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The Journal of Clinical Endocrinology & Metabolism 89(7):3449 –3454
Copyright © 2004 by The Endocrine Society
doi: 10.1210/jc.2003-031441
Dehydroepiandrosterone-Sulfate Inhibits Nuclear
Factor-␬B-Dependent Transcription in Hepatocytes,
Possibly through Antioxidant Effect
YASUMASA IWASAKI, MASATO ASAI, MASANORI YOSHIDA, TAKESHI NIGAWARA,
MACHIKO KAMBAYASHI, AND NOBUO NAKASHIMA
Departments of Clinical Pathophysiology (Y.I., T.N., M.K., N.N.) and Medicine (M.A., M.Y.), Nagoya University Graduate
School of Medicine and Hospital, Nagoya 466-8550, Japan
Dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEAS),
the representative sex steroid precursors, are postulated to
have antiinflammatory effects, although the molecular background remains unknown. In this study, we examined the
effects of these sex steroid precursors on cytokine-induced,
nuclear factor-␬B (NF-␬B)-mediated transcription. The HuH7
human hepatocyte cell line was stably transfected with an
NF-␬B-luciferase reporter gene or transiently transfected
with other representative response elements-luciferase fusion genes, and the effects of DHEA/DHEAS on proinflammatory cytokine-induced transcription were estimated by luciferase assay. The results showed that DHEA/DHEAS potently
inhibited TNF-␣-induced NF-␬B-dependent transcription in a
time- and dose-dependent manner. The effect was more obvious for DHEAS than for DHEA, and both steroids preferentially inhibited the cytokine-stimulated rather than basal NF␬B-mediated transcription. Similar effects were observed in
D
activator protein-1-dependent but not constitutive Rous sarcoma virus promoter-dependent transcription. Two major
downstream products of the sex steroid precursors, estradiol
and testosterone, had no effect, indicating that the observed
suppressive effect is not mediated by these metabolites. In
contrast, glucocorticoids showed inhibitory effects on both
basal and stimulated transcription and had an additive effect
with DHEAS, suggesting the independent mechanisms of action of these steroid hormones. Finally, DHEAS eliminated
hydroxyradical-induced activation of NF-␬B-dependent transcription as well. Altogether, these results suggest that DHEA/
DHEAS have an antiinflammatory effect in such a way that
they inhibit proinflammatory cytokine-stimulated, NF-␬Bmediated transcription, at least partly through their antioxidant properties. (J Clin Endocrinol Metab 89: 3449 –3454,
2004)
EHYDROEPIANDROSTERONE (DHEA) AND DHEAsulfate (DHEAS) are sex steroid precursors synthesized
in the zona reticularis of the adrenal gland in humans (1, 2). Both
steroids circulate in the peripheral blood at relatively high concentrations, i.e. approximately 10 –30 nm for DHEA and approximately 5–10 ␮m for DHEAS, the latter being the highest of
all endogenous steroid hormone levels. A percentage of each
steroid is subsequently transformed into biologically active androgens and estrogens in peripheral tissues (3), with DHEAS
usually recognized as a reservoir of DHEA.
Besides being the precursor of sex steroids in postmenopausal women, DHEA/DHEAS have long been postulated
to be multifunctional hormones with antiaging, antiinflammatory, immunomodulatory, antiatherogenic, anticancer,
and neurotropic effects (1–5). Nevertheless, the molecular
mechanisms of the effects of DHEA/DHEAS are still obscure, although a putative membrane receptor for DHEA/
DHEAS has recently been identified (6). Recent reports may
provide some clues, indicating that chronic DHEA treatment inhibits the function of a transcription factor, nuclear
factor-␬B (NF-␬B), in neuronal cells in vivo (7).
In this study, we examined the acute effects of DHEA/
DHEAS on NF-␬B-mediated gene expression in vitro using
the human hepatoma cell line HuH7. It is known that proinflammatory cytokines such as TNF-␣ or IL-1␤ have stimulatory effects on hepatocytes to produce acute phase reactants through an NF-␬B-dependent mechanism (8 –11). Here
we find that DHEA/DHEAS exert potent inhibitory effects
on the cytokine-induced activation of NF-␬B-dependent
transcription. Furthermore, our data suggest that these effects of DHEA/DHEAS are mediated, at least partly, by
eliminating the oxidative stress produced during the action
of cytokines.
Abbreviations: AP1, Activator protein-1; DHEA, dehydroepiandrosterone; DHEAS, DHEA-sulfate; FBS, fetal bovine serum; NF-␬B, nuclear
factor-␬B; ROS, reactive oxygen species.
JCEM is published monthly by The Endocrine Society (http://www.
endo-society.org), the foremost professional society serving the endocrine community.
Cell culture and transfection
Materials and Methods
Materials
DHEA, DHEAS, and hydrogen peroxide (H2O2) were obtained from
Wako Pure Chemicals (Osaka, Japan). TNF-␣ and IL-1␤ were purchased
from PeproTech (London, UK). A reporter plasmid vector pNF-␬B-Luc,
which contains five copies of the NF-␬B-binding motif, and other reporter plasmids, were obtained from Stratagene (La Jolla, CA). NF-␬B
p65 (Rel A) expression vector was a generous gift from Dr. Alcami
(Instituto de Salud Carlos III, Madrid, Spain).
HuH7 human hepatoma cells were maintained with DMEM supplemented with 10% fetal bovine serum (FBS) under a 5% CO2/95% air
atmosphere at 37 C. For transient transfection experiments, HuH7 cells,
plated in 24-well plates, were transfected with the test plasmids and
FuGene 6 transfection reagents (Roche Applied Science, Indianapolis,
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Iwasaki et al. • DHEAS Inhibits NF-␬B-Dependent Transcription
IN). Forty-eight hours after the start of transfection, the culture medium
was replaced with new medium containing 1% FBS, and the cells were
then treated with the test substances for a defined time period.
For stable transfection experiments, a mixed polyclonal HuH7 cell
line incorporated with NF-␬B-luciferase expression vector, designated
as HuH7NF, was established with TransIT LT-1 (PanVera, Madison,
WI). In each experiment, the HuH7NF cells were plated in 24-well plates
with medium containing 1% FBS, and the test substances (TNF-␣,
DHEA, DHEAS, dexamethasone in 1000⫻ concentration) were added
into the culture medium 24 – 48 h after plating of the cells. Finally, the
cells were harvested and applied for the luciferase assay (see Assays).
When H2O2 was used, cells were treated for 24 h with vehicle or
H2O2 (50 ␮m) and simultaneously with increasing doses of DHEAS
(0.1–100 ␮m).
Assays
For estimating DNA binding of NF-␬B, EMSA was carried out using
a LightShift Chemiluminescent EMSA kit (Pierce Biotechnology, Rockford, IL), following the manufacturer’s instructions. Briefly, the extracted nuclear protein of HuH7 cells (0.57 ␮g) was incubated with
double-stranded, 3⬘end-biotinylated oligonucleotides (50 fmol) encompassing the consensus NF-␬B binding sequence (sense, 5⬘-AGTTGAGGGGACTTTCCCAGGC-3⬘ biotin, antisense, 5⬘-GCCTGGGAAAGTCCCCTCAACT-3⬘ biotin) for 20 min, and the mixture was
subjected to 4% nondenaturing polyacrylamide gel electrophoresis. After electrophoresis at 160 V for 4 h, the biotinylated DNA was transferred
to nylon membranes by capillary transfer and cross-linked, and then the
biotin-labeled DNA was detected with digital imaging apparatus (LightCapture; Atto Bioscience & Technology, Tokyo, Japan).
Luciferase assay was performed as previously described (12), and
light output was measured for 20 sec at room temperature by a
luminometer (Lumat LB9501; Berthold Technologies, Bad Wildbad,
Germany).
Data analyses
Samples in each group of the experiments were in triplicate or quadruplicate. All data are expressed as mean ⫾ se. When statistical analyses were performed, data were compared by one-way ANOVA with
Fisher’s protected least significant difference test, and P ⬍ 0.05 vs. vehicle
alone or TNF-␣-stimulated was considered significant.
Results
The effects of DHEA/DHEAS on proinflammatory cytokinestimulated NF-␬B-dependent transcription
We first found that IL-1␤ and TNF-␣ potently stimulate
NF-␬B-dependent transcription in HuH7NF hepatic cells in
a dose- and time-dependent manner (Fig. 1). The dose-response studies exhibited the maximal effects of IL-1␤ and
TNF-␣ being obtained at 10 and 100 pm, respectively. Furthermore, the time-course studies showed that the effects of
IL-1␤ (100 pm) and TNF-␣ (100 pm) reached a plateau at 12
and 24 h, respectively.
We next studied the effects of DHEA/DHEAS on the NF␬B-mediated gene expression. The results revealed that
DHEA/DHEAS exert inhibitory effects on both basal and
TNF-␣-stimulated transcription (Fig. 2, A and B); the timecourse studies showed the maximal suppressive effects to be
obtained at 24 h in both basal and TNF-␣-stimulated groups,
and the dose-response studies showed more remarkable inhibitory effects of both sex steroid precursors on cytokinestimulated rather than basal NF-␬B transcriptional activity.
In this condition the effect was more obvious in the DHEAStreated than in the DHEA-treated group. EMSA analysis also
showed that the band corresponding to NF-␬B binding was
significantly less dense in the TNF-␣ (1 pm)/DHEAS (100
FIG. 1. Dose- and time-dependent effects of IL-1␤ and TNF-␣ on
NF-␬B-dependent transcription in HuH7NF hepatocytes. In a doseresponse study, cells were treated without (control, C) or with either
IL-1␤ (1–100 pM) or TNF-␣ (1–1000 pM) for 24 h (upper panels). In a
time-course study, cells were treated with either IL-1␤ (100 pM) or
TNF-␣ (100 pM) for up to 24 h (IL-1␤) or 36 h (TNF-␣). ⴱ, P ⬍ 0.05 vs.
control or the value at time zero; N.S., not significant.
␮m)-treated group compared with the only TNF-␣-treated
one (Fig. 3). Altogether, these data support the concept that
DHEAS/DHEAS have a negative effect on NF-␬B-mediated
transcription.
The above effects of DHEA/DHEAS were promoter specific and were not due to the general toxic/suppressive effect
of the hormones, because the same concentration of DHEAS
did not influence Rous sarcoma virus promoter-mediated
transcriptional activity (data not shown). Furthermore, estradiol or dihydrotestosterone did not mimic the effect (data
not shown), suggesting that the effects of DHEA/DHEAS are
not mediated by the two major downstream metabolites.
The effects of dexamethasone and DHEAS on basal or
TNF-␣-stimulated NF-␬B-dependent transcription
Because glucocorticoid hormones are known to inhibit
NF-␬B, we compared the effects of DHEAS and the synthetic
glucocorticoid dexamethasone on NF-␬B-dependent transcription in our experimental system. Dexamethasone (100
nm) potently inhibited basal as well as TNF-␣ (100 pm)stimulated transcription, in contrast to the above findings
that DHEAS preferentially inhibited TNF-␣-stimulated transcription (Fig. 4A). Furthermore, when dexamethasone (100
nm) and DHEAS (10 ␮m) were simultaneously used, a significant additive effect was observed (Fig. 4B). These results
Iwasaki et al. • DHEAS Inhibits NF-␬B-Dependent Transcription
J Clin Endocrinol Metab, July 2004, 89(7):3449 –3454 3451
FIG. 2. The effects of DHEA/DHEAS
on proinflammatory cytokine-stimulated, NF-␬B-dependent transcription
in HuH7NF hepatocytes. A, Timecourse effects of DHEA/DHEAS on
basal or TNF-␣-stimulated NF-␬B-dependent transcription. Cells were
treated with DHEA alone (100 nM) or
TNF-␣ (100 pM, 24 h) plus DHEA
(24 – 48 h) (upper panels). Similar experiments were performed with
DHEAS (10 ␮M; lower panels). ⴱ, P ⬍
0.05 vs. the value at time zero. B, Dosedependent effects of DHEA/DHEAS on
basal or TNF-␣-stimulated NF-␬B-dependent transcription in HuH7NF
cells. Cells were treated with DHEA
alone (1 nM to 10 ␮M) or TNF-␣ (100 pM)
plus DHEA (10 nM to 10 ␮M) for 24 h
(upper panels). Alternatively, cells were
treated with DHEAS alone (10 nM to
100 ␮M) or TNF-␣ (100 pM) plus DHEAS
(100 nM to 100 ␮M). ⴱ, P ⬍ 0.05 vs. control (C); ⫹, P ⬍ 0.05 vs. TNF-␣ alone;
N.S., not significant.
FIG. 3. The effect of DHEAS on the DNA binding of NF-␬B in
HuH7NF cells. Cells were treated with TNF-␣ alone or TNF-␣ plus
DHEAS for 24 h. The nuclear protein was extracted from the cells, and
then EMSA was performed (see Materials and Methods).
suggest that both glucocorticoid and DHEAS exert a negative
effect on the NF-␬B-dependent transcription, but through
different mechanism(s). More specifically, DHEAS appears
to exert a negative effect on NF-␬B not directly, but indirectly
through the cytokine-induced signaling pathway(s).
The molecular mechanisms of DHEAS on NF-␬Bdependent transcription
We then compared the effects of DHEAS on NF-␬Bdependent transcription enhanced either by TNF-␣ or
NF-␬B p65 overexpression. The results showed that, although DHEAS significantly inhibited TNF-␣ (100 pm)stimulated transcription, no suppression was observed
when NF-␬B-dependent transcription was stimulated
by p65 (Fig. 4C). These data again support the above
hypothesis that DHEAS exerts its negative effect on the
process of NF-␬B activation rather than on the NF-␬B
protein itself.
We tried to further clarify the molecular mechanism of
the DHEAS effects by using other representative transcription factor-mediated reporter genes. Among the constructs tested, DHEAS (10 ␮m) only inhibited activator
protein-1 (AP1; fos/jun)-mediated transcription (Fig. 4D).
These data not only confirm that the negative effect of
DHEAS is a promoter-specific event, but also raise the
possibility that DHEAS exerts its effect by antagonizing
the reactive oxygen species (ROS)-mediated transcription.
It is well recognized that NF-␬B- and/or AP1-dependent
transcriptions is radical-sensitive (13), and TNF-␣ is
known to exert its biological effect partly through generation of ROS (14).
Finally, to see whether DHEAS indeed inhibits ROS-mediated transcription, we examined the effect of the hormone
on hydroxyradical-stimulated NF-␬B-mediated transcription. Treatment of HuH7NF cells with H2O2 (50 ␮m) significantly stimulated NF-␬B-luciferase activity, whereas simultaneous treatment with DHEAS (0.1–100 ␮m) partly
eliminated the effect in a dose-dependent manner (Fig. 4E).
These data support the above hypothesis, suggesting that the
counteracting effect of DHEAS on NF-␬B-dependent transcription stimulated by TNF-␣ is partly brought about
through its antioxidant properties (Fig. 5).
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J Clin Endocrinol Metab, July 2004, 89(7):3449 –3454
Iwasaki et al. • DHEAS Inhibits NF-␬B-Dependent Transcription
FIG. 4. A, Effects of glucocorticoid on NF-␬B-mediated gene expression in HuH7NF cells. Cells were treated with dexamethasone (Dex; 100
nM, 12–24 h) alone or TNF-␣ (100 pM, 24 h) plus dexamethasone. ⴱ, P ⬍ 0.05 vs. the value at time zero. B, Combined effects of dexamethasone
and DHEAS. HuH7NF cells were treated with TNF-␣ (100 pM, 24 h), and also with vehicle, dexamethasone alone (100 nM), DHEAS alone (10
␮M), or both dexamethasone and DHEAS for 24 h. ⴱ, P ⬍ 0.05 vs. TNF-␣ alone. C, Differential effects of DHEAS on TNF-␣- or p65-stimulated
NF-␬B-dependent transcription. HuH7NF cells were treated with TNF-␣ (100 pM) alone or TNF-␣ plus DHEAS (10 ␮M) for 24 h (left).
Alternatively, HuH7 cells were transfected transiently with NF-␬B-luciferase and p65 expression vector (pCMV-p65), and then treated without
or with DHEAS (10 ␮M) for 24 h. ⴱ, P ⬍ 0.05 vs. TNF-␣ alone; N.S., not significant. D, Effects of DHEAS on the representative responsive
element-mediated transcription in HuH7 cells. Cells were transfected transiently with CRE-, AP1-, or GAS-luciferase reporter plasmids and
then treated with TNF-␣ (100 pM, 24 h) and either vehicle (C) or DHEAS (10 ␮M) for 24 h. ⴱ, P ⬍ 0.05 vs. TNF-␣ alone. CRE, cAMP-response
element; GAS, ␥-interferon-activating sequence. E, Effects of DHEAS on hydroxyradical-induced NF-␬B-dependent transcription in HuH7NF
cells. Cells were treated with vehicle (C), H2O2 (50 ␮M) alone, or H2O2 plus DHEAS (0.1–100 ␮M) for 24 h. ⴱ, P ⬍ 0.05 vs. control (C); ⫹, P ⬍
0.05 vs. H2O2 alone.
Discussion
The data in this study strongly suggest that DHEA/
DHEAS exert antiinflammatory effects by, at least partly,
inhibiting proinflammatory cytokine-induced NF-␬B activation in hepatocytes. This effect is more potent with DHEAS
than with DHEA and is thought to be caused by the direct
effect of the hormones rather than by their metabolites, such
as estradiol or testosterone. Although the molecular mechanism underlying the effect is not completely clear, DHEA/
DHEAS appear to eliminate the ROS-induced activation of
NF-␬B.
DHEA/DHEAS, well-known sex steroid precursors synthesized only in humans and a few higher primates, are
partly converted to estradiol or testosterone and act as sex
steroid hormones, especially in postmenopausal women (3).
Many lines of clinical evidence also prompt us to speculate
that the sex steroid precursors have antiinflammatory
and/or immunomodulatory effects (15, 16). Furthermore,
the physiological decline in the plasma concentrations of the
hormones antiparallels with the progression of the aging
process (17), indicating the presence of an antiaging effect as
well (18, 19). Nevertheless, the mechanisms whereby the
hormones produce a variety of effects are not completely
understood.
Our data provide a new concept showing that DHEA/
DHEAS inhibit NF-␬B-mediated transcription in the liver.
NF-␬B is a transcription factor composed of Rel family proteins such as p65 (Rel A), p50, or others and is known to play
a pivotal role in inflammation-induced gene expression (20).
Indeed, proinflammatory cytokines are known to induce the
expression of the acute phase reactant genes through NF-␬B
activation, with the resultant increase in a variety of proteins
including C-reactive protein, serum amyloid A, plasminogen
activator inhibitor-1, or coagulation factor VIII (8 –11). These
proteins may have some beneficial effects during acute inflammation, but chronically they cause deleterious effects
such as hypercoagulation, thrombosis, and/or atherosclerosis (21, 22). Furthermore, in obese patients, cytokines pro-
Iwasaki et al. • DHEAS Inhibits NF-␬B-Dependent Transcription
duced from adipocytes cause similar atherogenic events
through NF-␬B activation (23). In this context, the present
data suggest that DHEA/DHEAS exert an antiinflammatory
effect by interrupting the cascade of the cytokine-induced
inflammatory event in hepatocytes. In addition, because
NF-␬B is also involved in atherosclerotic events in the arterial
wall or in the progress of diabetic complication (24, 25),
DHEA/DHEAS may have a local antagonistic effect against
these disorders as well.
Interestingly, the inhibitory effects of DHEA/DHEAS on
NF-␬B-dependent transcription were much more remarkable when the transcription was stimulated by TNF-␣ or
IL-1␤, and little or no effect was observed on basal or p65
overexpression-stimulated transcription, in contrast to that
of glucocorticoids. In the latter case, the glucocorticoid receptor, when activated, is reported to interrupt NF-␬B function directly through protein-protein interaction (26), which
is confirmed by our data in that dexamethasone inhibits both
basal and stimulated NF-␬B-dependent transcription. Thus,
the differential mode of action between the two antiinflammatory hormones raises the possibility that DHEA/DHEAS
inhibit the pathway(s) through which cytokines activate NF␬B, rather than inhibit NF-␬B itself. In this sense, our current
data provide some clues, showing that DHEAS prevents
TNF-␣-induced AP1 activation and also inhibits H2O2-stimulated NF-␬B activation. Recent studies show that cellular
effects of cytokines are mediated, at least partly, through
generation of ROS (14), and both NF-␬B and AP1 are known
to be radical-sensitive transcription factors (27). Furthermore, DHEA/DHEAS are reported to have an inhibitory
effect on free radical generation (28 –31). Thus, it is tempting
to speculate that DHEA/DHEAS exert an antiinflammatory
action by eliminating free radical generation and subsequent
activation of NF-␬B-dependent transcription. In this case,
they may directly quench ROS (29), or they may induce
intrinsic antioxidants such as glutathione, thioredoxin, or
superoxide dismutase. It is also possible that DHEAS activates peroxisome gene expression through peroxisome proliferator-activated receptor ␣ and eliminates the intracellular
burden of O2 (32, 33). In addition, DHEA is reported to inhibit
the MAPK signaling pathway (34), possibly through activation of a protein tyrosine phosphatase (4). Because TNF-␣ is
known to activate NF-␬B partly through the MAPK pathway
(35), DHEA/DHEAS may exert their inhibitory effects by
antagonizing the activation of MAPK by the phosphatase. In
any event, further studies will clarify the precise mechanisms
underlying the variety of effects of DHEA/DHEAS at the
molecular level.
From a clinical standpoint, our data suggest that DHEA/
DHEAS have an antiatherogenic effect by interrupting the
link between the “inflammation-cytokine-atherosclerosis
axis” in the liver (Fig. 5). Atherosclerosis has recently been
recognized as an inflammatory event (36, 37), and oxidative
stress plays an important role in the aging process (13). The
decline in plasma DHEA/DHEAS thus promotes vascular
events through the increase in ROS and/or inflammation. In
this sense, the previously recognized diverse effects of
DHEA/DHEAS are likely to be explained by the common
antioxidant effect. Moreover, our data suggest that, in contrast to the effect of glucocorticoids, DHEA/DHEAS exert
J Clin Endocrinol Metab, July 2004, 89(7):3449 –3454 3453
FIG. 5. Possible molecular mechanisms of antiinflammatory effects
of DHEA, DHEAS, and glucocorticoid hormones. Proinflammatory
cytokines elicit genes encoding acute phase proteins and eventually
facilitate atherosclerosis. Glucocorticoid interrupts this axis through
direct inhibition of NF-␬B. In contrast, DHEA/DHEAS appear to
suppress this axis indirectly by eliminating the process of ROSmediated NF-␬B activation through its antioxidant property. CRP,
C-reactive protein; I␬B, inhibitory ␬B; GR, glucocorticoid receptor;
PAI-1, plasminogen activator inhibitor-1.
their effects mostly by eliminating cytokine-mediated deleterious effects, without an excessive immunosuppressive effect. Therefore, the replacement of DHEA/DHEAS may be a
preferential antiaging and antiinflammatory therapy for
long-term clinical use (1, 2, 18, 19).
Acknowledgments
We thank Ms. Tatsuyo Miura for her excellent technical assistance.
Received August 19, 2003. Accepted April 8, 2004.
Address all correspondence and requests for reprints to: Yasumasa
Iwasaki, M.D., Ph.D., Department of Endocrinology, Metabolism, and
Nephrology, Kochi University School of Medicine, Kohasu, Oko-cho,
Nankoku 783-8505, Japan. E-mail: [email protected].
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