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Molecular Cancer Research 4:275-281 (2006)
© 2006 American Association for Cancer Research

Signaling and Regulation

1'-Acetoxychavicol Acetate Inhibits RANKL–Induced Osteoclastic Differentiation of RAW 264.7 Monocytic Cells by Suppressing Nuclear Factor-{kappa}B Activation

Haruyo Ichikawa1, Akira Murakami2 and Bharat B. Aggarwal1

1 Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas and 2 Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan

Requests for reprints: Bharat B. Aggarwal, Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, Box 143, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-3503/6459; Fax: 713-794-1613. E-mail: aggarwal{at}mdanderson.org


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Osteoclastogenesis is commonly associated with various age-related diseases, including cancer. A member of the tumor necrosis factor superfamily, receptor activator of nuclear factor-{kappa}B (NF-{kappa}B) ligand (RANKL), has been shown to play a critical role in osteoclast formation and bone resorption. Thus, agents that suppress RANKL signaling have a potential to suppress bone loss. In this report, we investigated the effect of 1'-acetoxychavicol acetate (ACA), a component of Alpina galanga, on RANKL signaling and consequent osteoclastogenesis in RAW 264.7 cells, a murine monocytic cell line. Treatment of these cells with RANKL activated NF-{kappa}B, and coexposure of the cells to ACA completely suppressed RANKL-induced NF-{kappa}B activation in a time- and concentration-dependent manner. The suppression of NF-{kappa}B by ACA was mediated through suppression of RANKL-induced activation of I{kappa}B{alpha} kinase, I{kappa}B{alpha} phosphorylation, and I{kappa}B{alpha} degradation. Furthermore, incubation of monocytic cells with RANKL induced osteoclastogenesis, and ACA suppressed it. Inhibition of osteoclastogenesis was maximal when cells were simultaneously exposed to ACA and RANKL and minimum when ACA was added 2 days after RANKL. ACA also inhibited the osteoclastogenesis induced by human breast cancer MCF-7 cells, multiple myeloma MM1 cells, and head and neck squamous cell carcinoma LICR-LON-HN5 cells. These results indicate that ACA is an effective blocker of RANKL-induced NF-{kappa}B activation and of osteoclastogenesis induced by RANKL and tumor cells, suggesting its potential as a therapeutic agent for osteoporosis and cancer-associated bone loss. (Mol Cancer Res 2006;4(4):275–81)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Bone resorption is commonly associated with various age-related diseases, including cancer. Attempts to understand the process at the molecular level are under way on many fronts, in the hope that it can be modulated. Bone resorption is accomplished by osteoclasts, which are multinucleated cells belonging to the monocytic/macrophagic lineage and are formed by fusion of mononuclear precursors. This multistep differentiation process is under the control of the bone microenvironment, which includes stromal cells, osteoblasts, and local factors (1). One of the key factors mediating osteoclastogenesis is receptor activator of nuclear factor-{kappa}B (NF-{kappa}B; RANK) ligand (RANKL; ref. 2), a member of the tumor necrosis factor (TNF) family. It is also called osteoclast differentiation factor (3), TNF-related activation-induced cytokines (4), and osteoprotegrin ligand (5). RANKL, which is expressed on the surface of osteoblastic/stromal cells, is directly involved in the differentiation of monocyte-macrophages into osteoclasts (3, 5, 6). Mice with a disrupted RANKL gene show a lack of osteoclasts, severe osteopetrosis, and a defect in tooth eruption, indicating that RANKL is essential for osteoclast differentiation (7). In addition, RANKL induces osteoclast polarization and hypercalcemia (8, 9).

RANKL-induced osteoclastogenesis is mediated through the cell surface receptor RANK. Through the recruitment of the adapter proteins TNF receptor-associated factor 2, 3, 5, and 6 and NF-{kappa}B-inducing kinase (NIK), RANK activates NF-{kappa}B and c-Jun NH2-terminal kinase, p38 mitogen-activated protein kinase, and p44/p42 mitogen-activated protein kinase signaling pathways (10-13). Recently, molecular adapter Grb-2-associated binder-2 was also found to associate with RANK and to mediate RANK-induced activation of NF-{kappa}B, Akt, and c-Jun NH2-terminal kinase (14). Grb-2-associated binder-2 was found to be crucial in the differentiation of human progenitor cells into osteoclasts.

Various tumor cells induce osteoclastogenesis by secreting RANKL (15–24); therefore, the selective modulation of RANKL signaling pathways may have important therapeutic implications for cancer-induced bone loss as well as osteoporosis and osteoarthritis. Drugs that can suppress RANKL signaling are currently in clinical trial for osteoporosis. A single-dose placebo-controlled study of AMG 162, a fully human monoclonal antibody to RANKL, is under way in postmenopausal women (25).

In our own search for agents that would inhibit the RANKL signaling pathway, we have explored the world of natural products. Indeed, >70% of all cancer drugs approved by the Food and Drug Administration during the last two decades have been based on natural products (26). In this report, we describe the results of our study of the effects of 1'-acetoxychavicol acetate (ACA), a natural product derived from the rhizomes of an Asian ginger, Languas galanga Stuntz (Zingiberaceae). Because this agent suppresses NF-{kappa}B activation and NF-{kappa}B-regulated gene products (27, 28), we hypothesized that it would also suppress RANKL signaling and osteoclastogenesis. We found that ACA suppressed RANKL-induced NF-{kappa}B activation through inhibition of I{kappa}B{alpha} kinase (IKK) and inhibited osteoclastogenesis induced by RANKL and by various types of tumor cells.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
The goal of this study was to investigate the effect of ACA on RANKL-induced NF-{kappa}B activation pathway and on osteoclastogenesis induced by both RANKL and tumor cells. We used the murine monocytic cell line RAW 264.7 cell system, as it is a well-established model for osteoclastogenesis (29, 30).

ACA Inhibits RANKL-Induced NF-{kappa}B Activation
To determine the effect of ACA on RANKL-induced NF-{kappa}B activation in RAW 264.7 cells, we coincubated these cells with RANKL for different times in the absence and presence of ACA, prepared nuclear extracts, and assayed NF-{kappa}B activation by electrophoretic mobility shift assay. ACA completely abrogated RANKL-induced NF-{kappa}B activation (Fig. 1A ).


Figure 1
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  		FIGURE 1. RANKL induces NF-{kappa}B activation and ACA inhibits it in a time-dependent (A) and concentration-dependent (B) manner. A. RAW 264.7 cells (1 x 106) were left untreated (left) or coincubated with ACA (50 µmol/L; right) and RANKL (10 nmol/L) for the times indicated. Nuclear extracts were then prepared and assayed for NF-{kappa}B activation by electrophoretic mobility shift assay as described in Materials and Methods. B. RAW 264.7 cells (1 x 106) were coincubated with the indicated concentrations of ACA and then either untreated (left) or treated with RANKL (10 nmol/L; right). Nuclear extracts were then prepared and assayed for NF-{kappa}B activation by EMSA as described in Materials and Methods. Fold value is based on the medium control as 1.

 
To determine the minimum amount of ACA required for suppression of RANKL-induced NF-{kappa}B activation, cells were incubated with RANKL and various concentrations of ACA. ACA at 2.5 µmol/L inhibited RANKL-induced NF-{kappa}B activation by ~50% (Fig. 1B) and 10 µmol/L ACA almost completely inhibited it. Supershift assay of NF-{kappa}B-DNA probe binding showed that RANKL-activated NF-{kappa}B consisted of p65 and p50 subunits (data not shown). The specificity of the RANKL-induced formation of the NF-{kappa}B-DNA complexes was further confirmed by showing that binding was abolished by the presence of a 100-fold excess of unlabeled {kappa}B-oligonucleotides but not by the mutated oligonucleotide (data not shown).

ACA Inhibits RANKL-Induced I{kappa}B{alpha} Degradation
Activation of NF-{kappa}B by most agents requires degradation of its inhibitory subunit I{kappa}B{alpha}. To investigate the mechanism involved in the inhibition of NF-{kappa}B activation by ACA, we first checked the effects of ACA treatment on the levels of I{kappa}B{alpha} by Western blot analysis. The I{kappa}B{alpha} levels decreased within 15 minutes after treatment of cells with RANKL and returned to normal levels within 120 minutes (Fig. 2A, left ). In contrast, cells coincubated with ACA suppressed RANKL-induced I{kappa}B{alpha} degradation (Fig. 2A, right). This effect was specific, as actin levels did not change.


Figure 2
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  		FIGURE 2. ACA inhibits RANKL-induced I{kappa}B{alpha} degradation (A) and phosphorylation (B) through inhibition of IKK activity (C). A. RAW 264.7 cells (1 x 106) were coincubated with either RANKL alone (left) or in the presence of ACA (50 µmol/L) and RANKL (10 nmol/L) for the times indicated (right). Cytoplasmic extracts were prepared, fractionated on 10% SDS-PAGE, and electrotransferred to nitrocellulose membranes. Western blot analysis was done with anti-I{kappa}B{alpha}. B. RAW 264.7 cells (1 x 106) were incubated with either 50 µg/mL N-acetyl-Leu-Leu-norleucinal (ALLN) for 30 minutes or ACA (50 µmol/L) for 15 minutes or with RANKL (10 nmol/L) for 15 minutes or with the indicated combinations. Cytoplasmic extracts were prepared, fractionated on 10% SDS-PAGE, and electrotransferred to nitrocellulose membranes. Western blot analysis was done using either anti-phospho-specific-I{kappa}B{alpha} (top) or anti-I{kappa}B{alpha} (bottom). C. RAW 264.7 cells (3 x 106) were incubated with 50 µg/mL N-acetyl-Leu-Leu-norleucinal for 30 minutes and then coincubated with either RANKL (10 nmol/L) alone (left) or RANKL in the presence of ACA (50 µmol/L) for the indicated times (right). Whole-cell extracts were immunoprecipitated using antibody against IKK-{alpha} and analyzed by an immune complex kinase assay using recombinant glutathione S-transferase (GST)-I{kappa}B{alpha} as described in Materials and Methods. To examine the effect of ACA on the level of IKK proteins, whole-cell extracts were fractionated on 7.5% SDS-PAGE and examined by Western blot analysis using anti-IKK-{alpha} (middle) and anti-IKK-ß (bottom) antibodies.

 
ACA Inhibits RANKL-Induced I{kappa}B{alpha} Phosphorylation
The degradation of I{kappa}B{alpha} by most agents requires phosphorylation of inhibitory subunit I{kappa}B{alpha}, which occurs before its dissociation, ubiquitination, and degradation (31). We used N-acetyl-Leu-Leu-norleucinal, a proteasome inhibitor, to prevent I{kappa}B{alpha} degradation. Western blot analysis for phospho-I{kappa}B{alpha} (Fig. 2B) clearly indicates that N-acetyl-Leu-Leu-norleucinal alone increases the levels of I{kappa}B{alpha} phosphorylation in RAW 264.7 cells. However, these levels are further enhanced by RANKL and treatment of cells with ACA decreased I{kappa}B{alpha} phosphorylation induced by RANKL and N-acetyl-Leu-Leu-norleucinal. ACA alone did not lead to phosphorylation of I{kappa}B{alpha}.

ACA Inhibits RANKL-Induced IKK Activation
Because IKK phosphorylates I{kappa}B{alpha}, we next checked whether ACA alters the activity or the levels of IKK. Immunocomplex kinase assay on cells treated with RANKL showed a sharp increase in IKK activity as indicated by the phosphorylation of glutathione S-transferase-I{kappa}B{alpha} within 5 minutes. In contrast, cells pretreated with ACA could not phosphorylate glutathione S-transferase-I{kappa}B{alpha} on RANKL treatment (Fig. 2C, top). To check whether the apparent loss of IKK activity was due to the loss of IKK protein expression, the levels of the IKK subunits IKK-{alpha} and IKK-ß were tested by Western blot analysis. Results in Fig. 2C clearly show that ACA treatment did not alter the expression of IKK-{alpha} and IKK-ß.

ACA Inhibits RANKL-Induced Osteoclastogenesis of RAW 264.7 Cells
Next, we investigated the effect of ACA on RANKL-induced osteoclastogenesis. RAW 264.7 cells were incubated with different concentrations of ACA in the presence of RANKL and allowed to differentiate into osteoclasts. Figure 3A illustrates that RANKL induced osteoclasts at day 4. By contrast, ACA treatment impaired the generation of osteoclasts in a concentration-dependent manner (Fig. 3B). As little as 0.1 µmol/L ACA had a significant effect on RANKL-induced osteoclast formation. Under these conditions, the viability of cells was not significantly affected (data not shown).


Figure 3
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  		FIGURE 3. ACA inhibits RANKL-induced osteoclastogenesis. A. RAW 264.7 cells (1 x 104) were incubated with either medium or RANKL (5 nmol/L) or RANKL and ACA (0.5 µmol/L) for 5 days and then stained for TRAP expression. TRAP-positive cells were photographed. Original magnification, x100. B. RAW 264.7 cells (1 x 104) were incubated with either medium or RANKL (5 nmol/L) along with indicated concentration of ACA for 3, 4, or 5 days and then stained for TRAP expression. Multinucleated (three nuclei) osteoclasts were counted. C, cells exposed to medium alone. Columns, mean of three measurements; bars, SD. a, P < 0.005.

 
ACA Acts at Early Step in the Pathway Leading to RANKL-Induced Osteoclastogenesis
It normally takes up to 5 days for RAW 264.7 cells to differentiate into osteoclasts in response to RANKL. To determine how early in this pathway ACA acts, we treated the RAW 264.7 cells with RANKL, added ACA on different days, and then checked its effect on osteoclast formation. ACA inhibited osteoclastogenesis even when the cells were exposed 24 hours after the RANKL treatment. However, the inhibitory effect decreased significantly when cells were treated with ACA 2 days after RANKL treatment (Fig. 4 ).


Figure 4
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  		FIGURE 4. ACA inhibits RANKL-induced osteoclastogenesis 24 hours after stimulation. RAW 264.7 cells (1 x 104) were incubated with RANKL (5 nmol/L) and ACA (0.5 µmol/L) for the indicated intervals. Cells were cultured for 5 days after RANKL treatment and stained for TRAP expression. Multinucleated (three nuclei) osteoclasts were counted. C, cells treated with medium alone. Columns, mean of three measurements; bars, SD. a, P < 0.005.

 
ACA Inhibits Osteoclastogenesis Induced by Tumor Cells
Osteoclastogenesis is commonly associated with breast cancer (32, 33), multiple myeloma (34), and squamous cell carcinoma (35) through the secretion of RANKL (34). Whether ACA also inhibits tumor cell–induced osteoclastogenesis of RAW 264.7 cells, and ACA inhibited this response. As shown in Fig. 5 , all three tumor cell types induced osteoclastogenesis, and ACA inhibited it.


Figure 5
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  		FIGURE 5. ACA inhibits osteoclastogenesis induced by tumor cells. A to C. RAW 264.7 cells (1 x 104) were incubated in the presence of LICR-LON-HN5 cells (1 x 103; A) or MM1 cells (1 x 103; B) and MCF-7 cells (1 x 103; C) for 24 hours, then exposed to ACA (0.1 µmol/L) for 5 days, and thereafter stained for TRAP expression. Multinucleated (three nuclei) osteoclasts were counted in cocultures. Columns, mean of three measurements; bars, SD. a, P < 0.005.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
In the present study, we used a homogeneous, clonal population of murine monocytic RAW 264.7 cells to show a direct effect of ACA on osteoclast development induced by RANKL. The advantage of this system is that it does not contain any osteoblast/bone marrow stromal cells or cytokine like macrophage-colony-stimulating factor and allows us to focus on RANKL signaling in preosteoclast cells. In our study, we showed that ACA inhibited RANKL-mediated NF-{kappa}B activation by inhibiting IKK activity, thus preventing osteoclast formation. We also found that ACA inhibited the initial phase of cell differentiation and fusion induced by RANKL and the formation of multinucleated cells and that ACA suppressed osteoclastogenesis induced by breast, head and neck, and multiple myeloma tumor cells.

Our results indicate that RANKL activated NF-{kappa}B in osteoclastic precursor cells through the activation of IKK and subsequent I{kappa}B{alpha} phosphorylation and degradation. These results agree with those of Wei et al. (36). We also showed that ACA inhibited RANKL-induced IKK activation, leading to the suppression of NF-{kappa}B activation. The mechanism of NF-{kappa}B activation induced by RANKL differs from that of TNF. For instance, NIK, although required for RANKL-induced NF-{kappa}B activation (37), is dispensable for TNF-induced NF-{kappa}B activation (38).

A more recent study showed that IKKs are potent regulators of cytokine-induced osteoclastogenesis and inflammatory arthritis (34). Although the ability of ACA to suppress TNF-induced IKK activation has been established (27), ours is the first report to suggest that ACA can also suppress RANKL-induced IKK activation. How ACA inhibits RANKL-induced IKK activation is not clear. Numerous kinases have been implicated in the activation of IKK, including AKT, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1, 2, and 3, glycogen synthase kinase-3ß, and NIK (39). Previous work from our laboratory has shown that NIK is required for RANKL-induced NF-{kappa}B activation (40). Our laboratory has also shown that NIK-induced NF-{kappa}B activation is blocked by ACA (27). Thus, it is possible that ACA inhibits RANKL-induced IKK activation through inhibition of NIK. However, other potential mechanisms cannot be ruled out based on the data presented here.

The interaction of RANKL and RANK results in a cascade of intracellular events, including the activation of NF-{kappa}B and the protein kinase c-Jun NH2-terminal kinase (10, 11, 41). NF-{kappa}B signaling has been shown to play an important role in osteoclastogenesis (42). NF-{kappa}B p50–/– and p52–/– double knockout mice exhibit severe osteopetrosis caused by failure of osteoclast formation (38, 43). NF-{kappa}B is activated by RANKL in both RAW 264.7 cells and monocytes (5, 11, 44, 45), and it is required in vivo for osteoclast formation (43). Both p50 and p52 expression are essential for RANK-expressing osteoclast precursors to differentiate into tartrate-resistant acid phosphatase (TRAP)–positive osteoclasts in response to RANKL and other osteoclastogenic cytokines (46). Therefore, suppression of NF-{kappa}B activation would play an important role in osteoclast formation. In the present study, we found that suppression of NF-{kappa}B activation by ACA correlated with inhibition of osteoclastogenesis. Results presented in this study show that NF-{kappa}B activation was critical for RANKL-induced osteoclastogenesis.

Breast cancers commonly cause osteolytic metastases in bone, a process that is dependent on osteoclast-mediated bone resorption, but the mechanism responsible for tumor-mediated osteoclast activation has not yet been clarified. We showed in this study that ACA inhibited the induction of osteoclastogenesis by breast cancer cells (MCF-7). Besides blocking breast cancer–induced osteoclastogenesis, ACA also blocked osteoclastogenesis induced by head and neck squamous cell carcinoma (LICR-LON-HN5) and multiple myeloma (MM1).

ACA, derived from Asian ginger, should have minimum toxicity, as it is consumed routinely in Japan and other Asian countries; thus, it could be safely used in the treatment of secondary bone lesions associated with various cancers (17, 32-35), including breast cancer and those associated with nonmalignant diseases, such as postmenopausal osteoporosis, Paget's disease, and rheumatoid arthritis. Further studies are needed in animals to validate these findings.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Materials
ACA synthesized and supplied by Dr. Akira Murakami (Kyoto University, Kyoto, Japan) was prepared as 50 mmol/L solution in DMSO and then further diluted in cell culture medium. D-MEM/F-12, fetal bovine serum, 0.4% trypan blue vital stain, and antibiotic-antimycotic mixture were obtained from Invitrogen (Carlsbad, CA). Rabbit polyclonal antibodies to I{kappa}B{alpha} were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against phospho-I{kappa}B{alpha} was purchased from Cell Signaling Technology (Beverly, MA). Anti-IKK-{alpha} and anti-IKK-ß antibodies were kindly provided by Imgenex (San Diego, CA). Goat anti-rabbit horseradish peroxidase conjugate was purchased from Bio-Rad (Hercules, CA); goat anti-mouse horseradish peroxidase and BioCoat osteologic bone cell culture system from BD Biosciences (San Jose, CA); and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide from Sigma-Aldrich (St. Louis, MO). Protein A/G-Sepharose beads were obtained from Pierce (Rockford, IL). [{gamma}-32P]ATP was from ICN Pharmaceuticals (Costa Mesa, CA).

Cell Lines
The mouse macrophage cell line RAW 264.7 was obtained from American Type Culture Collection (Manassas, VA). RAW 264.7 cells were cultured in D-MEM/F-12 supplemented with 10% fetal bovine serum and antibiotics. This cell line has been shown to express RANK and differentiate into TRAP-positive, functional osteoclasts when cocultured with soluble RANKL (11). Moreover, RANKL has been shown to activate NF-{kappa}B in these cells (36). TRAP staining was done using a leukocyte acid phosphatase kit (387-A) from Sigma-Aldrich. MCF-7 (human breast adenocarcinoma) cells were obtained from American Type Culture Collection. These cells were cultured in RPMI 1640 with 10% fetal bovine serum. LICR-LON-HN5 (human squamous cell carcinoma) cells were obtained from Dr. M.J. O'Hare (Haddow Laboratories, Institute of Cancer Research, Sutton, Surrey, United Kingdom). MM1 cells (multiple myeloma) were cultured in RPMI 1640 with 10% fetal bovine serum. LICR-LON-HN5 and SCC4 cells were cultured in DMEM containing 10% fetal bovine serum, 100 µmol/L nonessential amino acids, 1 mmol/L pyruvate, 6 mmol/L L-glutamine, and 1x vitamins. Culture media were also supplemented with 100 units/mL penicillin and 100 µg/mL streptomycin.

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assays for NF-{kappa}B
Nuclear extracts were prepared as described previously (47). Briefly, 1.0 x 106 cells were washed with cold PBS and suspended in 0.5 mL hypotonic lysis buffer containing protease inhibitors for 30 minutes. The cells were then lysed with 5 µL of 10% NP40. The homogenate was centrifuged, and supernatant containing the cytoplasmic extracts was removed and stored frozen at –80°C. The nuclear pellet was resuspended in 20 µL ice-cold nuclear extraction buffer. After 1 hour of intermittent mixing, the extract was centrifuged, and supernatants containing nuclear extracts were secured. The protein content was measured by the Bradford method. If the samples were not used immediately, they were stored at –80°C. To determine NF-{kappa}B activation, we did electrophoretic mobility shift assay as described previously (47), with the following exceptions. Briefly, nuclear extracts prepared from RANKL-treated cells (1 x 106 cells/mL) were incubated with 32P-end-labeled 45-mer double-stranded NF-{kappa}B oligonucleotide (15 µg protein with 16 fmol DNA) from the HIV long terminal repeat, 5'-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3' (boldface indicates NF-{kappa}B-binding sites), for 30 minutes at 37°C, and the DNA-protein complex formed was separated from free oligonucleotide on 6.6% native polyacrylamide gels. A double-stranded mutated oligonucleotide, 5'-TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3', was used to examine the specificity of binding of NF-{kappa}B to the DNA. The specificity of binding was also examined by competition with the unlabeled oligonucleotide. The dried gels were visualized with a Storm820 and radioactive bands were quantified using ImageQuant software (Amersham, Piscataway, NJ).

Western Blot Analysis
To determine the levels of protein expression in the cytoplasm or nucleus, we prepared extracts (48) and fractionated them by 10% SDS-PAGE. After electrophoresis, the proteins were electrotransferred to nitrocellulose membranes, blotted with each antibody, and detected by enhanced chemiluminescence regent (Amersham). The bands obtained were quantified using NIH imaging software (NIH, Bethesda, MD).

IKK Assay
To determine the effect of ACA on RANKL-induced IKK activation, IKK assay was done by a method described previously (49). Briefly, the IKK complex from whole-cell extracts (400 µg protein) was precipitated with antibody against IKK-{alpha} followed by treatment with protein A/G-Sepharose beads. After 2 hours of incubation, the beads were washed with lysis buffer and assayed in a kinase assay mixture containing 50 mmol/L HEPES (pH 7.4), 20 mmol/L MgCl2, 2 mmol/L DTT, 20 mCi [{gamma}-32P]ATP, 10 mmol/L unlabeled ATP, and 2 µg substrate glutathione S-transferase-I{kappa}B{alpha} (amino acids 1-54). After incubation at 30°C for 30 minutes, the reaction was terminated by boiling with SDS sample buffer for 5 minutes. Finally, the protein was resolved on 10% SDS-PAGE, the gel was dried, and the radioactive bands were visualized with a PhosphorImager. To determine the total amounts of IKK-{alpha} and IKK-ß in each sample, the whole-cell protein (50 µg) was resolved on 7.5% SDS-PAGE, electrotransferred to a nitrocellulose membrane, and blotted with anti-IKK-{alpha} or anti-IKK-ß antibody.

Osteoclast Differentiation Assay
RAW 264.7 cells were cultured in 24-well dishes at a density of 1 x 104 cells per well and allowed to adhere overnight. D-MEM/F-12 was then replaced, and the cells were treated with 5 nmol/L (100 ng/mL) RANKL. At day 5, cultures were stained for TRAP expression as described (50) using an acid phosphatase kit, and the total number of TRAP-positive multinucleated osteoclasts (three nuclei) per well was counted.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Walter Pagel for his critical review of this article and Dr. Bryant Darnay for RANKL protein.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Grant support: Clayton Foundation for Research, Department of Defense/U.S. Army Breast Cancer Research Program grant BC010610, NIH on Lung Chemoprevention grant P01 CA91844, and NIH P50 Head and Neck Specialized Programs of Research Excellence grant (B.B. Aggarwal).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11/ 5/05; revised 2/14/06; accepted 3/ 1/06.


   References
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 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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