Marine algae Sargassum horneri bioactive factor stimulates osteoblastogenesis

kappa B-luciferase activity and receptor activator of nuclear factorkappa B-ligand-induced increase in pre-osteoclastic nuclear factorkappa B-luciferase activity. Conclusion This study demonstrates that an extract of S. horneri does have an effect on osteoblastogenesis and ost-eoclastogenesis in vitro.


Introduction
Bone is a dynamic tissue that preserves skeletal size, shape and structural integrity and regulates mineral homeostasis.Bone homeostasis is maintained through a balance between osteoblastic bone formation and osteoclastic bone resorption 1 .Aging and numerous pathologic processes have the capacity to disrupt this equilibrium, leading to conditions where the rate of bone resorption outpaces the rate of bone formation.This leads to osteoporosis that is induced with a decrease in bone mass 2 .Postmenopausal osteoporosis, a consequence of ovarian hormone deficiency, is the archetypal osteoporotic condition in women after menopause; it leads to bone destruction though complex and diverse metabolic and biochemical changes 2 .Osteoporosis is widely recognised as a major public health problem 2,3 .The most dramatic expression of this disease is represented by fractures of the proximal femur, the number of which increases with an increase in age 3 .
Chemical compounds in food and plants, which regulate bone homeostasis, have been be worthy of notice in maintaining bone health and preventing bone loss with increasing age [4][5][6][7] .
Among various marine algae, Sargassum horneri extract has been found to have an anabolic effect on bone calcification in rat femoral tissues both in vitro and in vivo 8 .S. horneri extract has been demonstrated to stimulate osteoblastic bone formation and inhibit osteoclastic bone resorption using rat femoraldiaphyseal and -metaphyseal tissues in vitro [9][10][11] .The intake of S. horneri extract may have a stimulatory effect on bone growth in young rats and a preventive effect on bone loss with increasing age in vivo 12 .Bone loss, which is induced in streptozotocindiabetic rats in vivo, is restored with the intake of S. horneri extract 13 .Thus, the intake of S. horneri extract may have a preventive effect on osteoporosis.Moreover, prolonged intakes of S. horneri extract (300 or 900 mg/day) for 8 weeks had an effect on bone metabolism in healthy humans; this was estimated by analysis of circulating bone metabolic markers 14 .
This study, therefore, was undertaken to determine the effect of S. horneri extract, which contains components with a molecular weight (MW) of <3000, on osteoblastogenesis and osteoclastogenesis in vitro.We found that S. horneri extract stimulates osteoblastogenesis and suppresses osteoclastogenesis in vitro.

Material and methods
The protocol of this study has been approved by the relevant ethical committee related to our institution in which it was performed.
Marine algae extract S. horneri (Turner) C. Agardh 15 , a marine algae, was seasonally gathered from the coast at Shimoda (Shizuoka Prefecture, Japan) and Miyako (Iwate Prefecture, Japan), and it was freezedried and powered.Gathered fresh marine algae was homogenised in distilled water and centrifuged at 5500 g in a refrigerated centrifuge for 10 min.The 5500 g supernatant fraction was pooled for freeze-drying.The powder of the water-solubilised extract was dissolved in ice-cold distilled water for subsequent use in the experiments.The water-solubilised extract from S. horneri was purified by membrane fractionation to collect active components with a MW of <3000 11 .

Cell culture
Preosteoblastic cell line MC3T3-E1, clone 14 (MC3T3), osteoclast precursor cell line RAW264.7,and primary mouse monocytes were purchased from the American Type Culture Collection (Manassas, VA) and cultured, as previously described 16 .
Osteoblast differentiation assay and Alizarin Red-S staining MC3T3 cells were plated and cultured for 72 hours in α-MEM (1.0 ml/ well) containing 10% FBS in 12-well dishes at a density of 1.0 × 10 5 cells/ well.The medium was aspirated and changed to mineralisation medium [α-MEM supplemented with 10% FBS, L-ascorbic acid (100 μg/ml) and 4 mM β-glycerophosphate], as previously described 16 .S. horneri extracts (containing components with MW <3000) were added as per the indicated dose, and the cells were replenished with fresh medium every 3 days.At 21 days, cells were rinsed with phosphate buffered saline, and mineralisation nodules were visualised by fixing the cells in 75% ethanol for 30 minutes at 4 o C, followed by staining with Alizarin Red-S (40 mM, pH 6.2) for 30 minutes at room temperature.Excess stain was removed by copious washing with distilled water.Plates were imaged using a flatbed scanner (Epson Perfection V600 Photo).For quantitation, 10% cetylpyridinium chloride solution was added to each well to elute the dye.After complete elution, absorbance of the eluted solution was measured at 570 nm using a microtitre plate reader.

Osteoclastogenesis assay and TRAP staining
Osteoclasts were generated as previously described 16 .Briefly, RAW264.7 cells were cultured in Dulbecco's Modification of Eagle's Medium (DMEM) containing 10% FBS and 1% penicillin plus streptomycin.The RAW264.7 osteoclast precursor cell line was cultured for 6 days in 96-well plates using α-MEM supplemented with 10% FBS and 100 IU/ml penicillin and 100 μg/ml streptomycin at a density of 1 × 10 4 cells/well.Osteoclast formation was induced by the addition of RANKL (30 ng/ml) after pre-incubation for 10 minutes with crosslinking anti-polyhistidine antibody (2.5 μg/ml).S. horneri extracts were added in the range of 5 to 100 μg/ml of medium.After 6 days of culture, the cells were fixed and stained for TRAP.TRAP+ cells with three or more nuclei were defined as osteoclasts and were quantitated under light microscopy; 5 wells/group were averaged.

Smad-4 luciferase assay
Osteoblastic cells (2 × 10 4 cells/well/ 0.1 ml) were cultured for 24 hours in α-MEM without 10% FBS and antibiotics in 96-well plates, and then the cells were transfected with Smad-4 promoter vector using lipofectamine™ 2000 Reagent (Invitrogen) 16 .Five hours later, the medium was changed to α-MEM containing 10% FBS plus 1% penicillin and streptomycin in the presence of either the vehicle or S. horneri extract (5-100 μg/ml of the medium).The cells were also cultured in presence of 10% FBS with or without TGF-β1 (1 ng/ml) or BMP-2 (0.5 μg/ml).The cells were cultured for 24 hours after addition of TGF-β1.BMP-2 was added after 18 hours of culture, and then the cells were cultured for 6 hours.Smad reporter was responsive to both TGF-β and BMPinduced Smad.

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Statistical analysis
Statistical significance was determined using GraphPad InStat version 3 for Windows XP (GraphPad Software Inc., La Jolla, CA).Multiple comparisons were performed by one-way analysis of variance (ANOVA) with Tukey-Kramer multiple comparisons post-test for parametric data.Gaussian distribution was assessed using Kolmogorov and Smirnov test; p < 0.05 was considered to be statistically significant.

Effect of S. horneri extract on osteoblastic mineralisation
The direct effect of S. horneri extract on bone formation and mineralisation is unclear.To address this issue, we investigated the action of S. horneri extract on osteoblast differentiation and mineralisation in vitro.MC3T3 preosteoblastic cells were differentiated into mineralising osteoblasts in vitro in the presence or absence of S. horneri extract (2.5, 5, 10 and 25 μg/ml of the medium) in mineralising medium for 21 days and stained for calcium deposition with Alizarin Red-S.S. horneri extract (10 and 25 μg/ml) was found to potently enhance mineralisation in MC3T3 cells (Figures 1A, 1B).S. horneri extract (25-100 μg/ml of the medium) did not have a significant effect on cell number of MC3T3-E1 cells with culture (data not shown).S. horneri extract did not appear to mediate a direct toxic effect on the cultures, as cells proliferated robustly over the culture period and were still alive and visibly attached to the plate at the end of the experiment as compared with those of the control group.

S. horneri extract suppresses the differentiation of RAW264.7 osteoclast precursors into mature osteoclasts
To examine the effect of S. horneri extract on osteoclast formation, RAW264.7 osteoclast precursors were differentiated into mature osteoclasts by stimulation with RANKL in the presence or absence of S. horneri extract, with a dose range of 5 to 100 μg/ml of the medium.The S. horneri extract (25-100 μg/ml of the medium) did not have a significant effect on the cell number of preosteoclasts (RAW267.4cells) with culture.S. horneri extract (100 μg/ml) did not have toxicity to the precursors suppressing their proliferation over 7 days of culture.S. horneri extract (25-100 μg/ml) significantly suppressed osteoclast formation induced by RANKL (Figure 5).

Discussion
In this study, we demonstrated that S. horneri extract stimulates osteoblastic

Research study
Licensee OA Publishing London 2012.Creative Commons Attribution License (CC-BY) differentiation and mineralisation and suppresses osteoclastogenesis in vitro.TGF-β1-and BMP-2-induced activation of Smad signalling plays an important role in the early commitment and differentiation of osteoblasts 17 .S. horneri extract was found to enhance BMP-2-or TGF-β1-induced Smad activation in a dose-dependent manner.S. horneri extract-induced Smad enhancement seems to be important to stimulate osteoblastic differentiation and mineralisation.NF-κB signalling has been shown to down-regulate osteoblast differentiation 18,21 .One major mechanism appears to involve the intersection of NF-κB with the Smad signalling pathway 18 .NF-κB signalling in osteoblasts intersects with and disrupts Smad signalling by promoting production of Smad7, an inhibitor of TGF-β-and BMP-induced R-Smad activation 21,22 .TNF-α further antagonizes BMP signalling by up-regulating Smad ubiquitination regulatory factor 1 (Smurf1), thereby promoting proteasomal degradation of bone morphogenetic signalling proteins 23 .Multiple suppressors of NF-κB activation are capable of rescuing the inhibitory effect of TNF-α on BMP-2 and/or TGF-β1-induced Smad activation 16,24,25 .In this study, S. horneri extract was found to suppress TNF-α-induced NF-κB activation in preosteoblastic MC3T3-E1 cells.This finding provides a possible mechanism by which S. horneri extract stimulates osteoblastic bone formation.

S. horneri extract may inhibit osteoclastogenesis through suppression of NF-κB activation.
The effect of S. horneri on mineralisation in MC3T3-E1 cells with culture for 21 days was observed at 10 μg/ ml.However, the effects of S. horneri on Smad activity in MC3T3-E1 cells and on NF-κB activity in RAW264.7 cells with culture for 24 hours were observed at 25 μg/ml.The effects of S. horneri on osteoclastogenesis in RAW264.7 cells with culture for 6 days were also revealed at 25 μg/ml.The effect of S. horneri on osteoblastogenesis and osteoclastogenesis may be observed with lower doses in the culture medium for longer periods.
The active components, which have a stimulatory effect on bone calcification and suppressive effect on bone resorption in bone tissues in vitro, have been reported to be present in S. horneri extract 11 .The active component in stimulating bone calcification has been reported to be of MW of >3000, while the active component of S. horneri extract in inhibiting osteoclastic cell formation in mouse bone

Figure 1 :
Figure 1: S. horneri extract promotes osteoblast differentiation and mineralisation in preosteoblastic MC3T3 cells in vitro.MC3T3 cells were cultured in the presence (+) or absence (-) of mineralising medium (MM), with a dose range of 2.5, 5, 10 or 25 μg/ml of the medium.Calcium deposition was visualised with Alizarin Red-S at 21 days of culture, and data shows the plates (a) and absorbance (b) for quantitation.Each data point was performed in duplicate.All wells within each experiment were from the same plate, but were digitally separated and reorganised for clarity with non-contiguous wells.Data are presented as mean ± SD of 4 replicate wells per dataset and are representative of 2 independent experiments.*p < 0.001 versus none; **p < 0.001 versus mineralisation medium only (grey bar); One-way ANOVA, Tukey-Kramer post-test.

Figure 2 :Figure 3 :
Figure 2: Effect of S. horneri extract on basal or BMP-2-induced Smad activity.MC3T3 cells were transfected with pGL3-Smad luciferase reporter plasmid, and then the cells were treated with S. horneri extract (5-100 μg/ml of the medium) to determine the effect on basal Smad-induced luciferase activity quantitated basal conditions or BMP-2 (0.5 μg/ml)-stimulated Smad activation were determined.*p < 0.001 versus basal (white bar); **p < 0.001 versus BMP2-stimulated (grey bar).Data are presented as mean ± SD of 5 replicate wells per data point and are representative of 2 independent experiments.One-way ANOVA, Tukey-Kramer post-test.