Downregulation of Runx2 by 1,25-Dihydroxyvitamin D3 Induces the Transdifferentiation of Osteoblasts to Adipocytes

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) indirectly stimulates bone formation, but little is known about its direct effect on bone formation. In this study, we observed that 1,25(OH)2D3 enhances adipocyte differentiation, but inhibits osteoblast differentiation during osteogenesis. The positive role of 1,25(OH)2D3 in adipocyte differentiation was confirmed when murine osteoblasts were cultured in adipogenic medium. Additionally, 1,25(OH)2D3 enhanced the expression of adipocyte marker genes, but inhibited the expression of osteoblast marker genes in osteoblasts. The inhibition of osteoblast differentiation and promotion of adipocyte differentiation mediated by 1,25(OH)2D3 were compensated by Runx2 overexpression. Our results suggest that 1,25(OH)2D3 induces the transdifferentiation of osteoblasts to adipocytes via Runx2 downregulation in osteoblasts.

The steroid hormone 1,25(OH) 2 D 3 is a key regulator of calcium homeostasis and skeletal health [5]. In bone, 1,25(OH) 2 D 3 regulates mineralization both in an indirect and a direct manner. 1,25(OH) 2 D 3 increases calcium absorption from the intestines, which indirectly stimulates bone formation [6]. It can also directly influence bone formation via the regulation of osteoblast differentiation and function. Various studies have demonstrated the direct effects of 1,25(OH) 2 D 3 on osteoblast differentiation and function in vitro. However, the results of these studies are highly controversial. Several studies have shown that 1,25(OH) 2 D 3 stimulates osteoblast differentiation by increasing alkaline phosphatase (ALP) activity and osteocalcin expression in human primary osteoblast-like cells [7][8][9]. However, several lines of evidence suggest that 1,25(OH) 2 D 3 negatively regulates osteoblast differentiation. For example, 1,25(OH) 2 D 3 downregulates osteocalcin expression in mouse calvaria 3T3 (MC3T3)-E1 osteoblasts [10]. Lieben et al. [11] have also reported that 1,25(OH) 2 D 3 impairs mineralization by upregulating the expression of mineralization inhibitors in mouse primary osteoblasts.
Osteoblasts, which are responsible for bone formation, are derived from mesenchymal stem cells by the action of several transcription factors, including Runx2, osterix, and β-catenin [12]. In particular, Runx2, a cell-specific member of the Runt family of transcription factors, is essential for mesenchymal cell differentiation into osteoblasts. Runx2 promotes the acquisition of an osteoblastic phenotype by mesenchymal stem cells by inducing the expression of genes encoding major bone matrix proteins, e.g., Col1a1, osteopontin, bone sialoprotein (BSP), and osteocalcin [11,12]. Runx2´/´mice exhibit a complete lack of intramembranous and endochondral ossification in vivo and Runx2´/´calvarial cells cannot differentiate into osteoblasts, even in the presence of osteogenic factors in vitro [12][13][14]. Multiple factors that play an important role in osteoblast differentiation via the regulation of Runx2 expression or activation have been identified.
Adipocytes as well as osteoblasts are derived from mesenchymal stem cells. Various transcription factors, such as CCAAT/enhancer binding protein-α (CEBP-α), CEBP-β, and peroxisome proliferator-activated receptor-γ (PPAR-γ), are essential for mesenchymal cell differentiation into adipocytes [15]. Increased adipose tissue in the bone marrow of osteoporotic patients and individuals with age-dependent bone loss may be associated with the transdifferentiation of osteoblasts to adipocytes [16]. While the effect of 1,25(OH) 2 D 3 on the transdifferentiation of skeletal muscle cells to adipose cells is known, it is not clear if 1,25(OH) 2 D 3 is involved in the transdifferentiation of osteoblasts to adipocytes in the bone marrow [17].
To elucidate the direct effect of locally produced 1,25(OH) 2 D 3 on bone formation, we investigated the effect of 1,25(OH) 2 D 3 on osteoblast differentiation, Runx2 expression, and the transdifferentiation of osteoblasts to adipocytes.

1,25-Dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) Inhibits Osteoblast Differentiation and Runx2 Expression in Primary Osteoblasts
To elucidate the direct effect of 1,25(OH) 2 D 3 on bone formation, we examined its role in osteoblast differentiation. When mouse primary osteoblasts were cultured in osteogenic medium including bone morphogenic protein 2 (BMP2), ascorbic acid, and β-glycerophosphate, bone nodule formation was dramatically induced (Figure 1a,b). However, supplementation with 1,25(OH) 2 D 3 significantly inhibited osteoblast differentiation induced by the osteogenic medium in a dose-dependent manner (Figure 1a,b). The negative effect of 1,25(OH) 2 D 3 on osteogenesis was confirmed by the expression of osteoblast differentiation-related genes. As shown in Figure 1c, 1,25(OH) 2 D 3 inhibited the expression of osteoblastic genes, such as Runx2, ALP, and BSP. Therefore, 1,25(OH) 2 D 3 may negatively regulate osteoblast differentiation by inhibiting the expression of Runx2 and its downstream targets ALP and BSP.

1,25(OH) 2 D 3 Induces the Transdifferentiation of Osteoblasts to Adipocytes
Intriguingly, 1,25(OH) 2 D 3 treatment during osteoblast differentiation resulted in lipid droplet formation with the inhibition of bone nodule formation. Therefore, we examined the effect of 1,25(OH) 2 D 3 on adipocyte differentiation during osteoblast differentiation. Osteoblasts were cultured in osteogenic medium either without or with 1,25(OH) 2 D 3 . Positive Oil Red-O staining confirmed the presence of lipid droplets, which were primarily observed in osteoblasts treated with high concentrations of 1,25(OH) 2 D 3 (Figure 2a,b). Additionally, 1,25(OH) 2 D 3 significantly increased the expression of adipocyte marker genes, including CEBP-α, PPAR-γ, and adipocyte protein 2 (aP2), compared with control cells. These results indicated that 1,25(OH) 2 D 3 can induce the transdifferentiation of osteoblasts to adipocytes during osteoblast differentiation.     Next, we assessed the time course of the effect of 1,25(OH) 2 D 3 on the transdifferentiation of osteoblasts to adipocytes. When 1,25(OH) 2 D 3 was added continuously from the start of osteoblast differentiation (days 1-9), adipocyte differentiation was dramatically enhanced, while osteoblast differentiation was significantly reduced ( Figure 3). Only the effect of 1,25(OH) 2 D 3 added from day 1 to day 6 was comparable to that of 1,25(OH) 2 D 3 added continuously from the start of osteoblast differentiation (i.e., days 1-9) ( Figure 3). However, the addition of 1,25(OH) 2 D 3 during the late stage of osteoblast differentiation (days 7-9) did not affect the transdifferentiation of osteoblasts to adipocytes. Interestingly, the effect of 1,25(OH) 2 D 3 on the transdifferentiation of osteoblasts to adipocytes was dependent on the addition of 1,25(OH) 2 D 3 during the initial three days of osteoblast differentiation ( Figure 3). Taken together, these results demonstrated that 1,25(OH) 2 D 3 primarily acts at the early stage of osteoblast differentiation to induce the transdifferentiation of osteoblasts to adipocytes. Next, we assessed the time course of the effect of 1,25(OH)2D3 on the transdifferentiation of osteoblasts to adipocytes. When 1,25(OH)2D3 was added continuously from the start of osteoblast differentiation (days 1-9), adipocyte differentiation was dramatically enhanced, while osteoblast differentiation was significantly reduced (Figure 3). Only the effect of 1,25(OH)2D3 added from day 1 to day 6 was comparable to that of 1,25(OH)2D3 added continuously from the start of osteoblast differentiation (i.e., days 1-9) (Figure 3). However, the addition of 1,25(OH)2D3 during the late stage of osteoblast differentiation (days 7-9) did not affect the transdifferentiation of osteoblasts to adipocytes. Interestingly, the effect of 1,25(OH)2D3 on the transdifferentiation of osteoblasts to adipocytes was dependent on the addition of 1,25(OH)2D3 during the initial three days of osteoblast differentiation ( Figure 3). Taken together, these results demonstrated that 1,25(OH)2D3 primarily acts at the early stage of osteoblast differentiation to induce the transdifferentiation of osteoblasts to adipocytes.

1,25(OH)2D3 Induces the Transdifferentiation of Osteoblasts to Adipocytes via the Regulation of Runx2 Expression
It has recently been reported that 1,25(OH)2D3 mediates the suppression of mineral incorporation via the upregulation of pyrophosphate (PPi) levels [11]; accordingly, we analyzed whether increased PPi levels due to 1,25(OH)2D3 are involved in the transdifferentiation of osteoblasts to adipocytes. The addition of PPi to cultured osteoblasts suppressed osteoblast differentiation, similar to treatment with 1,25(OH)2D3 (Figure 5a,b). However, PPi did not induce the formation of lipid droplets during osteoblastogenesis (Figure 5a,c). Therefore, these results indicated that increased PPi levels in response to 1,25(OH)2D3 are involved in the inhibitory effect of 1,25(OH)2D3 on mineralization, but not in the stimulatory effect of 1,25(OH)2D3 on the formation of lipid droplets.
Since 1,25(OH)2D3 inhibited the expression of Runx2 (Figure 1c), we next analyzed whether the inhibitory effect of 1,25(OH)2D3 on Runx2 expression regulates adipocyte differentiation during osteoblast differentiation. The overexpression of Runx2 restored the decrease in osteoblast differentiation as well as the increase in adipocyte differentiation regulated by 1,25(OH)2D3

1,25(OH) 2 D 3 Induces the Transdifferentiation of Osteoblasts to Adipocytes via the Regulation of Runx2 Expression
It has recently been reported that 1,25(OH) 2 D 3 mediates the suppression of mineral incorporation via the upregulation of pyrophosphate (PPi) levels [11]; accordingly, we analyzed whether increased PPi levels due to 1,25(OH) 2 D 3 are involved in the transdifferentiation of osteoblasts to adipocytes. The addition of PPi to cultured osteoblasts suppressed osteoblast differentiation, similar to treatment with 1,25(OH) 2 D 3 (Figure 5a,b). However, PPi did not induce the formation of lipid droplets during osteoblastogenesis (Figure 5a,c). Therefore, these results indicated that increased PPi levels in response to 1,25(OH) 2  results demonstrated that the downregulation of Runx2 by 1,25(OH)2D3 in osteoblasts results in the inhibition of osteoblast differentiation accompanied by the transdifferentiation of osteoblasts to adipocytes. However, because Runx2 overexpression did not completely reverse the effect of 1,25(OH)2D3 on adipocyte and osteoblast differentiation, there remains a possibility that 1,25(OH)2D3 regulates the transdifferentiation of osteoblasts to adipocytes through unknown pathways other than Runx2 regulation.  Transfected cells were treated with vehicle or 1,25(OH) 2 D 3 for 48 h. Luciferase activity was measured using a dual-luciferase reporter assay system. The data represent means and SD of triplicate samples. # p < 0.05; * p < 0.01; ** p < 0.001 as compared with controls.
Since 1,25(OH) 2 D 3 inhibited the expression of Runx2 (Figure 1c), we next analyzed whether the inhibitory effect of 1,25(OH) 2 D 3 on Runx2 expression regulates adipocyte differentiation during osteoblast differentiation. The overexpression of Runx2 restored the decrease in osteoblast differentiation as well as the increase in adipocyte differentiation regulated by 1,25(OH) 2 D 3 (Figure 5d,f). Moreover, the inhibitory effect of 1,25(OH) 2 D 3 on Runx2 expression was confirmed by the 1,25(OH) 2 D 3 -mediated suppression of Runx2 promoter activity (Figure 5g). Taken together, these results demonstrated that the downregulation of Runx2 by 1,25(OH) 2 D 3 in osteoblasts results in the inhibition of osteoblast differentiation accompanied by the transdifferentiation of osteoblasts to adipocytes. However, because Runx2 overexpression did not completely reverse the effect of 1,25(OH) 2 D 3 on adipocyte and osteoblast differentiation, there remains a possibility that 1,25(OH) 2 D 3 regulates the transdifferentiation of osteoblasts to adipocytes through unknown pathways other than Runx2 regulation.

Discussion
It is generally accepted that 1,25(OH) 2 D 3 stimulates bone formation by increasing calcium absorption from the intestines [6]. However, the role of 1,25(OH) 2 D 3 in bone formation mediated by osteoblasts is still largely unclear. Here, we showed that 1,25(OH) 2 D 3 directly suppressed osteoblast differentiation, potentially as a result of Runx2 inhibition.
Runx2 is a key transcription factor that initiates and regulates the early stage of osteoblast differentiation. An inhibitory effect of 1,25(OH) 2 D 3 on osteoblast differentiation was observed when 1,25(OH) 2 D 3 was added at the early stage, rather than the late stage of osteoblast differentiation. Furthermore, 1,25(OH) 2 D 3 suppressed the expression of Runx2 during osteoblast differentiation, which in turn inhibited the expression of downstream genes, such as ALP and BSP. Similarly, it has been reported that 1,25(OH) 2 D 3 suppresses Runx2 expression within 24 h in MC3T3 and rat osteosarcoma (ROS) 17/2.8 cells via the binding of vitamin D receptor (VDR) to vitamin D response element (VDRE) and Runx2 autonomously suppresses its own expression [18]. In contrast, Han et al. [19] reported that 1,25(OH) 2 D 3 increases Runx2 expression in vascular smooth muscle cells and induces vascular calcification. These findings suggest that 1,25(OH) 2 D 3 inhibits bone formation by suppressing Runx2 expression in osteoblasts, but the effect of 1,25(OH) 2 D 3 on bone mineralization may be dependent on the cell-type specificity of Runx2 expression regulated by 1,25(OH) 2 D 3 .
The direct effect of 1,25(OH) 2 D 3 on adipocyte differentiation is not yet clear. Several studies have reported a negative role of 1,25(OH) 2 D 3 in adipogenesis [20][21][22]. However, it has also been reported that 1,25(OH) 2 D 3 promotes the maturation of human subcutaneous preadipocytes via a PPAR-γ-independent pathway [23]. Previous studies have indicated that 1,25(OH) 2 D 3 does not directly induce critical early factors for adipocyte differentiation. 1,25(OH) 2 D 3 -mediated inhibition of osteoblast differentiation accompanied by an increase in lipid droplet formation suggests an important role of 1,25(OH) 2 D 3 in the transdifferentiation of osteoblasts to adipocytes. Previous results have also shown that 1,25(OH) 2 D 3 can induce the formation of lipid droplets in osteoblasts [24]. Similarly, we observed that 1,25(OH) 2 D 3 stimulated the formation of lipid droplets, even in osteogenic medium that lacked adipogenic stimulators. The formation of lipid droplets in osteoblasts was more highly stimulated in 1,25(OH) 2 D 3 -containing osteogenic medium than in adipogenic medium including insulin, dexamethasone, IBMX, and rosiglitazone. Therefore, the transdifferentiation of osteoblasts to adipocytes induced by 1,25(OH) 2 D 3 is involved in the regulation of osteoblastic genes, rather than the induction of adipogenic genes. Runx2 inhibits the late adipocyte maturation of human bone marrow precursor cells and Runx2-deficient osteoblasts spontaneously undergo adipocyte differentiation with the inhibition of osteoblast differentiation [25][26][27]. In our study, 1,25(OH) 2 D 3 inhibited Runx2 expression and the overexpression of Runx2 suppressed the formation of lipid droplets induced by 1,25(OH) 2 D 3 , suggesting that Runx2 downregulation mediated by 1,25(OH) 2 D 3 induces the transdifferentiation of osteoblasts to adipocytes. Recently, it has been reported that adipocytes support osteoclast differentiation and function via receptor activator of nuclear factor kappa-B ligand (RANKL) production [28]. It is also well known that 1,25(OH) 2 D 3 indirectly induces osteoclast differentiation via RANKL upregulation in osteoblasts [29,30]. 1,25(OH) 2 D 3 may primarily enhance RANKL in osteoblasts and secondarily enhance RANKL by increasing adipocyte differentiation. Although vitamin D supplementation indirectly increases bone mass by improving intestinal calcium absorption, the administration of high-dose vitamin D to older woman results in an increased fracture risk [31,32]. Vitamin D might have several roles in osteoblasts, including the inhibition of osteoblast differentiation, promotion of adipocyte differentiation, and the support of osteoclast differentiation, and these functions may result in unexpected deleterious effects of vitamin D on the skeleton.

Osteoblast Differentiation
Primary osteoblast precursor cells were isolated from neonatal mouse calvaria by digestion with 0.1% collagenase (Life Technologies, Carlsbad, CA, USA) and 0.2% dispase II (Roche Diagnostics GmbH, Mannheim, Germany). Isolated osteoblast precursor cells were cultured in α-Minimal Essential Medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. For osteoblast differentiation, primary osteoblast precursor cells were cultured in osteogenic medium containing BMP2 (100 ng/mL), ascorbic acid (50 µg/mL), and β-glycerophosphate (100 mM) for six to nine days. Cultured cells were fixed with 70% ethanol and stained with 40 mM alizarin red (pH 4.2). After nonspecific staining was removed with phosphate-buffered saline, alizarin red staining was visualized with a CanoScan 4400F (Canon Inc., Tokyo, Japan). Alizarin red-stained cells were dissolved with 10% cetylpyridinium (Sigma-Aldrich), and absorbance of the extracted solution was measured at 562 nm for quantification.

Luciferase Assay
Mouse myoblasts C2C12 cells were plated in 24-well plates at a density of 2ˆ10 4 cells/well one day before transfection. The Runx2 reporter plasmid was transfected into C2C12 cells using Attractene according to the manufacturer's instructions. Transfected cells were treated with vehicle or 1,25(OH) 2 D 3 (10´8 M) for two days. Luciferase activity was measured using a dual-luciferase reporter assay system (Promega, Madison, WI, USA) according to the manufacturer's instructions.

Retroviral Gene Transduction
The retrovirus packaging cell line Plat-E was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, puromycin (1 µg/mL), and blasticidin (10 µg/mL). To obtain viral supernatants, retroviral vectors were transfected into Plat-E using FuGENE 6 (Promega) according to the manufacturer's protocol. Viral supernatants were collected from culture medium at 48 h after transfection. Osteoblasts were infected with the retroviruses in the presence of 10 µg/mL polybrene (Sigma-Aldrich) for 6 h.

Statistical Analysis
All values are expressed as means˘standard deviation (SD). Statistical analyses were performed using two-tailed Student's t-tests. p-values less than 0.05 were considered statistically significant.

Conclusions
The downregulation of Runx2 mediated by 1,25(OH) 2 D 3 in osteoblasts stimulates the transdifferentiation of osteoblasts to adipocytes, and this may partially contribute to decreased bone mass by decreasing bone formation and increasing bone resorption.