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Molecular and Cellular Endocrinology 436 (2016) 59e67

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Molecular and Cellular Endocrinology


journal homepage: www.elsevier.com/locate/mce

Melatonin modulates glucocorticoid receptor mediated inhibition of


antioxidant response and apoptosis in peripheral blood mononuclear
cells
Amaresh Kumar Singh, Chandana Haldar*
Pineal Research Laboratory, Department of Zoology, Banaras Hindu University, Varanasi, 221005, India

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 31 March 2016
Received in revised form
18 July 2016
Accepted 20 July 2016
Available online 21 July 2016

Pineal melatonin is known for its immunomodulatory and anti-stress properties. It modulates stress
condition by regulating antioxidant responses and apoptosis in the immune cells. Stress causes increased
glucocorticoid level that acts through glucocorticoid receptor (GR) and is translocated into nucleus under
regulation of HSP90 based chaperone machinery. Melatonin inuences glucocorticoid and GR mediated
stress condition in rodents, but till date there are no reports which could suggest the effect of melatonin
treatment on GR mediated apoptosis and inhibition of Nrf-2/hemeoxygenase-1 (HO-1) induced antioxidant status in immunocompetent cells (peripheral blood mononuclear cells; PBMCs). Therefore, in the
present study, we considered GR mediated inhibition of Nrf2 and HO-1 along with anti-apoptotic Bcl-2
expression in PBMCs. The PBMCs were treated with synthetic glucocorticoid; dexamethasone (Dex) and
melatonin (Mel), to explore the effect of melatonin treatment in regulation of GR mediated apoptosis and
inhibition of antioxidant status in immune cells. It was noted that melatonin treatment retained GR into
cytoplasm by inhibiting the dissociation of HSP90 from GR-HSP90 complex and enhanced expression of
Nrf2/HO-1 and Bcl-2 expression. This led to increased HO-1 expression and elevated Bcl-2 led to
increased Bcl-2/Bax ratio that might ultimately enhanced the cellular antioxidant response and survival
under glucocorticoid mediated stress condition. Our observations suggest that the declined GR nuclear
translocation upon melatonin treatment might be responsible for the up-regulation of Nrf2 mediated
HO-1 activity and increased Bcl-2/Bax ratio in PBMCs to maintain the immune homeostasis under stress
condition.
2016 Published by Elsevier Ireland Ltd.

Keywords:
Glucocorticoid receptor
Melatonin
Nrf2
HO-1
PBMCs
Dexamethasone

1. Introduction
Stress is a constellation of events that acts as stimulus (stressor)
to activate stress response in the physiological system (Dhabhar
and McEwen, 2001). Stress has been reported to suppress or dysregulate immune function (Glaser and Kiecolt-Glaser, 2005) and
increase susceptibility to various infections (Cohen et al., 1991). The
detrimental effect of stress causes imbalance of the physiological
homeostasis by declining the immune function (such as the
reduction of immune cells activity, lymphocyte population, proliferation and NK cell activity) (Webster Marketon and Glaser, 2008)
along with decreased antioxidant response that leads to

* Corresponding author.
E-mail addresses: amareshsingh86@gmail.com (A.K. Singh), chaldar2001@
yahoo.com (C. Haldar).
http://dx.doi.org/10.1016/j.mce.2016.07.024
0303-7207/ 2016 Published by Elsevier Ireland Ltd.

immunocompromised state (Dohms and Metz, 1991). The increased


glucocorticoids (GC) in response to stress via HPA axis (Saplosky
et al., 2000) exerts its effects via intracellular GC receptor (GR)
and modulates the expression of different target genes (Stahn and
Buttgereit, 2008). The high GC and GR level has been associated
with declined immune responses i.e. stress mediated immunosuppression (Ashwell et al., 2000). The stress induced immunosuppression might be a result of decline antioxidant response and
increased apoptosis in immunocompetent cells/organs (Kannan
and Jain, 2000). The GR activation induces apoptosis by suppressing anti-apoptotic proteins Bcl-2 and enhancing Bax expression
(Distelhorst, 2002; Lepine et al., 2005). GR activation has also been
reported for declining antioxidant response (Kratschmar et al.,
2012). GC binding to GR in the cytoplasm and translocates GR
into the nuclear compartment. The GR nuclear translocation is
tightly regulated by HSP90 based chaperone machinery in which
HSP90 has been suggested to play an imperative role (Grad and

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A.K. Singh, C. Haldar / Molecular and Cellular Endocrinology 436 (2016) 59e67

Picard, 2007). HSP90 binds with GR and form HSP90-GR complex


that regulates functional activation and inactivation of GR (Freeman
and Yamamoto, 2002). It has also been reported that alteration in
HSP90/GR ratio and increased nuclear HSP90 attenuates activity of
GR activity (Ouyang et al., 2012).
Melatonin has been suggested to down-regulate GC and GR
mediated inhibition of immune responses (Gupta and Haldar,
2013). Seasonal variation in melatonin concentration inuences
GR expression in human mononuclear leucocytes (Blackhurst et al.,
2001). The activation of GR has been associated with suppressed
antioxidant response signaling via Nrf2 and down-regulates an
array of antioxidative enzymes like superoxide dismutase (SOD),
hemeoxygenase-1 (HO-1), catalase (CAT) etc. (Kratschmar et al.,
2012). The seasonal melatonin levels positively inuences antioxidative enzymes activity (SOD, CAT) in daily manner (Singh et al.,
2013). The down-regulation of Nrf2 signaling has been suggested to
increase apoptosis by inuencing apoptotic proteins (Pan et al.,
2013). However, there is no report available which could suggest
that how melatonin modulates GR mediated inhibition of Nrf2 and
thereby regulating antioxidant response and induction of apoptosis
in PBMCs in any ruminant species like goat. We hypothesized that
melatonin might be repressing the nuclear translocation of GR to
modulate antioxidative enzyme activity and to down regulate
apoptosis under glucocorticoid induced stress condition in PBMCs
of goat Capra hircus.
Therefore, in the present study, we considered GR mediated
inhibition of Nrf2 dependent HO-1 expression along with antiapoptotic Bcl-2 expression in the PBMCs. The PBMCs were treated
with dexamethasone (Dex) and melatonin (Mel) alone and in
combination to explore the effect of melatonin treatment in regulation of GR mediated inhibition of antioxidative enzymes activity
and apoptosis. The expression of GR; complete, nuclear and cytoplasmic fraction was noted along with nuclear HSP90 expression.
To note Nrf2 mediated antioxidant response, HO-1 and Bcl-2
expression was noted upon melatonin and dexamethasone treatment in vitro. The successive effect of dexamethasone and melatonin treatment was checked on apoptotic markers like Bax,
Caspase-3 in PBMCs along with Bcl-2/Bax ratio. The cell mediated
immune responses of PBMCs were noted by measuring IL-2
secretion and proliferative responses to mitogen Concanavalin-A
challenge in goat Capra hircus.
2. Material and method
All the experiments were performed in accordance with institutional practice and within the framework of the animals Act of
2007 of Govt. of India on animal welfare (Committee for the Purpose of Control and Supervision of Experiments on Animals;
CPCSEA).
2.1. Animal maintenance
In the present study, 7 adult goats (4e5 years; BW 35 2 kg)
were procured from Institute of Agricultural Sciences, Banaras
Hindu University, at Varanasi (latitude 25 180 N and longitude 83
10 E). The goats were maintained as per farm rule under ambient
condition (mean temp. 30 2  C; humidity 85 5%; day length
~12L:12D; for the months of AugusteSeptember). They were fed on
green leaves and grasses as per seasonal availability along with
usual roughages (hay, protein grains, mineral cake) and water ad
libitum. The health was monitored by recording the body temperature (normal rectal temperature: 102.5e103  F) and rumen
movement by authorized veterinarian doctor. Proper prophylactic
measures were adopted in terms of vaccination against enterotoxemia, foot and mouth diseases, and peste des petits ruminant

(PPR). The goats were treated with antihelmintics twice per year
and 0.5% solution of malathion (acaricidal baths) was sprayed
externally at 7e20 days as described by Chowdhury et al. (2002).
2.2. Experimental plan
The blood was collected from jugular vein to isolate PBMCs from
all the goats. The PBMCs were cultured in four groups, i.e. 1) control,
2) melatonin (Mel), 3) Dexamethasone (Dex), and 4) Dexamethasone (Dex) along with melatonin (Mel). The dexamethasone, a
synthetic glucocorticoid was used to mimic the glucocorticoid
induced immunosuppression under in vitro condition. Immunosuppressive dose of dexamethasone (2 mM) and an immunoproliferative dose of melatonin (500 pg/ml) were used for in vitro
treatment to PBMCs. To check the proliferative response and
apoptotic markers cells were incubated for 48 h, for GR nuclear
translocation experiments cells were treated for 4 h at 37  C in CO2
incubator. The proliferative response of PBMCs, IL-2 secretion,
expression of GR, HSP90, Nrf2, HO-1 and apoptotic marker proteins
(Bcl-2, Bax, Caspase-3) were checked after the treatments.(Schematic representation).

2.3. Blood collection


The blood samples were collected from the jugular vein in preheparinized tube from all the goats (n 7) by applying a minimal
stress after 2 h of sunset (at 20:00 h; IST) under dim red light (less
than 1 lx). The blood samples were processed for isolation of peripheral blood mononuclear cells.
2.3.1. Isolation of peripheral blood mononuclear cells
The PBMCs were isolated by density gradient centrifugation
(Boyum, 1968) and published elsewhere Singh et al. (2013).
Lymphocyte separation media was used according to manufacturer's instruction (HiSep LSM 1084, HiMedia, Mumbai, India).
Briey, the white band at the plasma-coll (Hisep, Himedia)
interphase was collected and washed twice with PBS and nally
suspended in complete media RPMI 1640 (Himedia, India) supplemented with 10% fetal bovine serum (FBS) and 100 units of
penicillin and streptomycin (Sigma Aldrich, USA).
2.3.2. MTT assay peripheral blood mononuclear cells (PBMCs)
proliferation
Cell-mediated immune function was assessed by measuring
PBMCs proliferation in response to T-cell specic mitogen,
Concanavalin-A (Con-A) by using a colorimetric assay based on the
reduction of tetrazolium salt (3-(4,5-Dimethylthiazol-2-yl)-2,5-

A.K. Singh, C. Haldar / Molecular and Cellular Endocrinology 436 (2016) 59e67

diphenyltetrazolium bromide (MTT) (Mosmann, 1983). The cell


counting was done by haemocytometer and viability was determined by trypan blue exclusion method. Viable cells (95%) were resuspended in complete RPMI1640 medium, and 1  106 cells/ml
were adjusted per well in at-bottom 24 well culture plate.
Concanavalin-A (Con A, Sigma-Aldrich, St. Louis, USA) was added to
the culture medium at the concentration of 5 mg/ml. Plates were
incubated at 37  C with 5% CO2 for 48 h. Four hours prior to the
completion of 48 h 100 ml of MTT (SRL, Bombay, India; 5 mg/ml in
complete media RPMI-1640) was added to each well. At the end of
48 h, 1 ml of acidied propanol (0.04 mol/L HCl in isopropanol) was
added to each well. The optical density (OD) of each well was
determined with a microplate reader (ELx-800, Biotek Instruments,
Winooski VT, USA) equipped with a 570 nm wavelength lter. Mean
OD values for each set of triplicate were used in subsequent statistical analysis. Response was calculated as percent stimulation
ratio (% SR) representing the ratio of absorbance of mitogen stimulated cultures to control cultures.

61

skimmed milk and 0.1% Tween-20 followed by horseradish


peroxidase conjugated secondary antibody (anti-rabbit goat IgG,
for GR, HSP90, Nrf2 HO-1, Bax, Bcl-2 Caspase-3 and lamin B1
diluted 1:500 and donkey anti-mouse IgG for b-actin, diluted
1:10,000), which were further detected using Super Signal West
Pico Chemiluminescent Substrate (P-34080, Thermo Scientic,
Rockford, USA). Bands were quantied by measuring of optical
density using the Scion Image Analysis Software (Scion Corporation, MD, USA). The values were expressed as the ratio of density of
the specic signal to b-actin signal and lamin B1 for nuclear
proteins.

2.4.2. Coimmunoprecipitation
The PBMCs (2  106) were incubated with and without melatonin in presence or absence of dexamethaonsone at 37  C in CO2
incubator. After 4 h of the hormonal treatment, cells were centrifuged at 1600 rpm for 3 mins and washed twice with cold PBS. The
coimmunoprecipitation was performed with commercially available Co-IP kit (Cat. No. 26,149; Thermon scientic, USA) following
Optical density of Challenged Con A manufacturer's protocol. Briey, Cells were lysed by adding lysis
% Stimulation ratio %SR
Optical density of Basal
buffer to the pellet and incubated for 5 min and then centrifuged at
 100
13000 g for 10 min at 4  C to collect supernatant and quantication of protein was done by Bradford's method (Bradford, 1976).
Four microliters of anti-GR-antibody was immobilized to the resin
in column. 1 mg protein was added to the column and incubated
with gentle mixing or rocking for overnight at 4  C. At the end,
2.3.3. Estimation of Interleukin-2 in culture supernatants
elution was performed by adding elution buffer, incubating for
The cultured cells were centrifuged to collect medium at
10 min at room temperature. After elution the samples, the ex1600 rpm for 15 min. The quantication of IL-2 secretion was
pressions were checked by western blotting.
estimated with sandwich ELISA following manufacturer's protocol
(Immunotech, Marseille Cedex, France) published elsewhere
2.5. Apoptotic index of PBMCs
(Kaushalendra, 2012). Intra-assay variation was between 3.3% and
7.2%, interassay variation was between 6.2% and 8.2%, sensitivity
Apoptotic cells were analyzed microscopically following Acriwas 5 pg/ml, and recovery was between 80% and 132%.
dine OrangeeEthidium Bromide (AOeEtBr) double staining
method as described elsewhere (Sharma and Haldar, 2009).
2.4. Western blot analyses
AOeEtBr dye of volume 0.01 ml (1) was admixed gently with
0.2 ml of the diluted sample (1  106 cells/ml in PBS). A drop of this
2.4.1. Expression of GR, HSP90, Nrf2, HO-1, Bax, Bcl-2 and Caspasemixture was placed underneath cover-slip on a clean slide and cells
3
were observed immediately under uorescence microscope (Leitz
For the expression pattern of receptor and proteins, cultured
MPV3, Wetzlar, Hesse, Germany) at 440e520 nm. A minimum of
PBMCs were lysed with lysis buffer and quantied by the Bradford's
200 cells was counted in every sample to observed cell death.
method (Bradford, 1976). Four hours after the treatment PBMCs
were isolated by centrifugation of the complete medium at
No: of apoptotic cells early late apoptotic
 100
1600 rpm for 15 min. The pellets were isolated was washed twice
Total no: of cells counted
with PBS (pH 7.4). The pellets were homogenized with buffer
containing 10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl2, 1 mM
DTT, 0.1 mM EDTA, and 0.1 mM phenylmethylsulfonyluoride
3. Statistical analyses
(PMSF) and incubated on ice for 15 min then centrifuged at 14000g
for 15 min for cytoplasmic extract. Further for nuclear proteins 10%
All statistical analyses were performed using SPSS version 17.0.
Nonidet P-40 (NP-40) solution was added and incubated for
The data were analyzed by performing one-way ANOVA followed
15e30 min and then centrifuged at 14000g for 15 min. Further,
by multiple comparisons by Tukey's HSD (Honest Signicant Difaliquots containing 50 mg protein were resolve on 10% SDS-PAGE gel
ference) multiple range tests. The values are expressed as
for GR, HSP90, and 12% SDS-PAGE for Nrf2, HO-1, Bax, Bcl-2 and
mean SE. A P-value of 0.05 was considered statistically
Caspase-3 respectively, followed by electro-transfer to nitrocellusignicant.
lose membrane (Bioscience, Keene NH, USA). Immunodetection
was carried out by using Glucocorticoid receptor (GR (H-300): sc8992, Santa Cruz Biotech, USA, diluted 1:250), HSP90 (#4874; cell
signaling, 1:500), Nrf2 (ab31163, Abcam 1:500), HO-1 (Thermo,
USA, 1:500), Caspase-3 (Caspase-3; H-277; sc-7148, Santa Cruz
Biotech, USA, diluted 1:200), Bax (N-20; sc-493, Santa Cruz Biotech,
USA, diluted 1:250) Bcl-2 (N-19; sc-492 Santa Cruz Biotech, USA,
1:200), b-actin antibody (A-2228, Sigma-Aldrich Chemicals, St.
Louis, USA, diluted 1:1000) and Lamin B1 antibody (ab-97775,
Abcam 1:500). All antibodies were diluted in phosphate buffer
saline (PBS; 0.1 M NaH2PO4, Na2HPO4, NaCl; pH 7.4) containing 5%

4. Results
4.1. Proliferative response of PBMCs
The proliferative response of PBMCs in terms of %SR was noted
towards a T-cell specic mitogen Concanavalin-A. % SR signicantly
(p < 0.01) increased upon melatonin treatment while a signicant
(p < 0.01) decline in %SR was noted upon dexamethasone treatment when compared to the control. Co-treatment of melatonin

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A.K. Singh, C. Haldar / Molecular and Cellular Endocrinology 436 (2016) 59e67

Fig. 1. Effect of in vitro melatonin and dexamethasone supplementation on (a) Blastogenic response (%SR) of peripheral blood mononuclear cells (PBMCs) stimulated with Con A and
(b) IL-2 concentration in the culture supernatant of PBMCs after in vitro supplementation with Mel, Dex, and Dex Mel. Values are expressed as mean, vertical bar on histograms
represents SEM, n 7. Signicance of difference; *p < 0.01, Con vs. all experimental groups and #p < 0.01, Dex vs. Dex Mel.

with dexamethasone signicantly (p < 0.01) increased %SR when


compared with dexamethasone treatment (Fig. 1a).
4.2. Enzyme linked immunosorbant assay of interleukin -2
A signicant (p < 0.01) increase IL-2 level was noted upon
melatonin treatment while dexamethasone treatment signicantly
(p < 0.01) reduced IL-2 level compared to control. Co-treatment of
melatonin with dexamethasone signicantly (p < 0.01) increased
IL-2 level, when compared to dexamethasone treatment alone
(Fig. 1b).
4.3. Expression of GR: complete, cytoplasmic and nuclear
A signicant (p < 0.01) declined level of GR was noted upon
melatonin treatment and dexamethasone treatment signicantly
(p < 0.01) increased GR expression compared to control. Cotreatment of melatonin with dexamethasone signicantly
(p < 0.01) increased GR expression compared to dexamethasone
groups alone (Fig. 2a). GR expression was signicantly (p < 0.01)
increased in cytoplasmic fraction whereas declined signicantly
(p < 0.01) in nuclear fraction upon melatonin treatment when
compared with control. Dexamethasone treatment signicantly
(p < 0.01) increased GR expression in nuclear compartment
compared to control while, combined treatment of melatonin and
dexamethasone signicantly (p < 0.01) reduces presence of nuclear
GR when compared to dexamethasone treatment (Fig. 2b, c).

4.6. Nrf2 and heme oxygenase 1 (HO-1) expression


A signicantly (p < 0.01) increased Nrf2 and HO-1 expression
was noted upon melatonin treatment while dexamethasone
treatment signicant (p < 0.01) declined Nrf2 and HO-1 expression
compared to control. The co-supplementation of melatonin and
dexamethasone signicantly (p < 0.01) increased Nrf2 and HO-1
expression when compared to dexamethasone treatment alone
(Fig. 4a, b).
4.7. Apoptotic markers
4.7.1. Bcl-2/Bax ratio
A signicantly (p < 0.01) increased level of Bcl-2/Bax ratio was
noted upon melatonin treatment and dexamethasone treatment
signicantly (p < 0.01) declined this ratio compare to control.
Melatonin co-treatment with dexamethasone further signicantly
(p < 0.01) improved the Bcl-2/Bax ratio in comparison to dexamethasone treatment (Fig. 5a).
4.7.2. Cleaved caspase -3 expression
Melatonin treatment signicantly (p < 0.01) decreased cleaved
caspase-3 expression, whereas dexamethasone treatment signicantly (p < 0.01) increases cleaved caspase-3 expression compared
to control. Co-treatment of melatonin and dexamethasone signicantly (p < 0.01) declined cleaved caspase-3 expression compared
to dexamethasone treatment (Fig. 5b).
4.8. Apoptotic index

4.4. Coimmunoprecipitation
The melatonin treatment inhibited the dissociation of GR-HSP90
complex and a high expression of GR and HSP90 was noted in
melatonin treated group compared to control and dexamethasone
treatment (Fig. 3a).

A signicantly (p < 0.01) increased apoptotic index (%) in PBMCs


was noted in Dexamethasone-treated group when compared with
control and a signicant (p < 0.01) decrease apoptotic index was
observed in PBMCs upon melatonin treatment. Co-treatment of
dexamethasone with melatonin signicantly (p < 0.01) declined
apoptotic index when compared with dexamethasone treated
group alone (Fig. 6a, b).

4.5. Heat shock protein 90 (HSP90)


5. Discussion
A signicantly (p < 0.01) increased HSP90 expression was noted
upon melatonin treatment while a signicantly (p < 0.01) declined
expression of HSP90 was observed in nuclear fraction upon dexamethasone treatment when compared to control. The melatonin
and dexamethasone combined treatment signicantly (p < 0.01)
increased nuclear HSP90 when compared to dexamethasone
treatment (Fig. 3b).

The present study demonstrates that melatonin retards GR


nuclear translocation by inhibiting dissociation of HSP90 from GRHSP90 complex and increases Nrf2/HO-1 dependent antioxidant
response and down-regulates apoptosis by increasing Bcl-2
expression under glucocorticoid induced stress condition in
PBMCs of goat Capra hircus. Glucocorticoids are multitasking

A.K. Singh, C. Haldar / Molecular and Cellular Endocrinology 436 (2016) 59e67

63

Fig. 2. Western blot analysis showing the effect of in vitro melatonin and dexamethasone supplementation on expression of (a) complete GR and differential GR expression (b) in
cytoplasmic fraction and (c) nuclear fraction of PBMCs. The data are expressed as % band intensity of GR expression in PBMCs. Left side of the panel shows western blots and right
side shows the representative histograms. Values are expressed as mean, vertical bar on histograms represents SEM, n 7. Signicance of difference; *p < 0.01, Con vs. all
experimental groups and #p < 0.01, Dex vs. Dex Mel.

Fig. 3. Co-IP analysis of (a) GR-HSP90 interaction in PBMCs following Mel, Dex, and Dex Mel treatment. Western blot analysis showing the effect of in vitro melatonin and
dexamethasone supplementation on the (b) expression of nuclear HSP90 following Mel, Dex, and Dex Mel treatment. Values are expressed as mean, vertical bar on histograms
represents SEM, n 7. Signicance of difference; *p < 0.01, Con vs. all experimental groups and #p < 0.01, Dex vs. Dex Mel.

molecules (Julia, 2006) that modulate various physiological functions including immune system (Saplosky et al., 2000). Glucocorticoid exhibits biological actions via its nuclear receptors;
glucocorticoid receptor (GR) which is a low afnity receptor
(Saplosky et al., 2000; De Kloet et al., 1998). Glucocorticoids are
clinically relevant and widely used as anti-inammatory and
immunosuppressive agent for numerous conditions associated
with the host defense (Fauci and Dale, 1974). Dexamethasone, a
synthetic glucocorticoid, is used to treat various clinical conditions
like chronic asthma (Barnes, 1995), rheumatoid arthritis (Buttgereit

et al., 2004), and in tissue/organ transplantation (Scherer et al.,


2007). In the present study, we used dexamethasone to demonstrate GR mediated inhibition of Nrf2 regulated HO-1 and Bcl-2
expression in immune cells under in vitro condition and cosupplemented melatonin with dexamethasone to explore its effect in regulation of cellular antioxidant response and regulation of
apoptosis in PBMCs.
The dexamethasone treatment reduced the proliferative responses of PBMCs towards mitogenic challenge and melatonin
treatment signicantly improved the proliferative responses alone

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A.K. Singh, C. Haldar / Molecular and Cellular Endocrinology 436 (2016) 59e67

Fig. 4. Western blot analysis showing the effect of in vitro melatonin and dexamethasone supplementation on the expression of (a) Nrf2 (b) HO-1 in PBMCs following Mel, Dex, and
Dex Mel treatment. b-actin expression was used as loading control. Left side of the panel shows western blots and right side shows the representative histograms. Values are
expressed as mean, vertical bar on histograms represents SEM, n 7. Signicance of difference; *p < 0.01, Con vs. all experimental groups and #p < 0.01, Dex vs. Dex Mel.

Fig. 5. Western blot analysis showing the effect of in vitro melatonin and dexamethasone supplementation on the expression of Bax, Bcl-2 and Caspase-3 in PBMCs. (a) Histogram
representing % Bcl-2/Bax ratio and (b) Histogram showing % band intensity of active Caspase-3. b-actin expression was used as loading control. Values are expressed as mean,
vertical bar on histograms represents SEM, n 7. Signicance of difference; *p < 0.01, Con vs. all experimental groups and #p < 0.01, Dex vs. Dex Mel.

and also in combination with dexamethasone. The declined proliferative response upon dexamethasone treatment could be due its
inhibitory effect on IL-2 secretion from immune cells. It has previously been reported that glucocorticoid treatment inhibits the
proliferation of immunocompetent cells (Ashwell et al., 2000) by
inhibiting IL-2 transcription and also affecting IL-2 mRNA stability
(McKay and Cidlowski, 1999). It compromises the activation of T
cells by down-regulating IL-2 R expression (Northrop et al., 1992).
On the contrary, melatonin due to its immunomodulatory property
(Conti et al., 2000) increases production of IL-2 from human PBMCs
at physiological dose (Garcia-Mauriiio et al., 1997). Furthermore,
melatonin avoids stress-induced immunosuppression by

increasing IL-2 secretion from T-helper cells (Lardone et al., 2009)


that usually serves to induce autocrine proliferation response
(Carrillo-Vico et al., 2004). It was also noted that melatonin treatment increased IL-2 secretion from caprine PBMCs and increased
the proliferative responses of these cells. The increased IL-2
secretion from immune cells like lymphocytes upon melatonin
treatment possibly helping cells to survive under glucocorticoid
induced stress condition. Melatonin treatment reduces glucocorticoids toxicity to improve hippocampal cell proliferation (Quiros
et al., 2008). A declined GR expression was noted upon melatonin
treatment in caprine PBMCs. It has been reported that melatonin
treatment reduces GR expression in rodent to modulate the

A.K. Singh, C. Haldar / Molecular and Cellular Endocrinology 436 (2016) 59e67

65

Fig. 6. (a): Morphological changes observed in germ cells from (A) control, (B) Melatonin, (C) Dex and (D) Dex Melatonin-treated PBMCs after staining with acridine orangeeethidium bromide (AOeEtBr) dye under uorescence microscope. Arrow indicates normal, dollar ($) indicates early apoptotic cells, # indicates late apoptotic cells and dead cells.
(b): Effect of in vitro melatonin and dexamethasone supplementation on apoptotic index of PBMCs of goat Capra hircus. Values are expressed as mean SEM, n 7. Signicance of
difference: *p < 0.01, Con vs. all experimental groups and #p < 0.01, Dex vs. Mel Dex. (For interpretation of the references to colour in this gure legend, the reader is referred to
the web version of this article.)

immune responsiveness of PBMCs (Gupta and Haldar, 2013).


Further, we checked the sub-cellular distribution of GR following
melatonin treatment and found that melatonin increased cytoplasmic retention of GR compared to nuclear fraction as evident
upon melatonin treatment in caprine PBMCs. Melatonin treatment
retarded GR nuclear translocation by inhibiting the dissociation of
HSP90 from GR-HSP90 complex as evident from our result. The

melatonin treatment inhibits thymic apoptosis by inhibiting GR


nuclear translocation (Quiros et al., 2008; Presman et al., 2006;
Sainz et al., 1999). It has been reported that GC binding to GR
sensitizes GR for its nuclear translocation, which is regulated by
HSP90 based machinery. The activity of GR has been suggested to
be modulated by the level of HSP90 (Presman et al., 2010). It has
been suggested that an increased HSP90/GR ratio inuences GR

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A.K. Singh, C. Haldar / Molecular and Cellular Endocrinology 436 (2016) 59e67

responsiveness towards its ligand and excess of HSP90 inhibits GR


binding to its DNA response element (Kang et al., 1999) which was
observed in our result that melatonin treatment increased HSP90 in
nuclear fraction compare to dexamethasone treatment in PBMCs.
The increased HSP90 might have attenuated GR mediated actions
as any alteration in HSP90/GR ratio and increased nuclear HSP90
has been suggested to attenuate activity of GR activity (Ouyang
et al., 2012). Apart from HSP90 other factors like immunophilins
FKBP52, FKBP51 and cyclophilin 40 also play a crucial role in
maturation and nuclear translocation of GR and stress condition
also inuences their binding to HSP90 in GR-HSP90 complex (Grad
and Picard, 2007). However, these factors have not been considered
in the present study.
The cytoplasmic retention of GR upon melatonin treatment
increased the Nrf2 mediated antioxidant response in PBMCs. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription
factor that regulates a series of genes known as antioxidant
response elements (AREs). ARE genes expresses cytoprotective and
detoxifying enzymes such as heme oxygenase-1 (HO-1). The HO-1
enzyme is responsible for the neutralizing of reactive oxygen species (ROS) to protect against oxidative stress (Carmona-Aparicio
et al., 2015). Dexamethasone treatment declined Nrf2 and HO-1
expression in PBMCs which is in line with the earlier ndings of
Ki et al. (2005), which suggest that ligand dependent GR activation
represses Nrf2-ARE gene expression. Furthermore, Kratschmar
et al. (2012), reported that activation of GR through glucocorticoids leads to suppression of Nrf2 dependent antioxidant response.
Melatonin modulates the antioxidant response by inuencing
antioxidative enzymes at transcription level (Mayo et al., 2002) by
activating Nrf2 (Jung et al., 2010) along with antioxidant enzyme
heme oxygenase-1 (HO-1) (Schmidt et al., 2004). Under oxidative
stress condition HO-1 is regulated by transcription factor Nrf2.
However HO-1 expression is also regulated by Nrf2 independent
pathways like FoxO1, activating protein-1 (AP-1) and p38 MAPK etc.
(Paine et al., 2010) and FoxO1 stimulates HO-1 promoter activity in
a dose dependent manner (Kang et al., 2014).
An increased Bcl-2/Bax ratio along with declined activation of
caspase-3 was noted upon melatonin treatment compared to
dexamethasone treatment in PBMCs. It has been reported that
dexamethasone treatment induces Bax expression in hamster
(Kaushalendra, 2012) and contrary melatonin treatment maintains
the balance between Bcl-2 and Bax that determines the cellular fate
(Singh and Haldar, 2014). Melatonin elicits anti-apoptotic effect by
down-regulation of Bax (Radogna et al., 2008) and/or the upregulating of Bcl-2 (Espino et al., 2010). Melatonin induced Nrf2
expression increased on Bcl-2 expression and decreased Bax
expression that led to increased Bcl-2/Bax. This increased Bcl-2/Bax
ratio might have rescued the cells from apoptosis which was
evident from apoptotic index observed following AO-EtBr stating in
PBMCs. Nrf2 ARE pathway has been suggested to be involved in
regulation of apoptosis (Pan et al., 2013). Nrf2-induces antiapoptotic Bcl-xL/Bcl-2 via binding to the promoter region of BclxL protein (Niture and Jaiswal, 2012) and inhibits apoptosis by
elevating Bcl-2/Bax ratio by increasing Bcl-2 expression (Pan et al.,
2013). Ji et al. (2013), suggested that knockdown of Nrf2 leads
declined/inhibited cellular proliferation.
In summary, melatonin treatment induced IL-2 secretion to
potentiate the proliferative capacity of the PBMCs under GR
mediated immunosuppressive condition and modulated antioxidant response and apoptosis in PBMCs by antagonistic effect on GR
nuclear translocation. The inhibition of GR nuclear translocation
upon melatonin treatment increased the Nrf2/HO-1 mediated
antioxidant response and anti-apoptotic Bcl-2 led to increased Bcl2/Bax ratio in PBMCs that rescued the cells from apoptosis.

6. Conclusion
It might be suggested that use of melatonin with synthetic
glucocorticoid (like dexamethasone) under clinical condition as
well as in regulation of GR mediated stress condition could be
benecial in reducing the side effects of glucocorticoids and GR
mediated immunosuppression, declined cellular antioxidant
response and apoptosis.
Acknowledgement
Authors thank to Department of Biotechnology (Grant No. BT/
PR14505/AAQ/01/445/2010) and UGC-CAS (R/Dev/CAS-JRF/Zool/
03848) for nancial support to Mr. Amaresh Kumar Singh and Instrument gift from Alexandar Von Humboldt foundation, Bonn,
Germany is gratefully acknowledged. Authors wish to thank Dr.
Sameer Gupta, UGC-Research fellow, for various help during
manuscript preparation.
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