7-HYB, a Phenolic Compound Isolated from Myristica fragrans Houtt Increases Cell Migration, Osteoblast Differentiation, and Mineralization through BMP2 and β-catenin Signaling

The seeds (nutmegs) of Myristica fragrans Houtt have been used as popular spices and traditional medicine to treat a variety of diseases. A phenolic compound, ((7S)-8′-(benzo[3′,4′]dioxol-1′-yl)-7-hydroxypropyl)benzene-2,4-diol (7-HYB) was isolated from the seeds of M. fragrans. This study aimed to investigate the anabolic effects of 7-HYB in osteogenesis and bone mineralization. In the present study, 7-HYB promotes the early and late differentiation of MC3T3-E1 preosteoblasts. 7-HYB also elevated cell migration rate during differentiation of the preosteoblasts with the increased phosphorylation of mitogen-activated protein kinases (MAPKs) including ERK1/2, p38, and JNK. In addition, 7-HYB induced the protein level of BMP2, the phosphorylation of Smad1/5/8, and the expression of RUNX2. 7-HYB also inhibited GSK3β and subsequently increased the level of β-catenin. However, in bone marrow macrophages (BMMs), 7-HYB has no biological effects in cell viability, TRAP-positive multinuclear osteoclasts, and gene expression (c-Fos and NF-ATc1) in receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis. Our findings suggest that 7-HYB plays an important role in osteoblast differentiation through the BMP2 and β-catenin signaling pathway. It also indicates that 7-HYB might have a therapeutic effect for the treatment of bone diseases such as osteoporosis and periodontitis.


Introduction
Bone is a dynamically mineralized connective tissue that is broken down and re-formed throughout life through complex events [1,2]. Bone metabolism is dependent on the balance between osteoblast-mediated formation and osteoclast-mediated bone resorption during the physiological process of bone remodeling in the adult skeleton [3,4]. Metabolic bone diseases such as osteoporosis and periodontitis are mainly characterized by defective or excessive bone formation, and these pathogenesis are caused by dysregulation in the commitment, differentiation, and survival of osteoblast lineages as well as the impaired differentiation and function of osteoclasts [5,6]. However, the limitation of safe and efficient drugs regulating the number and function of osteoblasts and osteoclasts makes it much more difficult to treat bone diseases [5,7,8]. In this context, it is important to identify potential compounds based on the molecular and cellular mechanisms underlying pathogenesis to translate this knowledge into efficient bone disease therapy.
In the present study, we examined intracellular signaling and mechanisms underlying the biological function of 7-HYB on the survival, migration, and differentiation of MC3T3-E1 preosteoblasts and bone marrow macrophages (BMMs) as an in vitro cell system. Our data present 7-HYB as a potential phenolic compound to treat bone diseases such as osteoporosis and periodontitis.

7-HYB Has No Effect on the Cell Toxicity in Preosteoblasts
((7S)-8 -(benzo [3 ,4 ]dioxol-1 -yl)-7-hydroxypropyl)benzene-2,4-diol (7-HYB) was isolated from the seeds of Myristica fragrans and the HPLC chromatogram and structure of 7-HYB are shown in Figure 1A,B. To test the effects of 7-HYB on the viability of preosteoblasts, the cells were treated with 0.1-100 µM 7-HYB for 24 h. 7-HYB did not affect cell viability except for 100 µM ( Figure 1C). For the following experiments, we used the dose of 7-HYB below 100 µM. mechanisms underlying pathogenesis to translate this knowledge into efficient bone disease therapy.
In the present study, we examined intracellular signaling and mechanisms underlying the biological function of 7-HYB on the survival, migration, and differentiation of MC3T3-E1 preosteoblasts and bone marrow macrophages (BMMs) as an in vitro cell system. Our data present 7-HYB as a potential phenolic compound to treat bone diseases such as osteoporosis and periodontitis.

7-HYB Promotes the Early Osteoblast Differentiation of Preosteoblasts
In order to investigate whether 7-HYB affects osteoblast differentiation, 7-HYB was treated with osteogenic supplement medium (OS) containing 50 µg/mL L-ascorbic acid (L-AA) and 10 mM β-glycerophosphate (β-GP) for seven days. Alkaline phosphatase (ALP) staining was observed to detect the early differentiation of preosteoblasts using a digital camera and colorimetric detector. The ALP staining showed that 7-HYB promoted the early osteoblasts differentiation in a dose dependent manner (Figure 2A). Using a light microscope, we also confirmed that 7-HYB increased ALP-stained cells in a dose dependent manner ( Figure 2B). In addition, 7-HYB also significantly elevated the ALP enzymatic activity in a dose dependent manner, which was similar to results of ALP staining ( Figure 2C).

7-HYB Promotes the Early Osteoblast Differentiation of Preosteoblasts
In order to investigate whether 7-HYB affects osteoblast differentiation, 7-HYB was treated with osteogenic supplement medium (OS) containing 50 μg/mL L-ascorbic acid (L-AA) and 10 mM β-glycerophosphate (β-GP) for seven days. Alkaline phosphatase (ALP) staining was observed to detect the early differentiation of preosteoblasts using a digital camera and colorimetric detector. The ALP staining showed that 7-HYB promoted the early osteoblasts differentiation in a dose dependent manner (Figure 2A). Using a light microscope, we also confirmed that 7-HYB increased ALP-stained cells in a dose dependent manner ( Figure 2B). In addition, 7-HYB also significantly elevated the ALP enzymatic activity in a dose dependent manner, which was similar to results of ALP staining ( Figure 2C).

7-HYB Enhances the Late Osteoblast Differentiation of Preosteoblasts
To further demonstrate the effects of 7-HYB in osteoblast differentiation, Alizarin red S (ARS) staining was performed to detect the late differentiation of preosteoblasts and we observed the

7-HYB Enhances the Late Osteoblast Differentiation of Preosteoblasts
To further demonstrate the effects of 7-HYB in osteoblast differentiation, Alizarin red S (ARS) staining was performed to detect the late differentiation of preosteoblasts and we observed the degree of matrix mineralization using a scanner and colorimetric detector at seven and 14 days. The mineralized nodule was formed at 14 days, ARS staining exhibited that 7-HYB promoted the late osteoblasts differentiation in a dose dependent manner ( Figure 3A,B). To confirm the observation, we visualized and quantified ARS staining. The results revealed that the mineralized nodule formation was significantly increased by 7-HYB in a dose-dependent manner ( Figure 3C,D). degree of matrix mineralization using a scanner and colorimetric detector at seven and 14 days. The mineralized nodule was formed at 14 days, ARS staining exhibited that 7-HYB promoted the late osteoblasts differentiation in a dose dependent manner ( Figure 3A,B). To confirm the observation, we visualized and quantified ARS staining. The results revealed that the mineralized nodule formation was significantly increased by 7-HYB in a dose-dependent manner ( Figure 3C,D).

7-HYB Increases Cell Migration in Osteoblast Differentiation of Preosteoblasts
We next asked whether cell migration could be regulated by 7-HYB during osteoblast differentiation. In wound healing migration assay, the induction of osteoblast differentiation increased cell migration rate, and the closure rate of the cells forward the wound area was significantly accelerated by the treatment of 7-HYB in a dose dependent manner ( Figure 4A,B). Under the same condition, we subsequently examined the involvement of mitogen-activated protein kinases (MAPKs) in 7-HYB-mediated cell migration. 7-HYB obviously increased the phosphorylation of ERK1/2, p38, and JNK ( Figure 4C). We next asked whether cell migration could be regulated by 7-HYB during osteoblast differentiation. In wound healing migration assay, the induction of osteoblast differentiation increased cell migration rate, and the closure rate of the cells forward the wound area was significantly accelerated by the treatment of 7-HYB in a dose dependent manner ( Figure 4A,B). Under the same condition, we subsequently examined the involvement of mitogen-activated protein kinases (MAPKs) in 7-HYB-mediated cell migration. 7-HYB obviously increased the phosphorylation of ERK1/2, p38, and JNK ( Figure 4C).

7-HYB Stimulates BMP2-Smad1/5/8-RUNX2 and β-catenin Signaling in Osteoblast Differentiation
To further elucidate the mechanisms underlying the stimulatory effects of 7-HYB on the differentiation of preosteoblasts, bone morphogenetic protein (BMP)-Smad1/5/8-RUNX2 signaling was examined during osteoblast differentiation. 7-HYB increased the level of BMP2 protein, the phosphorylation of Smad1/5/8 protein, and the expression of RUNX2, which is a key transcription factor during osteoblast differentiation ( Figure 5A,C). We also investigated β-catenin signaling during osteoblast differentiation. These result revealed increases in the phosphorylation of GSK3β and the level in response to the treatment of 7-HYB ( Figure 5B,D).  To further elucidate the mechanisms underlying the stimulatory effects of 7-HYB on the differentiation of preosteoblasts, bone morphogenetic protein (BMP)-Smad1/5/8-RUNX2 signaling was examined during osteoblast differentiation. 7-HYB increased the level of BMP2 protein, the phosphorylation of Smad1/5/8 protein, and the expression of RUNX2, which is a key transcription factor during osteoblast differentiation ( Figure 5A,C). We also investigated β-catenin signaling during osteoblast differentiation. These result revealed increases in the phosphorylation of GSK3β and the level in response to the treatment of 7-HYB ( Figure 5B,D).

7-HYB does not Affect the Cell Toxicity in Bone Marrow Macrophages (BMMs), Premature Osteoclasts, and Mature Osteoclasts
To assess the biological effects of 7-HYB on osteoclastogenesis, we firstly examined cell viability in BMMs, preosteoclasts, and mature osteoclasts. At concentrations ranging from 10 to 30 μM of 7-HYB, no cytotoxic effects were observed in the BMMs (Figure 6A), and under RANKL-

7-HYB Does Not Affect the Cell Toxicity in Bone Marrow Macrophages (BMMs), Premature Osteoclasts, and Mature Osteoclasts
To assess the biological effects of 7-HYB on osteoclastogenesis, we firstly examined cell viability in BMMs, preosteoclasts, and mature osteoclasts. At concentrations ranging from 10 to 30 µM of 7-HYB, no cytotoxic effects were observed in the BMMs (Figure 6A), and under RANKL-induced osteoclast differentiation at three days (premature osteoclasts) and five days (mature osteoclasts) ( Figure 6B,C). induced osteoclast differentiation at three days (premature osteoclasts) and five days (mature osteoclasts) ( Figure 6B,C).

7-HYB has no Biological Activities on TRAP-Positive Multinucleated Osteoclasts (MNCs) and Gene Expression in RANKL-Induced Osteoclastogenesis
We next investigated the effects of 7-HYB on osteoclastogenesis of BMMs. After BMMs were incubated with RANKL in the absence and presence of 7-HYB at concentrations of 10 and 30 μM for five days, osteoclast differentiation was detected by using TRAP assays. As shown in Figure 7A-C, 7-HYB did not affect TRAP staining, and TRAP-positive multinucleated osteoclasts (MNCs) compared to the number of 3-10 nuclei and 10 < nuclei. We further examined whether 7-HYB influenced osteoclast-related gene expression in RANKL-induced osteoclastogenesis. The results

7-HYB Has No Biological Activities on TRAP-Positive Multinucleated Osteoclasts (MNCs) and Gene Expression in RANKL-Induced Osteoclastogenesis
We next investigated the effects of 7-HYB on osteoclastogenesis of BMMs. After BMMs were incubated with RANKL in the absence and presence of 7-HYB at concentrations of 10 and 30 µM for five days, osteoclast differentiation was detected by using TRAP assays. As shown in Figure 7A-C, 7-HYB did not affect TRAP staining, and TRAP-positive multinucleated osteoclasts (MNCs) compared to the number of 3-10 nuclei and 10 < nuclei. We further examined whether 7-HYB influenced osteoclast-related gene expression in RANKL-induced osteoclastogenesis. The results also showed that 7-HYB did not affect gene expression of c-Fos and NF-ATc1 ( Figure 7D,E).

Discussion
Osteoblast lineage cells play a critical role in the bone formation mainly through three steps: Proliferation, when preosteoblasts increase numerically, differentiation, when preosteoblasts become osteoblasts, and matrix mineralization when mature osteoblasts form new bone matrix [2]. Dysregulation in these steps is one of pathogenesis in bone diseases such as osteoporosis [2]. In the

Discussion
Osteoblast lineage cells play a critical role in the bone formation mainly through three steps: Proliferation, when preosteoblasts increase numerically, differentiation, when preosteoblasts become osteoblasts, and matrix mineralization when mature osteoblasts form new bone matrix [2]. Dysregulation in these steps is one of pathogenesis in bone diseases such as osteoporosis [2]. In the present study, we first found the biological effects of 7-HYB in preosteoblast. 7-HYB potentiated the expression and enzymatic activity of ALP, and enhanced mineralized nodule formation during differentiation of preosteoblasts. It was reported that ALP activity is an early differentiation marker of osteoblast lineage cells, which induces and regulates specific osteoblast genes. Mature osteoblasts subsequently form matrix mineralization by calcium deposition [16][17][18]. Therefore, the findings of this study indicate that 7-HYB increases the early and late differentiation of preosteoblasts, leading to differentiation into mature osteoblasts responsible for bone formation.
During the recent years, several growth factors with positive effects on osteoblast linage cells have been identified [8]. Among these factors, BMPs have long been recognized for their function to increase the differentiation of osteoblast linage cells [19,20]. Especially, BMP2 induces bone formation in osteoblasts linage cells via interaction with BMP receptor IA (BMPRIA) or BMPRIB, and BMPRII [21]. In the present study, the treatment of 7-HYB enhanced the level of BMP2 protein. It was also reported that BMP2signaling activates Smad1/5/8 and forms complexes between Smad1/5/8 and Smad4. The complexes are translocated into nucleus and induce the transcription of RUNX2 that is a key transcription factor in the differentiation of osteoblast linage cells [21][22][23]. Thus, we also investigated BMP2 signaling molecules and our results demonstrated that 7-HYB obviously increases the phosphoryation of Smad1/5/8 and the expression of RUNX2. The signaling pathway also induces ALP expression during differentiation of osteoblasts lineage cells [24,25]. These data suggest that 7-HYB regulates osteoblast differentiation via BMP2 signaling.
Bone formation, remodeling, and maintenance are a physiologically complex process, and a series of events occur including cell migration of osteoblast lineage cells [37,38]. It is well known that the Wnt/β-catenin signaling pathway participates in the regulation of cell migration and differentiation [28,39,40]. The non-canonical BMP2 signaling pathway also is involved in the regulation of cell migration and differentiation in osteoblast lineage cells through the activation of MAPKs such as ERK1/2, p38, and JNK1/2 [41][42][43][44]. In the present study, we found that 7-HYB increased cell migration during the differentiation of preosteoblasts. In addition, 7-HYB activates MAPKs including ERK1/2, p38. Thus, these results suggest that 7-HYB promotes cell migration and differentiation through the BMP2 and Wnt/β-catenin signaling pathways.
In conclusion, we first demonstrated that 7-HYB isolated from the seeds of Myristica fragrans plays an important role in migration, differentiation, and mineralization in osteoblasts. Natural compounds have been used to treat a variety of disease and have increasingly attracted interest in the treatment and prevention of bone diseases [2,45,46]. Recently, there was the rapid evolution of laser technology in dentistry, and laser therapy has shown beneficial effects [47][48][49]. Thus, the combination of natural compounds and laser therapy could be a possible clinical approach. Furthermore, our finding provides that 7-HYB might be useful sources for development of new drugs that could be used to treat bone diseases such as osteoporosis and periodontal disease.

Nuclear Magnetic Resonance (NMR)
Nuclear magnetic resonance (NMR) experiments were performed on a JEOL ECX-500 spectrometer, operating at 500 MHz for 1 H and 125 MHz for 13 C NMR spectrum (JEOL Ltd., Akishima, Japan). All chemical shifts were referenced relative to the corresponding signals (δ H 3.31/δ C 49.15 for CD 3 OD). Electron ionization mass spectrometer (EI-MS) data were obtained using micromass spectrum (AUTOSPEC, Glasgow, UK). High performance liquid chromatography (HPLC) was performed using Agilent 1200 series (Agilent Technologies, CA, USA). The silica gel 60 (Merck 230-400 mesh, ASTM, Germany) and ODS-A (Merck ASTM, Germany) were used for column chromatography.

Western Blot Analysis
Western blot analysis was carried out as previously described [52]. Briefly, equal amounts of proteins (20 µg) transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA) were blocked for 1 h at room temperature and incubated overnight at 4 • C with the primary antibodies. The membrane incubated with diluted horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000, Jackson ImmunoResearch, West Grove, PA, USA) for 2 h at room temperature was detected using the ProteinSimple detection system (ProteinSimple Inc., Santa Clara, CA, USA).

Cell Migration Assay
Cell migration was accessed using an in vitro wound healing assay as previously described [53]. Briefly, the cells were wounded with a 200 µL pipette tip and cultured in the absence and presence of 7-HYB for 24 h at 37 • C in a humidified atmosphere of 5% CO 2 and 95% air. Cell migration was observed under light microscopy and cell migration rate was quantified.

Alkaline Phosphatase (ALP) Staining Assay
Cells were washed with 1 × PBS and then fixed in 10% formalin for 15 min at room temperature. After washing with distilled water, the cells were incubated with substrate solution for the reaction of ALP at 37 • C for 1 h, followed according to the manufacturer's protocol (Takara Bio Inc., Shiga, Japan) as previously described [50].

ALP Activity Assay
The cell lysates were performed according to the manufacturer's protocol using alkaline phosphatase activity colorimetric assay kit (Biovision, Milpitas, CA, USA) as previously described [50]. The absorbance was measured at 405 nm using the Multiskan GO microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Alizarin Red S (ARS) Staining
Cells were fixed in 10% formalin for 15 min and rinsed with distilled water. Cells were stained with 2% ARS (pH 4.2) (Sigma-Aldrich) for 10 min with gentle agitation. The level of ARS staining was observed using a scanner and colorimetric detector (ProteinSimple Inc., Santa Clara, CA, USA). After scanning the stained wells, stains were dissolved in 100% DMSO and the absorbance was measured at 590 nm using the Multiskan GO Microplate Spectrophotometer (Thermo Fisher Scientific).

Immunocytochemistry
Immunocytochemistry was performed as previously described [53]. Briefly, the cells were blocked with 3% BSA diluted in PBS for 1 h and incubated with specific primary antibodies overnight at 4 • C. Subsequently, the cells were incubated with an antirabbit secondary antibody labeled with Alexa-Fluor 488 (1:500 dilution, Invitrogen, Carlsbad, CA, USA) for 2 h at room temperature. Next, the cells were incubated with 4 ,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for 10 min at room temperature. The cells were washed three times, mounted, and viewed on a confocal microscope (K1-Fluo Confocal Laser Scanning Microscope, Republic of Korea).

Live Subject Statement
All mice used in this study were maintained in accordance with the National Institute of Toxicological Research of the Korea Food and Drug Administration guidelines for the humane care and use of laboratory animals. All protocols in the current study were approved by the Chungbuk National University Institutional Animal Care and Use Committee (IACUC) (CBNUA-792-15-01) and complied with the Korean National Institute of Health Guide for the Care and Use of Laboratory Animals.

Culture of Bone Marrow Macrophages, and Osteoclast Differentiation
Mouse bone marrow cells isolated from five-week-old mice were cultured dishes in α-MEM (WELGEME) containing 10% FBS), penicillin (100 units/mL), and streptomycin (100 µg/mL) at 37 • C overnight in a humidified atmosphere of 5% CO 2 and 95% air. The next day, adherent cells were discarded, and floating cells were further incubated with M-CSF (30 ng/mL) on Petri dishes. After three days, BMMs became adherent, and then the cells were incubated with RANKL (100 ng/mL) and M-CSF (30 ng/mL) until five days to induce osteoclast differentiation.

Tartrate-Resistant Acid Phosphatase (TRAP) Staining
BMMs were differentiated into osteoclasts for five days, fixed with 10% formalin for 30 min, and washed with 1 X PBS. Then, the cells were stained for TRAP according to the manufacturer's protocol (Takara Bio Inc.). The TRAP-positive multinucleated cells (MNCs) were counted as mature osteoclasts using a light microscope.

Quantitative Real-Time Polymerase Chain Reaction (PCR) Analysis
Total RNA was extracted using the RNAqueous ® kit and cDNA synthesized from RNA (1 µg) using the high-capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol. Quantitative real-time PCR was performed using a 7500 Real-Time PCR System (Applied Biosystems).

Statistical Analysis
The data were analyzed using Prism Version 5 program (GraphPad Software, Inc., San Diego, CA). All numeric values are presented as the means ± S.E.M. The statistical significance of data was determined using a Student's unpaired t test. A value of p < 0.05 was considered to indicate statistical significance.