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Dehydroepiandrosterone-Sulfate Inhibits Nuclear Factor-B-Dependent Transcription in Hepatocytes, Possibly through Antioxidant Effect

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

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: iwasaki{at}med.kochi-u.ac.jp.

	Abstract

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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 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-B-mediated transcription, at least partly through their antioxidant properties.

	Introduction

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DEHYDROEPIANDROSTERONE (DHEA) AND DHEA-sulfate (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 µM 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, 2, 3, 4, 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-1ß have stimulatory effects on hepatocytes to produce acute phase reactants through an NF-B-dependent mechanism (8, 9, 10, 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.

	Materials and Methods

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Materials

DHEA, DHEAS, and hydrogen peroxide (H₂O₂) were obtained from Wako Pure Chemicals (Osaka, Japan). TNF- and IL-1ß 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).

Cell culture and transfection

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, 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 1000x 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 µM) and simultaneously with increasing doses of DHEAS (0.1–100 µM).

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 µg) 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
The effects of DHEA/DHEAS on proinflammatory cytokine-stimulated NF-B-dependent transcription

We first found that IL-1ß 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-1ß and TNF- being obtained at 10 and 100 pM, respectively. Furthermore, the time-course studies showed that the effects of IL-1ß (100 pM) and TNF- (100 pM) reached a plateau at 12 and 24 h, respectively.

View larger version (38K): [in this window] [in a new window]   FIG. 1. Dose- and time-dependent effects of IL-1ß and TNF- on NF-B-dependent transcription in HuH7NF hepatocytes. In a dose-response study, cells were treated without (control, C) or with either IL-1ß (1–100 pM) or TNF- (1–1000 pM) for 24 h (upper panels). In a time-course study, cells were treated with either IL-1ß (100 pM) or TNF- (100 pM) for up to 24 h (IL-1ß) or 36 h (TNF-). *, P < 0.05 vs. control or the value at time zero; N.S., not significant.

View larger version (43K): [in this window] [in a new window]   FIG. 2. The effects of DHEA/DHEAS on proinflammatory cytokine-stimulated, NF-B-dependent transcription in HuH7NF hepatocytes. A, Time-course 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 µM; lower panels). *, P < 0.05 vs. the value at time zero. B, Dose-dependent 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 µM) or TNF- (100 pM) plus DHEA (10 nM to 10 µM) for 24 h (upper panels). Alternatively, cells were treated with DHEAS alone (10 nM to 100 µM) or TNF- (100 pM) plus DHEAS (100 nM to 100 µM). *, P < 0.05 vs. control (C); +, P < 0.05 vs. TNF- alone; N.S., not significant.

View larger version (59K): [in this window] [in a new window]   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).

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 µM) were simultaneously used, a significant additive effect was observed (Fig. 4B). These results 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).

View larger version (40K): [in this window] [in a new window]   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. *, 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 µM), or both dexamethasone and DHEAS for 24 h. *, 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 µM) 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 µM) for 24 h. *, 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 µM) for 24 h. *, 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 µM) alone, or H2O2 plus DHEAS (0.1–100 µM) for 24 h. *, P < 0.05 vs. control (C); +, P < 0.05 vs. H2O2 alone.

The molecular mechanisms of DHEAS on NF-B-dependent transcription

We then compared the effects of DHEAS on NF-B-dependent 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 µM) 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 µM) significantly stimulated NF-B-luciferase activity, whereas simultaneous treatment with DHEAS (0.1–100 µM) 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).

View larger version (45K): [in this window] [in a new window]   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 ROS-mediated NF-B activation through its antioxidant property. CRP, C-reactive protein; IB, inhibitory B; GR, glucocorticoid receptor; PAI-1, plasminogen activator inhibitor-1.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

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, 9, 10, 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 produced 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-1ß, 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, 29, 30, 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 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.

	Footnotes

 
Abbreviations: AP1, Activator protein-1; DHEA, dehydroepiandrosterone; DHEAS, DHEA-sulfate; FBS, fetal bovine serum; NF-B, nuclear factor-B; ROS, reactive oxygen species.

Received August 19, 2003.

Accepted April 8, 2004.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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