Conjugated linoleic acid inhibits osteoclast differentiation of RAW264.7 cells by modulating RANKL signaling.

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-kappaB 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-kappaB (NF-kappaB), and preexposure of the cells to CLA significantly suppressed RANKL-induced NF-kappaB activation, including phosphorylation of I-kappaBalpha, degradation of I-kappaBalpha, and nuclear translocation of p65. RANKL induced osteoclastogenesis in these monocytic cells, and CLA inhibited RANKL-induced tumor necrosis factor-alpha 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.

Abstract 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 boneresorbing 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-kB 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-kB (NF-kB), and preexposure of the cells to CLA significantly suppressed RANKL-induced NF-kB activation, including phosphorylation of I-kBa, degradation of I-kBa, and nuclear translocation of p65. RANKL induced osteoclastogenesis in these monocytic cells, and CLA inhibited RANKL-induced tumor necrosis factor-a 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. Supplementary key words lipids . monocytes/macrophages . transcription factors . cytokines . cell differentiation . inflammation . signaling pathways . receptor activator of nuclear factor-kB ligand Bone remodeling depends on a delicate balance between bone formation and bone resorption, wherein bone-forming osteoblasts and bone-resorbing osteoclasts play central roles (1). Tipping this balance in favor of osteoclasts leads to pathologic bone resorption, as seen in bone diseases such as autoimmune arthritis and postmenopausal osteoporosis (2).
Osteoclasts are bone-resorptive multinucleated cells (MNCs) derived from hematopoietic stem cells (3,4). Differentiation of osteoclasts is regulated by soluble or membrane-bound molecules expressed by osteoblasts and stromal cells in the bone microenvironment (5). One such factor, receptor activator of nuclear factor-kB ligand (RANKL), a member of the tumor necrosis factor (TNF) family (6)(7)(8)(9), 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) (10). 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 (11). 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 (10). 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 (12,13). In addition, several negative regulators of RANKL signaling have been reported (14)(15)(16). A typical example is osteoprotegerin, which functions as a soluble decoy receptor for RANKL, thereby attenuating excessive RANKL signaling. There-fore, 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 (17), reduction in the risk of atherosclerosis, hypertension, and diabetes, improvement in feed efficiency, promotion of energy metabolism, and positive effect on immune function (18,19). CLA is a collective term used to describe a set of 28 distinct positional and geometric isomers of linoleic acid (LA; 18:2) (18)(19)(20) and is most commonly found at positions cis-9,trans-11 (c9t11) and trans-10,cis-12 (t10c12), both of which possess biological activity (21). CLA isomers occur naturally in ruminant food products (beef, lamb, and dairy) (22). Our recent study showed that CLA significantly reduces body weight and body fat mass in mice, particularly with treadmill exercise (23).
Very recently, it was reported that dietary CLA may positively benefit bone mineral density in postmenopausal women (24). Increased whole body ash in young mice fed a diet with CLA supplementation has been reported (25). CLA increased the bone formation markers osteocalcin and alkaline phosphatase in a murine osteoblastic cell line (26). Anhydrous butterfat, a rich natural source of CLA, also stimulated the rate of bone formation in young growing chicks by modulating prostaglandin E 2 , which plays an important role in the local regulation of bone formation and bone resorption (27). 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 E 2 -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.

Materials
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-a 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-kBa; rabbit polyclonal antibodies against nuclear factor of activated T-cells (NFATc1), I-kBa, and nuclear factor-kB (NF-kB) 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. ( Cell cultures 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 mg/ml streptomycin (Sigma). Incubations were performed at 37jC in 5% CO 2 in humidified air. For osteoclast generation and all other experiments, a-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).
Fatty acid incorporation RAW264.7 cells were suspended in phenol a-MEM containing 10% FBS and plated at a concentration of 1 3 10 6 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 (28,29).
Total TRAP activity RAW264.7 cells were suspended in phenol a-MEM containing 10% FBS and plated at a concentration of 2 3 10 4 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 ml 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 ml of TRAP solution and was incubated at 37jC for 30 min. The reaction was stopped by adding 0.1 N NaOH, and absorbance was measured at 405 nm using a microplate reader.
TRAP-positive cell staining RAW264.7 cells were suspended in phenol a-MEM containing 10% FBS and plated at a concentration of 2 3 10 4 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 1 cells with more than three nuclei were counted as TRAP 1 ve MNCs.
Pit formation assay RAW264.7 cells were suspended in phenol a-MEM containing 10% FBS and plated at a concentration of 1 3 10 4 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 mM) or CLA (50 mM) 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.
TNF-a production in RAW264.7 cell culture RAW264.7 cells were suspended in phenol a-MEM containing 10% FBS and plated at a concentration of 1 3 10 5 cells/well into a 24-well culture dish (Corning) for 24 h. Then, they were treated with either CLA (100 mM) or LA (100 mM) for another 24 h before adding RANKL (50 ng/ml) to stimulate TNF-a secretion for 16 h. At the end of culture, medium was collected and analyzed for TNF-a using the ELISA kit according to the manufacturer's instructions.
Protein preparation and Western blot analysis RAW264.7 cells were cultured on 60 mm plates for 48 h with or without 100 mM LA or CLA. Cells were then treated with sRANKL (50 ng/ml) for 30 min. Cytosolic and nuclear proteins were prepared as described previously (30). In a separate culture, RAW264.7 cells were cultured for 48 h with or without 100 mM  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 mM 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 mg 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
Electrophoretic mobility shift assay (EMSA) was performed using 5 mg of nuclear extracts as described previously using a DNA probe (59-AGTTGAGGGGACTTTCCCAGGC-39) containing the NF-kB binding site (italics) (29).

Statistics
Results are expressed as means 6 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.

CLA incorporation in RAW264.7 cells
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 mM treatment for 24 h are shown in Table 1 as 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).
Effects of CLA on RANKL-stimulated, TRAP-positive osteoclast-like cell formation in RAW264.7 cells 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 RANKLstimulated 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 boneresorbing cells. CLA dramatically inhibited the RANKLstimulated 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).
Effects of CLA on bone resorption in RANKL-stimulated RAW264.7 cells 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).

Effects of CLA on osteoclast-specific gene expression
We previously showed that differentiated RAW264.7 cells express high levels of osteoclast-specific genes such as TRAP, cathepsin K, CTR, and MMP-9 (30). 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).
Effects of CLA on RANKL-induced TNF-a production of RAW264.7 cells TNF-a has the potential to induce osteoclast differentiation (31)(32)(33), and TNF-a together with soluble RANKL strongly promotes osteoclast differentiation and activation in vitro (33,34). Recently, TNF-a has become noteworthy as a target for the treatment of inflammatory bone diseases, and it is expected that understanding of molecules mediating TNF-a signals will provide clues to the development of new therapies for such diseases. Therefore, we examined whether CLA can modulate RANKL-stimulated TNF-a production in RAW264.7 cell cultures. Cultures treated with CLA showed significantly decreased production of TNF-a compared with cultures treated either with RANKL alone or together with LA (Fig. 5).

Effects of CLA on RANKL-stimulated activation of NF-kB and MAPKs
The interaction of RANKL with RANK leads to the activation of the signaling pathways of MAPK (p38 and JNK) and NF-kB. In an effort to elucidate the intracellular mechanisms underlying the inhibitory effect of CLA, we first focused on the NF-kB signaling pathway. NF-kB activation is regulated by three major steps: phosphorylation of I-kBa, I-kBa degradation, and nuclear translocation of p50/ p65 subunits. I-kBa phosphorylation, I-kBa degradation, and p65 NF-kB nuclear translocation were analyzed using cytosolic extracts and nuclear extracts from RANKLstimulated RAW264.7 cells. Treatment of RAW264.7 cells with RANKL significantly increased the phosphorylation of I-kBa, degradation of I-kBa, and nuclear translocation of p65 NF-kB (Fig. 6). Pretreatment of RAW264.7 cells with CLA for 48 h significantly reduced I-kBa phosphorylation, I-kBa degradation, and p65 NF-kB nuclear translocation compared with pretreatment with LA or untreated RANKL-stimulated controls (Fig. 6A, B). RANKL-stimulated NF-kB DNA binding by EMSA was also reduced significantly in CLA-pretreated RAW264.7 cells compared with LA-treated or untreated controls (Fig. 6C). The JNK and p38 MAPK pathways also play an important role in RANKLinduced osteoclast differentiation. Therefore, we next focused on the effect of CLA on the MAPK family members p38 MAPK and JNK. Western blot analysis using antiphospho-specific p38 with phosphorylated tyrosine and JNK antibodies with phosphorylated tyrosine and threonine was performed to determine the influence of CLA or LA on the activation of JNK and p38 MAPKs. As shown in Fig. 7, 45 min treatment of RAW cells with RANKL increased the phosphorylation of p38 and JNK. Pretreatment of RAW cells with CLA for 48 h significantly decreased the phosphorylation of p38 and JNK compared with cells pretreated with LA (Fig. 7). However, there was no significant difference between RANKL-stimulated untreated control and CLAtreated cells in the activation of JNK.
Effects of CLA on the expression of osteoclast-specific transcription factor c-Fos is considered one of the most important osteoclastspecific transcription factors (35,36). Very recently, another transcription factor, NFATc1, was determined as an Fig. 6. CLA inhibited RANKL (RL)-induced nuclear factor-kB (NF-kB) activation. RAW264.7 cells were treated with or without CLA (100 mM) or LA (100 mM) for 48 h, followed by treatment with RANKL (50 ng/ ml) for 30 min. Cytosolic and nuclear extracts were prepared and measured using the bicinchoninic acid kit. Equal amounts of proteins [15 mg of cytosolic extracts and nuclear extracts for Western blot and 5 mg of nuclear extracts for electrophoretic mobility shift assay (EMSA)] were analyzed. A: Western blot analysis using antibodies to phosphorylated (p) I-kBa, I-kBa, and NF-kB p65. Cytosolic extracts were used for pI-kBa and I-kBa analysis, and nuclear extracts were used for NF-kB p65 analysis. B: Relative expression of pI-kBa, I-kBa, and p65 NF-kB. The intensity of the bands was determined by densitometry, and values of pI-kBa and I- important transcription factor involved in osteoclastogenesis. The NFATc1 gene has been identified as the most strongly induced transcription factor gene after RANKLstimulated osteoclast differentiation in RAW264.7 cells and is considered to be the master regulator of osteoclastogenesis (16). We examined whether CLA modulates these transcription factors by Western blot analysis using RANKL-stimulated RAW264.7 cells. Pretreatment of RAW264.7 cells with CLA significantly decreased the RANKL-stimulated upregulation of these transcription factors compared with LA-treated or untreated controls (Fig. 8).

CLA is a strong inhibitor of osteoclastogenesis
In the past two decades, CLA had been associated with many potential health benefits (18). Recently, CLA was reported to enhance immunity and bone formation (24). Current evidence suggests that CLA may help to decrease bone loss by reducing prostaglandins in bone tissue (27,37) or by enhancing calcium absorption (38). Very recently, it was reported that CLA may have a beneficial role in preventing bone loss. Our recent findings also showed higher bone mineral density in young Balb/C male mice fed CLA compared with LA-enriched safflower oil (39). But it was not clear how CLA protected bone loss. The effects of CLA on osteoblastic factors have been primarily explored in animal studies and also in vitro (26,38). However, no study had been conducted to measure the effect of CLA on osteoclastogenesis. This is the first study to demonstrate the inhibitory effect of CLA on osteoclastogenesis.
In our in vitro study using RAW264.7 cells, we found that CLA dose-dependently inhibited RANKL-induced osteoclast differentiation. Although the inhibition of TRAPpositive MNC formation was most dramatic with CLA treatment, the reduction of total TRAP activity was not as dramatic but remained significant. This might be at-tributable to the presence of a number of TRAP-positive mononuclear cells in CLA-treated cultures. TRAP, MMP9, cathepsin K, and CTR are well-known markers of osteoclast differentiation (40). Significant but not dramatic re-  duction of these gene markers was also noted in CLAtreated RAW264.7 cells stimulated with RANKL, as TRAPpositive mononuclear cells are also positive for these genes. Thus, CLA not only significantly inhibits the differentiation of osteoclast precursors into TRAP-positive mononuclear cells but also prevents its fusion to form multinucleated active osteoclasts. Furthermore, TRAPpositive MNCs but not mononuclear cells are the active bone-resorbing cells. Indeed, a dramatic inhibition of bone resorption by CLA indicates that it strongly inhibits the formation of active TRAP-positive MNCs from its precursor cells to resorb bone. However, we found differences in the effect of CLA on TRAP-positive MNC formation and bone resorption. These variations in the effects of CLA might be attributable to the presence of both functional (with ruffled border) and nonfunctional (without ruffled border) multinucleated osteoclasts in the MNC count, whereas bone resorption represents primarily the effect of only functional multinucleated osteoclasts. These findings suggest that CLA not only inhibits osteoclast differentiation but also inhibits its activation as well.

NF-kB/MAPKs may be involved in the CLA-mediated inhibition of osteoclastogenesis
The differentiation and activation of osteoclasts depend on the signal through RANK stimulated with RANKL. Genetic studies using gene-targeting technology have clearly demonstrated that NF-kB and MAPKs, including JNK and p38, which are elicited by RANKL, are essential molecules for osteoclast differentiation (11,16,41). NF-kB knockout mice and transgenic mice expressing dominant-negative c-Jun or dominant-negative JNK1 exhibited osteopetrosis (11,42). In addition, it has been reported that the JNK inhibitor SP600125 and the p38 MAPK inhibitor SB203580 strongly inhibit osteoclastogenesis (11,16). Chung et al. (43) reported that CLA inhibits LPS-induced inflammatory events in RAW264.7 cells by negatively regulating inflammatory mediators and NF-kB activation. In contrast, NF-kB activation by the t10c12 CLA isomer was noted in human adipocytes (43). These variations might be attributable to isomer and/or tissue specificity. Interestingly, in our study, CLA inhibited RANKL-induced NF-kB activation in RAW264.7 cells. NF-kB is considered to be involved not only in the process of differentiation but also in the maturation of osteoclasts (44). Thus, the inhibitory action of CLA on osteoclastogenesis seems to involve the inhibitory effect of this agent on NF-kB activation. In this study, we also found that the phosphorylation of p38 MAPK was decreased after treatment with CLA, suggesting that the inhibition of the p38 MAPK pathway may also be involved in the inhibitory action of this agent. We also tried to determine the effect of CLA on the activation of the JNK pathway, but we could not find strong inhibition of RANKLstimulated JNK activation by CLA. However, JNK activation was significantly lower in CLA-treated cells compared with LA-treated controls. These findings clearly demonstrate that Fig. 9. Possible mechanisms of CLA action on osteoclast differentiation. Several possible inhibitory actions of CLA on osteoclast differentiation are shown. By inhibiting TNF-a production, CLA may suppress the production of RANKL. CLA inhibited the downstream molecules of RANKL signaling such as NF-kB, JNK, and p38. CLA also suppressed the production of important downstream regulators of osteoclast formation, including c-Fos and NFATc1. Thus, CLA inhibits osteoclast differentiation by modulating various cytokines, signaling molecules, and transcription factors. CATK, cathepsin K; MITF, microphthalmia associated transcription factor; OPG, osteoprotegerin.
CLA-mediated inhibition of osteoclastogenesis induced by RANKL reflects the blocking of specific components of these intracellular signals.

c-Fos/NFATc1 may be involved in the CLA-mediated inhibition of osteoclastogenesis
The binding of RANKL to RANK also results in a cascade of intracellular events that, in turn, increases the transcription of the c-Jun and c-Fos genes, and osteopetrosis is a feature of c-Fos-deficient mice (35,36,45). NFATc1 is also a key transcription factor for osteoclast formation. Stimulation of NFATc1 in bone marrow macrophage or RAW264.7 cells by RANKL demonstrates the necessity of NFATc1 in osteoclastogenesis in vitro (11). Most notably, the introduction of NFATc1 promotes the differentiation of bone marrow macrophage cells into TRAP 1 osteoclast-like cells even in the absence of sRANKL (11,16). The osteoclastogenic activity of NFATc1 was enhanced by overexpression of c-Jun and c-Fos but was inhibited by overexpression of either dominant-negative c-Jun or c-Fos, indicating that the partnership between c-Jun/c-Fos and the NFAT family is crucial for osteoclast differentiation (11). Interestingly, CLA inhibited the induction of both c-Fos and NFATc1 by RANKL in RAW264.7 cells, thereby attenuating the process of osteoclastogenesis. Figure 9 summarizes the effect of CLA on osteoclastogenesis. CLA seems to inhibit osteoclastogenesis by modulating various cytokines, signaling molecules, and transcription factors.
In summary, in this study, we present the novel action of CLA on osteoclastogenesis. Osteoclasts have a crucial role in physiologic bone remodeling and also in the pathologic bone loss that is closely associated with inflammatory bone diseases such as rheumatoid arthritis, periodontal disease, and postmenopausal osteoporosis (30). Our findings open up an additional avenue to search out a possible alternative to drugs to prevent the bone loss associated with excessive and progressive osteoclastic bone resorption. We believe that our findings provide clear evidence that CLA may become a novel dietary therapeutic agent after successful clinical trials not only against obesity but also against osteoporotic bone diseases.