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Endocrinology, doi:10.1210/en.2003-0764
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Endocrinology Vol. 145, No. 1 418-425
Copyright © 2004 by The Endocrine Society

Involvement of Bax Protein in the Prevention of Glucocorticoid-Induced Thymocytes Apoptosis by Melatonin

Esteban Hoijman, Luciana Rocha Viegas, María Inés Keller Sarmiento, Ruth E. Rosenstein and Adali Pecci

Departamento de Química Biológica (E.H., L.R.V., A.P.), Facultad de Ciencias Exactas y Naturales, Ciudad Universitaria, C1428EGA; and Laboratorio de Neuroquímica Retiniana y Oftalmología Experimental (M.I.K.S., R.E.R.), Departamento de Bioquímica Humana, Facultad de Medicina, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires 1428, Argentina

Address all correspondence and requests for reprints to: Adalí Pecci, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Pab. II, 4° P, Ciudad Universitaria (C1428EGA), Buenos Aires 1428, Argentina. E-mail: apecci{at}qb.fcen.uba.ar.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The antiapoptotic effect of melatonin has been described in several systems. In this study, the antagonistic effect of the methoxyindole on dexamethasone-induced apoptosis in mouse thymocytes was examined. Melatonin decreased both DNA fragmentation, and the number of annexin V-positive cells incubated in the presence of dexamethasone. Analysis of the expression of the members of the Bcl-2 family indicated that the synthetic glucocorticoid increased Bax protein levels without affecting the levels of Bcl-2, Bcl-XL, Bcl-XS, or Bak. This effect correlated with an increase in thymocytes bax mRNA levels. Dexamethasone also increased the release of cytochrome C from mitochondria. All of these effects were reduced in the presence of melatonin, which was ineffective per se on these parameters. In addition, the involvement of cAMP on glucocorticoid/melatonin antagonism was examined. Both melatonin and dexamethasone decreased the levels of this nucleotide in mouse thymocytes, indicating that the antagonistic action between both hormones involves a cAMP-independent pathway. In summary, the present results suggest that the antiapoptotic effect of melatonin on glucocorticoid-treated thymocytes would be a consequence of an inhibition of the mitochondrial pathway, presumably through the regulation of Bax protein levels.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
APOPTOSIS, OR PROGRAMMED cell death, is an intricately regulated process. It is well known that glucocorticoid-mediated thymus involution is a consequence of apoptosis provoked by the direct action of glucocorticoids on thymocytes. In vivo treatment with glucocorticoids induces 90% of cell death in the rat thymus, mainly of immature cells that are localized in the thymus cortex (1).

The molecular mechanism of apoptosis is a matter of an active debate. However, it is accepted that morphological changes observed during programmed cell death are the consequence of an activation of caspases cascade (2). At least two main signaling pathways have been postulated to participate in this process. The first one involves membrane receptors called death receptors (i.e. TNF receptor-1 and Fas/Apo-1) (3, 4), and the second one relies on the cell’s ability to sense changes in the ratio between the protein levels of the members of the Bcl-2 family. Bcl-2 prevents apoptosis induced by a wide range of stimuli, suggesting that different pathways of transduction signals converge at this point (for review, see Refs.5 and 6). Several authors have identified a variety of proteins related with Bcl-2, such as Bax, Bak, Bid, and the different Bcl-X isoforms, which can either promote or prevent apoptosis (7).

Many attempts have been made to isolate steroid-regulated genes involved in controlling cell death. In fact, a link between glucocorticoids and the Bcl-2 family has been demonstrated in several systems (8, 9, 10). On the other hand, a growing number of gene products seem to be implicated in blocking glucocorticoid-induced apoptosis; for example, the classic inhibitors of mitochondrion-dependent cell death bcl-2 and bcl-XL (11, 12) as well as the inhibitors of apoptosis proteins that inhibit the activity of some caspases (13).

Mitochondria plays a key role in the apoptotic pathway through the release of several factors from the intermembrane space to the cytoplasm, such as cytochrome C (14). It has been suggested that this pathway could be regulated by the relative levels and subcellular distribution of Bcl-2 family proteins (15). The antiapoptotic members (i.e Bcl-2 or Bcl-XL) are mostly associated to the outer membrane of mitochondria and inhibit cytochrome C release. On the other hand, the proapoptotic molecules such as Bax, Bad, or Bid are cytosolic proteins; they translocate to the mitochondria and trigger cytochrome C release on apoptosis induction (16). Although cytochrome C release has been demonstrated in apoptotic thymocytes, little is known about the involvement of mitochondrial events in the glucocorticoid-mediated cell death signal. Bcl-2 family proteins are factors involved in this process (12). Bcl-2 knockout mice show fulminant apoptosis in the thymus and hypersensitivity to glucocorticoids (17). On the other hand, thymus hyperplasia was observed in Bax knockout mice, whereas Bax overexpression increases thymus glucocorticoid-induced apoptosis (18).

The hormone melatonin (N-acetyl-5-methoxytryptamine), which is synthesized mainly in the pineal gland, is thought to be involved in several physiological functions, including the entrainment of seasonal and circadian rhythms (19). In humans, melatonin is believed to be involved in the regulation of sleep, seasonal disorders, depression, and aging. Besides, antitumoral properties of melatonin, as well as its involvement in the responsiveness of the immune system, have been described (for a review, see Refs.20 and 21). Most of the melatonin actions have been attributed to its interaction with membrane receptors acting through a cAMP- dependent pathway. On the other hand, several evidences suggest that melatonin, being a highly lipophilic molecule with pleiotropic non-receptor-mediated functions, may also influence peripheral tissues as direct targets. In this context, experimental data support that melatonin, as an antioxidant, can protect tissues against damage caused by free radicals (22). Moreover, it was shown that melatonin is able to bind and activate nuclear receptors of the retinoid acid-related orphan receptor (ROR) family (23).

Several reports support a link between melatonin and glucocorticoids. Melatonin or 2-Iodo-melatonin administration was associated with changes in the density and the affinity of cytosolic and nuclear forms of glucocorticoid receptors (GRs) in rat brain, pituitary, thymus, and liver (24), although melatonin does not seem to be a competitive glucocorticoid antagonist (25). Many lines of evidence indicate an effect of melatonin on thymus apoptosis. In this sense, it was shown that both melatonin and pineal grafts partially prevent thymic involution in very old mice (26), and that the methoxyindole decreases DNA fragmentation induced by glucocorticoids in cultured thymocytes of 25-d-old rats (27). In addition, an age-related decrease in 2-[125I] iodomelatonin binding to mouse thymus that correlates with thymus involution was demonstrated (28). Several hypotheses have been postulated to explain the antiapoptotic effect of melatonin in the thymus: a direct interaction of melatonin with thymus GRs, stimulation of IL-4 release, a direct genomic action that modulates the expression of apoptosis-inhibiting genes, an effect on nitric oxide synthase, and finally, the antioxidant action of melatonin (27, 29). Recently, it was demonstrated that melatonin down-regulates GR mRNA levels in thymocytes (30).

Although it was shown that glucocorticoids modulate the expression of several members of the Bcl-2 family proteins (8, 9, 10), their involvement as well as the participation of cytochrome C release in the antagonistic effect of melatonin on glucocorticoid-induced apoptosis is still lacking. Therefore, we considered it worthwhile to analyze the effect of melatonin on the levels of these pro- and antiapoptotic proteins in mouse thymocytes. In addition, the involvement of the cAMP pathway in the prevention of the dexamethasone-induced apoptosis by melatonin was studied.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and drugs
Dexamethasone, RU 38486, melatonin, 3-isobutyl-1-methylxanthine (IBMX), concanavalin A, and RPMI 1640 medium were purchased from Sigma (St. Louis, MO). Fetal calf serum was purchased from Life Technologies, Inc. (Carlsbad, CA). Hormones were dissolved in absolute ethanol; for in vitro assays x100 solutions were prepared. Stock solution of Concanavalin A (2 mg/ml) was prepared in water and added to RPMI 1640 medium just before incubations.

Animals and thymocytes preparation
CF-1 male mice (21 d old) were housed in a standard animal room, with food and water ad libitum, under controlled conditions of humidity and temperature (21 ± 2 C). Fluorescent lights were turned on automatically every 12 h, from 0600–1800 h. All animals were treated and cared for in accordance with standard international animal care protocols (31). Mice were killed by cervical dislocation at fixed hours (between 1100 and 1200 h) to avoid diurnal variations in serum melatonin levels. Thymuses were immediately removed and minced with scissors extensively into ice-cold RPMI 1640 medium. The cell suspension was filtered through Nytex, and viable cells were counted in a Neubauer hemocytometer in the presence of 0.04% Trypan-Blue. Aliquots of the cell suspension were used for the different assays as indicated below.

In vitro incubation of thymocytes
Thymocytes were incubated in plastic dishes in 1 ml of RPMI 1640 medium containing 10% charcoal-stripped fetal calf serum and concanavalin A (2 µg/ml), in the presence of 10-8 M dexamethasone with or without 10-8 M melatonin. The corresponding volume of ethanol was added to control cells. Cells were incubated at 37 C for different periods of time in a water bath under a normal atmosphere. After incubation, cells were gently resuspended, placed in a 1.5-ml tube, and centrifuged at 2000 rpm for 5 min at room temperature.

DNA isolation and apoptosis analysis
DNA fragmentation.
In each experiment, cell suspension was prepared from a pool of six thymuses, and DNA from 106 cells was isolated according to the method described by Herrmann et al. (32). Briefly, cells were gently resuspended in 0.1 ml lysis buffer (50 mM Tris, pH 7.5, 20 mM EDTA, and 1% Nonidet P-40) for 1 min. After centrifugation at 2000 rpm for 5 min at room temperature, the supernatant, containing the fragmented DNA, was collected in another tube. The pellet (nuclear DNA) was then resuspended in 0.1 ml lysis buffer. Both fractions were incubated at 56 C for 2 h in the presence of 5 µg/µl ribonuclease A and 1% sodium dodecyl sulfate (SDS). After ribonuclease A treatment, 5 µg/µl proteinase K was added to each sample and further incubated at 37 C overnight. DNA was precipitated by the addition of 75 µl of 10 M ammonium acetate and 450 µl absolute ethanol. After precipitation, DNA was resuspended in 100 µl water, electrophoresed in 1.5% agarose gels, and visualized under UV light. The negative was scanned, and the density was quantified with ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). The fragmentation index was defined as the ratio: fragmented DNA/(fragmented DNA + nuclear DNA), in which fragmented DNA was calculated as the area corresponding to fragments smaller than 2000 bp.

Flow cytometry.
An annexin V-fluorescein isothiocyanate apoptosis detection kit from Clontech Products (Palo Alto, CA) (catalog no. K2025–2) was used, following the instructions recommended by the manufacturer. In each experiment, cell suspension was prepared from a pool of six thymuses, and samples of 5 x 105 cells were analyzed by flow cytometry in a Cytoron Absolute cytometer (Ortho Diagnostic Systems, Johnson & Johnson, New Brunswick, NJ). Data were processed by a Wimdi 2.7 software (Windows Multiple Document Interface Flow Cytometry Application, The Scripps Research Institute, La Jolla, CA).

Confocal microscopy
For indirect immunofluorescence, cell suspension employed in each experiment was prepared from a pool of six thymuses, and 104 thymocytes were incubated with dexamethasone, melatonin, or both for 4 h at 37 C. After the treatments, a drop of the cell culture was extended on a glass cover slide, which was waved over a flame for 5 sec. Thymocytes were finally fixed with methanol for 5 min at -20 C. Cells were incubated with 100 nM Mitotracker Red CMXRos (Molecular Probes, M-7512, 579-nm excitation wavelength and 599-nm emission wavelength) in PBS for 10 min at room temperature in the dark, followed by permeabilization with 1% SDS in PBS for 5 min and washing three times with PBS. After this treatment, thymocytes were incubated with an anti-cytochrome-C antibody (Cytochrome C H-104, catalog no. sc-7159 from Santa Cruz Biotechnology, Inc., Santa Cruz, CA), diluted (1:50) in PBS containing 3% BSA (PBS-BSA) for 2 h at 37 C in a humid chamber and were washed three times with PBS-BSA. Then, cells were incubated with a Cy2-conjugated secondary antibody antirabbit IgG (492 nm excitation wavelength and 510 nm emission wavelength, catalog no. 711-225-152, Jackson ImmunoResearch, West Grove, PA) diluted 1:200 in PBS-BSA. Cells were finally washed five times with PBS and mounted on a glass slide by adding one drop of 50% glycerol in PBS. Fluorescence was detected with a Zeiss (Berlin, Germany) LSM 510 laser scanning confocal microscope, and images were analyzed with a LSM 510 Image Browser software.

Western blot
In each experiment, cell suspension was prepared from a pool of 16 thymuses, and protein extracts were prepared by lysing 2 x 107 cells in 5 vol lysis buffer (20 mM Tris-Cl, pH 8.0; 137 mM NaCl; 1% Nonidet P-40; and 10% glycerol) supplemented with protease inhibitors (10 µg/ml leupeptin, 1 mM phenylmethylsulfonylfluoride, and 2 µg/ml pepstatin A). The lysates were centrifuged at 4 C for 10 min at 13,000 rpm, and the pellets were discarded. Protein concentration in the supernatant was determined by the Bradford assay (33). After boiling for 5 min, 30 µg of protein was electrophoresed on a 15% SDS-polyacrylamide gel at 120 mV for 1.5 h. The proteins were transferred onto PVDF membranes in transfer buffer containing 20% methanol (vol/vol), 0.19 M glycine, 0.025 M Tris-base (pH 8.3) at 80 mV for 2 h at 4 C. Blots were blocked for 1 h in TBS (4 mM Tris-Cl, pH 7.5; 100 mM NaCl) containing 3% low-fat powdered milk and 0.05% Tween 20 at room temperature. Rabbit polyclonal anti-Bax (N-20), anti-Bcl-2 (N-19), anti-Bcl-x (S-18) (catalog nos. 493, 492, and 634, respectively, from Santa Cruz Biotechnology, Inc.), anti-Bak (catalog no. A3538, from Dako Corp, CA) diluted 1:300 in TBS, as well a mouse monoclonal anti-Glucocorticoid-Receptor antibody Bu-GR2 (Affinity Bioreagents, Golden, CO, diluted 1:500) were used as primary antibodies and incubated with the membranes overnight. Protein bands were visualized by incubating with a peroxidase-conjugated secondary antibody antirabbit IgG or antimouse IgG (1:3000, 1 h) followed by enhanced chemiluminescence with detection system, ECL (Amersham-Pharmacia, Little Chalfont, Buckinghamshire, UK). Densitometric analysis of protein levels was performed with ImageQuant software. The proper loading was evaluated by staining the membranes with Ponceau-S.

Isolation of total RNA and RT-PCR analysis
In each experiment, cell suspension was prepared from a pool of 16 thymuses, and 2 x 107 thymocytes were resuspended in denaturing solution (4 M guanidinium thiocyanate; 25 mM sodium citrate, pH 7; 0.1 M ß-mercaptoethanol, and 0.5% sarcosyl). Total RNA was extracted by the single-step method (34). For reverse-transcription, 4 µg of total RNA was used. The first cDNA strand was synthesized with superscript-reverse transcriptase (Life Technologies, Inc.) and 25 ng/µl oligo dT (Life Technologies, Inc.) as reverse complementary primer. For PCR amplification the following oligonucleotides: 5'-Bax: 5'-GGAATTCCAAGAAGCTGAGCGAGTGT-3' and 3'-Bax: 5'-GGAATTCTTCTTCCA GATGGTGAGCGAG-3' of bax coding region were used as forward and reverse primers, respectively. The reaction yielded a 394-bp length cDNA fragment.

All PCRs were normalized against glyceraldehyde-3-phosphate dehydrogenase (gapdh) expression. Primers gapdh forward 5'-TGATGACATCAAGAAGGTGGTGAAG-3' and gapdh reverse 5'-TCCTTGGAGGCCA TGTAGGCCAT-3', which specifically hybridize with gapdh mRNA, were used. The reaction yielded a 146-bp-length cDNA fragment. To achieve semiquantitative conditions, RT-PCRs were terminated, and the products were quantified when all the samples were in the linear range of amplification. The cDNA pool (2 µl), 1.25 U Thermus aquaticus polymerase (Life Technologies, Inc.) and amplification primers (20 pmol each) in 50 µl of PCR cocktail (x polymerase buffer, 2 mM MgCl2, 200 µM each deoxynucleotide triphosphate, 50,000 dpm [32P]deoxycytidine triphosphate) denatured 3 min at 96 C followed by 8, 15, 25, and 30 cycles of amplification by using a step program (96 C, 40 sec; 65 C (for gaphd), 58 C (for bax), 30 sec, and 72 C, 1 min), and a final extension at 72 C, 10 min. Ten microliters of PCR products were analyzed by electrophoresis in 8% nondenaturing polyacrylamide gels. Radioactivity was quantified with a phosphorimager (Fuji Photo Film Co. Ltd., Cypress, CA).

cAMP assessment
In each experiment, cell suspension was prepared from a pool of six thymuses, and 5 x 105 thymocytes were incubated for 30 min at 37 C with dexamethasone, melatonin, or both in the presence of 0.5 mM IBMX. After centrifuging for 10 min at 900 x g, pellets were resuspended in distilled water and boiled for 2 min. cAMP levels were assessed as previously described (35). Briefly, homogenates were centrifuged at 5000 x g for 5 min at 4 C, and cAMP content was assessed in the supernatants by RIA after acetylation with acetic anhydride/triethylamine. The acetylated products were incubated with [125I]cAMP (15,000–20,000 dpm, specific activity 140 mCi/mmol) and a rabbit antiserum, kindly supplied by the National Institute of Diabetes and Digestive and Kidney Disease, diluted 1:5000, and incubated overnight at 4 C. After adding 2 ml ethanol with 2% BSA, the antigen-antibody complexes were precipitated by centrifugation at 2000 x g for 15 min. The supernatants were separated by aspiration, and the radioactivity was measured in a {gamma}-counter. The range of the standard curves was 10–5000 fmol cAMP. Three independent experiments were performed in duplicate.

Statistical analysis
Statistical analysis was performed by the two-way ANOVA followed by Tukey’s or Dunnett’s test as stated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Figure 1AGo depicts the effect of 10 nM melatonin on genomic DNA fragmentation of thymocytes induced by 10 nM dexamethasone. After 3 h of incubation with the glucocorticoid, a significant increase in the DNA fragmentation index was observed (control = 0.19 ± 0.02; dexamethasone = 0.58 ± 0.04). When cells were incubated in the presence of both dexamethasone and melatonin, a significant reduction of this parameter was detected (dexamethasone + melatonin = 0.33 ± 0.03). In these conditions, melatonin showed no effect per se (melatonin = 0.28 ± 0.04). A similar pattern was obtained when the number of apoptotic cells was quantified by flow cytometry after staining with FITC-annexin V, as shown in Fig. 1BGo. In this case, the percentages of annexin-V-positive cells were: control = 19 ± 1, dexamethasone = 29 ± 1, dexamethasone + melatonin = 18 ± 1, and melatonin = 19 ± 2.



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  		FIG. 1. Effect of melatonin (Mel) on apoptosis of mouse thymocytes induced by dexamethasone (Dex). A, DNA fragmentation. Cells were incubated for 3 h with or without 10-8 M Dex or 10-8 M Mel. DNA density was quantified with Image Quant software, and the fragmentation index was defined as described in Materials and Methods. The gel is representative of three independent experiments. The right panel shows mean ± SEM values (n = 3). Dex significantly increased this parameter, whereas Mel reverted the effect of the glucocorticoid. **, P < 0.01 vs. control, Dex + Mel, and Mel, by Tukey’s test. B, Positive annexin V cells. Cells were incubated for 6 h with or without Dex (10-8 M) or Mel (10-8 M) and analyzed by flow cytometry as described in Materials and Methods. The histograms are representative of three independent experiments. The right panel shows mean ± SEM values (n = 3). *, P < 0.05 vs. control (n = 3), Dex + Mel, and Mel by Tukey’s test.

 
To evaluate the involvement of cytochrome C release in the antagonism melatonin/dexamethasone, the intracellular localization of this protein was analyzed by immunofluorescence confocal microscopy. In control conditions, cytochrome C was predominantly localized in mitochondria, as confirmed by its colocalization with Mitotracker Red CMXRos, a specific dye for this organelle (Fig. 2Go, A–C). When thymocytes were incubated with dexamethasone, the cytochrome C signal segregated from the mitochondrial dye (Fig. 2Go, D–F), suggesting its translocation to the cytosol. This effect of dexamethasone was blocked in the presence of melatonin (Fig. 2Go, G–I). Melatonin per se did not change the subcellular localization of cytochrome C (data not shown).



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  		FIG. 2. Cytochrome C release from mitochondria. Cells were incubated with Dex (10-8 M) (D–F), Dex (10-8 M) + Mel (10-8 M) (G–I) for 4 h at 37 C as described in Materials and Methods. Control: A–C. Then, they were treated with Mitotracker Red CMXRos to stain mitochondria, immunolabeled for Cytochrome C, and analyzed by laser fluorescence confocal microscopy as described in Materials and Methods. Each field was independently visualized with the appropiate wavelength for Mitochondrial Marker (red; A, D, and G) and for Cytochrome-C antibody plus Cy2 dye-conjugated secondary antibody (green; B, E, and H), and then the two images were overlaid (C, F, and I). Magnification, x2000. Bar, 2 µm. Results are representative of three independent experiments.

 
The relative abundance of the members of the Bcl-2 family is one of the key points in the control of cytochrome C release from mitochondria. Therefore, the effect of dexamethasone, melatonin, or both, on the levels of these proteins was examined. Figure 3Go shows representative Western blot analyses of some of these proteins. The levels of Bax were increased by dexamethasone (control: 2.03 ± 0.22 arbitrary units dexamethasone: 4.28 ± 0.48 arbitrary units), its effect being abolished by the specific antagonist RU 38486 [1.94 ± 0.25 arbitrary units, P < 0.001 vs. dexamethasone (n = 3), by Tukey’s test]. In the presence of melatonin, which showed no effect per se, Bax levels were reduced almost to control values (dexamethasone + melatonin, 2.52 ± 0.35 arbitrary units; melatonin, 2.24 ± 0.30 arbitrary units). The levels of Bcl-2, Bcl-XL, Bcl-Xs, and Bak did not significantly change as a consequence of any of these treatments (Fig. 3Go, B–D).



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  		FIG. 3. Effect of Dex and Mel on the levels of Bcl-2-related proteins. Cells were incubated for 2 h with or without Dex (10-8 M) or Mel (10-8 M). After incubation proteins were processed and Western blot analysis was performed as described with a rabbit polyclonal anti-Bax (A), anti-Bcl-2 (B), anti-Bcl-X(S/liter) (C), or anti-Bak (D), densitometric analysis of protein levels was performed with Image Quant software. The proper loading was evaluated by staining the membranes with Ponceau-S. The gels correspond to one representative experiment. Mean ± SEM values from three independent experiments are shown below each gel. a, P < 0.01 vs. control; b, P < 0.01 vs. Dex, by Tukey’s test.

 
We next examined bax mRNA levels by semiquantitative RT-PCR analysis. Dexamethasone provoked a 70% increase of this parameter, being this effect partially blocked by melatonin (Fig. 4Go). RU 38486 completely reduced the effect of dexamethasone (RU 38486: 2.21 ± 0.36 arbitrary units, P < 0.01 vs. dexamethasone (n = 3), by Tukey’s test), whereas melatonin showed no effect per se on bax mRNA levels (melatonin: 1.98 ± 0.22 arbitrary units). These results suggest that glucocorticoids could regulate bax expression, at least partially, in a melatonin-sensitive manner.



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  		FIG. 4. Effect of Dex and Mel on bax mRNA levels. Cells were incubated with 10-8 M Dex, 10-8 M Mel, or both for 1 h. After incubation, total RNA was obtained, and semiquantitative RT-PCR with specific primers for bax and gapdh coding region were performed as described in Materials and Methods. PCR products were resolved by electrophoresis in 8% nondenaturing polyacrylamide gel. Radioactivity was quantified with a phosphoimager (Fuji Photo Film Co. Ltd.). Dex significantly increased bax mRNA levels, and this effect was reverted in the presence of Mel. Data are mean ± SEM values (n = 3); **, P < 0.01 vs. control, by Tukey’s test.

 
The levels of GR in thymocytes incubated in the presence of dexamethasone, melatonin, or both were assessed. As shown in Fig. 5Go, neither dexamethasone nor melatonin affected this parameter.



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  		FIG. 5. Effect of Dex, Mel, or both on thymocytes GR levels. Cells were incubated and processed as described in Fig. 3Go. No changes in this parameter were observed after any of these treatments. Results are representative of three independent experiments. Data are mean ± SEM values (n = 3). Image corresponds to one representative experiment. No significant differences were detected by Tukey’s test.

 
Figure 6Go depicts the effect of melatonin, dexamethasone, or both on thymocytes cAMP accumulation in the presence of IBMX. Although melatonin decreased this parameter, the antagonistic action between both hormones was not observed. In fact, dexamethasone significantly decreased the nucleotide levels to a similar extent as that observed in the presence of both compounds.



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  		FIG. 6. Effect of Dex, Mel, or both on thymocytes cAMP accumulation. Cells were incubated for 30 min with or without 10-8 M Dex or 10 -8 M Mel in the presence 0.5 mM IBMX. cAMP content was assessed in the supernatants by RIA after acetylation. Both Dex and Mel significantly decreased this parameter. Data are mean ± SEM values from three independent experiments performed in duplicate. **, P < 0.01, by Dunnett’s test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present results indicate that melatonin, which prevented thymocyte apoptosis mediated by dexamethasone, also inhibits both the glucocorticoid-induced cytochrome C release and the increase in the levels of Bax protein and bax mRNA.

The antiapoptotic activity of melatonin was previously described in several systems, including cerebellar neurons (36) and rat brain astrocytes (37), among others. As for the thymus, a well-known model for apoptosis studies, our results are in agreement with a previous report by Sainz et al. (30), who showed that the daily administration of melatonin, during 40 d, reduces glucocorticoid-induced apoptosis in thymocytes from 65-d-old rats. As mentioned before, it was shown that preincubation with melatonin for 3 h significantly decreases the percentage of DNA fragmentation (assessed by a spectrophotometric assay) in thymocytes from 25-d-old rats, further incubated for an additional 6 h-period with dexamethasone (27). In our hands, the antiapoptotic effect of melatonin was also evident when it was coincubated with dexamethasone without preincubation and for only 3 h. Although our results confirm that melatonin reduces mouse genomic DNA fragmentation induced by dexamethasone, we extend this conclusion by showing that the methoxyindole also reduced the glucocorticoid-induced phosphatidylserine translocation to the outer layer of the cytoplasmatic membrane as shown by the annexin V assay.

During apoptosis, cytochrome C translocates from mitochondria to cytosol, where it triggers a cascade of events that involves caspases activation that, in turn, cleave multiple cytoplasmic and nuclear substrates. The link between apoptosis/mitochondria/glucocorticoids is a matter of active debate. As shown herein by immunofluorescence confocal microscopy, dexamethasone induced the segregation of the cytochrome C signal from the mitochondria. This result agrees with the demonstration that dexamethasone induces cytochrome C release in mouse thymocytes assessed by Western blotting (38). In addition, glucocorticoids induce loss of mitochondrial membrane potential in thymocytes and in T cell hybridoma cells (39, 40). In contrast, apoptosis induced by dexamethasone has no detectable effect on cytochrome C release in multiple myeloma cells (41). In this sense, Distelhorts (42) suggested that this step seems to occur downstream of the commitment to cell death, and may be induced by caspases activity. However, more experiments are still needed to clarify this point. Because melatonin prevented both apoptosis and cytochrome C release induced by dexamethasone, it seems possible that a target for melatonin in the glucocorticoid pathway could be located upstream from the mitochondrial dysfunction.

For the first time, the present results show an increase in thymocyte Bax levels induced by dexamethasone in vitro. This glucocorticoid action seems to be specific, because it was reverted by the specific antagonist RU 38486 and because the other tested members of the Bcl-2 family were unaffected by dexamethasone. Apoptosis of thymocytes induced by dexamethasone is an extensively examined process. It is thus surprising that the mechanism described herein remained largely unknown, although an increase in Bax-like (but not in Bcl-2-like) immunoreactivity was described in thymocytes after a treatment with hydrocortisone in vivo (43).

Because an increase in bax mRNA levels was also evident, the effect of dexamethasone on the Bax content in thymocytes is presumably mediated by a transcriptional mechanism. Moreover, in silico analysis showed the presence of putative GREs in the bax promoter (unpublished data). Although a direct link between the increase in Bax levels and apoptosis is still lacking in this system, it is tempting to infer a causal relationship between both phenomena. Several lines of evidence support this hypothesis. In effect, a correlation between dexamethasone-induced apoptosis and the increase in bax mRNA and protein levels in rat hippocampus granule cells was demonstrated (44). Moreover, the same cells from Bax knockout mice did not undergo apoptosis upon dexamethasone treatment (44). It has further been shown that the overexpression of Bax accelerates dexamethasone-mediated cell death in the thymus (18). In addition, as shown herein, melatonin reduced the increase in Bax levels and reverted the proapoptotic effect of dexamethasone. Taken together, these evidences support the consensus that Bax may play a key role in corticosteroid-induced apoptosis and suggest the involvement of this protein in the antagonism melatonin/ dexamethasone.

The ability of Bax to translocate to mitochondria in mouse thymocytes has been reported (16). The intracellular events that account for the redistribution of Bcl-2 family proteins are still unclear. However, the abundance of these proteins could be a necessary (although not sufficient) condition. It is possible that an increase in Bax levels could increase the probability of homo- or heterodimerization that seems necessary for the translocation (45). In this way, the increase in Bax levels observed after the incubation with dexamethasone could be an initial step in its destination to mitochondria.

So far, three molecular mechanisms have been described for the melatonin effect: (1) its antioxidant effect, and its specific interaction with (2) membrane and (3) nuclear receptors. Many evidences suggest that the antiapoptotic effect of melatonin in different tissues could be related to its antioxidant activity. In this sense, it was reported that 200 µM melatonin prevents cell death induced by hydroxyl radicals (OH{bullet}) in primary culture of mouse thymocytes (29). Based on the fact that the proapoptotic effect of glucocorticoids has been related to oxidative stress (46, 47), this hypothesis was examined in rat thymocytes by Sainz et al. (30). These authors concluded that melatonin does not regulate the mRNA levels of antioxidant enzymes in mouse thymocytes. High concentrations of melatonin are needed to effectively scavenge free radicals, thus it seems unlikely that an antioxidant effect could account for the antiapoptotic action of melatonin at a 10-nM concentration. However, because it was reported that nanomolar concentrations of melatonin increase mitochondrial electron transport chain activity and ATP production, counteracting mitochondrial oxidative stress, the possibility that melatonin increases mitochondrial membrane potential and, hence, may counteract the mechanism(s) involved in transition pore opening and cytochrome C release cannot be formally ruled out (48, 49). Sainz et al. (30) suggested that melatonin negatively regulates the expression of thymocyte GRs as previously described in other systems (36). Because, in our conditions, no changes in mouse thymocyte GR levels were found in the presence of melatonin, it seems unlikely that this mechanism may explain the effects of melatonin described herein.

Mel 1a melatonin membrane receptors have been described in rat T and B lymphocytes (50). Accordingly, the present results demonstrate that melatonin decreases cAMP accumulation in mouse thymocytes. As shown herein, dexamethasone also decreased thymocyte cAMP levels. Although an inhibitory effect of dexamethasone on the increase in cAMP levels induced by adrenergic agonists in rat thymic epithelial cells was reported (51), the effect per se of the glucocorticoid on this parameter in thymus has not been previously described. Because both melatonin and dexamethasone reduced cAMP levels, and no reversion of this effect was observed in the presence of both compounds, the role of cAMP as a mediator of melatonin/glucocorticoid antagonism seems unlikely.

Melatonin binds to purified thymocyte nuclei (52), suggesting the existence of functional nuclear receptors of the ROR family in the thymus. Through this receptor, melatonin regulates the expression of several genes in other tissues (53, 54). Because the effect of melatonin in thymus does not seem to involve membrane receptors or an antioxidant mechanism, ROR{alpha} receptors emerge as possible candidates to account for this effect. In fact, besides the glucocorticoid- responsive elements, several putative ROR{alpha}-responsive elements were found in bax promotor (unpublished data). In addition, the GR-interacting protein-1 has been shown to be a coactivator for ROR{alpha} (55). There are several possibilities to explain the antagonism between melatonin and glucocorticoids involving this type of receptors: 1) the ROR{alpha}/melatonin complex may affect the dexamethasone/receptor complex translocation to the nucleus; 2) it may modulate the binding of the dexamethasone/receptor complex to its DNA-responsive element, or 3) both complexes may bind to the bax promotor and regulate the expression of this gene in opposite directions. These mechanisms are currently under study.

The physiological relevance of the effect of melatonin described herein still remains an open question. The dose of melatonin used in this study (10 nM) is slightly above its normal plasma concentration measured during its nocturnal peak (1 nM). However, it was proposed that melatonin may accumulate in some tissues, and cellular structures such as cell membrane, cytosol, and nuclei to about 103 times higher than circulating levels (56). In addition, Martín et al. (48) reported intramitochondrial melatonin levels 100 times higher than those of plasma. Altogether, these data support a physiological role of melatonin in the prevention of thymocytes apoptosis.

In summary, the present results suggest the involvement of the Bax protein in the prevention of glucocorticoid- induced apoptosis in thymocytes by melatonin. Because glucocorticoids are well known as pleiotropic regulators, the existence of signals able to modulate their functions may contribute to prevent an all-or-none type of response, providing specific targets (such as the thymus) with a higher capacity to respond to a wide range of physiological demands. This is in accordance with reports on the effect of melatonin on age-related thymic involution (57). In this way, melatonin could well provide a "break" to adjust the timing of thymus apoptosis.


   Acknowledgments
 
The authors thank Dr. Carlos P. Lantos, Dr. Diego A. Golombek, and Dr. Cora B. Cymeryng for helpful discussion of this manuscript; Dr. Omar Pignataro for iodination of [125I]cAMP; and Dr. Tomás Santa Coloma and Dr. H. Eduardo Chuluyan for confocal microscopy and flow cytometry studies.


   Footnotes
 
This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), University of Buenos Aires, Fundación Antorchas and by the fellowship "Ramón Carrillo-Arturo Oñativia," Ministerio de Salud de la Nación, Argentina.

Abbreviations: gapdh, Glyceraldehyde-3-phosphate dehydrogenase; GR, glucocorticoid receptor; IBMX, 3-isobutyl-1-methylxanthine; ROR, retinoid acid-related orphan receptor; SDS, sodium dodecyl sulfate.

Received June 18, 2003.

Accepted for publication September 11, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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