Conjugated linoleic acid inhibits osteoclast differentiation of RAW264.7 cells by modulating RANKL signaling
2006; Elsevier BV; Volume: 47; Issue: 8 Linguagem: Inglês
10.1194/jlr.m600151-jlr200
ISSN1539-7262
AutoresMd Mizanur Rahman, Arunabh Bhattacharya, Gabriel Fernandes,
Tópico(s)NF-κB Signaling Pathways
ResumoBone destruction is a pathological hallmark of several chronic inflammatory diseases, including rheumatoid arthritis, periodontitis, and osteoporosis. Inflammation-induced bone loss of this sort results from increased numbers of bone-resorbing osteoclasts. Numerous studies have indicated that conjugated linoleic acid (CLA) positively influences calcium and bone metabolism. Gene-deletion studies have shown that receptor activator of nuclear factor-κB ligand (RANKL) is one of the critical mediators of osteoclastogenesis. In this report, we examine the ability of CLA to suppress RANKL signaling and osteoclastogenesis in RAW264.7 cells, a murine monocytic cell line. Treatment of these cells with RANKL activated nuclear factor-κB (NF-κB), and preexposure of the cells to CLA significantly suppressed RANKL-induced NF-κB activation, including phosphorylation of I-κBα, degradation of I-κBα, and nuclear translocation of p65. RANKL induced osteoclastogenesis in these monocytic cells, and CLA inhibited RANKL-induced tumor necrosis factor-α production and osteoclast differentiation, including osteoclast-specific genes such as tartrate-resistant acid phosphatase, cathepsin K, calcitonin receptor, and matrix metalloproteinase-9 expression and osteoclast-specific transcription factors such as c-Fos, nuclear factor of activated T-cells expression, and bone resorption pit formation. CLA also inhibited RANKL-induced activation of mitogen-activated protein kinase p38 but had little effect on c-Jun N-terminal kinase activation. Collectively, these data demonstrate for the first time that CLA inhibits osteoclastogenesis by modulating RANKL signaling. Thus, CLA may have important therapeutic implications for the treatment of bone diseases associated with enhanced bone resorption by excessive osteoclastogenesis. Bone destruction is a pathological hallmark of several chronic inflammatory diseases, including rheumatoid arthritis, periodontitis, and osteoporosis. Inflammation-induced bone loss of this sort results from increased numbers of bone-resorbing osteoclasts. Numerous studies have indicated that conjugated linoleic acid (CLA) positively influences calcium and bone metabolism. Gene-deletion studies have shown that receptor activator of nuclear factor-κB ligand (RANKL) is one of the critical mediators of osteoclastogenesis. In this report, we examine the ability of CLA to suppress RANKL signaling and osteoclastogenesis in RAW264.7 cells, a murine monocytic cell line. Treatment of these cells with RANKL activated nuclear factor-κB (NF-κB), and preexposure of the cells to CLA significantly suppressed RANKL-induced NF-κB activation, including phosphorylation of I-κBα, degradation of I-κBα, and nuclear translocation of p65. RANKL induced osteoclastogenesis in these monocytic cells, and CLA inhibited RANKL-induced tumor necrosis factor-α production and osteoclast differentiation, including osteoclast-specific genes such as tartrate-resistant acid phosphatase, cathepsin K, calcitonin receptor, and matrix metalloproteinase-9 expression and osteoclast-specific transcription factors such as c-Fos, nuclear factor of activated T-cells expression, and bone resorption pit formation. CLA also inhibited RANKL-induced activation of mitogen-activated protein kinase p38 but had little effect on c-Jun N-terminal kinase activation. Collectively, these data demonstrate for the first time that CLA inhibits osteoclastogenesis by modulating RANKL signaling. Thus, CLA may have important therapeutic implications for the treatment of bone diseases associated with enhanced bone resorption by excessive osteoclastogenesis. Bone remodeling depends on a delicate balance between bone formation and bone resorption, wherein bone-forming osteoblasts and bone-resorbing osteoclasts play central roles (1Manolagas S.C. Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis.Endocr. Rev. 2000; 21: 115-137Crossref PubMed Scopus (2046) Google Scholar). Tipping this balance in favor of osteoclasts leads to pathologic bone resorption, as seen in bone diseases such as autoimmune arthritis and postmenopausal osteoporosis (2Rodan G.A. Martin T.J. Therapeutic approaches to bone diseases.Science. 2000; 289: 1508-1514Crossref PubMed Scopus (1498) Google Scholar). Osteoclasts are bone-resorptive multinucleated cells (MNCs) derived from hematopoietic stem cells (3Burger E.H. Van der Meer J.W. van de Gevel J.S. Gribnau J.C. Thesingh G.W. van Furth R. In vitro formation of osteoclasts from long-term cultures of bone marrow mononuclear phagocytes.J. Exp. Med. 1982; 156: 1604-1614Crossref PubMed Scopus (255) Google Scholar, 4Kurihara N. Suda T. Miura Y. Nakauchi H. Kodama H. Hiura K. Hakeda Y. Kumegawa M. Generation of osteoclasts from isolated hematopoietic progenitor cells.Blood. 1989; 74: 1295-1302Crossref PubMed Google Scholar). Differentiation of osteoclasts is regulated by soluble or membrane-bound molecules expressed by osteoblasts and stromal cells in the bone microenvironment (5Suda T. Takahashi N. Martin T.J. Modulation of osteoclast differentiation.Endocr. Rev. 1992; 13: 66-80PubMed Google Scholar). One such factor, receptor activator of nuclear factor-κB ligand (RANKL), a member of the tumor necrosis factor (TNF) family (6Anderson D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. DuBose R.F. Cosman D. Galibert L. A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function.Nature. 1997; 390: 175-179Crossref PubMed Scopus (1969) Google Scholar, 7Wong B.R. Rho J. Arron J. Robinson E. Orlinick J. Chao M. Kalachikov S. Cayani E. Bartlett 3rd, F.S. Frankel W.N. et al.TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells.J. Biol. Chem. 1997; 272: 25190-25194Abstract Full Text Full Text PDF PubMed Scopus (922) Google Scholar, 8Yasuda H. Shima N. Nakagawa N. Yamaguchi K. Kinosaki M. Mochizuki S. Tomoyasu A. Yano K. Goto M. Murakami A. et al.Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL.Proc. Natl. Acad. Sci. USA. 1998; 95: 3597-3602Crossref PubMed Scopus (3609) Google Scholar, 9Lacey D.L. Timms E. Tan H.L. Kelley M.J. Dunstan C.R. Burgess T. Elliott R. Colombero A. Elliott G. Scully S. et al.Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4673) Google Scholar), plays an essential role in osteoclastogenesis. The binding of RANKL to its receptor RANK leads to the recruitment of TNF receptor-associated factor 6 (TRAF6) to the cytoplasmic domain of RANK, thereby resulting in the activation of distinct signaling cascades mediated by mitogen-activated protein kinases (MAPKs), including c-Jun N-terminal kinase (JNK) and p38 MAPK (p38) (10Boyle W.J. Simonet W.S. Lacey D.L. Osteoclast differentiation and activation.Nature. 2003; 423: 337-342Crossref PubMed Scopus (5050) Google Scholar). It has been shown that JNK1-activated c-Jun signaling in cooperation with nuclear factor of activated T cells (NFAT) is key to RANKL-regulated osteoclast differentiation (11Ikeda F. Nishimura R. Matsubara T. Tanaka S. Inoue J. Reddy S.V. Hata K. Yamashita K. Hiraga T. Watanabe T. et al.Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation.J. Clin. Invest. 2004; 114: 475-484Crossref PubMed Scopus (399) Google Scholar). Stimulation of p38 results in the downstream activation of the microphthalmia/microphthalmia transcription factor, which controls the expression of the genes encoding tartrate-resistant acid phosphatase (TRAP) and cathepsin K, indicating the importance of p38 signaling cascades (10Boyle W.J. Simonet W.S. Lacey D.L. Osteoclast differentiation and activation.Nature. 2003; 423: 337-342Crossref PubMed Scopus (5050) Google Scholar). RANKL also induces c-Fos by an as yet unknown mechanism. Indeed, the essential role of the TRAF6 and c-Fos pathways in osteoclastogenesis as determined by gene targeting studies is well documented (12Grigoriadis A.E. Wang Z.Q. Cecchini M.G. Hofstetter W. Felix R. Fleisch H.A. Wagner E.F. c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling.Science. 1994; 266: 443-448Crossref PubMed Scopus (1085) Google Scholar, 13Lomaga M.A. Yeh W.C. Sarosi I. Duncan G.S. Furlonger C. Ho A. Morony S. Capparelli C. Van G. Kaufman S. et al.TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling.Genes Dev. 1999; 13: 1015-1024Crossref PubMed Scopus (1096) Google Scholar). In addition, several negative regulators of RANKL signaling have been reported (14Simonet W.S. Lacey D.L. Dunstan C.R. Kelley M. Chang M.S. Luthy R. Nguyen H.Q. Wooden S. Bennett L. Boone T. et al.Osteoprotegerin: a novel secreted protein involved in the regulation of bone density.Cell. 1997; 89: 309-319Abstract Full Text Full Text PDF PubMed Scopus (4397) Google Scholar, 15Takayanagi H. Ogasawara K. Hida S. Chiba T. Murata S. Sato K. Takaoka A. Yokochi T. Oda H. Tanaka K. et al.T-cell-mediated regulation of osteoclastogenesis by signalling cross-talk between RANKL and IFN-gamma.Nature. 2000; 408: 600-605Crossref PubMed Scopus (1114) Google Scholar, 16Takayanagi H. Kim S. Koga T. Nishina H. Isshiki M. Yoshida H. Saiura A. Isobe M. Yokochi T. Inoue J. et al.Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts.Dev. Cell. 2002; 3: 889-901Abstract Full Text Full Text PDF PubMed Scopus (2022) Google Scholar). A typical example is osteoprotegerin, which functions as a soluble decoy receptor for RANKL, thereby attenuating excessive RANKL signaling. Therefore, agents that can suppress RANKL signaling can suppress osteoclastogenesis. Considerable attention over the last several years has focused on the possible beneficial effects of dietary conjugated linoleic acid (CLA), including anticarcinogenic and antitumorogenic effects (17Durgam V.R. Fernandes G. The growth inhibitory effect of conjugated linoleic acid on MCF-7 cells is related to estrogen response system.Cancer Lett. 1997; 116: 121-130Crossref PubMed Scopus (118) Google Scholar), reduction in the risk of atherosclerosis, hypertension, and diabetes, improvement in feed efficiency, promotion of energy metabolism, and positive effect on immune function (18Belury M.A. Dietary conjugated linoleic acid in health: physiological effects and mechanisms of action.Annu. Rev. Nutr. 2002; 22: 505-531Crossref PubMed Scopus (731) Google Scholar, 19Wahle K.W. Heys S.D. Rotondo D. Conjugated linoleic acids: are they beneficial or detrimental to health?.Prog. Lipid Res. 2004; 43: 553-587Crossref PubMed Scopus (443) Google Scholar). CLA is a collective term used to describe a set of 28 distinct positional and geometric isomers of linoleic acid (LA; 18:2) (18Belury M.A. Dietary conjugated linoleic acid in health: physiological effects and mechanisms of action.Annu. Rev. Nutr. 2002; 22: 505-531Crossref PubMed Scopus (731) Google Scholar, 19Wahle K.W. Heys S.D. Rotondo D. Conjugated linoleic acids: are they beneficial or detrimental to health?.Prog. Lipid Res. 2004; 43: 553-587Crossref PubMed Scopus (443) Google Scholar, 20Roche H.M. Noone E. Nugent A. Gibney M.J. Conjugated linoleic acid: a novel therapeutic nutrient?.Nutr. Res. Rev. 2001; 14: 173-187Crossref PubMed Scopus (121) Google Scholar) and is most commonly found at positions cis-9,trans-11 (c9t11) and trans-10,cis-12 (t10c12), both of which possess biological activity (21Pariza M.W. Park Y. Cook M.E. Mechanisms of action of conjugated linoleic acid: evidence and speculation.Proc. Soc. Exp. Biol. Med. 2000; 223: 8-13Crossref PubMed Scopus (270) Google Scholar). CLA isomers occur naturally in ruminant food products (beef, lamb, and dairy) (22Lawson R.E. Moss A.R. Givens D.I. The role of dairy products in supplying conjugated linoleic acid to man's diet: a review.Nutr. Res. Rev. 2001; 14: 153-172Crossref PubMed Scopus (144) Google Scholar). Our recent study showed that CLA significantly reduces body weight and body fat mass in mice, particularly with treadmill exercise (23Bhattacharya A. Rahman M.M. Sun D. Lawrence R. Mejia W. McCarter R. O'Shea M. Fernandes G. The combination of dietary conjugated linoleic acid and treadmill exercise lowers gain in body fat mass and enhances lean body mass in high fat-fed male Balb/C mice.J. Nutr. 2005; 135: 1124-1130Crossref PubMed Scopus (74) Google Scholar). Very recently, it was reported that dietary CLA may positively benefit bone mineral density in postmenopausal women (24Brownbill R.A. Petrosian M. Ilich J.Z. Association between dietary conjugated linoleic acid and bone mineral density in postmenopausal women.J. Am. Coll. Nutr. 2005; 24: 177-181Crossref PubMed Scopus (72) Google Scholar). Increased whole body ash in young mice fed a diet with CLA supplementation has been reported (25Park Y. Albright K.J. Liu W. Storkson J.M. Cook M.E. Pariza M.W. Effect of conjugated linoleic acid on body composition in mice.Lipids. 1997; 32: 853-858Crossref PubMed Scopus (982) Google Scholar). CLA increased the bone formation markers osteocalcin and alkaline phosphatase in a murine osteoblastic cell line (26Watkins B.A. Lippman H.E. Le Bouteiller L. Li Y. Seifert M.F. Bioactive fatty acids: role in bone biology and bone cell function.Prog. Lipid Res. 2001; 40: 125-148Crossref PubMed Scopus (151) Google Scholar). Anhydrous butterfat, a rich natural source of CLA, also stimulated the rate of bone formation in young growing chicks by modulating prostaglandin E2, which plays an important role in the local regulation of bone formation and bone resorption (27Watkins B.A. Shen C.L. McMurtry J.P. Xu H. Bain S.D. Allen K.G. Seifert M.F. Dietary lipids modulate bone prostaglandin E2 production, insulin-like growth factor-I concentration and formation rate in chicks.J. Nutr. 1997; 127: 1084-1091Crossref PubMed Scopus (158) Google Scholar). To date, studies have been carried out only to define whether CLA affects calcium and bone metabolism using animal models. However, it is not clear whether CLA protects bone loss as a result of the upregulation of osteoblastic factors and bone formation and/or as a result of the downregulation of osteoclastic factors and bone resorption. In light of the prostaglandin E2-decreasing ability of CLA, it is possible that CLA may have inhibitory effects on osteoclastogenesis. However, the effect of CLA on osteoclastogenesis has not yet been investigated. Hence, this study was designed to examine the effect of CLA on osteoclastogenesis using the mouse myeloid RAW264.7 cell line that differentiates in vitro into osteoclasts upon treatment with specific growth factors. All media components were purchased from GIBCO (Invitrogen Corp., Carlsbad, CA). Fatty acids (CLA c9t11, CLA t10c12, LA) were from Matreya, Inc. (State College, PA). Equal amounts of c9t11 and t10c12 isomers were used for all in vitro cultures. Murine sRANKL was from Pepro Tech, Inc. (Rocky Hill, NJ), reagents for mouse TNF-α ELISA were from BD Bioscience, and the RNA extraction reagent TRIZOL was from Invitrogen Life Technologies (Carlsbad, CA). The reverse transcription kit was from Promega Corp. (Madison, WI). The bicinchoninic acid kit for protein determination was from Pierce Chemical Co. (Rockford, IL). TRAP solution was No. 387 from Sigma Chemical Co. (St. Louis, MO). Mouse monoclonal antibodies against phosphorylated p38 (tyrosine 182), phosphorylated JNK (threonine 183 and tyrosine 185), and phosphorylated I-κBα; rabbit polyclonal antibodies against nuclear factor of activated T-cells (NFATc1), I-κBα, and nuclear factor-κB (NF-κB) p65; and goat polyclonal antibody against actin were from Santa Cruz Biotechnology (Santa Cruz, CA). Chicken polyclonal antibody against c-Fos was purchased from Abcam, Inc. (Cambridge, MA). [32P]ATP was from Perkin-Elmer-NEN (Boston, MA). Oligonucleotides for NF-κB were from Santa Cruz Biotechnology. T4 polynucleotide kinase and Sephadex G-25 M columns were from Promega. Poly [d(I-C)] was from Roche Diagnostics (Indianapolis, IN). All other chemicals were the highest grade available from Sigma Chemical Co. RAW264.7 cells were maintained in DMEM (Sigma) with 10% FCS. All media were supplemented with 2 mM glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin (Sigma). Incubations were performed at 37°C in 5% CO2 in humidified air. For osteoclast generation and all other experiments, α-MEM medium (Sigma) was used. Fatty acid concentrations used in in vitro experiments were optimized by fatty acid incorporation analysis using gas chromatography as described below and by cell viability analysis using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (data not shown). RAW264.7 cells were suspended in phenol α-MEM containing 10% FBS and plated at a concentration of 1 × 106 cells/well into a six-well culture dish (Corning) in the presence of different concentrations of LA or CLA for 24 and 48 h. At the end of culture, the plates were washed with PBS. The cells were then collected using a scraper and analyzed for fatty acid incorporation using gas chromatography as described previously (28Bhattacharya A. Rahman M. Banu J. Lawrence R.A. McGuff H.S. Garrett I.R. Fischbach M. Fernandes G. Inhibition of osteoporosis in autoimmune disease prone MRL/Mpj-Fas(lpr) mice by n-3 fatty acids.J. Am. Coll. Nutr. 2005; 24: 200-209Crossref PubMed Scopus (52) Google Scholar, 29Sun D. Krishnan A. Zaman K. Lawrence R. Bhattacharya A. Fernandes G. Dietary n-3 fatty acids decrease osteoclastogenesis and loss of bone mass in ovariectomized mice.J. Bone Miner. Res. 2003; 18: 1206-1216Crossref PubMed Scopus (231) Google Scholar). RAW264.7 cells were suspended in phenol α-MEM containing 10% FBS and plated at a concentration of 2 × 104 cells/well into a 48-well culture dish (Corning) in the presence of 50 ng/ml RANKL and incubated for 24 h. Then, different concentrations of LA or CLA were added to the cultures. The medium and factors were replaced after 3 days. After 5 days of culture, the medium was removed and the cell monolayer was gently washed twice with PBS. The cells were then lysed with 200 μl of 0.2% Triton X-100. TRAP activity in cell lysate was determined using TRAP solution. An aliquot of cell lysate was added to 150 μl of TRAP solution and was incubated at 37°C for 30 min. The reaction was stopped by adding 0.1 N NaOH, and absorbance was measured at 405 nm using a microplate reader. RAW264.7 cells were suspended in phenol α-MEM containing 10% FBS and plated at a concentration of 2 × 104 cells/well into a 48-well culture dish in the presence of 50 ng/ml RANKL and incubated for 24 h. Then, different concentrations of LA or CLA were added to the cultures. The medium and factors were replaced after 3 days. After 5 days of culture, the cells were fixed and stained for TRAP using the TRAP staining kit according to the manufacturer's instructions. TRAP+ cells with more than three nuclei were counted as TRAP+ve MNCs. RAW264.7 cells were suspended in phenol α-MEM containing 10% FBS and plated at a concentration of 1 × 104 cells/well on an Osteoclast Activity Assay Substrate plate (OCT USA, Inc.) in the presence or absence of 50 ng/ml RANKL and incubated for 24 h. Then, LA (50 μM) or CLA (50 μM) was added to the cultures. Half of the medium was replaced with a fresh one every 2 days. After 7 days of culture, the plates were washed in 6% sodium hypochlorite solution to remove the cells. The resorbed areas on the plates were captured with a digital camera attached to the microscope and analyzed by the Metaview Image Analysis System. RAW264.7 cells (1.0 × 104 cells/ml) were cultured in 60 mm tissue culture dishes (Corning) in the presence of RANKL (50 ng/ml). After 24 h of incubation, 100 μM LA or CLA was added to the culture. Medium was changed every other day. After 4 days of culture, total RNA was extracted using TRIZOL according to the manufacturer's instructions and RT-PCR analysis was performed as described previously (30Rahman M.M. Kukita A. Kukita T. Shobuike T. Nakamura T. Kohashi O. Two histone deacetylase inhibitors, trichostatin A and sodium butyrate, suppress differentiation into osteoclasts but not into macrophages.Blood. 2003; 101: 3451-3459Crossref PubMed Scopus (168) Google Scholar). The following primers were used: TRAP, 5′-AAATCACTCTTTAAGACCA-3′ and 5′-TTATTGAATAGCAGTGACAG-3′; cathepsin K, 5′-GGGCCAGGATGAAAGTTGTA-3′ and 5′-CCGAGCCAAGAGAGCATATC-3′; calcitonin receptor (CTR), 5′-CTGCTCCTAGTGAGCCCAAC-3′ and 5′-CAGCAATCGACAAGGAGTGA-3′; matrix metalloproteinase (MMP)-9, 5′-TCTGAGGCCTCTACAGAGTCT-3′ and 5′-CTCATGGTCCACCTTGTTCAC-3′. PCR products were separated on a 1.5% agarose gel and stained with ethidium bromide. As an internal control for RNA quantity, the same cDNA was amplified using primers specific for GAPDH mRNA. RAW264.7 cells were suspended in phenol α-MEM containing 10% FBS and plated at a concentration of 1 × 105 cells/well into a 24-well culture dish (Corning) for 24 h. Then, they were treated with either CLA (100 μM) or LA (100 μM) for another 24 h before adding RANKL (50 ng/ml) to stimulate TNF-α secretion for 16 h. At the end of culture, medium was collected and analyzed for TNF-α using the ELISA kit according to the manufacturer's instructions. RAW264.7 cells were cultured on 60 mm plates for 48 h with or without 100 μM LA or CLA. Cells were then treated with sRANKL (50 ng/ml) for 30 min. Cytosolic and nuclear proteins were prepared as described previously (30Rahman M.M. Kukita A. Kukita T. Shobuike T. Nakamura T. Kohashi O. Two histone deacetylase inhibitors, trichostatin A and sodium butyrate, suppress differentiation into osteoclasts but not into macrophages.Blood. 2003; 101: 3451-3459Crossref PubMed Scopus (168) Google Scholar). In a separate culture, RAW264.7 cells were cultured for 48 h with or without 100 μM LA or CLA. Cells were then treated with sRANKL (50 ng/ml) for 45 min. Whole cell lysates were then prepared using TNE buffer (10 mM Tris-HCl, pH 7.8, 0.15 M NaCl, 1 mM EDTA, 1% Nonidet P-40, 1 mM DTT, and protease inhibitor cocktail). In a separate culture, RAW264.7 cells were grown on 60 mm plates in the presence of sRANKL. After 24 h of incubation, 100 μM LA or CLA was added to the culture. Medium was changed every other day. After 4 days of culture, cells were washed twice with PBS and dissolved directly on the plate in TNE buffer. After centrifugation for 10 min, whole cell lysates were collected. Protein concentrations of the nuclear extracts, cytosolic extracts, and whole cell extracts were determined using a bicinchoninic acid protein assay kit. Fifteen micrograms of nuclear extracts and cytosolic extracts and 30 μg of whole cell extracts were subjected to SDS-PAGE. Proteins were transferred to immunoblot polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA) and subjected to Western blot analysis. Electrophoretic mobility shift assay (EMSA) was performed using 5 μg of nuclear extracts as described previously using a DNA probe (5′-AGTTGAGGGGACTTTCCCAGGC-3′) containing the NF-κB binding site (italics) (29Sun D. Krishnan A. Zaman K. Lawrence R. Bhattacharya A. Fernandes G. Dietary n-3 fatty acids decrease osteoclastogenesis and loss of bone mass in ovariectomized mice.J. Bone Miner. Res. 2003; 18: 1206-1216Crossref PubMed Scopus (231) Google Scholar). Results are expressed as means ± SEM. Data were statistically analyzed among all groups using one-way ANOVA, and P < 0.05 for F ratio was considered statistically significant. The Newman-Keuls multiple comparison test was used to test the differences between groups for significance. To determine whether in vitro added fatty acids incorporate into cells, we examined the fatty acid profile in LA/CLA-treated RAW264.7 cells using GC. LA/CLA incorporation was measured in RAW264.7 cells treated with different concentrations for different time points. Fatty acid incorporation data with 100 μM treatment for 24 h are shown in Table 1as an example. LA treatment increased the level of LA and arachidonic acid, whereas CLA-treated RAW264.7 cells showed incorporation of CLA fatty acid isomers (Table 1).TABLE 1Fatty acid incorporation in RAW264.7 cells treated with LA (100 μM) or c9t11 (50 μM) + t10c12 (50 μM) CLA for 24 hFatty AcidsUntreatedLAc9t11 + t10c12P14:02.62 ± 0.321.54 ± 0.092.13 ± 0.0816:021.53 ± 0.1819.11 ± 1.3418.17 ± 0.4016:1(n-9)6.46 ± 0.10a3.76 ± 0.24b2.71 ± 0.25b<0.0517:00.41 ± 0.010.47 ± 0.050.51 ± 0.0417:1(n-9)0.80 ± 0.050.79 ± 0.210.55 ± 0.0318:08.43 ± 0.32a14.27 ± 0.04b15.84 ± 0.55b<0.0518:1(n-9)18.49 ± 0.45a10.95 ± 0.24b10.47 ± 0.14b<0.00118:1(n-7)15.27 ± 0.00a10.02 ± 0.35b8.22 ± 0.15c<0.0518:2(n-6) (LA)2.34 ± 0.05a6.90 ± 0.64b2.94 ± 0.08a<0.001c9t11 CLANDND4.29 ± 0.20c9c11 CLANDND3.26 ± 0.02t9t11 CLANDND2.27 ± 0.11t10c12 CLANDND2.13 ± 0.0220:0ND0.28 ± 0.030.31 ± 0.0520:1(n-9)1.54 ± 0.46ab0.88 ± 0.15a1.65 ± 0.16b<0.0520:2(n-6)1.76 ± 0.05a0.66 ± 0.05b0.54 ± 0.12b<0.0120:3(n-6)ND0.68 ± 0.060.48 ± 0.0820:4(n-6) (arachidonic acid)6.58 ± 0.03a12.44 ± 1.19b7.22 ± 0.23a<0.00122:4(n-6)0.82 ± 0.01a3.04 ± 0.13b1.41 ± 0.01c<0.0122:5(n-6)ND2.30 ± 0.10ND22:5(n-3)1.31 ± 0.01a1.58 ± 0.14ab1.92 ± 0.01b<0.0122:6(n-3)3.67 ± 0.162.92 ± 0.343.10 ± 0.12CLA, conjugated linoleic acid; LA, linoleic acid. Values shown are means ± SEM of three independent experiments with triplicate cultures. Statistical differences were evaluated among all groups with one-way ANOVA. Values with different letters are significantly different. ND, not detected. Open table in a new tab CLA, conjugated linoleic acid; LA, linoleic acid. Values shown are means ± SEM of three independent experiments with triplicate cultures. Statistical differences were evaluated among all groups with one-way ANOVA. Values with different letters are significantly different. ND, not detected. As RAW264.7 cells differentiate into osteoclasts in the presence of RANKL, we examined the effect of CLA on the osteoclast differentiation of RAW264.7 cells in the presence of RANKL using TRAP staining. Both mononuclear osteoclast precursor cells and multinucleated osteoclasts are positive for TRAP. At first, we examined whether CLA has any effect on total TRAP activity (Fig. 1). CLA dose-dependently inhibited total TRAP activity in RANKL-stimulated RAW264.7 cells (Fig. 1). Next, we examined whether CLA has any effect on multinucleated osteoclast-like cell formation in RANKL-stimulated RAW264.7 cells (Fig. 2). Multinucleated osteoclasts are the active bone-resorbing cells. CLA dramatically inhibited the RANKL-stimulated osteoclast-like cell formation in RAW264.7 cells in a dose-dependent manner (Fig. 2). However, LA did not show any significant difference in total TRAP activity and osteoclast-like cell formation with different doses (Figs. 1, 2).Fig. 2CLA dose-dependently inhibited RANKL (RL)-stimulated TRAP+ve multinucleated cells (MNCs) in RAW264.7 cells. RAW264.7 cells were cultured with RANKL (50 ng/ml) for 24 h. Then, different concentrations of CLA or LA were added and cultured for another 4 days. Cells were then fixed and stained for TRAP. The number of TRAP+ MNCs was counted in each well. Values shown are means ± SEM of three independent experiments with triplicate cultures. Statistical differences were evaluated among all groups with one-way ANOVA. Values with different letters are significantly different: a versus b (P < 0.001), a versus c (P < 0.001), a versus d (P < 0.001), b versus c (P < 0.05), b versus d (P < 0.05), c versus d (P < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT) As we have already seen that CLA dramatically inhibits the formation of osteoclast-like MNCs, which are believed to be responsible for bone resorption, we further examined whether CLA has any effect on the ability of these mature osteoclasts to resorb bone. We used calcium phosphate-coated culture plates to stimulate RAW264.7 cells with RANKL to differentiate mature osteoclasts having bone-resorbing capacity. RANKL-stimulated RAW264.7 cells showed a number of resorption areas (Fig. 3A). Cultures treated with CLA showed significantly reduced numbers and areas of resorption pits compared with cultures treated with either RANKL alone or together with LA (Fig. 3). We previously showed that differentiated RAW264.7 cells express high levels of osteoclast-specific genes such as TRAP, cathepsin K, CTR, and MMP-9 (30Rahman M.M. Kukita A. Kukita T. Shobuike T. Nakamura T. Kohashi O. Two histone deacetylase inhibitors, trichostatin A and sodium butyrate, suppress differentiation into osteoclasts but not into macrophages.Blood. 2003; 101: 3451-3459Crossref PubMed Scopus (168) Google Scholar). To determine whether the inhibitory effect of CLA correlates with the expression of the osteoclast-specific genes, total RNA was prepared and analyzed by semiquantitative RT-PCR. RANKL-stimulated TRAP, cathepsin K, CTR and MMP-9 mRNA levels in RAW264.7 cells were significantly lower in CLA-treated cultures compared with LA-treated or untreated controls (Fig. 4). TNF-α has the potential to induce osteoclast differentiation (31Kanazawa K. Kudo A. TRAF2 is essential for TNF-alpha-induced osteoclastogenesis.J. Bone Miner. Res. 2005; 20: 840-847Crossref PubMed Scopus (88) Google Scholar, 32Kobayashi K. Takahashi N. Jimi E. Udagawa N. Takami M. Kotake S. Nakagawa N. Kinosaki M. Yamaguchi K. Shima N. et al.Tumor necrosis factor alpha stimulates osteoclast differentiation by a
Referência(s)