Role of MSX1 in Osteogenic Differentiation of Human Dental Pulp Stem Cells

Msh homeobox 1 (MSX1) encodes a transcription factor implicated in embryonic development of limbs and craniofacial tissues including bone and teeth. Although MSX1 regulates osteoblast differentiation in the cranial bone of young animal, little is known about the contribution of MSX1 to the osteogenic potential of human cells. In the present study, we investigate the role of MSX1 in osteogenic differentiation of human dental pulp stem cells isolated from deciduous teeth. When these cells were exposed to osteogenesis-induction medium, runt-related transcription factor-2 (RUNX2), bone morphogenetic protein-2 (BMP2), alkaline phosphatase (ALPL), and osteocalcin (OCN) mRNA levels, as well as alkaline phosphatase activity, increased on days 4–12, and thereafter the matrix was calcified on day 14. However, knockdown of MSX1 with small interfering RNA abolished the induction of the osteoblast-related gene expression, alkaline phosphatase activity, and calcification. Interestingly, DNA microarray and PCR analyses revealed that MSX1 knockdown induced the sterol regulatory element-binding protein 2 (SREBP2) transcriptional factor and its downstream target genes in the cholesterol synthesis pathway. Inhibition of cholesterol synthesis enhances osteoblast differentiation of various mesenchymal cells. Thus, MSX1 may downregulate the cholesterol synthesis-related genes to ensure osteoblast differentiation of human dental pulp stem cells.


Introduction
Msh homeobox 1 (MSX1) is a homeobox transcriptional factor involved in limb-pattern formation and craniofacial development and specifically in odontogenesis. Mouse Msx1 mutations cause craniofacial malformation and tooth agenesis [1]. Msx1-knockout mice show arrested tooth development at the bud stage and embryonic lethal defects [2]. Msx1 is expressed at high levels in craniofacial skeletal cells during early postnatal development [3], and transgenic mice expressing Msx1 under the control of the alpha (I) collagen promoter exhibit increased osteoblast number, cell proliferation, and apoptosis [4], suggesting Msx1 may have a role in craniofacial bone modeling. MSX1 is also expressed at high levels in the dental mesenchyme at the cap and bell stages [5] and may be a suppressor for cell differentiation that maintains mesenchymal cells in a proliferative state to ensure robust craniofacial and tooth development [6].

Stem Cells International
In addition, MSX1 is an upstream and downstream regulator for the bone morphogenetic protein BMP2/BMP4 signaling pathway [7,8]. Mutations in human MSX1 also cause cleft lip/palate and tooth agenesis [9,10]. However, the role of MSX1 in human craniofacial and tooth development has not been fully understood.
Dental pulp stromal cells isolated from whole pulp tissue can differentiate into osteoblasts, odontoblasts, endothelial cells, nerve cells, and adipocytes in vitro. Some of these cells identified by several cell surface antigens are referred to as dental pulp stem cells (DPSCs) [11,12]. DPSCs may play a role in dentinogenesis/osteogenesis in both developing and injured teeth. Furthermore, these cells are a promising source of cell-based regenerative therapies for dental, skeletal, vascular, and neuronal diseases [13,14]. Human DPSCs (hDPSCs) have not been fully characterized at the molecular level, but a previous reported showed that MSX1 is expressed at higher levels in hDPSCs than in bone marrow-derived mesenchymal stem cells and fibroblasts [15]. MSX1 may participate in the control of primary or secondary dentin formation and reparative dentin or osteodentin/bone formation in injured pulp tissue, in addition to the physiological role such as the maintenance of dental pulp stem/progenitor cells in healthy teeth. In the present study, we explored the role of MSX1 in pulpal mesenchymal cells using human DPSCs in culture.
Statins are a class of drugs that function as specific inhibitors of 3-hydoroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, a rate-limiting enzyme in cholesterol synthesis. Numerous studies have shown that statins exert bone anabolic effects in osteoblasts and osteogenic precursor cells [16,17]. Simvastatin enhances alveolar bone remodeling in the tooth extraction socket [18], enhances bone fracture healing [19], and reduces alveolar bone loss and tooth mobility in chronic periodontitis [20]. In addition, simvastatin enhances odontoblast/osteoblast differentiation of DPSCs and mesenchymal stem cells isolated from other tissues [17,21,22]. These studies indicate a close relationship between cholesterol synthesis and osteoblast differentiation.
Here, we demonstrated the role of MSX1 in osteoblast differentiation and cholesterol synthesis in hDPSCs using small interfering RNA (siRNA) against MSX1. DNA microarray analyses revealed that knockdown of MSX1 in hDPSCs undergoing osteogenic differentiation abolished the expression of various osteoblast-related genes but enhanced the expression of cholesterol synthesis-related genes. Our results suggest that MSX1 enhances osteoblast differentiation and calcification in hDPSCs through repression of cholesterol synthesis genes and induction of osteoblast-related genes.

Human DPSCs.
Extracted healthy deciduous teeth were collected from 6-12-year-old children following protocols approved by the ethical authorities at Hiroshima University (permit number: D88-2). Written informed consent was obtained from the subject or subject's parent. Pulp tissue specimens from deciduous teeth were minced and digested with 3 mg/mL collagenase type I (Life Technologies, Carlsbad, CA, USA) and 4 mg/mL dispase (Roche Diagnostics, Mannheim, Germany) in Dulbecco's modified Eagle's medium (DMEM; Sigma, St. Louis, MO, USA) for 1 h at 37 ∘ C. Single cell suspension was obtained by passing cells through a 70 m cell strainer (CORNING, Corning, NY, USA). The cells were incubated in DMEM supplemented with 20% fetal bovine serum (FBS; Biowest, Nuaillé, France) and 1% penicillin-streptomycin (Life Technologies) at 37 ∘ C in 95% air and 5% CO 2 [23]. Forming colonies were separated by incubation with Accutase (Funakoshi Co., Ltd., Tokyo, Japan), and isolated cells were transferred to passage cultures with DMEM supplemented by 10% FBS and 1% penicillinstreptomycin. The culture medium was changed every 2 days. Cells at passages 3-9 were used in subsequent experiments.
Human DPSCs were seeded at 5 × 10 4 cells/well in 24multiwell plates coated with type I collagen with 0.5 mL DMEM supplemented with 10% FBS. After 24 h, siRNA was transfected into cells with Lipofectamine 2000 (Life Technologies) and cells were incubated for an additional 48 h.
10 min and stained with 1% alizarin red S for 30 min. The cellmatrix layers were washed 6 times with sterile water.

Alkaline Phosphatase Activity.
Human DPSCs were washed twice with saline and homogenized ultrasonically with 1% NP-40 in saline. Alkaline phosphatase activity was determined using Lab Assay ALP (Wako, Osaka, Japan). DNA concentration was determined with the Quant-iT6 PicoGreen dsDNA Assay Kit (Life Technologies) to calculate alkaline phosphatase activity/ g DNA.

Reverse Transcription-Quantitative Polymerase Chain
Reaction (RT-qPCR). Total RNA was isolated and cDNA was synthesized as described [15]. The cDNA samples were amplified using Universal PCR Master Mix (Life Technologies) with primers (Table 1) and TaqMan probes were purchased from Roche Diagnostics (Basel, Switzerland). GAPDH primers/probe set was used for normalization. After amplification of DNA, expression levels were determined with the ABI prism 7900 HT sequence detection system (Life Technologies).

DNA Microarray.
After the induction of osteogenic differentiation for 4 days, total RNA was isolated from MSX1-knockdown and control hDPSCs using TRIzol (Life Technologies, Japan) and an RNeasy Mini Kit (Qiagen, Chatsworth, CA). DNA microarray analysis was performed using the SurePrint G3 Human GE 8 × 60 K v2 Microarray (Agilent Technologies, Santa Clara, CA, USA). Raw data were standardized by the global median normalization method using GeneSpring (Silicon Genetics, Redwood City, CA, USA). The raw data were deposited in the Gene Expression Omnibus database (GSE69992).

Statistical Analysis.
Results are expressed as mean ± SD. Differences between two groups were analyzed by two-way ANOVA with Tukey's post hoc test for multiple comparisons. In all analyses, < 0.05 indicated statistically significant differences between values.

Mesenchymal Stem Cell Markers Expressed in Cultured
hDPSCs. Human DPSCs from postnatal human primary teeth were used to explore the functional role of MSX1. These cells exhibited a fibroblastic shape (Figure 1(a)) and showed expression of mesenchymal stem cell surface markers CD73 (>90%), CD90 (>90%), CD105 (>10%), and CD166 (>30%) (Figure 1(b)) as expected from previous studies [24]. Relative mRNA level * * * * (b) Figure 2: Effects of MSX1 knockdown on calcification of hDPSCs. hDPSCs were transfected with either MSX1 siRNA (s8999 or s224066) or control siRNA and incubated for 2 days in growth medium before the cultures became confluent. Thereafter, the cultures were exposed to osteogenesis-induction medium. different siRNAs for MSX1 or control siRNA and then exposed to osteogenesis-induction medium. Both siRNA oligonucleotides targeting MSX1 (s8999 and s224066) abolished MSX1 mRNA expression at 48 h and subsequent matrix calcification on day 14 ( Figure 2). We selected MSX1 siRNA (s8999) for subsequent studies. Next, we examined the effect of MSX1 knockdown on alkaline phosphatase activity and the expression of osteoblast-related genes in hDPSCs after the onset of osteogenesis ( Figure 3). In hDPSCs transfected with control siRNA, alkaline phosphatase activity and RUNX2, BMP2, osterix (OSX), osteocalcin (OCN; also known as BGLAP), and alkaline phosphatase liver type (ALPL) mRNA levels increased on days 4-12 after the onset of differentiation. MSX1 mRNA levels also increased on days 4-12. However, MSX1 knockdown abolished the induction of alkaline phosphatase activity (Figure 3(a)) and the increases in ALPL, RUNX2, BMP2, OCN, and MSX1 mRNA levels, although it further increased OSX mRNA levels ( Figure 3(b)). It should be noted that the incubation with MSX1 siRNA abolished MSX1 expression at least until day 12 after the onset of osteogenic differentiation.

MSX1 Knockdown Abolishes Osteogenic
Next, we examined whether MSX1 knockdown might influence the expression of other master genes including a master regulator of chondrogenesis SOX9 and a master regulator of adipogenesis PPAR (Figure 4). In control hDPSCs, no significant changes in the expressions of SOX9 and PPAR were observed after the exposure to osteogenesis-induction medium. Under these conditions, MSX1 knockdown increased the expression of PPAR on days 4-8, although it had little effect on the expression level of SOX9.

MSX1 Knockdown Downregulated and Upregulated a Variety of Genes.
To characterize the effects of MSX1 knockdown on osteogenic differentiation, we performed DNA microarray analyses on day 4 after exposure to osteogenesisinduction medium. MSX1 knockdown decreased and increased mRNA levels of 2923 and 3480 genes, respectively, which were selected with cut-off values of >1.5-fold change and -test < 0.05. Tables 2 and 3 show lists of downregulated and upregulated genes in MSX1-knockdown hDPSCs, respectively.
To understand MSX1 actions in hDPSCs differentiating into osteoblasts, we performed a gene-set approach using the 2923 downregulated and 3480 upregulated genes. The WikiPathways analysis showed that the MSX1 knockdown downregulated various genes involved in focal adhesion, endochondral ossification, integrin-mediated cell adhesion, matrix metalloproteinases, calcium regulation, and insulin signaling (Table 4), whereas it upregulated genes involved in sterol regulatory element-binding protein (SREBP) signaling, cholesterol biosynthesis, adipogenesis, and fatty acid biosynthesis (Table 5). These findings revealed that MSX1 regulates various cellular processes in hDPSCs differentiating into osteoblasts.

Discussion
Previous studies showed that mouse MSX1 was implicated in craniofacial bone development [1,2,4]. In mouse embryos, MSX1 suppresses precocious differentiation and calcification in dental mesenchymal cells and maintains these cells in a proliferative state to ensure subsequent craniofacial and tooth development [6,26]. High levels of osteoblast number, cell proliferation, and apoptosis in MSX1 transgenic mice suggest that MSX1 modulates mouse craniofacial bone modeling [4]. However, the role of MSX1 in human cells remains poorly understood. In the present study, we demonstrated that MSX1 plays an essential role in osteogenic differentiation of hDPSCs. In human DPSC cultures, MSX1 knockdown resulted in suppressed expression of RUNX2, ALPL, BMP2, and OCN. These results demonstrate that MSX1 modulated the major signaling/transcriptional pathways regulating hard tissue differentiation to enhance osteogenic potential of hDPSCs. However, MSX1 knockdown unexpectedly increased the mRNA level of OSX, another transcriptional factor involved in osteoblast maturation. This indicates MSX1 does not activate the entire osteogenesis program, perhaps because MSX1 cooperates with other transcription factors to fully control osteogenesis. MSX1 knockdown enhanced PPAR expression under the osteogenesis-induced condition, suggesting that MSX1 negatively regulates adipogenic differentiation. MSX1 may direct hDPSCs into the osteoblast lineage by preventing them from differentiating into the adipogenic lineage. MSX1 knockdown also resulted in downregulation of various genes involved in focal adhesion, integrin-mediated cell adhesion, matrix metalloproteinases, calcium regulation, insulin signaling, and other processes. The extensive effect of MSX1 knockdown on the entire gene expression profile emphasizes a crucial role of MSX1 in hDPSCs undergoing differentiation into osteoblasts.
Bidirectional transcription of the Msx1 gene has been previously reported [27][28][29]. In embryonic and newborn mice, sense and antisense Msx1 transcripts are differently expressed during development. In 705IC5 mouse odontoblasts, overexpression of Msx1 antisense RNA decreased the expression of Msx1 sense transcript, whereas overexpression of Msx1 sense RNA increased Msx1 antisense transcript. Thus, expression of mouse Msx1 is controlled by the balance of the two transcripts. In our experiments, however, MSX1 antisense transcript was not detected during osteogenic differentiation of hDPSCs irrespective of siRNA knockdown of MSX1 (data not shown). The presence of MSX1 antisense transcript in humans has so far been reported only in the embryo. Therefore, Msx1 antisense RNA does not seem to be involved in the MSX1 expression in hDPSCs. Under these conditions, the expression of Msx1 sense transcripts was markedly depressed in hDPSCs after treatment with MSX1 siRNA, indicating that the knockdown experiments worked appropriately regardless of the presence or absence of the Msx1 antisense transcript.
Statins, drugs for hyperlipidemia, enhance osteogenic differentiation of various mesenchymal cells, including osteoblast precursor cells, mesenchymal stem cells, and DPSCs, by inhibiting the synthesis of farnesyl pyrophosphate, decreasing cellular cholesterol, and activating the Ras-PI3K-Akt/MAPK signaling pathway, thereby increasing the expression of BMP2 and RUNX2 [17], although the underlying mechanisms are still controversial. Statins also suppress osteoclast function and enhance mandibular bone formation in vivo [40]. Interestingly, a previous study showed that simvastatin induces odontoblast differentiation of hDPSCs in vitro and in vivo [22]. However, no studies have shown the involvement of transcription factor(s) in the control of cholesterol synthesis during osteoblast differentiation. Here we found for the first time that MSX1 suppresses the entire cholesterol synthesis pathway in osteoblast differentiating hDPSCs by repressing SREBP2 and other related genes. This suppression of cholesterol synthesis may facilitate osteoblast differentiation. It is also interesting to note that various mutations in the cholesterol synthesis pathway, including 7dehydrocholesterol reductase (DHCR7), cause craniofacial anomalies including cleft palate, suggesting the role of cholesterol synthesis in craniofacial development [41].
In conclusion, here we revealed for the first time that MSX1 is indispensable for osteoblast-like differentiation and calcification in hDPSCs derived from deciduous teeth. Furthermore, MSX1 was found to modulate a wide variety of genes, including cholesterol synthesis-related genes, during osteogenic differentiation of hDPSCs. We have not examined the effects of MSX1 siRNA in definitive teeth, although MSX1 may also function as a positive regulator of osteogenesis in definitive teeth as MSX1 mRNA levels are high in both definitive and deciduous teeth [15]. Our findings will provide new insights into the role of MSX1 in development and repair of teeth and may be useful in DPSC-based regenerative therapy.