Tissue-nonspecific alkaline phosphatase promotes Title the osteogenic differentiation of osteoprogenitor cells

Author(s) Nakamura, T; Nakamura-Takahashi, A; Kasahara, M; Alternative Yamaguchi, A; Azuma, T

Biochemical and biophysical research Journal communications, 524(3): 702-709

URL http://hdl.handle.net/10130/5424

This manuscript version is made available under the Right CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/

Description

Posted at the Institutional Resources for Unique Collection and Academic Archives at Tokyo Dental College, Available from http://ir.tdc.ac.jp/

Tissue-nonspecific alkaline phosphatase promotes the osteogenic differentiation of osteoprogenitor cells

Takashi Nakamura a, b, Aki Nakamura-Takahashi b, c, Masataka Kasahara b, c,

Akira Yamaguchi b, d, Toshifumi Azuma a, b, d,*

a. Department of Biochemistry, Tokyo Dental College, Tokyo 101-0061, Japan b. Tokyo Dental College Research Branding Project, Tokyo Dental College, Tokyo

101-0061, Japan c. Department of Pharmacology, Tokyo Dental College, Tokyo 101-0061, Japan d. Oral Health Science Center, Tokyo Dental College, Tokyo 101-0061, Japan

*Corresponding author: E-mail address: [email protected] (T. Azuma).

Abstract

Tissue-nonspecific alkaline phosphatase (TNAP) is expressed in the calcification sites of the skeletal tissue. It promotes hydroxyapatite crystal formation by degrading inorganic pyrophosphate (PPi) and increasing inorganic phosphate (Pi) concentration.

However, abnormalities in Alpl-/- mouse-derived osteoblasts are poorly understood, and the involvement of TNAP in osteoblast differentiation remains unclear. Therefore, in this study, we aimed to investigate the precise role of TNAP in osteoblast differentiation.

TNAP inhibition by levamisole, a reversible TNAP inhibitor, suppressed the expression of osteoblast differentiation marker genes in wild-type osteoblastic cells. Alpl overexpression increased the expression of master osteoblast transcription factor genes runt-related transcription factor 2 (Runx2) and Sp7 and the mature osteoblast and osteocyte marker genes, bone -carboxyglutamate protein 2 (Bglap2) and dentin matrix protein 1 (Dmp1), respectively in Alpl-deficient osteoblastic cells. TNAP regulated

Runx2 expression, which in turn regulated the expression of all other osteoblast markers, except Dmp1. Dmp1 expression was independent of RUNX2 but was dependent on extracellular Pi concentration in Runx2-deficient osteogenic cells. These results suggest that TNAP functions as an osteogenic differentiation regulator either by regulating

Runx2 expression or by controlling extracellular Pi concentration.

Keywords: Tissue-nonspecific alkaline phosphatase; Runx2; Bone; Osteoblast;

Osteocyte

Abbreviations: ALP: alkaline phosphatase; Bglap2: bone -carboxyglutamate protein 2; BMP2: bone morphogenetic protein 2; BP: bisphosphonate; Dmp1: dentin matrix protein 1; ECM: extracellular matrix; HPP: hypophosphatasia; LRP6: lipoprotein receptor-related protein 6; MV: matrix vesicle; Pi: inorganic phosphate; PPi: inorganic pyrophosphate; Runx2: runt-related transcription factor 2; TNAP: tissue-nonspecific alkaline phosphatase

1. Introduction

Alkaline phosphatase (ALP) is a glycosylphosphatidylinositol-anchored ectoenzyme that catalyzes the hydrolysis of various phosphate ester compounds to inorganic phosphate (Pi) and alcohol [1]. Human ALPs are classified into four forms: intestinal

ALP, placenta-like ALP, germ cell ALP, and tissue-nonspecific ALP (TNAP) expressed in various tissues, including the bone, liver, and kidney. These four forms are highly homologous with respect to the amino acids constituting their active sites [2].

Nevertheless, although the tissue-specific ALPs have been suggested to be involved in cell differentiation, material transport, and lipid metabolism, the precise physiological functions of these ALPs remain poorly understood [3–5] . Conversely, the TNAP is known to hydrolyze inorganic pyrophosphate (PPi), a calcification inhibitor, and lower cellular PPi concentration to promote calcification. TNAP is encoded by ALPL in humans and Alpl (Akp2) in mice. These genes are used as osteoblast markers and bone formation indicators, as they are strongly expressed in osteoblasts and matrix vesicles in the bone. Osteoblasts are osteogenic cells derived from mesenchymal stem cells and are induced to differentiate into mature osteoblasts by the transcription factor runt-related transcription factor 2 (RUNX2) [6]. Osteoblasts control bone calcification by secreting extracellular matrix (ECM) components such as type I collagen and osteocalcin and modulate the local Pi concentration via TNAP. As the osteoblasts differentiate, they

2+ release matrix vesicles (MVs) into the ECM. These MVs accumulate Ca and Pi, resulting in the formation of hydroxyapatite in lumen of MVs and initiation of mineralization. In the second phase of mineralization, MVs release the hydroxyapatite crystals, which propagate continuous mineral formation in the ECM. In this process,

TNAPs play an essential role in calcification by hydrolyzing PPi to Pi [7].

Hypophosphatasia (HPP) is a hereditary disease caused by ALPL mutation and is characterized by hypocalcification, rickets-like changes, reduced blood ALP level, PPi accumulation, and decreased phosphorus concentration [1,8]. Alpl-/- mice exhibit characteristics similar to those observed in infants with HPP, such as long bone and dental defects and skull fusion [9,10]. Administration of exogenous ALPs has been reported to ameliorate the manifestation of HPP significantly [1], increasing bone mass and mineral density and extending the life span of Alpl-/- mice [11–13]. However, abnormalities in Alpl-/- mouse-derived osteoblasts have not been well investigated, and the different osteogenic abilities of these osteoblasts remain controversial. Furthermore, it is not clear whether TNAP deficiency affects early osteoblast differentiation before mineralization occurs. Several studies have reported ALP functions other than pyrophosphate hydrolysis. Recently, Liu et al. reported that TNAP binds directly to the lipoprotein receptor-related protein 6 (LRP6) and promotes osteoblast differentiation through activation of the canonical Wnt pathway [14]; however, several aspects of this mechanism have not yet been elucidated. Therefore, in this study, we used Runx2-/- and

Alpl-/- mouse-derived mesenchymal cells and investigated the involvement of TNAP in osteoblast differentiation.

2. Material and Methods

2.1 Animals

Alpl+/+ and Alpl−/− mice were obtained by mating Alpl+/− mice with a mixed genetic background of 129/J and C57BL/6J [10]. Runx2+/+ and Runx2−/− mice were obtained by mating Runx2+/− mice with C57BL/6J background [6]. All the mice were housed in animal facilities approved by Tokyo Dental College. All the experiments were reviewed

and approved by the Animal Care and Use Committee of Tokyo Dental College (No.

300402, No. 300706).

2.2 Isolation of murine osteoblast precursor cells

Calvariae were isolated from newborn Alpl+/+ and Alpl-/- male mice and sequentially digested five times with 0.1% collagenase (FUJIFILM Wako, Osaka, Japan) and 0.2% dispase (Thermo Fisher Scientific, Waltham, MA) for 15 min at 37°C. The solution used for first digestion was discarded, and the solutions from the subsequent four digestion steps were collected by centrifugation and used as osteoblast precursors.

Runx2-deficient mesenchymal cells were isolated enzymatically from the dura mater of newborn Runx2-/- male mice.

2.3 Osteoblast differentiation

For osteoblast differentiation, calvarial cells were cultured in MEM with 10% fetal bovine serum, 5 mM -glycerophosphate, 100 g/ml magnesium ascorbate, and 100 ng/ml bone morphogenetic protein 2 (BMP2; Oriental Yeast, Tokyo, Japan) at 37°C in

5% CO2. Half the medium was changed every three days.

2.4 ALP staining

Cells were washed with 0.9% NaCl and fixed using 4% paraformaldehyde for 10 min.

The cells were then washed 3 times with 0.9% NaCl and reacted with a 0.1% Tris-HCl

(pH 9.5) solution containing 0.1 mg/ml Naphthol AS-MX phosphate (Sigma , St. Louis,

MO), 0.5% N, N-dimethylformamide, 2 mM MgCl2, and 0.6 mg/ml Fast blue BB salt

(Sigma) for 30 min at room temperature.

2.5 von Kossa staining

The cells were fixed with 4% paraformaldehyde, washed with deionized water, dried, incubated with 3% silver nitrate solution, and placed under ultraviolet light for 30 min.

Subsequently, they were washed with deionized water and fixed with 5% sodium thiosulfate for 1 min.

2.6 Immunoblotting

Cell lysates were separated using SDS-PAGE and transferred to a PVDF membrane.

The membranes were incubated overnight with primary antibodies against TNAP

(1:2000 dilution; AF2910; R&D Systems, Minneapolis, MN) and β-actin (1:5000 dilution; PM053; MBL, Nagoya, Japan) at 4°C. Then, the membranes were washed and incubated with peroxidase-conjugated secondary antibodies for 1 h at room temperature.

Signals were detected using a chemiluminescence substrate and the ImageQuant

LAS4000 mini Image Analyzer (GE Healthcare, Piscataway, NJ).

2.7 Lentiviral vector preparation and infection

Mouse Alpl and Runx2 cDNA were individually cloned into CSII-EF-MCS plasmids

(RIKEN BRC, Tsukuba, Japan). These plasmids were mixed with pCMV-VSVG-RSV-Rev and pCAG-HIVgp packaging plasmids and transfected into

293T cells. Virus particles were harvested 48 h post transfection, filtered through a

0.45-μm filter, pelleted using ultracentrifugation (50,000g at 4°C for 2 h), and re-suspended in HEPES-buffered saline. For lentivirus infection, cells were seeded in a

12-well plate at a density of 2.5 × 105 cells/well, and lentivirus-containing MEM with

10% FBS was added the next day. At 48 h after infection, the virus-containing medium was replaced with osteoblast differentiation medium.

2.8 Total RNA isolation and reverse transcription real-time PCR

Total RNA from the cultured cells was extracted using ISOGEN (Nippon Gene, Tokyo,

Japan) and reverse transcribed using High-Capacity cDNA Reverse Transcription kit

(Thermo Fisher Scientific) with random hexamers. Then, real-time PCR was performed using the Thunderbird Probe qPCR Mix (Toyobo, Osaka, Japan) and the ABI 7500 Fast

Real-Time PCR system (Thermo Fisher Scientific). 18S rRNA was used as an endogenous control. The gene-specific primers used are listed in Table S1.

2.9 Statistical analysis

The results were expressed as mean ± SD. Statistical analysis was performed using

Prism 7.0e (GraphPad Software, La Jolla, CA). Groups were compared using student′s t-tests or two-way ANOVA followed by Tukey’s post hoc test; p < 0.05 was considered to indicate statistical significance.

3. Results

3.1. Levamisole suppresses Alpl expression

To elucidate the role of TNAP in osteoblasts, we first observed the effect of levamisole, a TNAP inhibitor, on BMP2-induced osteoblast differentiation using ALP staining and von Kossa staining. Consistent with previously reported results, levamisole considerably suppressed ALP activity and calcification (Fig. 1A) [15]. As levamisole is a reversible inhibitor of ALP [16], the levamisole-treated cells rapidly regain ALP

activity after levamisole removal. However, the effect of continuous levamisole treatment on osteoblast ALP activity remains poorly understood. Our staining experiments revealed that osteoblasts without pre-treatment with levamisole showed high ALP activity, which was completely blocked by the addition of levamisole to the staining solution. We pre-treated osteoblasts with levamisole for 7 days, 1 day, or 1 h, rinsed the cells, and stained them for ALP. Levamisole treatment for 7 days completely blocked ALP activity whereas ALP inhibition due to levamisole treatment for 1 day or 1 h was reversible (Fig. 1B). We hypothesized that this continuous levamisole treatment for 7 days inhibited the osteogenic differentiation of cells. Therefore, we first examined the amount of TNAP mRNA and protein levels in osteoblasts continuously treated with or without levamisole 7, 14, and 21 days during osteoblast induction culture.

Levamisole reduced Alpl expression and ALP protein content of cells considerably (Fig.

1C,D).

[Insert Figure 1 here]

3.2. Levamisole suppresses osteoblast differentiation

Next, we investigated whether ALP activity is essential for osteoblast differentiation.

We examined the gene expression of several osteoblast differentiation markers. Runx2 and Sp7, the genes coding for master osteoblast transcription factors, and the differentiation marker gene bone -carboxyglutamate protein 2 (Bglap2) were suppressed by levamisole in a time-dependent manner. The expression of the osteocyte differentiation marker gene Dmp1 was also suppressed by levamisole treatment. These results suggest that levamisole-dependent suppression of osteoblast differentiation was mediated by Runx2 expression (Fig. 1E).

3.3. TNAP promotes osteoblast differentiation via RUNX2

Alpl-/- cells lacked ALP activity and the ability to form mineralized nodules [17].

Therefore, to further investigate the role of TNAP in osteoblast differentiation, Alpl-/- osteoblasts were infected with an Alpl-coding lentiviral vector. ALP activity was significantly recovered after infection with the Alpl-expressing lentivirus (Fig. 2A,B).

The expression of Runx2, Bglap2, and Dmp1 genes in Alpl-/- cells was lower than that in the wild-type cells (Fig. 2C). However, forced Alpl expression rescued the expression of

Runx2 in Alpl-/- osteoblasts, and this subsequently restored the expression of Bglap2 and

Dmp1. Furthermore, overexpression of Runx2 in Alpl-/- cells increased Bglap2 and

Dmp1 expression to some extent; however, the increased expression was not equivalent to that observed in the wild-type cells, and calcification ability was not recovered (Fig.

2D–F). These results indicate that TNAP promotes osteoblast differentiation by enhancing RUNX2-mediated osteoblast gene expression.

[Insert figure 2 here]

3.4. Pi produced by TNAP induces Dmp1 expression independent of RUNX2

We found that Runx2 expression was reduced in Alpl-/- cells, and Alpl overexpression increased Runx2 expression. Next, we investigated whether TNAP exerts its osteo-promoting effects on osteoblasts independent of RUNX2. As Runx2-/- mice do not form calcified bone [6], we used dura mater-derived cells instead of calvarial osteoblasts. These Runx2-/- mouse-derived dura mater cells did not have the characteristics of osteoblasts (Fig. 3A). However, when Runx2 was overexpressed (Fig.

3B), normal ALP activity, calcification ability (Fig. 3A), and osteoblast differentiation

marker gene expression (Fig. 3C) were observed. These results suggest that the dura mater cells have a potential to differentiate into osteo-lineage cells. Compared to uninfected Runx2-/- cells, the Runx2-/- cells infected with an Alpl-coding lentivirus showed no changes in the expression of the osteogenic cell marker genes except Dmp1

(Fig. 4A). Dmp1 expression in the Alpl-overexpressing Runx2-/- cells was significantly higher than that in the uninfected Runx2-/- cells (Fig. 4B). TNAP hydrolyzes phosphate esters to produce Pi, which has been reported to induce Dmp1 expression in wild-type osteoblasts, osteocytes, and PDL cells [18–20]. Therefore, we used Runx2-/- cells to investigate whether Pi induces Dmp1 expression in the absence of RUNX2. High

-/- extracellular Pi (10 mM) concentration promoted Dmp1 expression in Runx2 cells, whereas Bglap2 expression remained suppressed (Fig. 4C). As Dmp1 deletion results in defective osteocyte maturation [21], our results suggest that TNAP promotes osteoblast differentiation via RUNX2 and promotes young osteocyte differentiation by increasing extracellular Pi concentration.

[Insert figures 3 and 4 here]

4. Discussion

In this study, we confirm the involvement of TNAP in the regulation of osteogenic cell differentiation. First, prolonged TNAP inhibition abolished TNAP activity even after completion of TNAP inhibition. Although levamisole is a reversible TNAP inhibitor, osteoblasts treated with levamisole for 7 days showed considerably reduced ALP activity even after removal of levamisole. This prolonged loss of TNAP activity may be caused by suppression of Alpl expression, degradation of TNAP protein, or both.

Reverse transcription real-time PCR and immunoblotting revealed that prolonged

levamisole treatment completely reduced ALP protein levels and strongly suppressed

Alpl expression. Furthermore, prolonged levamisole treatment suppressed the expression of Runx2 and Sp7, which encode the key transcription factors for osteoblast differentiation; Bglap2, which encodes a mature osteoblast marker; and Dmp1, which encodes an osteocyte marker. These results indicate that TNAP may play an important role in osteogenic cell differentiation. However, few studies have reported the mechanism underlying the precise regulatory role of TNAP in osteoblast differentiation.

Therefore, we performed lentivirus-based Alpl overexpression in wild-type osteoblasts and assessed the expression of osteoblast differentiation marker genes.

Alpl-overexpressing cells showed increased expression of Runx2, Sp7, Bglap2, and

Dmp1. In addition, forced Alpl expression in Alpl-/- cells restored calcification and osteoblast differentiation marker gene expression. These data indicate that TNAP functions as an osteoblast differentiation facilitator. Human TNAP encoded by the

ALPL gene has several isoforms, and the one expressed in the bone tissue is called bone alkaline phosphatase (BAP). BAP is used as an index of bone formation in clinical examinations and is considered useful as an indicator of osteoblast activity.

Enzyme replacement therapy in patients with HPP has been confirmed to improve bone mineralization and survival, and this further supports our finding that TNAP facilitates osteoblast differentiation [1]. Furthermore, successful systemic administration of Alpl- expressing viral vectors has been reported in animal models [11,13]. Although hypo-mineralization in patients with HPP and Alp-/- mice is considered to be caused by the insufficient Pi supply associated with TNAP deficiency, some reports have revealed that osteoblasts derived from patients with HPP show reduced osteocalcin expression

[22].

Furthermore, Alpl-overexpressing osteoblasts were reported to show increased bone mineralization in vivo [12], consistent with our in vitro results. On the basis of these results and the report that the transcription factor MSX2 controls BMP2-induced osteoblast differentiation without RUNX2 [23], we investigated whether TNAP regulates osteoblast differentiation in a RUNX2-independent manner. As calvariae are not formed in Runx2-/- mice because of lack of bone formation, we used dura mater cells instead. As observed in wild-type calvarial osteoblasts, BMP2-dependent osteoblast differentiation and calcification were observed in Runx2-/- dura mater cells infected with

Runx2 lentivirus. This suggests that dura mater cells are osteo progenitor cells. In

Runx2-/- dura mater cells, ALP-dependent upregulation of Sp7 and Bglap2 did not occur, suggesting that the effect of TNAP on osteoblast differentiation was exerted only in the presence of RUNX2. Although Runx2-/- mice lack osteoblasts, ALP is expressed in the dura mater and mid-shaft region of long bones [6]. Such ALP may contribute to the induction of osteoblast differentiation in wild-type mice.

Liu et al. demonstrated that TNAP promotes osteoblast differentiation by inducing

Runx2 expression [14] and that TNAP is involved in the regulation of mesenchymal stem cell senescence [24]. In our study, TNAP overexpression increased Runx2 expression, whereas TNAP deficiency decreased Runx2 expression. In TNAP-deficient

Alpl-/- cells, the forced expression of Runx2 resulted in a limited increase of Bglap2 expression (Fig. 2F). Interestingly, TNAP expression in Runx2-/- cells increased the expression of Dmp1, a selective differentiation marker for osteocytes. Next, we

-/- examined whether the Dmp1 expression in Runx2 cells is regulated by extracellular Pi.

Our results show that increase in extracellular Pi could induce Dmp1 expression even in the absence of Runx2 expression, indicating that TNAP-generated Pi controls osteocyte

differentiation in a RUNX2-independent manner. However, this observation is limited to young osteocytes because these osteocytes retain ALP activity and is not valid for bone-embedded mature osteocytes with no ALP activity.

Bisphosphonate (BP), a well-known antiresorptive agent, inhibits ALP [25], and BP administration reduces serum bone ALP protein levels [26]. Previous studies showed that long-term BP administration caused atypical femur fractures [27]. As BPs reduce bone remodeling, they might “freeze” the skeleton, allowing the accumulation of microcracks over time, thereby leading to fatigue fractures [28]. Hence, it is possible that prolonged BP treatment inhibited TNAP-mediated osteoblast differentiation, resulting in atypical femur fractures. Jo et al. [29] reported that ALP protein is abundant in the patients with ankylosing spondylitis characterized by excessive osteoblast function and that ALP may be responsible for the increased osteoblast function in such patients. Although TNAP is used as a marker for bone formation, TNAP abnormalities may be involved in the onset of skeletal disorders.

Since the substrate specificity of ALPs is low [2], various molecules are considered to be candidate substrates for promoting TNAP-mediated osteoblast differentiation.

Further extensive research is warranted to understand the molecular mechanisms underlying the TNAP-mediated differentiation of osteogenic cells. Nevertheless, our study provides novel insights into the role of TNAP in regulating osteoblast/osteocyte differentiation.

Acknowledgements

We thank Dr. Jose Luis Millán and Dr. Sonoko Narisawa (Sanford-Burnham Medical

Research Institute) for providing the Alpl-/- mice, Toshihisa Komori (Nagasaki

University) for providing the Runx2-/- mice, Dr. Hisataka Yasuda for providing BMP2, and Dr. Hiroyuki Miyoshi for providing the lentivirus plasmids.

Author contribution

T.N. designed the study, performed the experiments, analyzed the data, and drafted the manuscript. A.N.-T. and M.K. performed the experiments using Alpl-/- mice. A.Y. and

T.A. advised and drafted the manuscript.

Funding

This work was supported by Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (grant number: 18K09047) and Tokyo

Dental College Branding Project for Multidisciplinary Research Center for Jaw Disease from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and a grant from the Nakatomi Foundation.

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Figure legends

Fig. 1 Levamisole suppresses osteogenic differentiation in murine calvarial cells.

(A) Effect of levamisole on alkaline phosphatase (ALP) activity and mineralization.

Primary osteoblasts from wild-type mice were treated with levamisole for 7 and 21 days, followed by ALP and von Kossa staining, respectively. (B) Changes in ALP activity due to the difference in time of levamisole addition. The black horizontal bar indicates the time point for 1 mM levamisole addition. From top to bottom: no addition, 7 days, last 1 day, last 1 hour, added to ALP staining solution. (C) Alpl expression was observed at days 0, 7, 14, and 21. (D) Immunoblotting analysis for tissue-nonspecific ALP (TNAP) expression. (E) Effect of levamisole on the expression of osteoblast marker genes. Open bars, vehicle; closed bars, 1 mM levamisole.

Fig. 2 Runt-related transcription factor 2 (RUNX2)-induced osteogenic differentiation is decreased in Alpl-/- calvarial cells.

(A) Alpl-/- osteoblasts were infected with or without Alpl lentivirus, cultured with bone morphogenetic protein (BMP2; 100 ng/ml) for 7 or 21 days. The cells were subjected to alkaline phosphatase (ALP) or von Kossa staining. (B) Alpl expression in Alpl-/- calvarial cells infected with lentiviral vectors. (C) Expression of osteogenic marker genes in Alpl-/- osteoblasts infected with Alpl lentivirus. After BMP2 stimulation, Runx2 and Sp7 were analyzed on day 0, bone -carboxyglutamate protein 2 (Bglap2) on day 7, and dentin matrix protein 1 (Dmp1) on day 14. (D) Alpl-/- osteoblasts were infected with or without Runx2 lentivirus and subjected to ALP or von Kossa staining (E) Runx2 expression in Alpl-/- calvarial cells infected with lentiviral vectors. (F) Expression of osteogenic marker genes in Alpl-/- osteoblasts infected with Runx2 lentivirus.

Fig. 3 Runx2-/- dura mater-derived cells infected with runt-related transcription factor 2 (Runx2) lentivirus differentiate into osteoblasts.

(A) Runx2-/- dura mater cells were infected with or without Runx2) lentivirus, cultured with bone morphogenetic protein (BMP2; 100 ng/ml) for 7 or 21 days, and subjected to alkaline phosphatase (ALP) or von Kossa staining (B) Reverse transcription real-time

PCR analysis of Runx2 expression in Runx2-/- dura mater cells infected with lentiviral vectors. (C) Expression of osteogenic marker genes in Runx2-/- osteoblasts infected with

Runx2 lentivirus. After BMP2 stimulation, Runx2 and Sp7 were analyzed on day 0, bone

-carboxyglutamate protein 2 (Bglap2) on day 7, and dentin matrix protein (Dmp1) on day 14.

Fig. 4 Tissue-nonspecific alkaline phosphatase (TNAP) promotes osteoblast differentiation in the presence of runt-related transcription factor 2 (RUNX2) but induces dentin matrix protein (Dmp1) expression in a Pi-dependent manner.

(A) Alpl expression in Runx2-/- dura mater cells infected with lentiviral vectors. (B)

Expression of osteogenic marker genes in Runx2-/- osteoblasts infected with Alpl lentivirus. After BMP2 stimulation, Runx2 and Sp7 were analyzed on day 0, bone

-carboxyglutamate protein 2 (Bglap2) on day 7, and Dmp1 on day 14. (C) Expression

-/- of osteogenic marker genes in Runx2 osteoblasts cultured with 10 mM Pi (as sodium phosphate buffer). (D) Effect of Alpl overexpression or 10 mM Pi treatment on alkaline phosphatase (ALP) activity and mineralization in Runx2-/- dura mater cells. ALP and von Kossa staining were performed on days 7 and 21, respectively.