4-Hydroxyderricin inhibits osteoclast formation and accelerates osteoblast differentiation

4-Hydroxyderricin (4-HD) is a major polyphenol of Angelica keiskei (Japanese name Ashitaba), exhibiting anti-allergic, anti-diabetic, anti-oxidant, and antitumor effects. The present study was designed to evaluate the effects of 4-HD on bone formation and maintenance by using cultured osteoclasts and osteoblasts. 4-HD did not affect cell proliferation of stromal ST2 cells and preosteoblast MC3T3-E1 cells at concentrations of 1–10 μM. This compound inhibited the formation of multinucleated osteoclasts from mouse splenic cells, and we identified a molecular pathway of osteoclast differentiation mediated by 4-HD, which led to inhibition of the expression of receptor activator of nuclear factor-κB ligand and macrophage-colony stimulating factor in ST2 cells. By contrast, 4-HD enhanced indices of osteoblast differentiation, such as alkaline phosphatase activity and calcium deposition by osteoblastic MC3T3-E1 cells, at concentrations of 1–10 μM. Furthermore, we found that 4-HD at 1 μM attenuated H2O2 levels in MC3T3-E1 cells. Our findings indicate that 4-HD may have critical effects on bone formation and maintenance.


Introduction
Both the formation and maintenance of bone are controlled by bone-resorbing osteoclasts and boneforming osteoblasts. Osteoclasts are multinucleated giant cells with the ability to resorb mineralized tissues. They are formed from hematopoietic cells of the monocyte/macrophage lineage (Udagawa et al. 1990). The development of osteoclasts in culture is strictly dependent on support provided by osteoblasts and/or stromal cells (Udagawa et al. 1990). The formation and activation of osteoclasts are controlled by the combined actions of receptor activator of nuclear factor-jB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Here, we formed multinucleated osteoclasts from splenic cells by coculture with stromal ST2 cells that had been stimulated by activated vitamin D 3 (Hagiwara et al. 2008Notoya et al. 2007). Bone formation involves a complex series of events that include the proliferation and differentiation of osteoprogenitor cells, resulting in the formation of a mineralized extracellular matrix. The deposition of calcium and the sequential expression of type I collagen, alkaline phosphatase, and osteocalcin are known as markers of osteoblastic differentiation. Several model systems have been developed for studying the proliferation and differentiation of bone-forming cells in vitro and the molecular biology of the mineralization process, such as preosteoblastic cells from mouse calvariae (MC3T3-E1 cells) and osteoblast-like cells from rat calvariae (Bredford et al. 1993;Hagiwara et al. 1996;Liu et al. 1994;Stein et al. 1990). An imbalance of activities between osteoclasts and osteoblasts leads to bone metabolic diseases such as osteoporosis and osteopetrosis.
4-Hydroxyderricin (4-HD) ( Fig. 1) is more abundant as aglycone forms, with relative abundances of 1.5% in the stem exudates of Angelica keiskei (Japanese name, Ashitaka). A number of studies have shown that this compound possess biological properties, including antidiabetic (Li et al. 2016;Zhang et al. 2015;Enoki et al. 2007), anti-inflammatory (Yasuda et al. 2014;Yadav et al. 2011) and antitumor (Sumiyoshi et al. 2015;Akihisa et al. 2011;Kimura et al. 2004;Okuyama et al. 1991) activities. However, little information is available on the effects of 4-HD on bone metabolism. The present study was designed to evaluate the in vitro effects of 4-HD on the formation and maintenance of bone by using cultured mouse cells. Our results indicate that 4-HD is useful in the prevention and treatment of osteoporosis.

Formation of osteoclastic cells
Multinucleated osteoclastic cells were formed from spleen cells by co-culture with ST2 cells (RIKEN Cell Bank, Tsukuba, Japan) that had been stimulated by 100 nM 1a, 25-dihydroxy-vitamin D 3 [1a, 25(OH) 2 vitamin D 3 ] (Wako Pure Chemical Industries, Ltd., Osaka, Japan). ST2 cells (2 9 10 4 cells/well) in 48-well plates (area of each well, 0.75 cm 2 ) were pre-cultured with 100 nM 1a, 25(OH) 2 vitamin D 3 for 1 day to express RANKL. Spleen cells were collected from the splenic tissues of 6-weeks-old male ddY mice (Sankyo Labo Service, Tokyo, Japan). Erythrocytes contaminating the spleen cell fraction were eliminated by adding 0.83% ammonium chloride in 10 mM Tris-HCl (pH 7.4) to the cell pellet. Mouse spleen cells (1 9 10 5 cells/well) were spread on ST2 cell layers in 48-well plates and cultured in a-Modified minimum essential medium (a-MEM: Technologies, Inc. Grand Island, NY, USA), supplemented with 10% fetal bovine serum (Moregate BioTech, Bulimba, Australia), 50 units mL -1 penicillin and 50 lg mL -1 streptomycin (Life Technologies, Inc., Grand Island, NY, USA), in a humidified atmosphere of 5% CO 2 in air at 37°C for 7 days. Cultures were maintained at 37°C in a humidified atmosphere of 5% CO 2 in air. Fresh medium, 100 nM 1a, 25(OH) 2 vitamin D 3 and 4-HD were supplied at 2-days intervals. The Institutional Animal Care and Use Committee of Toin University of Yokohama approved all animal protocols and procedures.
Multinucleated osteoclastic cells formed were fixed in 3.7% formaldehyde for 5 min and then in a mixture of ethanol and acetone (1:1; v:v) for 1 min. These cells were then stained for tartrate resistant acid phosphatase (TRAP) activity (Udagawa et al. 1990). TRAP activity is a marker of multinucleated osteoclasts. TRAP-positive multinucleated cells (five or more nuclei) were counted under a microscope (IX70; Olympus, Tokyo, Japan). Preosteoblastic MC3T3-E1 cells were obtained from RIKEN Cell Bank (Tsukuba, Japan). Cells were maintained in a 55-cm 2 dish in a-Modified minimum essential medium (a-MEM), supplemented with 10% fetal bovine serum, 50 units mL -1 penicillin and 50 lg mL -1 streptomycin, in a humidified atmosphere of 5% CO 2 in air at 37°C. After reaching 70% confluence, cells were detached by treatment with 0.05% trypsin, replated in either 55-cm 2 dishes or 12-well plates (area of each well, 3.8 cm 2 ) at a density of 1 9 10 4 cells/cm 2 , and grown in a-MEM supplemented with 10% fetal bovine serum, 50 units mL -1 penicillin, 50 lg mL -1 streptomycin, 5 mM b-glycerophosphate, and 50 lg mL -1 ascorbic acid. Fresh medium and 4-HD were supplied to cells at 2-days intervals. MC3T3-E1 cells formed nodules, and mineralization of nodules was observed after cultivation for 2-3 weeks.

Measurement of intracellular reactive oxygen species
Intracellular reactive oxygen species were measured using the oxidant-sensitive probe 2 0 , 7 0 -dichlorofluorescin diacetate (DCFH-DA). MC3T3-E1 cells (4 9 10 4 cells/dish) in 3.5-cm dishes were cultured for 48 h with subsequent 48-h incubation with 4-HD. Thereafter, cells were incubated with 50 lM DCF-DA for 30 min followed by a 30-min incubation with 500 lM H 2 O 2 and washed with Hank's Balanced Salt Solution. Fluorescence emission was detected by confocal laser scanning microscopy at excitation and emission wavelengths of 488 and 505 nm, respectively. Images were analyzed using a confocal scanning system (TCP SP2; Leica, Tokyo, Japan).
Measurement of alkaline phosphatase activity MC3T3-E1 cells were subcultured in 12-well plates (3.8 cm 2 /well) in a-MEM containing 10% fetal bovine serum, 5 mM b-glycerophosphate, and 50 lg mL -1 ascorbic acid. After the cells had reached confluence (day 3), 4-HD was added to cultures at various concentrations for 9 days. Cells were washed with 10 mM Tris-HCl, pH 7.2, and were sonicated in 1 mL of 50 mM Tris-HCl (pH 7.2) containing 0.1% Triton X-100 and 2 mM MgCl 2 for 15 s with a sonicator (Ultrasonic Disruptor UD-201; Tomy Co., Tokyo, Japan). Alkaline phosphatase activity was determined using an established technique with pnitrophenyl phosphate as the substrate. Protein concentrations were determined using BCA protein assay reagent (Pierce Chemical Co., Rockford, IL, USA) with bovine serum albumin as a standard.
Quantitation of calcium deposition MC3T3-E1 cells were subcultured in a-MEM containing 10% fetal bovine serum, 5 mM b-glycerophosphate, and 50 lg mL -1 ascorbic acid. After the cells had reached confluence (day 3), 4-HD was added at various concentrations to the culture medium and cells were subcultured for 11 days. The amount of calcium, deposited as hydroxyapatite in the cell layer, was measured as follows: Layers of cells in 12-well plates (3.8 cm 2 /well) were washed with PBS and incubated overnight with 1 mL of 2 N HCl with gentle shaking. Ca 2? ions in the samples were quantitated by the ocresolphthalein complexone method with a Calcium C kit (Wako Pure Chemical Industries). This kit is specific for Ca 2? ions and has a detection limit of 1 lg mL -1 . The solution of Ca 2? ions (20 mg dL -1 ) provided in the kit was used as the standard solution.

Statistical analysis
Numerical data have been expressed as mean ± S.D. values of the results from three to four cultures, and the significance of differences was analyzed by using ANOVA (Dunnett's test) or Tukey-kramer. Statistical significance was set at P \ 0.05. Experiments were repeated independently in triplicate and the results were qualitatively identical in every case. Results from representative experiments are shown.

Toxicity of 4-HD
We evaluated the toxicity of 4-HD for MC3T3-E1 cells and ST2 cells by using the MTT assay. As shown in Fig. 2, the viability of MC3T3-E1 cells and ST2 cells was significantly increased and decreased at some concentrations. However, its disparity is not great. Furthermore, exposure of MC3T3-E1 cells and ST2 cells to 4-HD at 10 lM did not affect cell morphological features (data not shown).

Effects of 4-HD on the formation of multinucleated osteoclasts
Multinucleated osteoclastic cells were formed from mouse splenic cells in co-culture with ST2 cells which had been stimulated by 1a, 25(OH) 2 vitamin D 3 . Figure 3a shows representative results for the detection of TRAP activity in multinucleated osteoclastic cells treated with 4-HD at the indicated  Fig. 2 Effects of 4-HD on osteoblastic cell viability. MC3T3-E1 cells and ST2 cells (each 2.5 9 10 3 cells/well; 96-well plates) were exposed to 4-HD at various concentrations (1-10 lM) and were subcultured for indicated periods. After treatment with 4-HD, the cells were treated with MTT (50 lg/ well) for 4 h, and the absorbance at 570 nm was measured. The values represent the mean ± S.D. of results from three wells. Data are representative of results from three separate experiments. *P \ 0.05 versus vehicle (V) and **P \ 0.01 versus V concentrations. Formation of TRAP-positive multinucleated osteoclastic cells was dose-dependently inhibited by the addition of 4-HD (Fig. 3b). Exposure of 10 lM 4-HD completely inhibited the formation of multinucleated osteoclastic cells relative to control cultures treated with the vehicle alone.

Effects of 4-HD on cultured osteoblasts
To assess the effects of 4-HD on the differentiation and mineralization of MC3T3-E1 cells, we added 4-HD to the culture medium of post-proliferative cells and assayed alkaline phosphatase activity (a middlestage marker of osteoblastic differentiation) and calcium deposition (Fig. 5). 4-HD significantly increased the activity of alkaline phosphatase in MC3T3-E1 cells on day 9 when used at 10 lM  The RNeasy Mini Kit was used to extract RNA from ST2 cells that had been exposed to 4-HD for 2 days. The PCR conditions are described in the Materials and methods. Data are representative of results from two separate experiments. Vehicle was represented as 1 (Fig. 5a). Furthermore, as demonstrated in Fig. 5b, 4-HD dose-dependently enhanced the deposition of calcium by MC3T3-E1 cells on day 11. Exposure of MC3T3-E1 cells to 10 lM 4-HD increased the deposition of calcium by approximately 270% on day 11, relative to control cultures treated with the vehicle alone (Fig. 5b).
It has been reported that hydrogen peroxide (H 2 O 2 ) suppressed the differentiation of osteoblasts. Therefore, we examined the effects of 4-HD on H 2 O 2 levels in MC3T3-E1 cells (Fig. 6). As shown in Fig. 6, 4-HD at 1 lM decreased H 2 O 2 levels in cells.

Discussion
We screened natural polyphenols for the ability to regulate the proliferation, differentiation, and function of cultured osteoclasts and osteoblasts in order to identify factors that may cause, prevent, or treat bone metabolic diseases such as osteoporosis and osteopetrosis. We had previously reported that genistein attenuates osteoclastogenesis by decreasing the levels of receptor activator NF-jB ligand mRNA in osteogenic/stromal cells (Yamagishi et al. 2001). Quercetin ) and curcumin (Notoya et al. 2006) have been reported to inhibit cultured osteoblast metabolism. In addition, quercetin ) and carnosic acid  have been found to inhibit osteoclastogenesis. Recently, we showed that the olive polyphenols oleuropein and hydroxytyrosol (Hagiwara et al. 2011) and apigenin (Goto et al. 2015) inhibit the formation of osteoclasts and attenuate bone loss in OVX mice. Thus, polyphenols regulate bone metabolism in culture via osteoclasts and osteoblasts. Effects of 4-HD on alkaline phosphatase activity and mineralization of osteoblasts. MC3T3-E1 cells were cultured in 12-well plates (3.8 cm 2 /well) with a-MEM containing 10% fetal bovine serum, 5 mM b-glycerophosphate, and 50 lg mL -1 ascorbic acid. After the cells reached confluence (day 3), 4-HD was added at various concentrations (1-10 lM) to the culture medium. Fresh medium with test compound was supplied at 3-days intervals. a Alkaline phosphatase activity was measured at day 9 as described in the method. b Deposition of Ca 2? ions was measured at day 11. Quantitative analysis of Ca 2? ions was performed as described in the method. All values represent the mean ± S.D. of the results from three wells. Data are representative of results from three separate experiments. *P \ 0.05 versus vehicle (V) and **P \ 0.01 versus V In this study, we attempted to clarify the potential effects of 4-HD on bone metabolism. We found that 4-HD attenuated osteoclast formation a part through the decrease in expression of RANKL and M-CSF mRNAs and induced osteoblast differentiation markers such as alkaline phosphatase activity and calcium deposition by MC3T3-E1 osteoblasts. These findings indicate that 4-HD has properties of down-regulation of osteoclastogenesis and up-regulation of osteoblastogenesis in vitro.
It is well known that polyphenols have antioxidant properties (Rice-Evans et al. 1995). Recent reports have suggested that reactive oxygen species (ROS) play an important role in the regulation of cell proliferation, differentiation and metabolism. In particular, ROS inhibit the formation of bone by osteoblastic cells (Hosoya et al. 1998;Lee et al. 2006;Mody et al. 2001). Oxidative stress resulting in increased levels of intracellular ROS has been reported to suppress bone metabolism. Arai et al. (2007) reported that mineralization of MC3T3-E1 cells was reduced by half after a single exposure to H 2 O 2 within the non-toxic concentration range. In addition, there have been some reports that H 2 O 2 suppresses differentiation markers such as alkaline phosphatase activity, type I collagen gene expression, and the mineralization of osteoblastic cells (Hosoya et al. 1998;Lee et al. 2006;Mody et al. 2001). We also reported that carnosic acid attenuated H 2 O 2 levels and osteoblastic differentiation in osteoblastic MC3T3-E1 cells . In the present study, 4-HD significantly reduced intracellular H 2 O 2 level of MC3T3-E1 cells. These results suggest that 4-HD induces osteoblastic differentiation a part through the decrease in intracellular H 2 O 2 level of MC3T3-E1 cells.
Ashitaba, a perennial herb growing mainly along the Pacific coast of Japan, has been used in traditional food and medicine (Baba et al. 1998). Our results using cultured cells suggest that 4-HD may be effective on bone maintenance, if 4-HD was absorbed into the plasma. It was reported that 4-HD was quickly absorbed into the plasma after oral administration of Ashitaba extract in mice (Nakamura et al. 2012). The concentration of free 4-HD reached 1.2 ± 0.3 lM in plasma at 2 h after oral administration of Ashitaba extract at 200 mg/kg body weight. This concentration is enough to be effective on inhibition of osteoclast formation and induction of osteoblast differentiation from our results.
In conclusion, polyphenol 4-HD extracted from Angelica keiskei (Ashitaba) markedly inhibited the formation of multinucleated osteoclasts in culture and induced osteoblastic differentiation. These findings suggest that 4-HD may provide insights into the development of tools useful for the prevention and treatment of osteoporosis.