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Jan 31, 2011 - Caffeine could also increase the cyclooxygenase-2 (COX-2) protein expression and ... markedly in recent years, and it likely induces problems ... 5-week-old ICR mice and flushing the bone marrow cavity with a-minimum essential ... 22 days, to observe calcium deposition, cultures were washed once with ...
Caffeine Enhances Osteoclast Differentiation from Bone Marrow Hematopoietic Cells and Reduces Bone Mineral Density in Growing Rats Shing Hwa Liu,1 Chinliang Chen,2 Rong Sen Yang,3 Yuan Peng Yen,1 Ya Ting Yang,3 Chingmin Tsai2 1 Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan, 2Department of Bioscience Technology, Chung Yuan University, Jhongli, Taiwan, 3Department of Orthopaedics, College of Medicine, National Taiwan University, Taipei, Taiwan

Received 26 May 2010; accepted 8 November 2010 Published online 31 January 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.21326

ABSTRACT: Caffeine-containing beverage consumption has been associated with low bone mass and increased fracture risk in some, but not most, observational studies. The effects of caffeine on bone metabolism are still controversial. We investigated the effects of caffeine on the differentiation of bone progenitor cells and bone mineral density (BMD) by in vitro and in vivo experiments. Low-concentration caffeine (0.005–0.1 mM) did not affect the bone marrow cell viability and alkaline phosphatase activity during osteoblast differentiation from bone marrow stromal cells, but it effectively enhanced the osteoclastogenesis from bone marrow hematopoietic cells and the bone resorption activity by pit formation assay. Moreover, caffeine effectively enhanced the receptor activator of NF-kB ligand (RANKL), but reduced the osteoprotegerin protein expressions in osteoblast MC3T3-E1 cells. Caffeine could also increase the cyclooxygenase-2 (COX-2) protein expression and prostaglandin (PG)E2 production in cultured neonatal mouse calvariae. In animal study, BMD in lumbar vertebra, femur, or tibia was significantly lowered in growing rats supplemented with 0.2% caffeine in diets for 20 weeks compared with the control group. The calcium contents in tibia and femur of caffeine-treated rats were also lower than that in the control group. The osteoclastogenesis of bone marrow cells isolated from caffeine-treated rats was markedly enhanced as compared with the control group. Taken together, these results suggest that caffeine may reduce BMD in growing rats through the enhancement in osteoclastogenesis. Caffeine may possess the ability to enhance a COX-2/PGE2-regulated RANKL-mediated osteoclastogenesis. ß 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:954–960, 2011 Keywords: caffeine; osteoclastogenesis; osteoblastogenesis; bone mineral density

Caffeine is probably the most commonly used pharmacologically active substance in the world. Caffeine exists in some common beverages (coffee and tea), cocoa products, and some medications. Moreover, the consumption of high caffeine content energy drinks has increased markedly in recent years, and it likely induces problems with increased caffeine intoxication.1 These energy drinks vary widely in both caffeine content (ranging from 50 to 505 mg/can or bottle) and caffeine concentration (ranging from 2.5 to 171 mg/fluid ounce).1 It has been suggested that the moderate daily caffeine intake of up to 400 mg/day (equivalent to 6 mg/kg body weight/day in a 65-kg person) for the healthy adult population is not associated with adverse effects induced by caffeine on the central nervous and cardiovascular systems, bone status, increased incidence of cancer, and effects on male fertility.2 Caffeine-containing beverage consumption was associated with reduced bone mass and increased fracture risk in some, but not most, observational studies.3,4 Heaney4 suggested that no evidence exists that caffeine has a harmful effect on bone status or on calcium economy in individuals who ingest the currently recommended daily allowances of calcium. An animal study showed no significant differences in serum and urinary biochemical markers of bone metabolism, and bone

Shing Hwa Liu, Chinliang Chen, Rong Sen Yang, and Yuan Peng Yen contributed equally to this work. Correspondence to: Chingmin Tsai (T: 886-3265-3510; F: 8863265-3599; E-mail: [email protected]) ß 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

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histomorphometry between coffee-diet rats and control-diet rats after 140 days of treatment.5 However, Rapuri et al.6 indicated that intakes of caffeine in amounts >300 mg/day (approximately 514 g, or 18 oz, brewed coffee) accelerate bone loss at the spine in elderly postmenopausal women. Wink et al.7 also showed that if young, rapidly growing rats are exposed to caffeine, disruption of osteoblasts and retarded bone development occur. An in vitro study showed that caffeine may enhance the rate of osteoblast apoptosis and has potential deleterious effect on the osteoblast viability.8 Thus, the effects of caffeine on bone metabolism seem to remain controversial. The dosage of caffeine may be an important determinant factor, but no conclusive results were found in those consuming low dosage caffeine. The effects of caffeine on bone cell differentiation and bone mass need to be further clarified. We investigated the effects of caffeine on the differentiation of bone marrow progenitor cells and bone mineral density (BMD) by in vitro and in vivo experiments. The effects of low and non-toxic doses of caffeine (0.005–0.1 mM) on the osteoblastogenesis and osteoclastogenesis were examined.

MATERIALS AND METHODS Animals ICR male mice (5-week-old) and Wister rats (3-week-old) were purchased from the BioLASCO Taiwan Co., LTD (Taipei, Taiwan). Animals were housed in individual stainless steel cages in a room kept at 23  18C and 60  5% relative humidity with a 12-h light-dark cycle. Food and water were available ad libitum. The animal study was approved by the Animal House Management Committee of Chung Yuan University. The animals were maintained in accordance with the guidelines

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for the care and use of laboratory animals as issued by the Animal Center of the National Science Council. Cell Culture Bone marrow cells were prepared by removing femurs from 5-week-old ICR mice and flushing the bone marrow cavity with a-minimum essential medium (aMEM; Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS). In some experiments, MC3T3-E1 cells, a mouse osteoblast-like cell line, were used. Cells were cultured in a-MEM supplemented with 10% FBS, 100 units/ml penicillin, and 100 mg/ml streptomycin at 378C in a humidified atmosphere of 5% CO2 in air. Neonatal Mouse Calvaria Tissue Culture The heads of 6-day-old ICR mice from two litters were provided by the Laboratory Animal Center of National Taiwan University College of Medicine. The raising, feeding, and surgical processes were approved by the Committee of Animal Study in National Taiwan University. The frontoparietal bones were removed and washed free of blood and adherent brain tissue in Hanks’ balanced salt solution, and then divided along the sagittal suture. The bones were cultured in aMEM medium supplemented with 10% FBS and 100 U/ml each of penicillin/ streptomycin. Bones were cultured in a humidified atmosphere of 5% CO2 at 378C. Culture media were changed every 3 days. Cell Number Assay Cells were seeded in 96-well dishes with 2  104 cells/well in complete medium for 24 h. Then cells were incubated in fresh complete medium in the presence or absence of caffeine (0.005– 0.1 Mm; Sigma, St. Louis, MO) for 24 h. Viability cells were measured with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide (MTT) (Sigma). Annexin-V FITC Staining Annexin V/propidium iodide (Clontech, Mountain View, CA) was used to quantify numbers of apoptotic cells. Cells were washed twice with PBS and stained with annexin V and PI for 20 min at room temperature. The level of apoptosis was determined by measuring the fluorescence of the cells by flow cytometer (Becton Dickinson, Rockville, MD). Data acquisition and analysis were performed by the CellQuest program (Becton Dickinson). Osteoblastogenesis Osteoblast differentiation was induced by culturing bone marrow cells in osteoblastogenic medium that was primary culture medium supplemented with 108 M dexamethasone, 5 mg/ml ascorbic acid, and 10 mM b-glycerophosphate. After 7 days, the osteoblastogenesis of mesenchymal stem cells (MSCs) were confirmed by detecting alkaline phosphatase activity. After 22 days, to observe calcium deposition, cultures were washed once with PBS, and stained for 5 min with Alizarin Red S stain. Osteoclastogenesis Bone marrow cells were seeded in 24-well plates in the presence of mouse recombinant soluble receptor activator of NF-kB ligand (RANKL, 50 ng/ml) and murine macrophage conlonystimulating factor (M-CSF, 20 ng/ml; R&D Systems, Minneapolis, MN). The culture medium was replaced every 3 days with a fresh complete medium containing the appropriate reagents. After 5 days, cells were washed and subjected to a tartrate-resistant acid phosphatase (TRAP) assay, which was used to measure osteoclast formation. Osteoclast-like TRAP-

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positive cells in each well were scored by counting the number of TRAP-positive multinucleated cells containing 3 nuclei. Moreover, pit formation assay was used to detect the bone resorption activity. Protein Extraction and Immunoblotting MC3T3-E1 cells were washed with PBS and lysed with RIPA buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EGTA, 0.1% SDS, 1 mM NaF, 1 mM Na3VO4, 1 mM phenylmethanesulphonyl fluoride [PMSF], 1 mg/ml aprotinin, and 1 mg/ml leupeptin). In this buffer, NaF and PMSF were the phosphatase inhibitor and serine protease inhibitor, respectively. The cell suspension was then left on ice for 20 min, and then centrifuged at 10,000g for 20 min at 48C; the supernatant was used for the experiment. An equal amount (40 mg) of protein was separated by 10% SDS-polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene difluoride membranes (0.2 mm) using transfer buffer (192 mM glycine, 25 mM Tris, 20% methanol, and pH 8.3) followed by blocking in TBST buffer (20 mM Tris, 150 mM NaCl, 0.01% Tween 20, and pH 7.5) supplemented with 5% non-fat powdered milk. The membranes were then probed with the primary antibodies (anti-RANKL, anti-osteoprotegerin [OPG], anti-cyclooxygenase [COX]-2, anti-a-tubulin, and anti-b-actin; Santa Cruz Biotech., Santa Cruz, CA) for overnight at 48C followed by incubation with a secondary goat anti-rabbit or anti-mouse antibody conjugated with horseradish peroxidase. The blots were developed using an enhanced chemiluminescence reagent detection system according to the manufacturer’s protocol. The extents of protein expressions in terms of relative band intensity were quantified by scanning densitometry using a GS-670 imaging densitometer (BioRad, Hercules, CA). The data are representative of at least three independent experiments. PGE2 Production PGE2 levels in cell culture supernatants were determined using the PGE2 enzyme immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, MI). Bone Resorption Pit Formation Assay Bone marrow cells were cultured for 5 days in the presence of RANKL (50 ng/ml) and M-CSF (20 ng/ml) on BD BioCoat Osteologic Multitest slides, which consisted of submicron synthetic calcium phosphate thin films coated onto various culture vessels (Becton Dickinson). Then, cells were removed and the formed resorption pits were observed under a microscope. BMD and Bone Calcium Content Thirty male Wistar rats (3-week-old) were assigned into three groups randomly, 10 each, and fed diets containing 0% (control), 0.1% caffeine, and 0.2% caffeine for 20 weeks. The BMD of lumbar vertebra, femur, and tibia were determined by a small animal dual-energy X-ray absorptiometry research scanner (pDEXA, Norland Stratec Medizintechinik GmbH, Birkenfeld, Germany). The calcium content of femur and tibia were also measured after animals were euthanized. Bone tissues were dried for 16 h at 1208C, weighed, and then dissolved in nitric acid solution, followed by 100 dilution with distilled water. Calcium levels were determined by Raichem1 colorimetric assay (Hemagen Diagnostics, Inc., San Diego, CA). Statistics Data are expressed as mean  SD for three or four independent experiments. Statistical analysis was performed using one-way JOURNAL OF ORTHOPAEDIC RESEARCH JUNE 2011

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ANOVA followed by Dunnett’s test for each paired experiment. p Values < 0.05 were considered significant.

RESULTS Caffeine-Enhanced Osteoclast Differentiation from Bone Marrow Cells The cell viability of bone marrow cells was not affected by the treatment with 0.005–0.1 mM caffeine for 24 h (p > 0.05; Fig. 1A). Caffeine (0.01–1 mM) did also not induce cell apoptosis (p > 0.05; Fig. 1B). Caffeine (0.001– 1 mM) did also not induce cytotoxicity in MC3T3-E1 cells (MTT assay, data not shown). Caffeine (0.005–0.1 mM) in medium affected neither the alkaline phosphatase activity (p > 0.05; Fig. 1C), nor the bone mineralization stained by Alizarin Red S (data not shown) during osteoblast differentiation from bone marrow stromal cells. Low-dose caffeine (0.005 and 0.01 mM) effectively enhanced osteoclast differentiation from bone marrow hematopoietic cells (p < 0.05; Fig. 2A). The activity of bone resorption by pit formation assay (Fig. 2B) was markedly enhanced by the treatment of low-dose caffeine (0.005 and 0.01 mM). Caffeine (0.001–1 mM) effectively enhanced the RANKL expression and reduced the OPG expression in MC3T3-E1 cells (Fig. 3A; RANKL: caffeine 0.01 mM, 187.3  27.3, 0.1 mM, 223.7  20.8% of control; OPG: caffeine 0.01 mM, 28.3 W 5.8, 0.1 mM, 31.2 W 8.8%

of control; n ¼ 3, p < 0.05). Moreover, caffeine (0.01– 1 mM) also induced protein expressions of RANKL, COX-2, and OPG increased the PGE2 production in cultured neonatal mouse calvariae (Fig. 3B; RANKL: caffeine 0.01 mM, 255.1  42.2, 0.1 mM, 286.7  59.3% of control; OPG: caffeine 0.01 mM, 33.3  6.8, 0.1 mM, 56.7  9.8% of control; COX-2: caffeine 0.01 mM, 440.0  30.6, 0.1 mM, 966.7  120.2% of control; n ¼ 3, p < 0.05). Caffeine Reduced BMD and Bone Calcium Contents in Growing Rats BMD of lumbar vertebra, femur, and tibia were significantly lowered in growing rats supplemented with 0.2% caffeine in diets for 20 weeks compared to the control group (p < 0.05; Fig. 4A). Moreover, the calcium contents in tibia and femur of rats supplemented with 0.2% caffeine in diets were lower than that in control group (p < 0.05; Fig. 4B). Caffeine supplementation (0.2% in diet) did not affect osteoblast differentiation from bone marrow cells in growing rats detected by alkaline phosphatase activity (p > 0.05; Fig. 5A) and bone mineralization stained by Alizarin Red S (data not shown). Moreover, the osteoclast differentiation of bone marrow cells in growing rats supplemented with 0.2% caffeine was markedly enhanced as compared with control group (p < 0.05; Fig. 5B).

Figure 1. In vitro effects of caffeine on cell viability, apoptosis, and alkaline phosphatase activity. Bone marrow cells were treated with or without caffeine (0.005–1 mM) for 24 h. Cell viability (A) and apoptosis (B) were determined by MTT assay and annexin V/propidium iodide staining, respectively. Data are mean  SD (n ¼ 4). No significant difference was observed among the groups (p > 0.05). In C, alkaline phosphatase activity was detected in cells during osteoblast differentiation with or without caffeine (0.005–0.1 mM) treatment. All data are mean  SD (n ¼ 4). No significant difference was observed among groups (p > 0.05). JOURNAL OF ORTHOPAEDIC RESEARCH JUNE 2011

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Figure 2. In vitro effect of caffeine on osteoclast formation and bone resorption of osteoclasts. (A) Bone marrow stem cells were treated with or without caffeine (0.005 and 0.01 mM) for 5 days under differentiation medium for osteoclast formation. The number of TRAP-positive multinucleated cells was scored. Data are mean  SD (n ¼ 4). p < 0.05 as compared with control. (B) Cells were treated with or without caffeine (0.005 and 0.01 mM) for 5 days, and pit formation assay was used to detect bone resorption by osteoclasts. White holes are the resorption pits formed by osteoclasts on thin calcium phosphate bone plates. Scale bar ¼ 1 mm.

Figure 3. Effects of caffeine on RANKL, OPG, and COX-2 protein expressions in MC3T3-E1 osteoblastic cells and neonatal mouse calvaria. (A) MC3T3-E1 cells were treated with or without caffeine (0.001–1 mM) for 4 days. Protein expressions of RANKL and OPG were determined by western blot analysis. The numbers below represent the fold increase in proteins relative to the untreated group after normalization to the loading control. (B) Neonatal mouse calvariae were treated with or without caffeine (0.005–0.1 mM) for 48 h. The PGE2 production was determined by EIA kit. Data are mean  SD (n ¼ 4). p < 0.05 as compared with control. JOURNAL OF ORTHOPAEDIC RESEARCH JUNE 2011

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Figure 4. In vivo effect of caffeine on BMD and calcium contents in rat bones. Wistar rats were fed diets containing 0% (control), 0.1% caffeine, and 0.2% caffeine for 20 weeks. (A) Initial and final BMDs of lumbar vertebra (A), tibia (B), and femur (C) determined by DEXA. Data are mean  SD (n ¼ 10). p < 0.05 as compared with control. (B) Calcium contents in tibia (a) and femur (b) isolated from control and caffeine-treated rats. Data are mean  SD (n ¼ 10). p < 0.05 as compared with control.

DISCUSSION We found that low-concentration caffeine (0.005– 0.01 mM) did not affect the cell viability and the differentiation of osteoblasts from bone marrow MSCs, but could significantly enhance the differentiation of osteoclasts from bone marrow hematopoietic stem cells (HSCs) and bone resorption activity. These in vitro

results indicate that caffeine is capable of enhancing osteoclastogenesis. Our animal study results also showed that caffeine (22 and 44 mg caffeine/day by diet intake for 20 weeks) significantly reduces BMD and bone calcium content and enhances osteoclastogenesis of HSCs in growing rats. These findings imply that enhancement of osteoclastogenesis may cause

Figure 5. In vivo effects of caffeine on osteoblastogenesis and osteoclastogenesis in rats. Wistar rats were fed diets containing 0% (control) and 0.2% caffeine for 20 weeks. The differentiations of osteoblasts and osteoclasts from bone marrow stem cells isolated from control and caffeine-treated rats were measured. Data are mean  SD (n ¼ 4). p < 0.05 as compared with control. JOURNAL OF ORTHOPAEDIC RESEARCH JUNE 2011

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the reduction in BMD in growing rats fed caffeinesupplemented diets. Some studies indicated that the viability of osteoblasts was significantly decreased at concentrations >0.5 mM caffeine,9 and the formations of osteoblast and mineralization were significantly decreased in the presence of 10 mM caffeine.8 These findings indicated that high concentrations of caffeine may possess the cytotoxicity to osteoblasts and its precursor cells. In contrast, we found that non-cytotoxic concentrations of caffeine (0.005– 0.1 mM) did not affect cell viability and osteoblast differentiation, but effectively enhanced osteoclastogenesis. Moreover, a study in fast-growing rats showed that rat pups from lactating dams received a 20% protein diet with caffeine experience disruption of osteoblasts and retarded bone development.7 This finding infers that caffeine intake in early life exerts effects on osteoblasts and bone development.7 Conversely, our results showed that caffeine supplementation (0.2% in diet) in weaned rats for 20 weeks significantly enhanced osteoclast differentiation, but did not affect osteoblast differentiation from bone marrow cells. The significance of these different effects on osteoblasts in rats exposed to caffeine before and after weaning needs to be clarified. Bone remodeling is an incorporated interaction between resorption and formation, which plays an important role in bone homeostasis. Various factors modulate bone remodeling, including prostaglandins (PGs), 1,25-dihydroxyvitamin D3, parathyroid hormone (PTH), nitric oxide, sex hormones, calcitonin, and growth factors and cytokines.10,11 Bone resorption-related osteoclastogenesis involves important molecules including RANKL, RANK, and OPG.12,13 RANKL expressed by osteoblasts binds to RANK (a receptor of RANKL) expressed by osteoclast progenitors; their interaction stimulates the osteoclast progenitors differentiation into mature osteoclasts in the presence of M-CSF. Moreover, OPG produced by osteoblasts acts as a decoy receptor for RANKL and inhibits osteoclast formation by interrupting the RANK-RANKL interaction. Conversely, PGs are abundant in bone and are potent regulators of bone cell function.14,15 Stimulated PG production by osteoblasts requires both induction of COX-2 expression and availability of arachidonic acid substrate. PGs can increase osteoclast formation by enhancing induction of the RANKL expression in osteoblasts. Okada et al.16 reported that COX-2 protein expression and its associated PG production are necessary for bone resorption responses to 1,25 (OH)2D3 and PTH. COX-2-mediated PGE2 expression is also required for lipopolysaccharide (bacterial endotoxin)-induced inflammatory bone resorption.17 Bradykinin potentiates cytokine-induced PGs biosynthesis in osteoblasts by enhanced expression of COX-2, resulting in increased RANKL expression.18 Compressive force induces osteoclast differentiation by increasing RANKL expression and M-CSF production and decreasing OPG production via a COX-2/ PGE2 pathway in osteoblasts.19 We found that lower

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concentrations of caffeine are capable of increasing the protein expressions of RANKL in cultured osteoblastic MC3T3-E1 cells and neonatal mouse calvariae and decreasing the expression of OPG in osteoblasts. Moreover, COX-2 protein expression and PGE2 production in neonatal mouse calvariae could also be increased by caffeine. These results indicate that caffeine may be able to enhance a COX-2/PGE2-regulated RANKL-mediated osteoclastogenesis. In conclusion, our in vitro study shows that lowconcentration caffeine triggers a COX-2/PGE2-regulated RANKL enhancement in osteoblasts, resulting in increased osteoclast formation. Our in vivo animal study indicates that caffeine is capable of reducing the BMD in growing rats enhanced osteoclastogenesis.

ACKNOWLEDGMENTS This work was supported by a research grant from the National Science Council of Taiwan (NSC96-2313-B-033-001) and the Department of Health, Executive Yuan of Taiwan (DOH93TD-1002).

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15. Fracon RN, Teo´filo JM, Satin RB, et al. 2008. Prostaglandins and bone: Potential risks and benefits related to the use of nonsteroidal anti-inflammatory drugs in clinical dentistry. J Oral Sci 50:247–252. 16. Okada Y, Pilbeam C, Raisz L, et al. 2003. Role of cyclooxygenase-2 in bone resorption. J UOEH 25:185–195. 17. Coon D, Gulati A, Cowan C, et al. 2007. The role of cyclooxygenase-2 (COX-2) in inflammatory bone resorption. J Endod 33:432–436.

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18. Brechter AB, Lerner UH. 2007. Bradykinin potentiates cytokine-induced prostaglandin biosynthesis in osteoblasts by enhanced expression of cyclooxygenase 2, resulting in increased RANKL expression. Arthritis Rheum 56:910– 923. 19. Sanuki R, Shionome C, Kuwabara A, et al. 2010. Compressive force induces osteoclast differentiation via prostaglandin E(2) production in MC3T3-E1 cells. Connect Tissue Res 51: 150–158.