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* Departamento de Morfología y Biología Celular and
Departamento de Biología Funcional, Facultad de Medicina, Oviedo, Spain; and
Department of Cell and Structural Biology, University of Texas Health Science Center at San Antonio, Texas, USA
1Correspondence: Departamento de Morfología y Biología Celular, Facultad de Medicina, Julian Claveria 33006 Oviedo, Spain. E-mail: carro{at}sci.cpd.uniovi.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: antioxidant enzymes • glucocorticoids • apoptosis • DNA fragmentation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5
6
7
8
9
10)
. The most recently described
properties of melatonin are 1) its antioxidant capability,
acting both as a free radical scavenger (11
, 12)
and a
stimulator of activity (13
, 14)
and mRNA levels for
several antioxidant enzymes (8
, 15
, 16)
and 2)
its antiapoptotic effects in thymocytes (17
, 18)
and
neuronal cells (16
, 19
, 20)
. The antioxidant and
antiapoptotic properties suggest possible clinical applications of this
mediator particularly in the prevention of free radical damage and
apoptosis in neurodegenerative diseases (21
22
23)
.
Most T cells synthesize their receptors once they are in the thymus.
Thymocytes may thereafter proceed in one of the following ways: they
may 1) be selected positively and then mature in the thymic
medulla, where they are insensitive to apoptosis-inducing factors,
2) be selected negatively and die in a typical programmed
cell death process (24)
, or 3) remain
unselected in the thymic cortex (the vast majority of them) extremely
sensitive to multiple apoptosis-inducing factors (e.g., oxidative
stress, DNA damaging agents, radiation, and especially glucocorticoids)
(24)
. Concentrations of glucocorticoids in a high
physiological range readily induce apoptosis in nonselected thymocytes.
Cohen et al. (24)
proposed that these hormones are also
responsible for the physiological apoptosis occurring in thymus with
aging. The rise in thymus weight by adrenalectomy seems to support this
(25)
. It is because of these observations that the
induction of apoptosis by dexamethasone in thymocytes from the thymic
cortex is a standard in vitro model for the study of
apoptosis.
Here we investigated the mechanism by which melatonin inhibits cell death induced by dexamethasone in thymocytes. To do this, we analyzed the effect of this hormone on cell death induced by other agents, examining its influence on the expression of several genes related to programmed cell death. We conclude that regulation of the expression of the glucocorticoid receptor (GcR)2 gene could be the main mediator of the antiapoptotic effect of melatonin in the thymus.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture
After decapitation thymus was quickly removed and immersed in
Hanks' balanced salt solution (HBSS) containing glucose (1 g/l) to
preserve cellular viability. Thymocytes were obtained by pressing the
thymus against a sterile stainless steel screen. After several washes
with HBSS and recovery of the cells by centrifugation at 1000 rpm for 5
min, cells were filtered through a smaller screen. Thymocytes were
maintained in RPMI 1640 culture medium supplemented with 10% fetal
bovine serum (FBS), sodium bicarbonate (2 g/l), and 1%
antibiotic-antimitotic mixture (100 µg/ml penicillin, 100 µg/ml
streptomycin, and 25 µg/ml amphotericin) at 37°C and 5%
CO2. Cells were counted with a NEUBAUER camera
and cultured in plastic plates (NUNC, Roskilde, Denmark).
IM-9 cells were cultured in RPMI 1640 culture medium supplemented with 10% FBS. Cells were grown in suspension in T-75 flasks at 37°C and 5% CO2.
DNA electrophoresis
Cells were cultured in 35 mm plates at a density of 3 x
106 cells/ml and collected by centrifugation
after scraping the plates. They were homogenized in 1 ml of
homogenization buffer (0.1 M NaCl, 0.01 M EDTA pH 8, 0.3 M Tris-HCl pH
8, and 0.2 M sucrose) and processed as described Tilly et al.
(26)
. To analyze DNA fragmentation, 15 µg of DNA were
electrophoresed in 2% agarose gel in TAE buffer (2%) and 0.5 µg of
ethidium bromide. Gel was run at 5 v/cm, visualized under UV, and
photographed with a POLAROID camera.
Quantification of DNA fragmentation
Cells were collected and recovered in Eppendorf tubes by
centrifugation at 13,000 x g for 2 min. The pellet was
washed in phosphate-buffered saline (PBS) and cells were lysed with 400
µl of a lysis buffer (10 mM Tris pH 7.5, 1 mM EDTA, and 0.2% of
Triton X-100) by incubation in ice water for 20 min. High and low
molecular weight DNAs were obtained as described by Collota et al.
(27)
and quantified following a colorimetric assay
described by Burton (28)
. Absorbance was measured at 600
nm in a spectrophotometer and percentage of fragmentation was
calculated as follows:
% fragmentation = supernatant ODr600/supernatant ODr600 + pellet ODr600
Morphometric analysis
Cells cultured at a density of 3 x
106 cells/ml were collected and recovered by
centrifugation at 1000 x g for 5 min. They were washed
several times in PBS and fixed in 500 µl of 3% glutaraldehyde in 0.1
M phosphate buffer, pH 7.3, for 2 h at 4°C. Fixed cells were
then embedded in a small volume of 1% agar, dehydrated in increasing
concentrations of acetone, and embedded in SPURR resin (EMS, Fort
Washington, Pa.). Semithin (1 µM) sections were obtained at four
different levels separated by 30 µM from three blocks of each
experimental group and stained with 0.2% toluidine blue. Apoptotic
and nonapoptotic cells were counted in 15–20 areas in each section at
100x. A minimum of 500 cells was counted in each group. The percentage
of apoptotic cells was calculated in relation to the total number of
cells.
RNA isolation and Northern blot analysis
Thymus tissue (400 mg) or cells cultured in 100 mm plates at a
density of 50 x 106 cells/plate were used.
Total RNA was extracted according to the method described by
Chomczynski and Sacchi (29)
.
(poly(A)+-RNA was obtained from total RNA with a
purification mRNA kit (Pharmacia Biotech, Piscataway, N.J.). After
electrophoresis in a 1% agarose gel, RNA was transferred to a nylon
membrane (HYBON-N+, Amersham Life Sciences,
Little Chalfont, U.K.) and hybridized with the following probes: a 1.6
kb HindIII/ECO RI fragment from the rat catalase cDNA clone
pTZCTL (30)
; a 0.6 kb EcoRI fragment from the
rat copper-zinc superoxide dismutase (Cu-Zn SOD) cDNA clone, pUC13
(31)
; a 0.8 kb SalI fragment from the rat
glutathione peroxidase (GPx) cDNA clone, LK 440 (32)
; a
2.4 kb BamHI fragment from the GcR cDNA clone pSP65
(33)
; a 0.85 kb ECO RI/HindIII fragment from
the human bcl-2 cDNA clone pBluescript (34)
; a
2 kb BamHI fragment from the mouse p53 cDNA clone
pBR322 (35)
; and a 2.1 kb fragment from the human
ß-actin cDNA clone, pHFBA-1 (36)
, which was used to
normalize the remainder of the mRNA values. Autoradiographies shown
belong to a representative experiment.
Statistical analysis
Data result from three independent experiments. Results are
shown as the mean ± standard error. Statistic analysis was
performed with analysis of variance, followed by a Student Newman-Keuls
test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; this is
prevented by melatonin in other systems by increasing the levels of
mRNA for antioxidant enzymes (15
, 16)
. To determine
whether the antiapoptotic action of melatonin in dexamethasone-treated
thymocytes (17)
correlates with the increase of
antioxidant enzymes, we measured the levels of mRNAs for GPx, Cu-Zn
SOD, and catalase in these cells.
Three groups of cells were used: one group received no treatment; a
second group was treated with 10-7 M
dexamethasone; and a third group was treated with
10-7 M dexamethasone plus
10-7 M melatonin (preincubation of 3 h and
coincubation with the dexamethasone for 6 additional hours). Both
morphometric analysis of apoptotic cells and DNA electrophoresis
confirmed that melatonin prevented cell death induced by dexamethasone
(data not shown). Dexamethasone decreased the levels of mRNA for GPx
(almost 40%) and increased the levels of mRNA for Cu-Zn SOD (45%)
whereas melatonin prevented both of these changes (Fig. 1
A, B). No mRNA for catalase was detected.
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Melatonin did not prevent apoptosis induced by etoposide
To test whether melatonin was acting on the general machinery of
apoptosis, programmed cell death was induced in the same experimental
model, with etoposide causing apoptosis by inhibiting DNA topoisomerase
II (38)
.
Morphometric analysis, quantification of DNA fragmentation, and DNA
electrophoresis gel pattern showed no differences between the group of
cells treated with 50 µM etoposide for 4 h and cells also
treated with 10-7 or 10-9
M melatonin (preincubation of 3 h and coincubation with etoposide
for an additional 4 h) (Table 1
and Fig. 2
A). When cells were treated with 10 µM etoposide for
18 h with or without administration of melatonin (3 h pretreatment
and coincubation for an additional 18 h), the same results were
obtained (Table 2
and Fig. 2B
).
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Melatonin increases mRNA levels for bcl-2 in thymus in
vivo but does not affect these levels in cultured thymocytes
A nuclear receptor for melatonin has been reported
(39)
; this receptor may be involved in the regulation of
several mRNAs (9
, 40)
. To test whether melatonin was
regulating the expression of the apoptosis-involved genes
bcl-2 and p53, Northern analysis with the cDNA of
these two genes was performed in both in vivo and in
vitro experiments.
Four groups of animals (n=8) were used for the in vivo experiment. The first group, killed at 25 days of age, was used as a control (young controls). The second group was intraperitoneally injected daily with 50 µg/kg melatonin from 25 to 65 days of age. The third group was injected daily with a high dose of melatonin (500 µg/kg). The vehicle alone was administered to a last group (old controls), also for 40 days.
mRNA for Bcl-2 decreased with aging and both doses of melatonin
prevented this decrease. mRNA for p53 also decreased with age, but
melatonin did not prevent this fall (Fig. 3
A, B).
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Three groups of cells were used for the in vitro
experiment: one group was left untreated; a second group was treated
with 10-7 M dexamethasone; and the last group
was treated with 10-7 M dexamethasone plus
10-7 M melatonin. The mRNA for Bcl-2 did not
change either after dexamethasone or dexamethasone plus melatonin
treatment (Fig. 3C, D
).
Melatonin decreases the levels of mRNA for GcR
Given 1) that the only positive result in the
antiapoptotic effect of melatonin in thymocytes was obtained when
apoptosis was induced by dexamethasone; 2) the
glucocorticoid-opposite effects of melatonin in the mRNA levels for
antioxidant enzymes; and 3) the classically described
steroid-related effects of melatonin, we surmised that melatonin may be
regulating the GcR in this system. Northern analysis with the cDNA for
GcR was performed in both in vivo and in vitro
experiments.
The experimental designs were identical to those described above. The
levels of mRNA for GcR in vivo increased with age (GcRI,
20%; GcRII, 30%), as expected, since GC are known to up-regulate
their own receptor in T lymphocytes and melatonin prevented this
increase (Fig. 4
A, B). In the in vitro experiment, dexamethasone
increased the levels of GcR mRNA (GcRI, 45%, and GcRII, 35%) and
melatonin prevented this rise (Fig. 4C, D
).
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Melatonin has a direct effect on GcR expression
As GC up-regulate their own receptor in thymus, the effects of
melatonin seem to be opposite to the effects of these hormones both in
terms of effects on the mRNA for antioxidant enzymes and on the mRNA
for GcR. To determine whether melatonin reduces the action of GC in
some way or is directly regulating its receptor expression, the effect
of melatonin on the mRNA levels for GcR was studied in the IM-9 cells,
where this receptor is down-regulated by its ligands (41)
.
As expected, dexamethasone decreased the levels of mRNA for GcR
(GcRI, 35%; GcRII, 20%). Melatonin did not have the opposite effect
of dexamethasone; on the contrary, the group treated with dexamethasone
and melatonin showed mRNA levels for GcR below that of the group
treated only with dexamethasone (GcRI, 70%; GcRII, 60% vs. control)
(Fig. 5
A, B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
Glucocorticoids have been related to oxidative stress. Beaver and
Waring (42)
reported glutathione concentrations to
decrease after dexamethasone administration, and Behl et al.
(37)
have shown that GC enhance oxidative stress-induced
cell death in neurons. Because melatonin has important antioxidant
properties (43)
and its increase of mRNA for antioxidant
enzymes has been proposed as its mechanism to prevent apoptosis in
other systems (16)
, we studied the effect of melatonin on
these mRNAs. The present findings suggest that, for this system,
melatonin does not strictly regulate antioxidant enzymes mRNAs, but
seems to exert an action opposite to that of GC.
Our finding on the decrease of GPx and increase of SOD by GC may well
explain why these hormones enhance the oxidative damage induced by
other substances, as a balance in the activity of these antioxidant
enzymes is necessary to counteract cellular oxidative attack. Thus, the
rise in SOD activity would increase intracellular levels of hydrogen
peroxide (H2O2) as
consequence of the dismutation of the superoxide anion radical
(O2-·). In the presence of
transition metals (most often Fe2+)
H2O2 generates the highly
toxic hydroxyl radical (.OH), which has
devastating actions within cells. When an increase in SOD activity
occurs simultaneously with a reduced GPx activity (which converts
H2O2 into nontoxic
products), the results can be disastrous in terms of free radical
damage, consistent with the results of Peled-Kamar et al.
(44)
. These authors reported that the overexpression of
Cu-Zn SOD in transgenic mice produces increased cell death in the
thymus after administration of lipopolysaccharides, which show a rise
in the production of H2O2
and high levels of lipid peroxidation. The melatonin prevention of the
imbalance in the mRNA for antioxidant enzymes caused by GC may be
considered another facet of the antioxidant defenses of the organism.
Melatonin has also been shown to restore Zn2+
levels in the thymus of old animals (45)
and to antagonize
the formation of the Ca2+–calmodulin complex
(6)
. It has been reported that DNA fragmentation, a
hallmark of programmed cell death, is caused by a
Ca2+- and Mg2+-dependent
endonuclease that is inhibited by Zn2+
(46)
; this enzyme could well be inactivated by melatonin.
This does not seem to occur in the thymus, however. First, when
morphometrical analysis of dexamethasone-induced apoptotic cells in
thymocytes was performed, we found melatonin to prevent the
morphological changes associated with programmed cell death. The fact
that these markers precede and are independent from DNA fragmentation
(47)
implies that melatonin acts at an early step of
apoptosis in thymocytes. Second, if melatonin inhibits the
Ca2+- and Mg2+-dependent
endonuclease, it should do so regardless of the mechanism used to
induce apoptosis; in the present study, melatonin was totally unable to
prevent the fragmentation of DNA when it was induced by etoposide.
Bcl-2 and p53 are proto-oncogenes closely related
to the process of programmed cell death (48
, 49)
. Bcl-2
protein has been reported to inhibit all processes of apoptosis in
thymocytes except the deletion of the negatively selected thymocytes
(50)
. Melatonin has been reported to regulate the mRNA for
several proteins (7
, 8)
possibly via a nuclear receptor
(9
, 40)
. Although melatonin increased the levels of mRNA
for Bcl-2 in vivo, this was not found in vitro,
suggesting that the in vivo increase may have been an
indirect consequence of melatonin administration rather than a direct
effect on the expression of this oncogene. When animals are exposed to
short photoperiods (rising melatonin levels), thymus weight also
increases, mainly the medulla (51)
. Given that
bcl-2 mainly is expressed in the medulla (52)
,
higher expression of this oncogene after chronic melatonin
administration in vivo may have been a consequence of
greater cellularity in this area. On other hand, since melatonin did
not inhibit apoptosis induced by etoposide, it seems unlikely that
bcl-2 mediated this effect, inasmuch as bcl-2 has
been reported to inhibit apoptosis induced by dexamethasone, etoposide,
radiation, or exposure to antibodies (53)
.
Interleukin-2 (IL-2) and IL-4 mediate melatonin antiapoptotic effects
in bone marrow stem cells (4)
. These interleukins also
inhibit apoptosis induced by dexamethasone in thymocytes where IL-2
increases mRNA for Bcl-2 (54)
. Although we did not measure
interleukins, these are mitogenic factors in fetal and adult thymocytes
(55)
, and we have found a decrease of cellular
proliferation in thymus after melatonin administration
(3)
. Nor did we find an accumulation of mRNA for Bcl-2 in
thymocytes treated with melatonin. Bearing in mind that thymocytes only
produce IL-2, we noted that our data do not support interleukins as
mediators of the antiapoptotic effect of melatonin in thymocytes.
It seems that the effects of melatonin in the thymus are contrary to those of GC in terms of its effect on both GcR and antioxidant enzymes mRNA levels. These data suggest two possibilities: 1) melatonin may reduce the action of GC (by altering binding to the GcR, translocation of the GC-GcR complex into the nucleus, or binding of the GC-GcR complex to its response element in DNA); and 2) melatonin may regulate expression of the GcR itself. In the IM-9 cell line, GC down-regulate the expression of their receptor. The fact that melatonin magnifies the action of GC in this line, reducing the levels of mRNA for GcR, indicates that melatonin is not acting via any of the mechanisms mentioned in the first item. This suggests that melatonin regulates expression of the GcR gene (affecting transcription, translational efficiency, or mRNA stability), causing a reduction of the GcR and mediating through this decrease its antiapoptotic effects when apoptosis is induced by dexamethasone in thymocytes.
Melatonin has been reported to have a nuclear receptor (RZR-
and
ß/ROR-1, 2, and 3) that is an orphan of the nuclear receptor
superfamily (39)
. It has also been shown that this hormone
binds purified cell nuclei from thymus (56)
, suggesting
the existence of its nuclear receptor in this tissue. Through this
receptor, melatonin has been reported to regulate the expression of
several genes (9
, 40)
. It is known that several nuclear
receptors may bind to and activate each other's response elements,
albeit with lower efficiency; they can also form heterodimers
(57)
. These and other characteristics make the study of
the mechanisms of action of molecules having nuclear receptors quite
complex. Melatonin is a highly soluble lipophilic molecule with a
possible nuclear receptor, which makes it a candidate as a
transcription factor similar to other steroids. Its receptor
theoretically may be able to interact with other nuclear receptors or
nuclear receptor response elements, making melatonin mechanisms of
action complex and varied. This complexity may be enhanced by the fact
that expression of Mel 1a melatonin receptor has been reported in T and
B lymphocytes from rat thymus and spleen (58)
, rendering
it impossible to rule out a regulation throughout a phosphorylation
cascade pathway. Hereby we demonstrate melatonin regulation of the GcR;
however, a possible regulation of the melatonin receptor by GC and
melatonin regulation of other steroid and nuclear receptors in general
should be investigated further.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication December 14, 1998. Revised for publication March 15, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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