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Endocrinology Vol. 141, No. 8 2904-2913
Copyright © 2000 by The Endocrine Society

ARTICLES

Dexamethasone Suppresses Tumor Necrosis Factor-{alpha}-Induced Apoptosis in Osteoblasts: Possible Role for Ceramide1

Han Jung Chae, Soo Wan Chae, Jang Sook Kang, Byung Gwan Bang, Seoung Bum Cho, Rae Kil Park, Hong Seob So, Yong Kwang Kim, Hyung Min Kim and Hyung Ryong Kim

Department of Dental Pharmacology and Wonkwang Dental Research Institute, School of Dentistry, Wonkwang University (H.J.C., J.S.K., B.G.B., S.B.C., H.R.K.); Department of Pharmacology and Institute of Cardiovascular Research, School of Medicine, Chonbuk National University (H.J.C., S.W.C.); Department of Microbiology and Immunology, School of Medicine (R.K.P., H.S.S.), and Department of Oriental Pharmacy, College of Pharmacy (Y.K.K., H.M.K.), Center of Oriental Medicinal Science, Wonkwang University, Chonbuk 570–749, South Korea

Address all correspondence and requests for reprints to: Hyung-Ryong Kim, D.D.S., Ph.D., Department of Dental Pharmacology, Wonkwang University, School of Dentistry, Iksan, Chonbuk 570–749, South Korea. E-mail: hrkimdp{at}wonnms.wonkwang.ac.kr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ceramide has been proposed as a second messenger molecule implicated in a variety of biological processes, including apoptosis. Recently, it has been reported that tumor necrosis factor-{alpha} (TNF-{alpha}) activates the release of ceramide and that ceramide acts as a mediator for the TNF-{alpha}-induced stimulation of the binding affinity of nuclear factor-{kappa}B (NF-{kappa}B), a ubiquitous transcription factor of particular importance in immune and inflammatory responses. In this study we demonstrate that dexamethasone, which reduces the production of ceramide, significantly inhibits TNF-{alpha}-induced activation of NF-{kappa}B, c-Jun N-terminal kinase, also known as stress-activating protein kinase, caspase-3-like cysteine protease, redistribution of cytochrome c, and apoptosis in MC3T3E1 osteoblasts. Compared with TNF-{alpha}-induced JNK activation, ceramide elicits a more rapid activation of JNK within 30 min. C2-ceramide activates NF-{kappa}B and caspase-3 like protease to the same degree and with kinetics similar to those of TNF-{alpha}. This study provides evidence that the release of ceramide may be required as a second messenger in TNF-{alpha}-induced apoptosis. These results also suggest a regulatory role for dexamethasone in TNF-{alpha}-induced apoptosis via inhibition of ceramide release. Therefore, our in vitro results suggest that therapies targeted at the inhibition of ceramide release may abrogate inflammatory processes in TNF-{alpha}-related diseases, including rheumatoid arthritis and periodontitis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROGRAMMED CELL death, or apoptosis, is the process by which cells are induced to activate their own death or cell suicide. Apoptosis occurs in a wide variety of cell types and is recognized to have a major impact on the development of numerous systems. Histologically, the term apoptosis refers to the characteristic morphology of cells undergoing programmed cell death. Apoptotic cells appear shrunken, with extensive membrane blebbing and nuclear fragmentation. The final point in apoptosis involves the fragmentation of the cells into membrane-bound vesicles containing cellular remnants of protein and fragmented chromatin, referred to as apoptotic bodies. These membrane-bound vesicles are eventually phagocytosed by macrophages without the involvement of an inflammatory reaction (1, 2, 3).

In the bone microenvironment, there is a dynamic balance of resorption and formation, which maintains skeletal homeostasis. The cells responsible for these functions, osteoclasts and osteoblasts, require many different mediators, including osteotropic hormones, and proinflammatory cytokines (4). Of the latter, tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-1ß are mediators of inflammatory bone loss, whereas interferon-{gamma} has been reported to selectively inhibit bone resorption. TNF-{alpha} has been shown to play an important role in the local control of bone remodeling (5).

A novel signal transduction pathway, which is involved in mediating apoptotic effects of TNF-{alpha}, has been identified in several cell types (6, 7). This apoptotic pathway, referred to as the sphingomyelin pathway, is initiated by activation of a neutral sphingomyelinase that hydrolyzes membrane’s sphingomyelin to ceramide. Ceramide functions as a second messenger molecule and can stimulate a membrane-bound serine/threonine kinase, termed ceramide-activated protein kinase (8). Activation of this pathway appears to occur early in TNF-{alpha} action, within seconds or minutes, and is closely coupled to the receptor complex, as this event can be reconstituted in a cell-free system.

Glucocorticoids are well known as antiinflammatory and immunosuppressive drugs that have been successfully used as repressors of immune response and inflammatory processes (9, 10). Some properties of glucocorticoids are attributed to its effects on transcription factors. One important mechanism is inhibition of nuclear factor-{kappa}B (NF-{kappa}B) activation. Because NF-{kappa}B activates many immunoregulatory genes in response to proinflammatory stimuli, the inhibition of its activity would be one major component of the antiinflammatory activity of glucocorticoids (11, 12). Another possible mechanism is inhibition of c-Jun-N-terminal kinase, known as stress-activating protein kinase (JNK/SAPK), a member of the mitogen-activated protein kinase family that has been defined as being involved in the signaling pathways that lead to apoptosis (13, 14).

Mechanistically, dexamethasone blocked apoptotic signal transduction pathways, including NF-{kappa}B, JNK/SAPK, caspase-3-cysteine protease activation, and cytochrome c redistribution, in TNF-{alpha}-treated MC3T3E1 osteoblasts.

In this study we observed that dexamethasone, a potent antiinflammatory agent, prevents TNF-{alpha}-induced apoptosis by inhibiting ceramide production in MC3T3E1 osteoblasts. Our results have suggested that NF-{kappa}B, JNK/SAPK, caspase-3-cysteine protease activation, and cytochrome c redistribution are related to dexamethasone protective effects. Furthermore, this study strongly suggested that the inhibition of ceramide is responsible for the antiapoptotic activity of dexamethasone against TNF-{alpha} in MC3T3E1 osteoblasts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Recombinant mouse TNF-{alpha} was obtained from Genzyme Corp. (Cambridge, MA). Dexamethasone, C2-ceramide, dihydro-C2- ceramide, Hoechst 33258, and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO). N-Acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (AC-DEVD-AMC) and N-acetyl-Tyr-Val-Ala-Asp-7-amino-4-methylcoumarin (AC-YVAD-AMC) were obtained from Calbiochem-Behring Corp. (La Jolla, CA). Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) was purchased from Kamiya Bio Co. (Seattle, WA). A genomic DNA purification kit was obtained from Promega Corp. (Madison, WI). The JNK1 antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Glutathione-S-transferase (GST)-c-Jun N-terminal protein was purchased from Stratagene (La Jolla, CA). Cytochrome c antibody was purchased from PharMingen (San Diego, CA). [{gamma}-32P]ATP and [{alpha}-32P]CTP were purchased from NEN Life Science Products (Boston, MA). All culture wares were purchased from Nunc, Inc. (Naperville, IL). Other culture reagents, including {alpha}MEM, HBSS, and FBS, were bought from Life Technologies, Inc. (Gaithersburg, MD).

Cell culture
The clonal murine osteoblast cell line, MC3T3E1, was cultured in {alpha}MEM, supplemented with 10% FBS, penicillin, and streptomycin. The cultures were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37 C. Before treatment, MC3T3E1 osteoblasts were washed once with {alpha}MEM and then various agents were added to the cells (1 x 105 cells/cm2) in {alpha}MEM containing 10% FBS, antibiotics, and glutamine. Lyophilized TNF-{alpha} was reconstituted in 0.1% bovine serum (10 µg/ml). C2-ceramide or dihydro-C2-ceramide was dissolved in ethanol and added to the cells as an ethanolic solution (final concentration of ethanol not exceeding 0.1%). Cells in the presence or absence of dexamethasone were incubated with different concentrations of TNF-{alpha} or C2-ceramide for specific times.

MTT cell viability assay
TNF-{alpha} cell viability was determined for each concentration of dexamethasone tested or for each pretreated hours of dexamethasone. Cell viability was determined by the MTT-dye reduction microassay according to the method of Green et al. (15). Briefly, after 48-h incubation, 10 µl MTT were added for 3 h to the 96-well microplates, and the absorbance was read at 540 nm on a Titer-Tek multiscan MicroElisa reader. Cell viability was calculated as the ratio of optical densities in wells with and without TNF-{alpha} in the presence or absence of dexamethasone.

Agarose gel electrophoresis for DNA fragmentation
To assess DNA fragmentation, we prepared genomic DNA from MC3T3E1 osteoblasts and analyzed it by a slight modification of the electrophoretic method described previously (16). The cells were then lysed by incubation in digestion buffer (150 mM NaCl, 25 mM EDTA, 100 µg/ml proteinase K, and 0.2% SDS). The genomic DNA was extracted using a genomic DNA purification kit (Promega Corp.) and was analyzed by 1.5% agarose gel electrophoresis.

Quantitation of DNA fragmentation
DNA fragmentation was essentially assayed as previously reported (17). Briefly, after incubation, cells were scraped off the culture plates, resuspended in a 250 µl TE buffer (10 mM Tris and 1 mM EDTA, pH 8.0), and incubated with an additional volume of lysis buffer [5 mM Tris, 20 mM EDTA (pH 8.0), and 0.5% Triton X-100] for 30 min at 4 C. After lysis, intact chromatin (pellet) was separated from DNA fragments (supernatant) by centrifugation for 15 min at 13,000 x g. After addition of 5% trichloroacetic acid (300 µl), samples were boiled for 15 min. The fragmented DNA contents were quantified using the diphenylamine reagents. The percentage of fragmented DNA was calculated as the ratio of the DNA content in the supernatant to the amount in the pellet.

In addition, morphological evaluation of apoptotic cell death was performed as previously described with some modification (18). Coverslips were fixed for 5 min in 3.7% paraformaldehyde in PBS. After air-drying, coverslips were stained for 10 min in Hoechst 33258 (10 µg/ml), mounted in 50% glycerol containing 20 mmol/liter citric acid and 50 mmol/liter orthophosphate, and stored at -20 C before analysis. Nuclear morphology was evaluated using a Carl Zeiss IM 35 fluorescent microscope (New York, NY) at excitation and emission wavelengths of 440 and 460 nm, respectively. Apoptotic cells were identified as those whose nucleus exhibited brightly staining condensed chromatin or nuclear fragmentation.

Fluorogenic substrate assay for caspase activity
Cytosolic cell extracts were prepared by lysing the cells in a buffer containing 1% Nonidet P-40, 200 mM NaCl, 20 mM Tris-HCl (pH 7.4), 10 µg/ml leupeptin, and aprotinin (0.27 mM trypsin inhibitor/U·ml). Caspase-1- or caspase-3-like activity was determined by incubation of cell lysate (containing 25 µg total protein) with 50 µM of the fluorogenic substrate, AC-YVAD-AMC or AC-DEVD-AMC, respectively, in 200 µl cell-free system buffer [comprising 10 mM HEPES (pH 7.4), 220 mM mannitol, 68 mM sucrose, 2 mM NaCl, 2.5 mM KH2PO4, 0.5 mM EGTA, 2 mM MgCl2, 5 mM pyruvate, 0.1 mM phenylmethylsulfonylfluoride (PMSF), and 1 mM dithiothreitol]. The release of fluorescent 7-amino-4-methyl coumarin was measured for 1 h at 2-min intervals by spectrofluorometry.

Cytochrome c measurements
Mitochondrial fractions were prepared from 1 x 107 MC3T3E1 osteoblasts by differential centrifugation in buffer containing 250 mM sucrose as described previously (19). Protein samples of 25 µg were loaded on SDS-15% polyacrylamide gels, subjected to electrophoresis, and then electrophoretically transferred to nitrocellulose membranes. Western blots were probed with primary monoclonal anticytochrome c antibody (PharMingen, San Diego, CA) and secondary antimouse horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and then developed with enhanced chemiluminescence (Amersham Pharmacia Biotech, Aylesbury, UK).

Ceramide quantification
Total lipids were extracted by the method of Bligh and Dyer (20). Samples were homogenized in the solvents. The lipid was dissolved in CHCl3:CH3OH. Ceramide levels were measured using diacylglyceride kinase as described. Baseline ceramide levels were 25.2/1.36 pmol/nmol phospholipids. The solvent system to separate phosphatidic acid and ceramide phosphate was chloroform/pyridine/formic acid (60:30:8, vol/vol/vol). Ceramides were purified by diisopropyl ether/1-butanol partition followed by Sephadex G-50 gel filtration. The purified ceramides were quantified by the modified resorcinol method (21) as nanomoles of lipid-bound sialic acid. High performance TLC analysis of ceramides was performed using 10 x 20 cm precoated silica gel 60 high performance TLC plates (Merck, Darmstadt, Germany). The plates were developed in chloroform/methanol/0.25% aqueous CaCl2·2H2O (60:40:9) and were stained with resorcinol.

Immunoprecipitation and kinase assays
Cells were lysed in a modified radioimmune precipitation buffer [25 mM Tris-HCl, pH 8.0, containing 137 mM NaCl, 10% (vol/vol) glycerol, 0.1% SDS, 0.5% (vol/vol) deoxycholate, 1% (vol/vol) Nonidet P-40, 2 mM EDTA, 1 mM Pefabloc, 1 mM sodium vanadate, 5 mM benzamidine, 5 µg/ml aprotinin, and 5 µg/ml leupeptin] on ice for 30 min. Cell debris was removed by centrifugation at 15,000 rpm for 10 min. Supernatants were then incubated with anti-JNK1 for 2 h at 4 C. The immunocomplexes were precipitated with Pansorbin (Calbiochem, La Jolla, CA) and washed extensively with a lysis buffer [50 mM LiCl/100 mM Tris-HCl (pH 7.6)/0.1% (vol/vol) Triton X-100/1 mM dithiothreitol]. The pellets were left as a 1:1 suspension in an assay buffer, and 20 µl (0.3 mg/ml) of GST-c-Jun were added. Kinase reactions were initiated by the addition of 15 µl {gamma}-32P-labeled Mg/ATP solution (50 mM MgCl/500 µM ATP/10 µCi [{gamma}-32P]ATP) and were performed at 30 C for 30 min. Reactions were stopped by the addition of Laemmli sample buffer and boiling for 5 min. Samples were separated by SDS-PAGE [12% (wt/vol) gel] and after drying were subjected to autoradiography. Quantification was performed with a PhosphorImager analyzer (BSA, Fuji Photo Film Co., Ltd., Tokyo, Japan).

Electrophoretic mobility shift assay (EMSA)
Nuclei from TNFs or dexamethasone-treated MC3T3E1 osteoblasts was extracted according to modification of the procedure described by Dignam et al. (22). The cells were lysed with a hypotonic buffer [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.2 mM PMSF, 0.5 mM dithiothreitol, 10 µg/ml aprotinin, 20 µM pepstatin A, and 100 µM leupeptin]. After centrifugation, the nuclear pellets were resuspended in an extraction buffer [20 mM HEPES (pH 7.9), 25%(vol/vol) glycerol, 0.4 M KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.2 mM PMSF, and 0.5 mM dithiothreitol]. The nuclear proteins were recovered after centrifugation at 15,000 x g, quantified using a bicinchoninic acid protein assay kit (Sigma), and used to carry out EMSA. To measure the activation of transcription factors including NF-{kappa}B, the oligonucleotide probes of NF-{kappa}B containing the IgG chain-binding site (NF-{kappa}B, 5'-CCG GTT AAC AGA GGG GGC TTT CCG AG-3') (23) were used. Two complementary strands of the oligonucleotides were annealed and labeled with [{alpha}-32P]deoxy-CTP using a random primer labeling kit (Rediprime, Amersham Pharmacia Biotech). Nuclear extracts (5 µg) were reacted with 2–5 ng of the radiolabeled NF-{kappa}B (50,000–100,000 cpm/ng). The reaction was performed in the presence of 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM dithiothreitol, and 4% glycerol (final volume, 25 µl) at room temperature for 30 min. The reaction products were subjected to 4% PAGE in 0.5 x TBE buffer [50 mM Tris-HCl (pH 8.5), 50 mM borate, and 1 mM EDTA]. Gels were dried under a vacuum for 1 h. DNA-binding activity for NF-{kappa}B was measured using a PhosphorImager analyzer (BAS, Fuji Photo Film Co., Ltd.).

Statistical analysis
Differences between groups were tested for statistical significance using ANOVA. P < 0.05 was selected as the level of significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dexamethasone induces cell resistance to the TNF-{alpha}-induced cytotoxicity
To explore whether dexamethasone modifies TNF-{alpha}induced responses in MC3T3E1 osteoblasts, the cells were pretreated with different concentrations of dexamethasone, a glucocorticoid hormone, and thereafter tested with 20 ng/ml TNF-{alpha}. Figure 1AGo shows the effect of dexamethasone (1 nM to 10 µM) on the cytotoxicity exerted by 20 ng/ml TNF-{alpha} on MC3T3E1 osteoblasts after 48 h. The synthetic glucocorticoid induced an evident increase in cell viability after 48 h of incubation with TNF-{alpha}. This effect became significant at the concentrations of more than 100 nM dexamethasone and reached maximum at 1 µM. Figure 1BGo also showed that pretreatment with dexamethasone for more than 16 h was required for maximum inhibition of TNF-{alpha}-induced cytotoxicity. This confirms that pretreatment with glucocorticoid protects against cytotoxicity induced by TNF-{alpha} in MC3T3E1 osteoblasts.



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  		Figure 1. Dexamethasone reduces TNF-{alpha}-induced cytotoxicity in MC3T3E1 osteoblasts. A, MC3T3E1 osteoblasts were pretreated with various doses of dexamethasone (0.001, 0.01, 0.1, 1, or 10 µM) for 16 h and then further exposed to TNF-{alpha} (20 ng/ml) for 48 h. Cell viability was measured using a MTT assay. B, Pretreatment of dexamethasone for more than 16 h was required to achieve maximum inhibition of TNF-{alpha}-induced cytotoxicity. After pretreatment with dexamethasone (1 µM) for various periods (0, 2, 4, 8, 16, or 32 h), TNF-{alpha} (20 ng/ml) was treated to MC3T3E1 osteoblasts for 48 h. Then cell viability was measured using a MTT assay. Results of four experiments are expressed as the mean ± SEM. *, P < 0.05 vs. without dexamethasone.

 
Dexamethasone inhibits TNF-{alpha}-induced apoptosis in MC3T3E1 osteoblasts
Many studies have demonstrated that dexamethasone has a regulatory function in stress response-induced apoptosis in several kinds of cells (24, 25). First, we analyzed DNA fragmentation from these cells using an agarose gel electrophoresis for evidence of apoptosis. Oligosomal DNA fragmentation consistent with the onset of apoptosis was evident in cells exposed to TNF-{alpha} (20 or 40 ng/ml). In contrast, incubation of MC3T3E1 osteoblasts with 20 ng/ml TNF-{alpha} in the presence of 1 µM of the synthetic glucocorticoid analog, dexamethasone (16-h pretreated), revealed that dexamethasone suppressed DNA fragmentation measured by diphenylamine reaction (Fig. 2BGo). Furthermore, morphological alterations, i.e. chromatin condensation induced by TNF-{alpha}, were significantly reduced by dexamethasone (16-h pretreated; Fig. 2CGo). It is evident that dexamethasone repressed TNF-{alpha}-induced apoptosis in MC3T3E1 osteoblasts up to 50%.



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  		Figure 2. Dexamethasone attenuates TNF-{alpha}-induced DNA fragmentation in MC3T3E1 osteoblasts. A, MC3T3E1 osteoblasts (1 x 106 cells/ml) were treated with TNF-{alpha} (0, 10, 20, or 40 ng/ml) for 48 h. B, MC3T3E1 osteoblasts were treated for 48 h with or without 20 ng/ml TNF-{alpha} in the absence or presence of dexamethasone (16 h pretreated). Then a DNA fragmentation assay was performed with a diphenylamine reagent. C, MC3T3E1 osteoblasts were treated as described in B and stained with Hoechst 33258 (2.5 µg/ml in PBS). The nucleus condensation of the apoptotic cell was analyzed using a fluorescence microscope. The results of four experiments are expressed as the mean ± SEM. *, P < 0.05 vs. control; #, P < 0.05 vs. TNF-{alpha} treated.

 
Dexamethasone inhibits TNF-{alpha}-induced caspase-3 activation
In the first set of experiments aimed at assaying caspase-1 or -3 activity in TNF-{alpha}-induced cell death in MC3T3E1 osteoblasts, the fluorescence intensity of the caspase protease cleavage product AMC was monitored at various incubation periods of TNF-{alpha}. Caspase-3-like protease activity was increased in a time-dependent manner and reached a maximum level 48 h after TNF-{alpha} treatment, whereas caspase-1-like activity was not affected by treatment with TNF-{alpha} (Fig. 3AGo). To further assess the influence of dexamethasone on TNF-{alpha}-induced caspase activation, caspase-3-like protease was measured in MC3T3E1 homogenates in the presence of either 20 ng/ml TNF-{alpha} alone or TNF-{alpha} in the presence of 16-h pretreated dexamethasone (1 µM). As shown in Fig. 3BGo, TNF-{alpha}-induced activation of caspase-3-like activity was significantly reduced by the pretreatment with dexamethasone (1 µM). In addition, to examine the role of caspase activation in TNF-{alpha}-induced apoptosis of osteoblasts, MC3T3E1 osteoblasts were pretreated with the pan-caspase inhibitor Z-VAD-FMK, a specific caspase-1 inhibitor (Ac-YVAD-CHO), or a specific caspase-3 inhibitor DEVD-CHO. As shown in Fig. 3CGo, pretreatment with 100 µM Z-VAD-FMK and 100 µM DEVD-CHO of MC3T3E1 osteoblasts largely prevented the DNA fragmentation that was seen after 20 ng/ml TNF-{alpha} treatment. However, a caspase-1-like protease inhibitor (Ac-YVAD-CHO), had no inhibitory effect on TNF-{alpha}-induced DNA fragmentation. Our data suggest that caspase-3-like cysteine protease activity is required to induce apoptosis in MC3T3EI osteoblasts and that dexamethasone, a glucocorticoid hormone, has a regulatory function on TNF-{alpha}-induced apoptosis via the inhibition of caspase-3-like cysteine protease activity in MC3T3E1 osteoblasts.



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  		Figure 3. Dexamethasone inhibits TNF-{alpha}-induced activation of caspase3-cysteine protease in MC3T3E1 osteoblasts. A, MC3T3E1 osteoblasts were treated with TNF-{alpha} (20 ng/ml) for various periods as indicated. *, P < 0.05 vs. control. B, MC3T3E1 osteoblasts were treated with TNF-{alpha} (20 ng/ml) in the presence or absence of dexamethasone (1 µM; 16 h pretreated) for 48 h. Then caspase activity was measured as described in Materials and Methods. Results of four experiments are expressed as the mean ± SEM. *, P < 0.05 vs. control; #, P < 0.05 vs. TNF-{alpha} treated. C, MC3T3E1 osteoblasts were treated with TNF-{alpha} (20 ng/ml) in the presence or absence of Z-VAD-CHO, Ac-DEVD-CHO, or Ac-YVAD-CHO (100 µM) for 48 h. Then genomic DNA was prepared and analyzed using agarose gel electrophoresis as described in Materials and Methods. M, Marker; 1, control; 2, TNF-{alpha}; 3, TNF-{alpha} plus Z-VAD-CHO; 4, TNF-{alpha} plus Ac-DEVD-CHO; 5, TNF-{alpha} plus Ac-YVAD-CHO.

 
Dexamethasone inhibits TNF-{alpha}-induced cytochrome c release into cytoplasm
Cytochrome c release into cytoplasm is recognized as one of the final signaling pathways leading to cell death. Cytosolic cytochrome c cooperates with Apaf-1 and dATP to activate caspase-9, which, in turn, activates caspase-3 (13, 14). The steps involved in TNF-{alpha} signaling through cytochrome c have not been investigated in osteoblasts. Therefore, the amounts of cytochrome c in mitochondria and cytosolic fractions were measured using a Western blot analysis. Induction of apoptosis was associated with cytochrome c release into the cytosol as determined by immunoblotting (Fig. 4AGo). In this study we observed that dexamethasone had an inhibitory effect on TNF-{alpha}-induced cytochrome c release in MC3T3E1 osteoblasts (Fig. 4BGo). Thus, the inhibition of apoptosis by dexamethasone is associated with reduced release of cytochrome c into the cytoplasm in MC3T3E1 osteoblasts.



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  		Figure 4. Dexamethasone inhibits TNF-{alpha}-induced cytochrome c release into the cytoplasm in MC3T3E1 osteoblasts. A, MC3T3E1 osteoblasts were treated with TNF-{alpha} (20 ng/ml) for various periods as indicated. B, MC3T3E1 osteoblasts were treated with TNF-{alpha} (20 ng/ml) for 48 h in the presence or absence of dexamethasone (1 µM; 16 h pretreated). Then mitochondrial and cytoplasmic fractions were prepared and separated on SDS-PAGE and transferred onto a nitrocellulose membrane. Cytochrome c was visualized by Western blot analysis.

 
Dexamethasone blocks TNF-{alpha}-induced NF-{kappa}B activation
NF-{kappa}B, a transcription factor, was reported to be an essential component in the signal transduction pathway of TNF-{alpha}-induced apoptosis (26). Therefore, we examined whether NF-{kappa}B could be activated in TNF-{alpha}-treated cells and whether the activation of NF-{kappa}B transcription factors could be affected by dexamethasone. The binding of nuclear extracts from TNF-{alpha}-treated cells to the oligonucleotide of NF-{kappa}B consensus binding sequences was observed using an electrophoretic gel mobility shift analysis (Fig. 5AGo), but the activation of NF-{kappa}B was clearly eliminated by pretreatment with 1 µM dexamethasone (Fig. 5BGo). These results suggest that the inhibition of TNF-{alpha}-induced apoptosis by dexamethasone may occur via prevention of NF-{kappa}B activation in MC3T3E1 osteoblasts.



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  		Figure 5. Dexamethasone blocks TNF-{alpha}-induced NF-{kappa}B activation in MC3T3E1 osteoblasts. A, MC3T3E1 osteoblasts were treated with TNF-{alpha} (20 ng/ml) for various periods as indicated. B, MC3T3E1 osteoblasts were treated with TNF-{alpha} (20 ng/ml) for 1 h in the presence or absence of dexamethasone (1 µM; 16 h pretreated). Then the nuclear extracts were prepared. An electrophoretic gel mobility shift assay was performed with {alpha}-32P-labeled oligonucleotide containing the NF-{kappa}B consensus sequence in the presence of the nuclear proteins.

 
Dexamethasone blocks TNF-{alpha}-induced JNK/SAPK activation
JNK/SAPK is activated by a wide variety of cell stresses and has been involved in cell survival/death signals (27, 28). In this study pretreatment with dexamethasone significantly suppressed JNK1 activity in TNF-{alpha}-treated MC3T3E1 osteoblasts. First, we found that 20 ng/ml TNF-{alpha} increased the phosphotransferase activity of JNK1 toward c-Jun protein at 0.5 and 1 h. This increase in JNK1 activity returned to the basal level around 12 h after treatment with TNF-{alpha} (Fig. 6AGo). Then we tested whether treatment with dexamethasone, an antiinflammatory glucocorticoid hormone, had a suppressive effect on TNF-{alpha}-induced JNK activation in MC3T3E1 osteoblasts. As shown in Fig. 6BGo, 1Go µM dexamethasone (16-h pretreated) reduced TNF-{alpha}-stimulated JNK activation to the basal level. This reduction of JNK1 activation in the system may result in the inhibition of TNF-{alpha}-induced apoptosis, as JNK1 is postulated to be involved in the cell death program of MC3T3E1 osteoblasts.



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  		Figure 6. Dexamethasone blocks TNF-{alpha}-induced JNK/SAPK activation in MC3T3E1 osteoblasts. A, MC3T3E1 osteoblasts were treated with TNF-{alpha} (20 ng/ml) for various periods as indicated. B, MC3T3E1 osteoblasts were treated with TNF-{alpha} (20 ng/ml) for 1 h in the presence or absence of dexamethasone (1 µM; 16 h pretreated). Endogenous JNK activity was examined by immunocomplex assays as described in Materials and Methods.

 
Dexamethasone blocks TNF-{alpha}-induced ceramide release
TNF-{alpha} induced a rapid elevation of endogenous ceramide levels to approximately 200% of control levels within 45 min after stimulation. Comparable peak levels (204% of control) were observed 45 min after treatment with TNF-{alpha}, and then the ceramide concentration decreased gradually to the basal level at 90 min (Fig. 7AGo). Furthermore, it is demonstrated that pretreatment with dexamethasone (1 µM) reduced TNF-{alpha}-stimulated ceramide release significantly (Fig. 7BGo). These results suggest that the decrease in ceramide might be necessary for the inhibition of cell death to occur after dexamethasone pretreatment in TNF-{alpha}-stimulated cells.



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  		Figure 7. Dexamethasone inhibits TNF-{alpha}-induced ceramide release in MC3T3E1 osteoblasts. A, MC3T3E1 osteoblasts were treated with 20 ng/ml TNF-{alpha} for the indicated periods. Then the ceramide concentration was measured. B, MC3T3E1 osteoblasts were treated for 30 min with or without 20 ng/ml TNF-{alpha} in the presence or absence of dexamethasone (1 µM; 16 h pretreated). Then the ceramide concentration was measured as described in Materials and Methods. The data are expressed as a percentage of the ceramide level. The results of four experiments are expressed as the mean ± SEM. *, P < 0.05 vs. control; #, P < 0.05 vs. TNF-{alpha} treated.

 
Exogenous ceramide induces apoptosis and dexamethasone does not prevent the cell death
MC3T3E1 osteoblasts were cultured in the absence or presence of various concentrations of C2-ceramide (1–100 µM) for 48 h. In Fig. 8AGo, treatment with C2-ceramide (50 and 100 µM) induced DNA fragmentation in MC3T3E1 osteoblasts. Oligosomal DNA fragmentation, consistent with the onset of apoptosis, was evident after 48 h in C2-ceramide-treated cells. Next, to know whether dexamethasone regulates apoptosis in C2-ceramide-stimulated MC3T3E1 osteoblasts, we analyzed DNA from these cells using agarose gel electrophoresis (Fig. 8BGo). However, dexamethasone did not block exogenous ceramide-induced apoptosis in this system. These data show that the synthetic glucocorticoid has no inhibitory effect on exogenous ceramide-induced cell death, suggesting that dexamethasone has its protective effects on TNF-{alpha}induced apoptosis via the inhibition of ceramide release in MC3T3E1 osteoblasts.



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  		Figure 8. Dexamethasone has no effect on C2-ceramide-induced apoptosis in MC3T3E1 osteoblasts. A, MC3T3E1 (1 x 107 cells/dish) cells were incubated for 48 h with various concentrations of C2 ceramide (10–100 µM). Then genomic DNA was purified and subjected to agarose gel electrophoresis as described in Materials and Methods. M, Marker; 1, control; 2, 10 µM C2-ceramide; 3, 20 µM C2-ceramide; 4, 50 µM C2-ceramide; 5, 100 µM C2-ceramide. B, MC3T3E1 osteoblasts were treated with C2-ceramide (100 µM) for 48 h in the presence or absence of dexamethasone (1 µM; 16 h pretreated). Then genomic DNA was prepared and analyzed by agarose gel electrophoresis as described in Materials and Methods.

 
Ceramide induces the activation of caspase-3-like cysteine protease, NF-{kappa}B, and JNK/SAPK
In this study we documented that caspase-3-like cysteine protease activity is required in TNF-{alpha}-induced apoptosis and suggested that ceramide release plays an important role in apoptosis. Activation of caspase-1 or caspase-3 in C2ceramide-treated cells was measured using a fluorometric assay with the substrates Ac-YVAD-AMC and Ac-DEVD-AMC, respectively. In MC3T3E1 osteoblasts, maximal caspase-3 activity was observed after treatment with 100 µM C2-ceramide for 48 h (Fig. 9AGo). Although caspase-1 was not activated by the treatment of C2-ceramide, C2-ceramide induced a marked increase in caspase-3 protease activity that peaked at 48 h (3.5-fold above basal levels).



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  		Figure 9. Ceramide induces the activation of caspase-3-like cysteine protease, NF-{kappa}B, and JNK/SAPK in MC3T3E1 osteoblasts. The cells were treated with 100 µM C2-ceramide for the indicated periods and harvested. Caspase-1 or -3-like activity (A), electrophoretic gel mobility shift assay for NF-{kappa}B (B), and JNK activity (C) were measured as described in Materials and Methods. *, P < 0.05 vs. control.

 
Recent studies have indicated that TNF-{alpha} is a potent activator of the NF-{kappa}B transcription factor in various cell lines (9, 11). In this system, the early phase activation of NF-{kappa}B that occurred after ceramide stimulation was analyzed using an electrophoretic gel mobility shift assay (Fig. 9BGo). Signals were detected after 15 min of stimulation with 100 µM C2ceramide. The patterns of NF-{kappa}B bands were also quite similar between TNF-{alpha} and C2-ceramide stimulations. Nuclear protein from cells treated with 100 µM C2-dihydroceramide (generally used as a negative control in studies of C2ceramide) did not react with NF-{kappa}B probe (data not shown). In addition, analysis of the time period of C2-ceramide on MC3T3E1 osteoblasts suggests that TNF-{alpha} stimulates JNK/SAPK activation via ceramide release. C2-ceramide induced a transient increase in JNK activity that peaked at 10–30 min and decreased 1 h poststimulation (Fig. 9CGo). C2-ceramide-induced JNK activation was more rapid than TNF-{alpha}-induced JNK activation, which peaked between 30 min and 1 h and returned to basal activity 12 h poststimulation (Fig. 6AGo).

Dexamethasone does not regulate ceramide-induced activation of caspase-3-like cysteine protease, NF-{kappa}B, or JNK/SAPK
In this study we measured the effects of dexamethasone on ceramide-induced apoptosis. As shown in Fig. 10AGo, caspase-3-like cysteine protease activity following ceramide exposure with or without dexamethasone increased continuously up to 48 h. It is consistent with the data in Fig. 9AGo. Next, we examined, using the gel mobility shift assay, whether NF-{kappa}B binding activity in ceramide-treated cells was regulated by dexamethasone. However, pretreatment with dexamethasone did not inhibit and, in fact, enhanced NF-{kappa}B binding affinity in 100 µM C2-ceramide-treated MC3T3E1 osteoblasts (Fig. 10BGo). In addition, the activation of JNK/SAPK was not affected by pretreatment with dexamethasone (1 µM). Taken together, these data suggest that the effect of dexamethasone on attenuation of TNF-{alpha}-induced apoptosis lies upstream of ceramide production, and the protective effect may be mediated by inhibition of ceramide production.



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  		Figure 10. Dexamethasone does not affect the ceramide-induced activation of caspase-3-like cysteine protease, NF-{kappa}B, or JNK/SAPK in MC3T3E1 osteoblasts. A, MC3T3E1 osteoblasts were treated with C2-ceramide (100 µM) for 48 h in the presence or absence of dexamethasone (1 µM; 16 h pretreated). Then caspase-3 like cysteine protease activity was measured as described in Materials and Methods. B, MC3T3E1 osteoblasts were treated with C2-ceramide (100 µM) for 20 min in the presence or absence of dexamethasone (1 µM; 16 h pretreated). Then nuclear extracts were prepared and assayed for NF-{kappa}B binding as described in Materials and Methods. C, MC3T3E1 osteoblasts were treated with C2-ceramide (100 µM) for 30 min in the presence or absence of dexamethasone (1 µM; 16 h pretreated). Then the endogenous JNK activity was examined by immunocomplex assays as described in Materials and Methods. The results of four experiments are expressed as the mean ± SEM. *, P < 0.05 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Glucocorticoids are among the most potent and clinically important antiinflammatory drugs. Under some conditions, such as acute infections, the increase in adrenal levels seems to be an important defense mechanism against the noxious action of inflammatory cytokines. In our model, dexamethasone treatment enhanced the resistance of MC3T3E1 osteoblasts to the cytotoxic activity of TNF-{alpha}, a multifunctional cytokine with an important role in immune response and inflammation, such as rheumatoid arthritis and periodontal diseases. In addition to the glucocorticoid-induced antiinflammatory effects, glucocorticoids such as corticosterone are used as immunosuppressive drugs, and in this context are known as classical inducers of apoptotic cell death in primary osteoblasts (29). However, MC3T3E1 osteoblasts are resistant to dexamethasone-induced apoptosis, and dexamethasone prevents TNF-{alpha}-induced apoptosis in MC3T3E1 osteoblasts. The protective effect of dexamethasone was dose dependent and reached its maximum at micromolar concentrations. This is in line with a few other reports documenting that glucocorticoids suppressed apoptosis in various cell types including osteoblasts (24, 25, 30, 31, 32). The precise mechanisms by which glucocorticoids affect bone remodeling, especially apoptosis, are not well known, and the changes they induce in bone cells and tissues are to some extent controversial, probably because of differences in the type of steroid administered and the type of cell used (30, 33, 34, 35).

Thus, we set out to further characterize the biochemical pathways leading to signaling molecules directly involved in dexamethasone-induced protective effects in TNF-{alpha}-induced apoptosis. The pathway leading to cytotoxicity/apoptosis is known to involve the release of cytochrome c by mitochondria and the downstream caspase-3-cysteine protease activation (36, 37). Our experiments show that caspase-3 processing and the release of cytochrome c precede apoptosis by TNF-{alpha}, thus indicating a temporal and perhaps casual relationship. Furthermore, the release of cytochrome c into the cytoplasm is blocked by dexamethasone, suggesting that the synthetic glucocorticoid agent propagates an inhibitory signal that determines mitochondrial cytochrome c release.

Glucocorticoids decrease the transcription of the genes involved in inflammation, and these genes have no identifiable glucocorticoid response element in their promoter region (38). These data suggest that some other mechanism must mediate the inhibitory effect of the hormones. Although the molecular mechanism of the glucocorticoid on chronic inflammation is not well understood, there is increasing evidence that the glucocorticoid inhibits the action of transcription factors such as NF-{kappa}B (11, 12). NF-{kappa}B was first identified as a regulator of the expression of the {kappa} light chain gene in murine B lymphocytes (39), but has subsequently been found in many different cells. Although there is no direct evidence indicating that activation of NF-{kappa}B stimulated by TNF-{alpha} causes the apoptosis of MC3T3E1 osteoblasts, many studies indicate that the regulation of NF-{kappa}B is one of the most apoptotic signaling pathways (26, 40, 41).

Dexamethasone reduced the activity of JNK significantly in MC3T3E1 osteoblasts. JNK is a member of the mitogen-activated protein kinase family and phosphorylates serine at the amino-terminal trans-activation domain of c-jun. Similar to other mitogen-activated protein kinases, JNK is activated through the dual phosphorylation at a Thr-Pro-Tyr motif. Many studies have shown that JNK is required to induce apoptosis in various cells (42, 43, 44). As dexamethasone reduced the level of JNK/SAPK activity and attenuated TNF-{alpha}-induced apoptosis, it is possible that the inability to maintain JNK activity after cell stress inhibits susceptibility to apoptosis. The study certainly does not exclude the possibility of involvement of other factors affected by dexamethasone in the inhibition of apoptosis, and further studies are underway to define the involvement of JNK/SAPK or NF-{kappa}B in the mechanism.

The signal transduction pathway used by TNF-{alpha} to induce ceramide biosynthesis is a subject of great importance. Ceramide is considered an important aspect of the apoptotic pathway and plays a role in such fundamental biological processes as differentiation and cell death (45, 46). Wolff et al. (47) recently reported results using HL-60 cells, in which TNF-{alpha} caused early and reversible sphingomyelinase hydrolysis at 30–60 min. Furthermore, synthetic ceramide analogs and sphingomyelinase mimicked the action of TNF-{alpha} in the initiation of apoptosis in these cells.

Ceramide is formed during cell death triggered by TNF-{alpha} and might serve as a mediator in the apoptosis in MC3T3E1 osteoblasts. Our studies show that TNF-{alpha}-induced cell death is significantly reduced by the synthetic glucocorticoid that inhibits the production of ceramide from MC3T3E1 osteoblasts. However, ceramide-induced apoptosis of mouse osteoblasts is not affected by dexamethasone in the osteoblasts. These phenomena suggest that the production of ceramide may be an important trigger of apoptosis in TNF-{alpha}-exposed cells. In a parallel with that, ceramide-induced activation of JNK/SAPK, NF-{kappa}B transcription factor, and caspase-3 cysteine protease was not inhibited by the pretreated dexamethasone, although the glucocorticoid abrogated the activation of signal transduction pathways in TNF-{alpha}-exposed osteoblasts. Furthermore, exogenous ceramide induced NF-{kappa}B activation at the same rate as TNF-{alpha}. The addition of ceramide stimulated JNK/SAPK activation before TNF-{alpha} did in MC3T3E1 osteoblasts. NF-{kappa}B and JNK/SAPK activation is generally accepted as a crucial step in the activation of apoptosis in various model systems, the interruption of the apoptotic signaling pathway by the pretreated dexamethasone seems to occur via the inhibition of ceramide release in MC3T3E1 osteoblasts.

With respect to the findings that dexamethasone either blocks the activity of NF-{kappa}B transcription factors or inhibits that of JNK/SAPK, one can speculate that glucocorticoid may inhibit apoptosis in osteoblasts by blocking NF-{kappa}B and JNK/SAPK activities, which are required for the apoptotic pathway. On the other hand, glucocorticoids also promote apoptosis in other systems, which may be mediated at least in part by similar mechanisms (30, 48). For example, TNF-{alpha} is able to elicit pro- and antiapoptotic signals at the same time (49), and each signal depends on the activation of NF-{kappa}B as well as JNK/SAPK (50, 51). It will be of interest to investigate whether and, if so, which set of specific cell transcription factors and stress-related kinases determines whether glucocorticoids display either pro- or antiapoptotic activity. In this context, we have shown for MC3T3E1 osteoblasts that dexamethasone suppressed the activation of NF-{kappa}B transcription factors and JNK/SAPK, cytochrome c release into the cytoplasm, and subsequent activation of caspase-3-like cysteine proteases. This suggests that these signal transduction mechanisms may be involved in the inhibition of ceramide release and the resultant protective effects displayed by the synthetic glucocorticoid, dexamethasone, in osteoblasts.


   Acknowledgments
 
We thank Kimberley Hutchins, an English instructor at Wonkwang University, for editing this manuscript.


   Footnotes
 
1 This work was supported by Korea Research Foundation Grant 1999–015-FP0046 in 1999 and partially by Wonkwang University in 2000. Back

Received December 3, 1999.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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