells transfected with wild-type WNT1 plasmid, the WNT1 c.110 T>C, WNT1 c.505G>T, and WNT1 c.884C>A mutation groups showed downregulated mRNA and protein expression levels of BMP2 and upregulated mRNA and protein expression levels of RANKL ( Fig. 2A-E). The ELISA results showed that after induction for 14 days, the content of osteocalcin in the osteogenic differentiation medium in the wild-type WNT1 group was significantly higher than that in the Vector group and also higher than that in the mutation groups (Fig. 2F). The ALP staining results showed that the ALP content in the osteogenic differentiation medium in the wild-type WNT1 group was significantly higher than that in the Vector group and also higher than that in the mutation groups (Fig. 2G), suggesting that the c.110 T>C and c.505G>T mutation sites of WNT1 inhibited the expression of osteocalcin and ALP during osteogenic induction and affected the osteogenic differentiation of the cells. For further evaluation of WNT1 expression in the mutation and wild-type groups and the possible mechanism underlying the effects of WNT1 mutations on the differentiation of osteoblasts, MC3T3-E1 cells were transfected with WNT1 and c.110 T>C, c.505G>T, and c.884C>A plasmids. Next, the expression levels of proteins related to the WNT1/β-catenin signaling pathway were validated in transiently transfected cells. WB and IF assays revealed that wild-type WNT1 plasmids were able to upregulate WNT1 expression at the protein level in MC3T3-E1 cells compared with that in the Vector group, but mutation plasmids downregulated WNT1 expression at the protein level in MC3T3-E1 cells compared with that in the wildtype WNT1 group. The highest expression occurred in the wild-type cells (Figs. 3A, B; 4; and 5A). Compared with wild-type WNT1 cells, cells transfected with WNT1 c.110 T>C, WNT1 c.505G>T, and WNT1 c.884C>A mutant plasmids showed downregulated expressions of non-p-β-catenin and p-GSK-3β (Figs. 3C, D; 4; and 5B, C), demonstrating that WNT1 c.110 T>C and c.505G>T mutations might inhibit osteoblast proliferation and the expression of BMP2 by weakening the WNT1/β-catenin signaling pathway. Fig. 2 Effects of WNT1 mutation on the mRNA and protein expression levels of BMP2 (osteoblast marker) and RANKL (osteoclast marker) in the MC3T3-E1 cells. The mRNA expression levels of BMP2 (A) and RANKL (B) were determined using quantitative real-time PCR. C The protein expression levels of BMP2 and RANKL were determined using western blotting. D, E Semi-quantitative results of BMP2 and RANKL proteins, respectively, using Image-Pro software. F Osteocalcin expression levels in the osteogenic differentiation medium on day 14 in different groups by ELISA. G Alkaline phosphatase expression levels in different groups by ALP staining. Bars represented 1000 μm * P < 0.05 and ** P < 0.01 indicate significant differences

Discussion
In a previous study, four new heterozygous WNT1 mutations (c.110 T>C, c.505G>T, c.385G>A, and c.506G>A) were found to be associated with OI in four independent pedigree peripheral blood samples [6]. Of these, a missense mutation (WNT1 c.110 T>C) located in exon 2 caused the conversion of isoleucine to threonine. Further, a missense mutation in exon 3 (WNT1 c.505G>T) allowed the substitution of glycine by cysteine. However, currently, the physiological significance of these mutations remains unclear. Therefore, in the present study, we specifically analyzed the effects of WNT1 c.110G>T and c.505G>T mutations on osteoblast differentiation in MC3T3-E1 cells for the first time. The results revealed that WNT1 c.110 T>C and c.505G>T mutations had effects on osteoblast proliferation and the WNT1/β-catenin signaling pathway. Wild-type WNT1 could induce the expression of osteoblast differentiation markers such as BMP2, osteocalcin, and ALP; suppress the expression of the osteoclast differentiation marker RANKL; and activate the WNT1/βcatenin signaling pathway; however, these effects were considerably impaired in the presence of the WNT1 c.110 T>C and c.505G>T mutations.
In healthy humans, bone resorption of osteoclasts and bone formation of osteoblasts contribute to the maintenance of homeostasis. The disruption of the balance between bone resorption and bone formation of osteoblasts leads to the development of bone diseases such as osteoporosis, which is a critical feature in patients with OI [14,15]. Research has been shown that BMP2 and RANKL induce osteoblast and osteoclast differentiation, respectively [16][17][18][19][20], and that osteoblast cell viability in WNT1-deficient mice is reduced and associated with the fracture phenotype [21]. In this regard, our study found that WNT1 c.110G>T and c.505G>T mutations inhibited the cell viability, weakened the mRNA and protein expression levels of BMP2, and enhanced the mRNA and protein expression levels of RANKL, indicating that WNT1 c.110G>T and c.505G>T mutations can lead to osteoblast phenotype dysplasia, inhibit bone formation, and easily lead to an increased risk of osteoporosis and fracture. A study has also revealed that WNT1 mutations could lead to changes in the bone structure by deactivating the canonical WNT pathway [12]. Although some mutant forms have induced the activation of the WNT1 signaling pathway, the expressions of the downstream target genes of the WNT signaling pathways are dysregulated and osteoblast mineralization is impaired [11]. The canonical WNT/β-catenin signaling pathway is activated by a combination of WNT and the Frizzled/LRP5/6 complex, which mediates the activation of β-catenin and then activates downstream gene expression. Furthermore, a functional study of essential receptors in the WNT signaling pathway has revealed that loss-offunction mutations in LRP5 can lead to reduced bone formation and decreased bone mass [22]. However, when there is no extracellular WNT, GSK-3β adds a phosphate group to β-catenin, resulting in the hydrolysis of β-catenin, decreased expression of β-catenin in cells  Semi-quantitative results of IF for the WNT1, non-p-β-catenin, and p-GSK-3β protein expression levels in Fig. 4. * P < 0.05 and ** P < 0.01 indicate significant differences and, ultimately, inhibition of the WNT/β-catenin signaling pathway. When the WNT signal is present, GSK-3βmediated β-catenin hydrolysis is inhibited and downstream target genes of β-catenin can be activated [23].
Thus, the present study evaluated the effects of c.110G>T and c.505G>T mutations on WNT1 expression. The WB results revealed that the WNT1 c.110G>T and c.505G>T mutations decreased the levels of p-GSK-3β and non-p-β-catenin compared with that in the wildtype WNT1 group, suggesting that WNT1 c.110G>T and c.505G>T mutations are associated with decreased WNT1 activation and decreased activation of the canonical WNT1/β-catenin signaling pathway. However, the precise mechanisms require further study for better understanding.

Conclusion
This study is the first to confirm the effect of WNT1 c.110 T>C and c.505G>T mutations on osteoblast differentiation via the WNT1/β-catenin signaling pathway, and consequently, a possible cause of OI at the cellular level. These findings suggest a new molecular mechanism for OI development.