Effects of PIN on Osteoblast Differentiation and Matrix Mineralization through Runt-Related Transcription Factor

Styrax Japonica Sieb. et Zucc. has been used as traditional medicine in inflammatory diseases, and isolated compounds have shown pharmacological activities. Pinoresinol glucoside (PIN) belonging to lignins was isolated from the stem bark of S. Japonica. This study aimed to investigate the biological function and mechanisms of PIN on cell migration, osteoblast differentiation, and matrix mineralization. Herein, we investigated the effects of PIN in MC3T3-E1 pre-osteoblasts, which are widely used for studying osteoblast behavior in in vitro cell systems. At concentrations ranging from 0.1 to 100 μM, PIN had no cell toxicity in pre-osteoblasts. Pre-osteoblasts induced osteoblast differentiation, and the treatment of PIN (10 and 30 μM) promoted the cell migration rate in a dose-dependent manner. At concentrations of 10 and 30 μM, PIN elevated early osteoblast differentiation in a dose-dependent manner, as indicated by increases in alkaline phosphatase (ALP) staining and activity. Subsequently, PIN also increased the formation of mineralized nodules in a dose-dependent manner, as indicated by alizarin red S (ARS) staining, demonstrating positive effects of PIN on late osteoblast differentiation. In addition, PIN induced the mRNA level of BMP2, ALP, and osteocalcin (OCN). PIN also upregulated the protein level of BMP2 and increased canonical BMP2 signaling molecules, the phosphorylation of Smad1/5/8, and the protein level of Runt-related transcription factor 2 (RUNX2). Furthermore, PIN activated non-canonical BMP2 signaling molecules, activated MAP kinases, and increased β-catenin signaling. The findings of this study indicate that PIN has biological roles in osteoblast differentiation and matrix mineralization, and suggest that PIN might have anabolic effects in bone diseases such as osteoporosis and periodontitis.


Introduction
Osteoporosis is a systemic metabolic bone disease characterized by low bone mass and strength with fragility fractures and low-trauma fractures, which has recently become a major clinical problem [1]. It is also recognized as a multifactorial chronic systemic disease [2]. Bone fragility is caused by inadequate bone formation and excessive bone resorption, leading to decreased bone mass and micro architectural deterioration of bone tissue [1]. Osteoblasts, specialized cells derived from mesenchymal stem cells (MSCs), are essential for bone formation and maintenance, and osteoblast dysfunction causes pathological fragility and diseases such as osteoporosis and periodontitis [3]. Rovert et al. reported the anabolic effects of osteogenic compounds on human mesenchymal progenitor-derived osteoblasts and suggested the possibility of bone tissue engineering and bone diseases [4]. Anabolic treatments for osteoporosis increase the differentiation and maturation of osteoblast by altering bone metabolism [5,6]. However, safe and efficient drugs in improving osteoblast dysfunction and bone regeneration are limited, and it is much more difficult to treat bone diseases [3,[6][7][8]. In recent years, there interest has grown in natural compounds to treat bone diseases due to low adverse effects and suitably long-term treatment when compared with chemically synthesized drugs [9,10]. Therefore, it is important to identify potential compounds to prevent and treat bone diseases.
Styrax Japonica Sieb. et Zucc. is a member of the Styracaceae family, which is a deciduous tree grown in the South Korea and Japan, including Central America, Mexico, and the Mediterranean region [11,12]. It has been used as traditional medicine in inflammatory diseases as well as in soap and cough medicine [13]. Previous studies on natural compounds isolated from the stem bark of S. Japonica demonstrated that norlignan and styraxlignolide significantly enhance anti-complement activity, Triterpenoid has biological effects via the expression of matrix metalloproteinases-1 and type 1 procollagen in skin diseases, and Styraxoside A has anti-inflammatory effects in RAW 264.7 Cell [14][15][16]. Pinoresinol glucoside (PIN) is isolated from the stem bark of S. Japonica belonging to lignans that have important pharmacological activities [11]. Previous phytochemical studies indicated that pinoresinol-4,4 -di-O-beta-D-glucoside from Valeriana officinalis induced calcium mobilization and cell migration through the activation of lysophosphatidic acid (LPA) receptor subtype in Mouse Embryo Fibroblasts [17]. Pinoresinol-4-O-β-D-glucoside isolated from Artemisia selengensis does not affect interleukin-6 (IL-6) production in TNF-α stimulated MG-63 cells [18]. However, effects of PIN isolated from the stem bark of S. Japonica in pre-osteoblast have not been defined yet.
In the present study, we investigate the underlying mechanism and biological effects of PIN on cell migration, osteoblast differentiation, and matrix mineralization in pre-osteblasts.

PIN Increases Cell Migration during the Differentiation of Pre-Osteoblasts
Pinoresinol glucoside (PIN) was isolated from the stem bark of Styrax Japonica Sieb. et Zucc ( Figure 1A,B). To examine the effects of PIN on cell toxicity, pre-osteoblasts were treated with PIN for 24 h and cell viability was analyzed using an 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. At concentrations ranging from 0.1 to 100 µM, PIN did not influence cell viability of pre-osteoblasts ( Figure 2A). In order to examine whether PIN affects cell migration during the differentiation of pre-osteoblasts, we induced osteoblasts differentiation using osteogenic supplement medium (OS) containing 50 µg/mL l-ascorbic acid (L-AA) and 10 mM β-glycerophosphate (β-GP) in the absence and presence of 10 and 30 µM PIN for 24 h. The migratory effect of PIN was evaluated by wound healing assay, which revealed that PIN significantly promoted the closure rate of the cells in front of the wound area in a dose-dependent manner ( Figure 2B,C).

PIN Increases the Early Differentiation of Pre-Osteoblasts
In order to investigate whether PIN affects osteoblast differentiation, 10 and 30 µM PIN were treated with OS for 7 days, and alkaline phosphatase (ALP) enzymatic activity was measured to detect the early differentiation of pre-osteoblasts. We found that ALP activity was significantly increased in response to treatment with PIN in a dose-dependent manner during osteoblast differentiation ( Figure 3A). Consistent with the assay results, ALP staining was observed a digital camera and colorimetric detector, which showed that PIN treatment promoted the osteoblast differentiation ( Figure 3B). Using a light microscope, the effects of PIN on osteoblast differentiation were also confirmed on light microscopy observations ( Figure 3C

PIN Increases Mineralized Nodule Formation during Differentiation of Pre-Osteoblasts
We subsequently investigated whether PIN influences the late differentiation of pre-osteoblasts based on the degree of matrix mineralization using Alizarin red S (ARS) staining. After we induced osteoblasts differentiation using OS in the absence and presence of PIN (10 and 30 µM), we observed the formation of mineralized nodule at 7 and 14 days using a scanner and colorimetric detector. The matrix mineralization was formed at 14 days, which revealed that PIN treatment promoted the late osteoblasts differentiation of pre-osteoblasts in a dose-dependent manner ( Figure 4A,B). Using a light microscope, PIN-induced mineralized nodule formation was also visualized ( Figure 4C). Consistent with the observations, effects of PIN on late osteoblast differentiation were statistically validated by the quantification of ARS staining ( Figure 4D).

PIN Increases BMP2 Expression and Activates Its Signaling during Differentiation of Pre-Osteoblasts
To further investigate the molecular mechanisms underlying the effects of PIN on the differentiation of pre-osteoblasts, we examined bone morphogenetic protein (BMP)-Smad1/5/8-Runt-related transcription factor 2 (RUNX2) signaling using RT-PCR and western blotting. Ten and 30 µM PIN upregulated the mRNA level of BMP2 and its target osteoblast genes, ALP and osteocalcin (OCN) ( Figure 5A). PIN also enhanced protein level of BMP2, followed by the phosphorylation of Smad1/5/8 and the expression of RUNX2 (a key transcription factor in osteoblasts ( Figure 5B-E)). Immunofluorescence observation using confocal microscopy also confirmed an increase in the phosphorylation of Smad1/5/8 in response to PIN treatment ( Figure 5F).

PIN Increases Non-Canonical BMP2 Signaling and β-Catenin Sigaling during Differentiation of Pre-Osteoblasts
We subsequently investigated whether PIN influences non-canonical BMP2 signaling using western blotting. We found that 10 and 30 µM PIN increased the activities of the non-canonical BMP2-mediated MAPKs pathways, including ERK1/2 and p38 ( Figure 6A-D). Under same condition, we also investigated β-catenin signaling since β-catenin also plays an important role in osteoblast differentiation and bone formation. These resulted in the absence and presence of 10 and 30 µM PIN during osteoblast differentiation, and revealed that PIN increased in the phosphorylation of GSK3β and the protein level of β-catenin in response to the treatment of PIN ( Figure 6E-H). We also validated that PIN-induced osteoblast differentiation is mediated by BMP2 and β-catenin signaling (Supplementary Figure S1).

Discussion
Bone formation contains complex process, including the proliferation of osteoblast lineage cells, the mobilization and differentiation of pre-osteoblasts, and the maturation of osteoblasts [9]. After bone destruction, the recovery process of bone tissues, including cell migration, differentiation, and bone matrix mineralization, is also dependent on osteoblasts [19,20]. Thus, osteoblasts regulate bone homeostasis from birth until death, and the dysregulation of osteoblasts causes etiology of bone diseases such as osteoporosis [9,21]. In the present study, we found that PIN increased the cell migration of osteoblasts. The migration of osteoblasts from the bone marrow, periosteum, surrounding tissues, and circulating blood is required to form bone tissue through the synthesis, secretion, and mineralization of the bone matrix [19,22,23]. We also demonstrated that PIN promotes the expression and enzymatic activity of ALP and the formation of mineralized nodules. It is well known that ALP activity is an early marker of osteoblast differentiation, and when mature osteoblasts form matrix mineralization by calcium deposition it is a late and mature marker of osteoblast differentiation [21,24,25]. Based on the literature and our results, the findings of this study suggest that PIN is responsible for bone formation and bone homeostasis through cell migration, differentiation, and mineralization in osteoblasts.
A complex network activated by specific osteogenic signals controlled osteoblast differentiation [26,27]. In the present study, we demonstrated that PIN increased the expression of BMP2, the phosphorylation of Smad1/5/8, and the expression of RUNX2. BMP2, an osteogenic factor, activates the Smad1/5/8 and RUNX2 pathways to produce bone-specific proteins, including osterix, collagen type I, ALP, and OCN through RUNX2, a key transcriptional factor in osteoblast differentiation [28][29][30][31]. We also investigated BMP2 signaling target genes, and our results demonstrated that PIN increases the mRNA levels of ALP and OCN on osteoblast differentiation. These findings indicate that PIN increases osteoblast differentiation by regulating the BMP2 signaling pathway.
In conclusion, our results first demonstrated that PIN isolated from isolated from the stem bark of S. Japonica has biological effects on cell migration, differentiation, and mineralization through BMP2 and β-catenin signals in osteoblasts. Plant-derived natural compounds are becoming increasingly relevant in the treatment and prevention of bone diseases because natural compounds have been used to treat many diseases and have shown fewer adverse reactions than chemically synthesized medicines [9,43,44]. Therefore, the findings of this study suggest that PIN might be a potential source in the development of anabolic drugs to prevent and treat bone diseases such as osteoporosis and periodontal disease.

Plant Material
The stem bark of Styrax Japonica Sieb. et Zucc. was collected at Jeju (Korea) in July 2002. A voucher specimen (00250) was verified by Dr. Tae-Jin Kim, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Korea.

Extraction and Isolation of Compound
The dried stem bark of Styrax Japonica Sieb. et Zucc. (12.0 kg) was extracted with MeOH. The MeOH extract (1.4 kg) was suspended with 2000 mL of distilled water and solvent partitioned using same volume of Hx and EtOAc. The EtOAc soluble fraction (78.0 g) was separated into five fractions (SJE 1~SJE 5) by chromatography on a silica gel column eluted with a gradient of CHCl 3 and MeOH (10:1 to 1:1). The fraction SJE 4 (19.3 g) was re-chromatographed using ODS-A column with 25% aqueous ACN to collect five fractions (SJE 4-1~SJE 4-5). The subfraction SJE 4-2 (8.2 g) was purified by medium pressure liquid chromatography (MPLC) on a Lichroprep RP-18 (25-40 µm, Merck, Darmstadt, Germany) eluted with 30% aqueous ACN to obtain active compound (1.3 g, 98.7% purity). The purity of active compound was established by HPLC. The chemical structure of active compound was determined on the basis of spectroscopic data and comparison of those before literatures and identified as a PIN [45].

MTT Assay
Cell viability was detected using MTT (Sigma-Aldrich) solution [47]. Absorbance was measured at a wavelength of 540 nm using the Multiskan GO Microplate Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Western Blot Analysis
Western blot analysis was carried out as previously described [48]. Briefly, equal amounts of lysate (20 µg

Cell Migration Assay
Cell migration was accessed using an in vitro wound healing assay [46]. Briefly, the cells were wound with a 200 µL pipette tip and cultured for 24 h at 37 • C in a humidified atmosphere of 5% CO 2 and 95% air. Cell migration was observed using a light microscope, and the migration images were compared to quantify cell migration rate.

Alkaline Phosphatase (ALP) Activity Assay
Osteoblast differentiation was induced using OS containing 50 µg/mL L-AA and 10 mM β-GP with PIN (10 and 30 µM) for 7 days. The cell lysates were performed according to the manufacturer's protocol using alkaline phosphatase activity colorimetric assay kit (Biovision, Milpitas, CA, USA) [46].

ALP Staining Assay
Osteoblast differentiation was induced using OS containing 50 µg/mL L-AA and 10 mM β-GP with PIN (10 and 30 µM) for 7 days. Cells were washed with 1 × PBS and then fixed in 10% formalin for 10 min at room temperature. After washing with distilled water, the cells were incubated with substrate solution for the reaction of ALP, followed according to the manufacturer's protocol (Takara Bio Inc., Tokyo, Japan) as previously described [46].

Alizarin Red S (ARS) Staining
Osteoblast differentiation was induced using OS containing 50 µg/mL L-AA and 10 mM β-GP with PIN (10 and 30 µM) for 7 and 14 days. Cells were fixed in 10% formalin for 10 min at room temperature, washed with distilled water, and were stained with 2% ARS (pH 4.2) (Sigma-Aldrich) for 10 min. The level of ARS staining was observed using a scanner and colorimetric detector (ProteinSimple Inc., Santa Clara, CA, USA). To quantify and validate, stains were dissolved using 100% DMSO and measured at a wavelength of 590 nm using the Multiskan GO Microplate Spectrophotometer (Thermo Fisher Scientific).

Immunofluorescence
Immunofluorescence was performed [49]. Briefly, the cells were fixed, permeabilized, blocked, and incubated overnight at 4 • C with the specific primary antibodies. After washing, the cells were incubated with an anti-rabbit secondary antibody labeled with Alexa-Fluor 568 (1:500 dilution, Invitrogen, Carlsbad, CA, USA) for 2 h at room temperature in the dark and counterstained with 4 ,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for 10 min at room temperature in the dark. The cells were washed three times, mounted on glass slides, and observed using a confocal microscope (K1-Fluo Confocal Laser Scanning Microscope, Korea).

Statistical Analysis
The data were analyzed using Prism Version 5 program (GraphPad Software, Inc., San Diego, CA, USA). 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.