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. 2022 Jan 26;240(6):1152–1161. doi: 10.1111/joa.13612

ERα/β/DMP1 axis promotes trans‐differentiation of chondrocytes to bone cells through GSK‐3β/β‐catenin pathway

Xue Yan 1, Deng‐Yun Fan 1, Ya‐Lei Pi 1, Ya‐Nan Zhang 1, Peng‐Jiu Fu 1, Hui‐Feng Zhang 1,✉
PMCID: PMC9119614  PMID: 35081258

Abstract

Estrogen‐induced premature closing of the growth plate in the long bones is a major cause of short stature after premature puberty. Recent studies have found that chondrocytes can directly trans‐differentiate into osteoblasts in the process of endochondral bone formation, which indicates that cartilage formation and osteogenesis may be a continuous biological process. However, whether estrogen promotes the direct trans‐differentiation of chondrocytes into osteoblasts remains largely unknown. Chondrocytes were treated with different concentrations of 17β‐estradiol, and Alizarin Red staining and alkaline phosphatase activity assay were used to detected osteogenesis. Specific short hairpin RNA and tamoxifen were used to block the estrogen receptor (ER) pathway and osteogenic marker genes and downstream gene expression were detected using real‐time quantitative polymerase chain reaction, western blot, and immunohistochemistry staining. The findings showed that 17β‐estradiol promoted the chondrocyte osteogenesis in vitro, even at high concentrations. In addition, blocking of the ERα/β pathway inhibited the trans‐differentiation of chondrocytes into osteogenic cells. Furthermore, we found that dentin matrix protein 1 (DMP1), which is a direct downstream molecular of ER, was involved in 17β‐estradiol/ER pathway‐regulated osteogenesis. As well, glycogen synthase kinase‐3 beta (GSK‐3β)/β‐catenin signal pathway also participates in ERα/β/DMP1‐regulated chondrocyte osteogenesis. The GSK‐3β/β‐catenin signal pathway was involved in ERα/β/DMP1‐regulated chondrocyte osteogenesis. These findings suggest that ER/DMP1/GSK‐3β/β‐catenin plays a vital role in estrogen regulation of chondrocyte osteogenesis and provide a therapeutic target for short stature caused by epiphyseal fusion.

Keywords: chondrocytes, epiphyseal fusion, estrogen, growth plate, osteoblasts, trans‐differentiation

Here, we demonstrate that 17β‐estradiol promotes the chondrocyte osteogenesis in vitro and obstruction of the estrogen receptor (ER)α/β pathway inhibits the trans‐differentiation of chondrocytes into osteogenic cells. Dentin matrix protein 1 (DMP1), which is a direct downstream molecular of ER, was involved in 17β‐estradiol/ER pathway‐regulated osteogenesis. The glycogen synthase kinase‐3 beta (GSK‐3β)/β‐catenin signal pathway also participates in osteogenesis regulated by ERα/β/DMP1 chondrocytes.

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1. INTRODUCTION

Endochondral bone formation on the epiphyseal growth plate plays a key role in bone development (Matsushita et al., 2020). This is a complex process that includes cartilage formation and the replacement of cartilage and bone tissue (Debnath et al., 2018). This process consists of a complex network of nutritional, cellular, paracrine and endocrine factors (Lui et al., 2014). In the growth spurt period of puberty, the proliferation and differentiation of chondrocytes, the secretion of extracellular matrix, the calcification of hypertrophy, the invasion and differentiation of osteoblasts, and the formation of blood vessels are repeated in the growth plate, which promotes the elongation of long bones and leads to the increase of height (Shim, 2015). However, the premature differentiation of chondrocytes may interfere with or hinder this repetitive process, leading to growth restriction (Nilsson et al., 2005). After the fusion of the epiphyzes, the growth plate is closed in late puberty, which often results in a slowdown in growth rate or even a stop of growth (Murray & Clayton, 2013). Therefore, elucidating the detailed mechanisms of puberty growth and epiphyseal fusion may help to develop new strategies for the treatment of short stature.

Estrogen receptors (ERs) include two subtypes, ERα and ERβ, and estrogen works by binding to them (Eyster, 2016). Studies have shown that estrogen promotes puberty growth spurts by inducing the growth hormone‐IGF‐I axis during puberty (Felice et al., 2013; Shim, 2015). Both ERα and ERβ are expressed in the resting zone, proliferation zone, and hypertrophic zone of the growth plate and participate in the secretion of GH (Börjesson et al., 2013). Studies have shown that exhaustion of the growth plate chondrocyte proliferation capacity or premature osteogenic transformation and estrogen‐promoted growth plate aging will lead to epiphyseal fusion (Shim, 2015; Weise et al., 2001). A large number of studies have shown that the combination of high‐dose estrogen and ER promotes the transformation of a variety of cells, including chondrocytes, to bone formation (Gao et al., 2015; Jing et al., 2015; Shi et al., 2017; Wu et al., 2020). Low‐dose estrogen is important for pubertal growth spurts in early puberty, and the high‐dose estrogen in growth plate cartilage is essential for the fusion of the epiphyzes in late puberty (Börjesson et al., 2010). In a recent breakthrough study, it was reported that the direct trans‐differentiation of chondrocytes into osteoblasts usually occurs in the process of endochondral bone formation, which indicates that cartilage formation and osteogenesis may be a continuous biological process, rather than two independent processes (Jing et al., 2018). However, whether estrogen promotes the premature osteogenic transformation of chondrocytes leading to late puberty epiphyseal fusion remains to be elucidated.

Dentin matrix protein 1 (DMP1), a key bone matrix protein, plays a vital role in the maturation of osteoblasts and odontoblasts (Qin et al., 2007). Studies have found that, as a secreted protein, DMP1 is expressed in large amounts in long bone tissues but in lower amounts in the cartilage (Weng et al., 2017). The full‐length DMP1 protein is divided into two functional fragments, a 37‐kD N‐terminal and a 57‐kD C‐terminal under the action of protease (Sun et al., 2010). Inactivating mutations in DMP1 can cause autosomal recessive hypophosphatemic rickets, in which bone cells mainly overproduce FGF23, leading to renal phosphate depletion, rickets, and osteomalacia (Liu et al., 2019). By using tracing lines display, Li et al. (2020) found that there is a promotion of cell trans‐differentiation from chondrocytes into bone cells in Dmp1 KO mice. Although the effect of DMP1 on bone mineralization is known, the effect of DMP1 on estrogen‐regulated epiphyseal closure remains unclear.

Here, we demonstrate that 17β‐estradiol promotes the chondrocyte osteogenesis in vitro, even at high concentrations. Additionally, obstruction of the ERα/β pathway inhibited the trans‐differentiation of chondrocytes into osteogenic cells. Furthermore, we found that DMP1, which is a direct downstream molecular of ER, was involved in 17β‐estradiol/ER pathway‐regulated osteogenesis. As well, the glycogen synthase kinase‐3 beta (GSK‐3β)/β‐catenin signal pathway also participates in osteogenesis regulated by ERα/β/DMP1 chondrocytes. Our results suggest that blocking the ERα/β/DMP1 pathway may restrict chondrocyte osteogenesis and provide a therapeutic target for short stature caused by epiphyseal fusion.

2. MATERIALS AND METHODS

2.1. Cell lines and transfection

Human articular chondrocytes (CP‐H096) were purchased from Procell Life Science & Technology. Cells were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F‐12 supplemented with 10% fetal bovine serum (Clark Bio) and 1% penicillin/streptomycin (Solarbio). Cells were grown in a humidified atmosphere of 95% air and 5% CO2. The transfection was performed according to the manufacturer's protocols using Lipofectamine 2000 (Invitrogen).

2.2. Alkaline phosphatase activity assay

The alkaline phosphatase (ALP) activity assay was performed as previously described (Gao et al., 2015). In brief, cells were washed twice with phosphate‐buffered saline (PBS) and fixed with 10% formalin. After washing with PBS, the cells were stained with BCIP/NBT ALP color kit in the dark. The cells were then lysed with radio immunoprecipitation assay (RIPA) buffer and centrifuged at 10,000 g for 5 min. The ALP activity assay kit was used to measure ALP activity in the clear supernatant. The total protein concentration was determined by the Bradford protein assay, and ALP activity was normalized to total protein concentration.

2.3. Alizarin Red staining

Alizarin Red S staining was performed as previously described (Gao et al., 2015). Briefly, chondrocytes were treated with E2 and then incubated in the differentiation culture for the designated time. The cells were then fixed in ice‐cold 10% formalin for 20 min and stained with 40 mM Alizarin Red S (pH 4.4; Sigma Chemical) for 45 min at room temperature. To estimate matrix calcification, the stain was dissolved in 10% cetylpyridinium chloride by shaking for 15 min. The absorbance of the released Alizarin Red S was measured at 562 nm, and photographs were taken for later analysis.

2.4. Western blotting

Western blotting was performed as described previously (Yang et al., 2017, 2019). Cells were lysed in RIPA lysis buffer (50 mM Tris‐HCl, pH 7.5, 150 mM NaCl, 1% NP‐40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate [SDS]) on ice for 30 min. Total protein (15 mg) was then separated by 8%–10% SDS‐polyacrylamide gel electrophoresis, and the protein was transferred to the polyvinylidene fluoride membrane. The following antibodies were used in the western blotting (with β‐actin as the loading control): anti‐ERα (1:1000), anti‐ERβ (1:1000), anti‐DMP1 (1:1000), anti‐Akt (1:1000), anti‐p‐Akt (1:1000), anti‐ERK (1:1000), anti‐p‐ERK (1:1000), anti‐GSK‐3β (1:1000), anti‐p‐GSK‐3β (1:1000) anti‐β‐catenin (1:1000), and anti‐β‐acting protein (1:2000). Use horseradish peroxidase‐conjugated secondary antibody at 1:5000. The image was analyzed using ECL (Enhanced Chemiluminescence) Fuazon Fx (Vilber Lourmat).

2.5. Isolation of RNA and polymerase chain reaction

Quantitative reverse transcription PCR (RT‐qPCR) was performed as previously described (Ma et al., 2021; Yang et al., 2015). Trizol (Invitrogen) was used to lyse cultured cells, and total RNA was extracted from the lysate according to the manufacturer's instructions. Nanodrop 2000 (Thermo Fisher) was used to determine the quality of RNA. M‐MLV First Strand Kit (Life Technologies) was used to synthesize cDNA. Platinum SYBR Green qPCR Super Mix UDG Kit (Invitrogen) was used to perform qRT‐PCR on mRNA. The ABI 7500 FAST system (Life Technologies) was used for real‐time PCR. The primers are shown in Table S1.

2.6. Chromatin immunoprecipitation assay

The chromatin immunoprecipitation (ChIP) assay was performed as previously described (Zhang, et al., 2019). Briefly, cells were fixed with formaldehyde, after which cross‐linked chromatin was prepared and sonicated to an average size of 300–600 bp. The sample was diluted 10 times and then treated with Protein A‐Sepharose/Salmon Sperm DNA for 30 min at 4°C. The DNA fragments were immunoprecipitated with anti‐ERα or ERβ anti‐immunoglobulin G (IgG) antibodies at 4°C overnight. After the cross‐linking was reversed, the ERα or ERβ occupancy rate on the DMP1 promoter was checked using qRT‐PCR. The ChIP primer sequence is summarized in Table S1.

2.7. Immunofluorescence staining

The cells were fixed with 4% formaldehyde for 15 min and washed with PBS. After preincubated with 10% normal goat serum (710 027; KPL), the cell smears were incubated with primary antibody anti‐Runx2 and anti‐DMP1 at 37°C for 2 h. The secondary antibody was the fluorescence‐labeled rabbit IgG antibody. Lastly, the cell smears were incubated in 4′,6‐diamidino‐2‐phenylindole for 10 min for nuclear counterstaining. Images were acquired using confocal microscopy (DM6000CFS; Leica) and digitized with LAS AF software.

2.8. Statistical analysis

All data are expressed as means ± standard deviation. Each experiment was repeated at least three times independently. Student's t‐test was used to assess the difference in means between the two groups. p < 0.05 was considered statistically significant.

3. RESULTS

3.1. 17β‐estradiol promotes the trans‐differentiation of chondrocytes into osteogenic cells

Previous studies have shown that estrogen partially promotes bone marrow mesenchymal stem cells osteogenesis (Gao et al., 2015). To confirm whether 17β‐estradiol (E2) plays a role in chondrocyte osteogenesis, chondrocytes were treated with different concentrations of 17β‐estradiol (E2) and stained with Alizarin Red to detected osteogenesis. The results show that as the concentration of E2 increased, the osteogenesis of chondrocytes also increased and reached the peak value at 10−6 M/L of E2 (Figure 1a). In addition, the results of the calcium deposition and ALP activity assays further supported these observations (Figure 1b,c). Studies have found that high concentrations of E2 (10−6) inhibit cell proliferation (Shi et al., 2017), but we have found that it can still promote cell calcification. Furthermore, E2 treatment significantly promoted expression of osteogenic gene marker ALP, Runx2, and OCN in a concentration‐dependent fashion (Figure 1d). These data demonstrate that 17β‐estradiol promotes the trans‐differentiation of chondrocytes into osteogenic cells in high concentration conditions.

FIGURE 1.

FIGURE 1

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17β‐estradiol promotes chondrocyte osteogenesis. Chondrocytes were treated with different concentrations (10−8, 10−7, 10−6, and 10−5) of 17β‐estradiol (E2) for 7 days to induce osteogenesis. (a) Alizarin Red staining was used to detect osteogenesis (scale bar = 100 μm). (b) Statistical analysis of E2 on mineralization during chondrocytes osteogenic differentiation. **p < 0.01, ***p < 0.001 versus the corresponding controls. (c) Alkaline phosphatase (ALP) activity assay was used to detect during chondrocytes osteogenic differentiation. *p < 0.05, **p < 0.01 versus the corresponding controls. (d) Chondrocytes were treated with different concentrations E2, after which the expression of osteogenic genes ALP, Runx2, and OCN mRNA was measured using quantitative reverse transcription PCR. *p < 0.05, **p < 0.01 versus control

3.2. Blocking the ERα/β pathway inhibits chondrocyte osteogenesis

To investigate whether ERα and ERβ play a vital role in E2‐induced trans‐differentiation of chondrocytes into osteogenic cells, we first knocked down ERα and ERβ with shRNAs. As shown in Figure 2a–c, both shERα‐1# and shERα‐2# or shERβ‐1# and shERβ‐2# transfection reduced ERα and ERβ mRNA and protein expression in chondrocytes. Next, chondrocytes were transfected with shERα or shERβ and treated with E2. The ALP activity results show that knocked down shERα or shERβ alone could reduce the promotion of E2 treatment, and this process will enhance transfection of both shERα and shERβ, simultaneously (Figure 2d). Additionally, similar results can be observed in osteogenic marker genes detection (Figure 2e–g). Because tamoxifen (TAM) is an important inhibitor of ERα and ERβ (Ghasemi et al., 2019; Zhang, et al., 2019b), we aimed to determine whether TAM treatment neutralized promotion of E2 in chondrocyte osteogenesis. As shown in Figure 2h, TAM treatment clearly inhibited the ALP activity promotion by E2. Furthermore, examination of the osteogenic marker genes further supported these observations (Figure 2i). Together, these data demonstrate that blocking of ERα/β pathway may offset the promotion of E2 in chondrocyte osteogenesis.

FIGURE 2.

FIGURE 2

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Blocking the ERα/β pathway inhibits the transformation of chondrocytes to osteogenesis. (a) Chondrocytes transfected with shERα‐1#, shERα‐2#, shERβ‐1#, shERβ‐2#, and control vectors, and quantitative reverse transcription PCR (RT‐qPCR) detected the mRNA expression of ERα and ERβ. *p < 0.05, **p < 0.01, ***p < 0.001 versus the corresponding controls. (b) Cells treated as (a) and western blot detected protein levels of ERα and ERβ. (c) Statistical analysis of results in (b); *p < 0.05, **p < 0.01, ***p < 0.001 versus the corresponding controls. (d–g) Chondrocytes were treated with E2 (10−7) and transfected with indicated vector to knock down ERα or ERβ or both, followed by ALP activity assay (d) or RT‐qPCR (e–g) to detected osteogenic differentiation. *p < 0.05, **p < 0.01 versus con + pLKO; # p < 0.01 versus E2 +pLKO; # p < 0.01 versus E2 + pLKO. $ p < 0.01 versus E2 + shERα; $ p < 0.05 versus E2 + shERα; & p < 0.05 versus E2 + shERβ. (h) Chondrocytes were treated with E2 (10−7) and different concentrations of tamoxifen (TAM), followed by ALP activity assay to detected osteogenic differentiation. *p < 0.05, ***p < 0.001 versus Con + Sham; # p < 0.05 versus E2 + Sham; $ p < 0.05 versus E2 + 1 μM. (i) Chondrocytes were treated with E2 (10−7) and TAM (5 μM), and then RT‐qPCR detected the expression of osteogenic genes ALP, Runx2, and OCN mRNA. *p < 0.05, **p < 0.01 versus corresponding controls. ALP, alkaline phosphatase; ER, estrogen receptor

3.3. DMP1 participates in 17β‐estradiol‐regulated osteogenesis

To explore how the estrogen/ER pathway is involved in regulating chondrocyte osteogenesis, we measured the expression of osteogenic‐related genes in chondrocytes after transfection of shERβ and treatment with E2. As shown in Figure 3a, only DMP1 was downregulated in E2‐treated chondrocytes and was upregulated after ERβ depletion. Chondrocytes were then treated with different concentrations of E2, and mRNA and protein expression levels were measured. The results show that both mRNA and protein of DMP1 were reduced in a concentration‐dependent manner (Figure 3b–d). To investigate whether TAM reversed the E2‐inhibited DMP1 expression, chondrocytes were treated with TAM alone or both TAM and E2 simultaneously. We found that TAM significantly promoted the DMP1 expression that depression by E2 (Figure 3e,f). These findings suggested that DMP1 was involved in 17β‐estradiol regulation of chondrocyte osteogenesis.

FIGURE 3.

FIGURE 3

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DMP1 mediates 17β‐estradiol‐regulated chondrocyte osteogenesis. (a) Chondrocytes were transfected with oeERβ and then treated with or without TAM. Candidate gene on osteogenesis was detected by quantitative reverse transcription PCR (RT‐qPCR). *p < 0.05 versus the corresponding controls. (b–d) Chondrocytes were treated with different concentrations of 17β‐estradiol (E2) after which RT‐qPCR and western blot were used to detect the DMP1 mRNA (b) or protein (c, d) expression. **p < 0.01, ***p < 0.001 versus the corresponding controls. (e, f) Chondrocytes were treated with TAM or TAM + E2, and western blot analysis was used to measure expression of DMP1. ***p < 0.001 versus the corresponding controls. DMP1, dentin matrix protein 1; ER, estrogen receptor; TAM, tamoxifen

3.4. ERα/β restrains the expression of DMP1 through transcription

To study whether ERα/β directly regulates DMP1 expression by transcription, we first predicted the binding sites of ERα and ERβ in the 2‐kb 5′‐promoter region of DMP1. The results show that there is one potential binding site for ERα and three for ERβ in this region. (Figure 4a,b). ChIP‐PCR results demonstrated that ERα and ERβ were bound predominantly to the region −1 to −182 bp, but ERβ was also bound to the region −651 to −873 upstream of the transcription start site within the DMP1 promoter (Figure 4c). As expected, depletion of ERα or ERβ reversed the expression of DMP1 inhibition by E2, and these effect will be enhanced while knocked down of both ERα and ERβ simultaneously (Figure 4d). Furthermore, double immunofluorescence staining revealed that E2 promoted Runx2 expression while inhibiting the DMP1 protein level, and these effects are reversed by knock down of both ERα and ERβ (Figure 4e–g). These findings demonstrate that DMP1 is transcriptionally depressed by ER and participates in E2‐stimulated chondrocyte osteogenesis.

FIGURE 4.

FIGURE 4

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ERα/β regulates the expression of DMP1 through transcription. (a, b) Potential binding site of ERα or ERβ in the DMP1 promoter. (c) ChIP‐qPCR was used to examine the binding of the ERα/β to the DMP1 promoter region in chondrocytes. *p < 0.05, **p < 0.01 versus corresponding controls. (d) Chondrocytes were treated with E2 (10−6) and transfected with indicated vector, after which quantitative reverse transcription PCR was used to detect DMP1 mRNA expression. (e) Chondrocytes were treated as in (d), and double immunofluorescence staining was used to detect the expression of Runx2 (green) and DMP1 (red). Blue is 4′,6‐diamidino‐2‐phenylindole (DAPI). (scale bar = 50 μm). (f, g) Statistical analysis of fluorescence intensity of DMP1 and Runx2. (f, g) Data are presented as the mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus con + pLKO; # p < 0.01 versus E2 + pLKO; # p < 0.01 versus E2 + pLKO. $ p < 0.01 versus E2 + shERα; $ p < 0.05 versus E2 + shERα; & p < 0.05 versus E2 + shERβ. ChIP, chromatin immunoprecipitation; DMP1, dentin matrix protein; 1ER, estrogen receptor; SEM, standard error of mean

3.5. GSK‐3β/β‐catenin signal pathway participates in ER/DMP1‐regulated chondrocyte osteogenesis

To explore which signal pathway is involved in DMP1‐regulated chondrocyte osteogenesis, we first knocked down DMP1 and examined the signal pathway molecular expression. As shown in Figure 5a,b, depletion of DMP1 upregulated p‐GSK‐3β and β‐catenin expression but did not affect the AKT and ERK molecular pathways. Previous studies have shown that β‐catenin plays an important role in chondrocyte osteogenesis regulated by DMP1 (Li et al., 2020). In addition, the RT‐qPCR results also confirmed that DMP1 depletion significantly promoted expression of β‐catenin and osteogenic marker genes, and these effects were reversed in treatment with E2 (Figure 5c). To verify whether β‐catenin participates in ERα/β/DMP1 axis regulated chondrocyte osteogenesis, we performed Alizarin Red staining. As shown in Figure 5d,e, depletion of β‐catenin alone inhibited the mineralization that E2 promoted. However, these effects could be reversed with simultaneous knock down of DMP1. Taken together these findings demonstrate that the GSK‐3β/β‐catenin signaling pathway may be involved in the function of ERα/β/DMP1 in chondrocyte osteogenesis.

FIGURE 5.

FIGURE 5

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GSK‐3β signal pathway participates in ERα/β/DMP1‐regulated trans‐differentiation of chondrocytes into osteogenic cells. (a) Chondrocytes were transfected with pKLO and shDMP1, after which western blot analysis was used to measure the indicated proteins level. (b) Quantitative analysis of indicated protein expression in (a). (c) Chondrocytes were treated with E2 after transfection with shDMP1, followed by quantitative reverse transcription PCR to measure the expression of ALP, OCN, Runx2, and β‐catenin. *p< 0.05 versus corresponding controls. (d, e) Chondrocytes were treated with E2 and then transfected with shβ‐catenin or shDMP1 or both vectors for 7 days. Alizarin Red staining was used to detected osteogenesis (scale bar = 100 μm). *p < 0.05 and **p < 0.01 versus corresponding controls. ALP, alkaline phosphatase; DMP1, dentin matrix protein 1; ER, estrogen receptor; GSK‐3β, glycogen synthase kinase‐3 beta

4. DISCUSSION

The principal finding of this study is that high concentration of 17β‐estradiol, which usually disturbs cell proliferation, will facilitate the trans‐differentiation of chondrocytes into osteogenic cells in vitro. Inhibition of ER by shRNA or TAM neutralized the promotion of E2 in chondrocyte osteogenesis. In addition, DMP1 was found to be involved in 17β‐estradiol‐regulated trans‐differentiation of chondrocytes into osteogenic cells. Mechanistically, DMP1 was transcriptionally regulated by ER and participated in E2‐stimulated chondrocyte osteogenesis. In addition, the GSK‐3β/β‐catenin signal pathway was involved in ERα/β/DMP1‐regulated chondrocyte osteogenesis. These findings suggest that ER/DMP1/GSK‐3β/β‐catenin plays a vital role in regulation of chondrocyte osteogenesis and thus may provide a therapeutic target for short stature caused by epiphyseal fusion.

During the growth spurt period of puberty, longitudinal bone growth occurs on the growth plate through the formation of intrachondral bone (Nilsson et al., 2014). This process often includes the proliferation and differentiation of chondrocytes, the secretion of extracellular matrix, the calcification of hypertrophic areas, the invasion and differentiation of osteoblasts, and the formation of blood vessels repeated in the growth plate (Nilsson et al., 2005; Shim, 2015). It is generally believed that as the growth plate cartilage undergoes procedural aging, it will eventually lead to bone elongation and epiphysis fusion, at which point growth ceases (Bello & Garla,; Eerden et al., 2003). However, the cellular mechanism of epiphyseal senescence is still unclear. Animal experiments have shown that exposure to estrogen accelerates the decrease in growth plate thickness and the decrease in the number of hypertrophic chondrocytes (Börjesson et al., 2013; Nilsson et al., 2014). This view is supported by precocious puberty (PP) leading to short stature after premature puberty (Shim, 2015). Treatment of idiopathic central PP with gonadotropin‐releasing hormone agonists can reduce the secretion of estrogen and delay growth plate closure (Choi et al., 2019). However, with age, estrogen will accelerate the loss of cartilage cells in the resting area, and this decrease in cell number does not seem to be due to apoptosis (Nilsson et al., 2014). For decades, it has been generally believed that terminal hypertrophic chondrocytes trans‐differentiate into osteoblasts at the cartilage junction of the growth plate, and undergo apoptosis before forming (Emons et al., 2011; Shim, 2015). However, recent studies on long bones have shown that chondrocytes can be directly transformed into bone cells (Jing et al., 2015). A large number of studies have shown that the combined effect of estrogen and ER can promote the transformation of a variety of cells, including chondrocytes, to bone formation (Gao et al., 2015; Jing et al., 2015; Shi et al., 2017; Wu et al., 2020). In the present study, we found that high concentrations of 17β‐estradiol usually interfere with cell proliferation but can still promote the trans‐differentiation of chondrocytes into osteoblasts in vitro. The inhibition of ER by shRNA or TAM neutralized the promotion of E2 in chondrocyte osteogenesis. Therefore, we suspect that the continuous induction of estrogen during the development of puberty interferes with the normal ossification process of chondrocytes in the growth plate. Especially in late puberty, high doses of estrogen may stimulate the direct conversion of chondrocytes into bone cells to accelerate the closure of the epiphysis and cause the cessation of growth.

The Wnt/β‐catenin pathway is a key pathway involved in chondrocyte maturation and cartilage development in longitudinal bone growth (Kinsley et al., 2015; Yuan et al., 2016). Studies have found that the cartilage‐specific deletion of Ctnnb1, which encodes β‐catenin, leads to a decrease in the proliferation and differentiation of chondrocytes in the growth plate (Golovchenko et al., 2013). Indirubin‐3′‐oxime accelerates chondrocyte maturation and longitudinal bone growth by activating the Wnt/β‐catenin pathway (Choi et al., 2019). In addition, the inactivation of glycogen synthase kinase 3 (GSK3) causes β‐catenin instability and increases the longitudinal growth of the tibia in vitro (Zhou et al., 2015). GSK‐3β is a key enzyme in the β‐catenin signaling pathway, and activated GSK‐3β (Ser9) promotes β‐catenin to enter the nucleus and regulate the expression of downstream genes (Yan et al., 2020). In this study, the depletion of DMP1 downregulated the expression of p‐GSK‐3β and β‐catenin but did not affect the AKT and ERK pathway molecules. DMP1 knockdown reduced the expression of β‐catenin and osteogenic marker genes induced by E2. Consumption of DMP1 or β‐catenin alone inhibits the mineralization promoted by E2. However, these effects may be enhanced when both DMP1 and β‐catenin are knocked down at the same time.

5. CONCLUSIONS

In conclusion, we found that E2 promotes the trans‐differentiation of chondrocytes into osteoblasts, and these effects are offset by blocking the ER pathway. In addition, DMP1 is regulated by ER transcription and participates in E2‐stimulated chondrocyte osteogenesis. Our results show that ER/DMP1/GSK‐3β/β‐catenin plays a vital role in the regulation of chondrocyte osteogenesis and provides a therapeutic target for short stature caused by epiphyseal fusion.

CONFLICT OF INTEREST

The authors declare that they have no competing interests.

AUTHOR CONTRIBUTIONS

Xue Yan and Deng‐Yun Fan organized the article writing and critically modified the manuscript. Ya‐Nan Zhang modified the manuscript. Xue Yan, and Hui‐Feng Zhang drafted the manuscript and were responsible for the acquisition of data; Ya‐Lei Pi participated in the data analysis; Peng‐Jiu Fu contributed to the literature search. Hui‐Feng Zhang check and correct language expression. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Supporting information

Table S1

Click here for additional data file. (18.8KB, docx)

Yan, X. , Fan, D.‐Y. , Pi, Y.‐L. , Zhang, Y.‐N. , Fu, P.‐J. & Zhang, H.‐F. (2022) ERα/β/DMP1 axis promotes trans‐differentiation of chondrocytes to bone cells through GSK‐3β/β‐catenin pathway. Journal of Anatomy, 240, 1152–1161. Available from: 10.1111/joa.13612

Funding information

The study was funded by Intra‐hospital Fund of the Second Hospital of Hebei Medical University (No. 202015).

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

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Supplementary Materials

Table S1

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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