Article pubs.acs.org/jnp
Magnolol Inhibits RANKL-Induced Osteoclast Differentiation of RAW 264.7 Macrophages through Heme Oxygenase-1-Dependent Inhibition of NFATc1 Expression Sheng-Hua Lu,† Tso-Hsiao Chen,‡ and Tz-Chong Chou*,†,§,⊥,∥ †
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan Division of Nephrology, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan § Institute of Medical Sciences, Tzu Chi University, Hualien, Taiwan ⊥ Department of Biotechnology, Asia University, Taichung, Taiwan ∥ China Medical University Hospital, China Medical University, Taichung, Taiwan ‡
ABSTRACT: Magnolol (1) isolated from Magnolia of f icinalis exhibits many beneficial effects such as anti-inflammatory and antioxidant activity. The aim of this study was to evaluate the effects of magnolol (1) on RANKL-induced osteoclast differentiation and investigate the underlying molecular mechanisms. Treatment with magnolol (1) significantly inhibited osteoclast differentiation of RAW 264.7 macrophages and bone-resorbing activity of osteoclasts in the RANKL-induced system. Moreover, RANKL-activated JNK/ ERK/AP-1 and NF-κB signaling, ROS formation, and NFATc1 activation were attenuated by magnolol (1). A novel finding of this study is that magnolol (1) can increase heme oxygenase-1 (HO-1) expression and Nrf2 activation in RANKL-stimulated cells. Blocking HO-1 activity with tin protoporphyrin IX markedly reversed magnolol (1)-mediated inhibition of osteoclast differentiation, NFATc1 nuclear translocation, and MMP-9 activity, suggesting that HO-1 contributes to the attenuation of NFATc1-mediated osteoclastogenesis by magnolol (1). Therefore, the inhibitory effect of magnolol (1) on osteoclast differentiation is due to inhibition of MAPK/c-fos/AP-1 and NF-κB signaling as well as ROS production and up-regulation of HO-1 expression, which ultimately suppresses NFATc1 induction. These findings indicate that magnolol (1) may have potential to treat bone diseases associated with excessive osteoclastogenesis.
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RANKL-mediated up-regulation of the MAPK-dependent cfos/AP-1 pathway and NF-κB signaling2,8 ultimately activates the transcription of transcription factor nuclear factor of activated T cells-c1 (NFATc1) and its regulated osteoclastspecific gene expression such as cathepsin K, TRAP, MMP-9, and calcitonin receptor.9 Furthermore, RANKL-induced reactive oxygen species (ROS) generation also plays an important role in MAPK activation and osteoclast differentiation.10 Excessive osteoclast formation has been observed in various bone diseases including rheumatoid arthritis, periodontal disease, and osteoporosis.2 Therefore, suppressing RANKL-induced osteoclast differentiation-related signaling pathways is a therapeutic target for preventing osteoclastogenesis. Heme oxygenase (HO), a rate-limiting enzyme in heme catabolism, leads to the generation of carbon monoxide (CO), free iron, and biliverdin. There are three isoforms of HO: HO1, HO-2, and HO-3. Only HO-1 is inducible and is activated by a variety of cellular stress and stimuli including inflammation,
one homeostasis is highly regulated by the balance between the activities of bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoclasts are multinucleated giant cells formed by fusion mononuclear progenitors of monocytes/ macrophage lineage through a differentiation process that is mainly modulated by receptor activator of nuclear factor-κB (RANKL) and macrophage-colony-stimulating factor (MCSF).1,2 The secretion of acid through a vascular-type proton pump to dissolve hydroxyapatite mineral and release of proteindegrading enzymes such as tartrate-resistant acid phosphatase (TRAP) and matrix metalloproteinase (MMP) to resorb matrix protein are main factors resulting in the bone resorption of osteoclasts.1,3 It has been demonstrated that RANKL-induced osteoclast differentiation is modulated by multiple pathways. Binding of RANKL to its receptor, RANK, promotes osteoclast differentiation through recruitment of TNF receptor-associated factor 6 (TRAF 6), which subsequently activates downstream signaling pathways including nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK).4−6 Moreover, the activator protein-1 (AP-1) complex containing c-fos is a critical transcription factor for interaction with specific sites in the promoter region of genes required for osteoclastogenesis.7 © XXXX American Chemical Society and American Society of Pharmacognosy
Received: September 9, 2014
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DOI: 10.1021/np500663y J. Nat. Prod. XXXX, XXX, XXX−XXX
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Magnolol (1) Inhibited RANKL-Induced Signaling Pathways. MAPKs (p38, ERK, and JNK) play an important role in regulation of several fundamental biological processes and inflammatory responses in response to extracellular stimuli.19,20 It has been confirmed that binding of RANKL to RANK activates MAPK signaling, which ultimately promotes osteoclastogenesis-related gene expression.2,21 Furthermore, inhibitors of MAPKs (p38, ERK 1/2, JNK) attenuate osteoclast formation.22−24 To clarify the molecular mechanisms of magnolol’s inhibitory effect on osteoclast differentiation, we next analyzed the impact of magnolol (1) treatment on MAPK signaling. Under stimulation of RANKL, the phosphorylation of MAPKs was markedly increased and reached a maximal expression at 15 min. However, treatment with magnolol (1) strongly inhibited JNK phosphorylation and moderately inhibited ERK phosphorylation but did not affect phosphorylation of p38 (Figure 2A). Previous studies have reported that magnolol (1) inhibits lipopolysaccharide (LPS)-induced MAPKs (p38, ERK, and JNK) phosphorylation in RAW 264.7 macrophages.25 Therefore, magnolol (1) inhibition of RANKL-induced osteoclast differentiation may be uniquely associated with down-regulation of JNK and ERK phosphorylation. The importance of MAPK/c-fos/activator protein-1 (AP-1) signaling in RANKL-induced osteoclastogenesis has been demonstrated.26,27 ERK-1/2 and JNK can phosphorylate ELK-1, resulting in its activation. ELK-1 in turn phosphorylates the transcription factor SRF (serum response factor), which induces c-fos expression. Phosphorylated c-fos can dimerize with phosphorylated c-jun to generate AP-1, which can migrate to the nucleus to affect gene transcription.28 Notably, AP-1 (containing c-fos) is essential for a robust autoamplification of NFATc1, a major transcription factor accounting for osteoclastogenesis, through selective recruitment of NFATc1 to its own promoter region.1 By contrast, in c-fos-deficient cells, RANKL-induced NFATc1 expression was completely decreased,29 and the c-fos knockout mice exhibit a serious osteopetrotic phenotype because of a lack of osteoclast differentiation.30 In this study, the nuclear c-fos expression was dramatically suppressed by magnolol (1) compared to that in RANKL-stimulated alone cells (Figure 2B). Just as we expected, on blocking JNK activation with its specific inhibitor, SP600125, RANKL-induced osteoclast differentiation was significantly impaired (Figure 4B). NF-κB is another important transcription factor causing osteoclastogenesis by promoting an initial induction of NFATc1 and the transcription of genes encoding osteoclastogenesis-related inflammatory mediators.1,31 Previous work has indicated that suppressing NF-κB activation blocks osteoclastogenesis and prevents inflammatory bone destruction in vivo.32 Our data showed that magnolol (1) significantly decreased RANKL-induced nuclear translocation of phospho-p65 NF-κB in a dose-dependent manner (Figure 2B), which is consistent with early findings on LPS-stimulated macrophages.25 These results suggest that magnolol (1)-mediated inhibition of osteoclast differentiation is at least partly mediated through suppression of JNK/ERK/AP-1 and NF-κB signaling. Magnolol (1) Inhibited RANKL-Induced ROS Production. ROS have been regarded as an essential mediator for regulating RANKL-induced osteoclast differentiation.10,33 A recent study has reported that NADPH oxidase and mitochondria were the major sources of intracellular ROS in response to RANKL.34 Importantly, oxidative stress also activates the MAPK pathway.35 In addition, H2O2-induced
pro-inflammatory cytokines, bacterial compounds, and chemicals that produce oxidative stress, whereas HO-2 and HO-3 are constitutively expressed. Cumulative evidence has indicated that HO-1 exerts a cytoprotective effect against cellular insults through its antioxidative, anti-inflammatory, and antiapoptotic properties.11,12 Induction of HO-1 by transient gene vector or natural bioactive components inhibits osteoclastogenesis.13,14 By contrast, down-regulation of HO-1 by RNA interference enhances osteoclast formation.13 These findings support that agents that induce HO-1 may possess a protective effect against diseases involving bone loss. Magnolol (1) can be extracted from the traditional Chinese herb Magnolia off icinalis Rehder & E.H. Wilson (Magnoliaceae), which is widely used in oriental medicine. Magnolol (1) has been reported to exhibit several beneficial effects such as anti-inflammatory activity by suppressing NF-κB activation.15,16 Moreover, magnolol (1) is able to reduce alveolar bone resorption in a model of ligature-induced periodontitis.17 However, the underlying molecular mechanisms by which magnolol (1) regulates osteoclast differentiation have not yet been investigated. In this study, we examined the effects of magnolol (1) on RANKL-induced osteoclastogenesis of RAW264.7 macrophages and further studied the mechanisms involved, especially determining whether the actions of magnolol (1) on osteoclast formation are mediated by an HO-1-dependent pathway.
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RESULTS AND DISCUSSION Magnolol (1) Suppressed Osteoclast Differentiation and Bone-Resorbing Activity. Excessive bone resorption caused by osteoclasts is critical for the development of bone loss-related diseases such as osteoporosis and periodontitis.2 It is known that RAW 264.7 macrophages can differentiate into osteoclasts under the stimulation of RANKL. Currently, TRAP staining is an acceptable method to assess osteoclast formation.18 As expected, RANKL greatly induced osteoclast differentiation from RAW 264.7 macrophages, evidenced by an increased numbers of TRAP-positive multinuclear osteoclasts and TRAP activity. However, treatment with magnolol (1) dose-dependently reduced RANKL-enhanced numbers and TRAP activity of multinucleated TRAP-positive cells (Figure 1A). The inhibition of magnolol (1) on osteoclastogenesis was not due to cytotoxicity, as MTT analysis showed that magnolol (1) (5−20 μM) did not affect the cell viability (data not shown). As mature osteoclasts possess an ability to resorb bone, a pit formation assay was used to determine whether magnolol (1) affects the bone-resorbing activity of osteoclasts. In line with the trend of osteoclast formation, the calculated resorption area in the cells treated with RANKL and magnolol (1) was significantly less than that of RANKL-treated only cells (Figure 1B). These findings indicate that magnolol (1) is capable of suppressing osteoclastogenesis and bone-resorbing activity in the RANKL-induced system. B
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Figure 1. Effects of magnolol (1) on RANKL-induced osteoclast differentiation and bone-resorbing activity. RAW 264.7 cells were treated with indicated concentrations of magnolol (1) in the absence or presence of RANKL (50 ng/mL) for 5 days. Then, the cells were fixed and stained with TRAP. The TRAP-positive multinucleated cells (TRAP+ MNCs) were visualized under light microphotography (scale bar = 100 μm). The TRAP+ MNCs that contained three or more nuclei were counted. The TRAP activity was assessed by spectrophotometry as described in the Experimental Section (A). RAW 264.7 cells were incubated with different concentrations of magnolol (1) for 6 days in the absence or presence of RANKL (50 ng/ mL). The resorption area was analyzed and expressed as a percentage of the values of RANKL-treated alone cells (B). Data are expressed as the mean ± SD (n = 5); *p < 0.05, ***p < 0.001 versus RANKL-treated only cells.
MMP-7 expression, which is responsible for the cleavage of RANKL to form soluble RANKL with more potent activity,20 is
due to JNK and ERK activation in an AP-1-dependent manner.36 Our results indicated that magnolol (1) treatment C
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Figure 2. Effects of magnolol (1) on RANKL-induced signaling pathways. Magnolol (1) was added to cells 2 h before RANKL addition and further incubated for 15 min. Then, the protein samples were prepared for total and phosphorylated MAPKs analysis (A). After treatment with RANKL for 1 h, the nuclear proteins were prepared for p-NF-kB p65 and c-fos analysis (B). The figures are representative of at least three independent experiments. Data are expressed as the mean ± SD; *p < 0.05 versus RANKL-treated alone cells.
dose-dependently inhibited RANKL-induced H2O2 and O2− production, as measured by 2′,7′-dichlorfluorescein (DCF) fluorescence and dihydroethidium (DHE) immunofluorescence, respectively (Figure 3A and B). On the basis of previous studies showing that magnolol significantly inhibited ethanolinduced superoxide anion formation via suppressing NADPH oxidase activity in Raw 264.7 cells and mitochondrial-derived superoxide anion production in osteoblastic MC3T3-E1 cells,37,38 the antioxidant activity of magnolol may be attributed to reduced NADPH oxidase activity and mitochondrial ROS generation. Therefore, suppressing ROS formation is another mechanism to attenuate RANKL-induced osteoclast differentiation by magnolol (1). Magnolol (1) Enhanced HO-1 Expression and Nrf-2 Activation. HO-1-dependent signaling is an important adaptive mechanism to preserve homeostasis and enhance cellular resistance to oxidative stress.39 The protective effects of HO-1 may be attributed to its metabolites (biliverdin and CO) derived from the degradation of prooxidative heme.11 The expression of HO-1 is mainly modulated by the transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2). Normally, Nrf2 is combined with Kelch-like ECH-associated
protein 1 (Keap1), a repressor of Nrf2, and retains the complex in the cytosol. Upon activation, Nrf2 is dissociated from Keap1 and translocates into the nucleus, where it activates the transcription of phase II cytoprotective genes including HO-1 through binding the antioxidant response element (ARE) sequence in the promoter region of target genes.40 Several lines of evidence have demonstrated that induction of HO-1 inhibits RANKL-induced osteoclastogenesis,13,41 by inhibiting osteoclast signaling pathways. However, whether magnolol (1) affects HO-1 expression was previously unknown. A novel finding of this study is that treatment of RAW 264.7 cells with magnolol (1) significantly increased HO-1 protein expression and enhanced the nuclear accumulation of Nrf2 compared to that in RANKL-stimulated alone cells (Figure 4A). To further determine the role of HO-1 in magnolol-mediated inhibition of osteoclast differentiation, the tin protoporphyrin IX (SnPP), an HO-1 inhibitor, was added. As shown in Figure 4B, blocking HO-1 activity markedly reversed the attenuation of osteoclast differentiation by magnolol (1), evidenced by a significant increase of TRAP-positive multinucleated cells (Figure 4C) and TRAP activity (Figure 4D). Accordingly, the inhibitory effect of magnolol (1) on RANKL-induced osteoclast differentiation is D
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antibodies used in this study include antiphospho-nuclear factor kappa B (p-NF-κB), phosphorylated p38, extracellular signal regulated kinase (ERK), and c-Jun N-termainl kinase (JNK) (Cell Signaling, Beverly, MA, USA), anti-β-actin (Abcam, Cambridge, MA, USA), anti-HO-1, NFATc1 (Santa Cruz Biotechnology, CA, USA), and anti-Nrf2 (GeneTex, CA, USA). Other chemical agents were obtained from Sigma Chemical Company. Cell Viability Assay. RAW 264.7 macrophages were obtained from Bioresource Collection and Research Center, Hsinchu, Taiwan. Cells (2 × 105 cells/well) were seeded in 24-well plates and grown in DMEM (Sigma, St Louis, MO, USA) supplemented with 10% heatinactivated FBS (Gibco, NY, USA). The cell viability was determined by measuring the reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide (MTT) to formazan, which is dependent on mitochondrial activity. After cells were incubated with different concentrations of magnolol (1) or vehicle (0.1% DMSO) for 24 h, the MTT reagent was added for 1 h at 37 °C. Then, the medium was removed, and the platelet was washed twice with phosphatebuffered saline (PBS). The intracellular insoluble formazan was dissolved in DMSO, and the absorbance of each well was measured at 570 nm. TRAP Staining and Activity. RAW 264.7 cells (5 × 105 cells/ well) were seeded in 24-well plates for 24 h, and the medium was replaced followed by an additional incubation for 5 days in α-minimal essential medium (α-MEM) (Sigma, St. Louis, MO, USA) containing 10% FBS, RANKL (50 ng/mL) (Peprotech, Rocky Hill, NJ, USA), and the indicated drugs. Then, the cells were fixed with 10% formalin for 10 min for TRAP staining. Cells were washed three times with distilled water, and the images of TRAP-positive multinucleated cells (MNCs ≥ three nuclei) were captured and counted under a light microscope (Leica, Vertrieb Deutschland, Germany) to evaluate the osteoclast differentiation. For the TRAP activity assay, the osteoclasts differentiated from RAW 264.7 macrophages treated with RANKL for 5 days were fixed with10% formalin for 10 min and ethanol/acetone (1:1) for 1 min. Subsequently, the cells were incubated in 50 mM citrate buffer (pH 4.6) containing 10 mM tartrate and 5 mM pnitrophenylphosphate (pNPP) for 1 h. The reaction mixtures were transferred to new well plates containing an equal volume of 0.1 N NaOH to stop the reaction, and the absorbance of each well was measured at 410 nm by using a spectrophotometer. The TRAP activity was expressed as the percent of RANKL-treated alone group. Pit Formation Assay. RAW 264.7 cells (1 × 105 cells/well) were seeded on Corning Osteo Assay Surface wells (Corning, NY, USA) according to the manufacturer’s instructions. Cells were treated with different concentrations of magnolol (1) and RANKL (50 ng/mL) for 6 days in α-MEM medium. After the incubation, cells were removed with 1 N NaOH for 20 min, and the wells were washed twice with PBS. The resorption areas were examined by light microscopy (Leica, Vertrieb Deutschland, Germany) and analyzed by Metamorph imaging analysis (Metamorph Imaging System, Universal Imaging Corp., Downingtown, PA, USA). The resorption area was expressed as the percent of RANKL-treated alone group. Measurement of Matrix Metalloproteinase Activity. After RAW 264.7 cells were cultured with RANKL (50 ng/mL) for 5 days in the absence or presence of magnolol (1), the supernatant samples were collected and applied to an 8.0% sodium dodecyl sulfatepolyacrylamide gel (SDS-PAGE) copolymerized with 0.1% gelatin. Then, the gels were washed with washing buffer (Triton 2.5% X-100) for 1 h at room temperature followed by incubation with shaking overnight at 37 °C in reaction buffer (2 M Tris-HCl, pH = 8.0, 1 M CaCl2, 1% NaN3). The gels were stained with a solution containing 0.1% commassie brilliant blue staining solution. The formation of a clear zone against the blue background on the polyacrylamide gels indicated MMP-9 activity. Measurement of ROS Production. The cell-permeable nonfluorescent compound 2′,7′-dichlorfluorescein-diacetate (H2DCF-DA) and dihydroethidium (DHE) were used for assessing the intracellular ROS formation and superoxide anions, respectively. Cells were treated with the indicated drugs or vehicle for 2 h and then stimulated with RANKL for 10 min followed by incubation with 50 μM DCFH-DA or
Figure 3. Effects of magnolol (1) on RANKL-induced ROS formation. RAW 264.7 cells were pretreated with different concentrations of magnolol (1) for 2 h followed by stimulation with RANKL (50 ng/ mL) or vehicle for 10 min. Formation of H2O2 (A) and superoxide anion (B) was determined as described in the Experimental Section. The profiles are representative of three similar experiments. *p < 0.05, **p < 0.01 versus RANKL-treated alone cells.
regulated by up-regulation of HO-1 expression resulting from Nrf2 activation. HO-1 Is Involved in Magnolol-Mediated Suppression of NFATc1 Activation. During RANKL-induced osteoclastogenesis, NFATc1 expression is up-regulated, which leads to the expression of osteoclastogenesis-related genes such as TRAP and MMP-9.42 Increased MMP-9 activity has been observed in osteoclasts and is thought to promote bone resorption.43 We then asked whether magnolol (1) can affect NFATc1 expression and MMP-9 activity and whether HO-1 is involved in the actions of magnolol (1). The Western blot and immunofluorescent staining assays showed that RANKLinduced nuclear expression of NFATc1 and MMP-9 activity was markedly inhibited by magnolol (1). However, addition of SnPP significantly attenuated the above inhibitory effects of magnolol (1) (Figure 5A−C). On the basis of these results, it is highly likely that magnolol-mediated inhibition of osteoclast formation is attributed to down-regulation of NFATc1 via an HO-1-dependent pathway. In conclusion, we demonstrated that magnolol (1) is capable of suppressing osteoclast differentiation via multiple pathways, including up-regulation of HO-1 expression, inhibition of RANKL-induced JNK/ERK/c-fos/AP-1 signaling, NF-κB activation, and ROS formation, which in turn attenuates NFATc1 induction and osteoclastogenesis-related gene expression. The present study indicates that magnolol (1) has potential as a therapeutic drug for bone diseases associated with excessive osteoclastogenesis.
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EXPERIMENTAL SECTION
Test Compound and Reagents. Magnolol (1) (Sigma, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO) followed by dilution, and the final concentration of DMSO was fixed at 0.1%. The purity of magnolol (1) (>95%) was determined by HPLC assay. The E
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Figure 4. Effects of magnolol (1) on the Nrf2/HO-1 pathway in RANKL-treated RAW 264.7 cells. Cells were treated with RANKL (50 ng/mL) for 48 h in the absence or presence of indicated concentrations of magnolol (1), and the cell protein was prepared for HO-1 expression assay. After cells were treated with RANKL (50 ng/mL) for 3 h, the nuclear protein was collected for determining the Nrf2 level (A). RAW 264.7 cells were treated with magnolol (1) (20 μM) or in combination with SnPP (10 μM) or SP600125 (10 μM) in the presence or absence of RANKL (50 ng/mL) for 5 days. Then, the cells were fixed and stained for TRAP (B), and the TRAP+ MNCs that contained three or more nuclei were counted (C) and the TRAP activity (D) was determined by spectrophotometry as described in the Experimental Section. The profiles are representative of at least three independent experiments. Data are expressed as the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001 versus RANKL-treated alone cells. #p < 0.05 versus RANKL + magnolol (1) (20 μM) group. 40 μM DHE for 30 min at 37 °C in the dark. The cells were washed twice with PBS and resuspended in cold PBS. The quantification of
fluorescence intensity and the fluorescence images were measured by using a Fluoroskan Ascent FL (Thermo Electron Co., Vantaa, Finland) F
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Figure 5. Effects of magnolol (1) on RANKL-induced nuclear accumulation of NFATc1 and MMP-9 activity. Cells were incubated with magnolol (1) (20 μM) or in combination with SnPP (10 μM) followed by addition of RANKL (50 ng/mL) for 24 h. The nuclear NFATc1 expression (A) and nuclear translocation of NFATc1 (B) were determined. The nuclei of cells were visualized by DAPI staining (blue), and NFATc1 (green) was detected. The merged photos indicated the nuclear translocation of NFATc1. Cells were incubated with indicated drugs and stimulated with RANKL for 5 days. The MMP-9 activity was determined by using gelatin zymographic assays (C). Data are expressed as the mean ± SD (n = 5). *p < 0.05, **p < 0.01 versus RANKL-treated alone cells. #p < 0.05 versus RANKL + magnolol (1) (20 μM) group. vortex-mixed for 10 s. Then, the nuclei were pelleted by centrifugation at 15000g for 10 min and finally resuspended in hypertonic buffer (20 mM HEPES, pH 7.9, 25% (v/v) glycerol, 420 mM NaCl, 1.5 mM MgCl2, 1 mM DTT, 0.2 vmM ETDA, 1 mM PMSF) for 30 min on ice. The supernatants containing nuclear proteins were collected by centrifugation at 15000g for 2 min. The nuclear protein or total cell lysates were separated on 12% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were subsequently blocked
with 485 nm excitation and 538 nm emission settings and a fluorescence microscope (Leica DMI6000B, Wetzlar, Germany), respectively. Western Blotting. After washing with PBS, the total cell lysates were prepared with radio-immunoprecipitation assay (RIPA) buffer. To extract nuclear protein, cells were first suspended in hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 1 mM DTT, 1.5 mM MgCl2, 1 mM PMSF, 0.1% Triton X-100) for 15 min on ice and G
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by tris-buffered saline (TBS) containing 5% powered nonfat milk for 1 h and then incubated with various primary target antibodies overnight at 4 °C. The membrane was washed and incubated with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse IgG (Abcam, Cambridge, MA, USA) for 30 min at room temperature. The immunoreactive bands of the target protein were visualized with enhanced HRP Substrate Luminol Reagent (Millipore, Billerica, MA, USA). The intensity of blotted protein was measured densitometrically with ImageJ image analysis software (National Institutes of Health, USA). Immunofluorescence Staining. Cells plated on four-well glass coverslips were treated with the indicated drugs for 24 h and fixed with methanol for 5 min. The nonspecific binding sites were blocked with 4% BSA in PBST for 30 min. Then, NFATc1 antibody (Santa Cruz Biotechnology, CA, USA) was added overnight at 4 °C diluted 1:100 with 1% BSA in PBS followed by a fluorescein isothiocyanate (FITC)coupled secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:100 with 1% BSA in PBST. In addition, cells were also stained with a proper dilution of 4′,6′-diamidino-2-phenylindole (DAPI) for 2 min to counterstain DNA in the nuclei. After extensive washings with PBS, the coverslips were mounted onto the glass slides and photographed with a fluorescence microscope. Statistical Analysis. Data are presented as the mean ± SD. A oneway ANOVA with a post hoc Bonferroni test was used for statistical analysis. Statistical significance was defined as p < 0.05.
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AUTHOR INFORMATION
Corresponding Author
*Tel: 886-3-8561825, ext. 5620. Fax: 886-3-8573710. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was supported by a research grant from the National Science Council of Taiwan, Republic of China (NSC 97-2320B-016-008-MY3).
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