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Molecular and Cellular Gynecological Endocrinology Laboratory, Department of Reproductive Medicine and Child Development, Division of Obstetrics and Gynecology, University of Pisa, Pisa 56100, Italy
Address all correspondence and requests for reprints to: Tommaso Simoncini, M.D., Ph.D., Molecular and Cellular Gynecological Endocrinology Laboratory, Department of Reproductive Medicine and Child Development, Division of Obstetrics and Gynecology, University of Pisa, Via Roma 57, 56100 Pisa, Italy. E-mail: t.simoncini{at}obgyn.med.unipi.it.
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Introduction |
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Several epidemiological studies indicate an inverse correlation between DHEA/DHEAS plasma concentration and mortality, particularly due to cardiovascular disease. The analysis of the Rancho Bernardo population was the first to correlate DHEAS levels with cardiovascular risk in males over 50 yr of age (5). Afterward, a number of studies extended this observation to young men (6) as well as to premenopausal (7) and postmenopausal women (8). Low DHEAS levels have been also associated with cardiovascular events (9), with the extent of angiographic coronary stenosis (10), as well as with allograft vasculopathy (11), suggesting a role for DHEAS in delaying coronary disease.
However, other studies have questioned the association between DHEA/DHEAS and cardiovascular disease, and a later reanalysis of the Rancho Bernardo cohort showed a much weaker correlation (12). The prospective PAQUID study has recently renewed the interest in this issue, showing higher mortality in male patients with lower DHEAS concentrations (13, 14).
Animal studies indicate a protective role of DHEA for atherosclerotic disease in primates (15) and rabbits (16, 17), but in vitro studies explaining the mechanisms are largely missing.
Steroid hormones regulate vascular function in part through general metabolic modifications, but mostly through actions exerted directly on the vessel wall (18, 19). Endothelial cells are primary targets of steroids that regulate endothelial function through transcriptional as well as rapid nontranscriptional mechanisms (18). For instance, estradiol stimulates nitric oxide (NO) synthesis via the induction of endothelial NO synthase (eNOS) expression (20) as well as through nongenomic enhancement of its activity (21).
The mechanisms of action of DHEA are not clear. Part of the effects of DHEA depends on the conversion to estrogens and androgens and on the recruitment of the respective receptors. This is also true in the vessels, as shown by a report indicating that the reduction of atherosclerotic lesions by DHEA in rabbits may be partially mediated by conversion to estrogens (22). However, there is suggestive evidence that DHEA may have a dedicated receptor. This has been suggested also at the vascular level, where DHEA binds with high affinity to nonhuman endothelial cell membranes, without being displaced by structurally related steroids (23). DHEA binding to endothelial cell membranes is associated with G
i2 and Gi3 and with eNOS activation (23).
As endothelial-derived NO plays antiinflammatory and antiatherogenic actions (24), we tested the hypothesis that DHEA may exert direct regulatory actions on the vessel wall through the induction of endothelial NO synthesis. We therefore characterized the genomic and nongenomic actions of DHEA in vitro, in human endothelial cells, as well as in vivo, in Wistar rats.
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Materials and Methods |
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Animal treatments
Fertile female Wistar rats, weighing 175–199 g (Harlan, S. Pietro al Natisone, Italy), were kept under 14 h of illumination per day (0600–2000 h) and had free access to standard rat chow and tap water. After 14 d the rats were either sham-operated or ovariectomized in the same estrous cycle stage. Ovariectomized rats were treated with DHEA for 14 d (0.5 or 1 mg/kg·d). DHEA was dissolved in pure ethanol and administered orally after dilution in 0.9% NaCl. All fertile rats were in the same stage of the estrous cycle at the beginning of the treatment. Animals were euthanized by decapitation under pentobarbital anesthesia (30 mg/kg), and the abdominal aorta was obtained. Aortas were snap-frozen in dry ice and stored at -80 C. Animals were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (www.nap.edu/readingroom/books/labrats).
eNOS activity assay
Endothelial cells were harvested in 320 mM sucrose, 20 mM HEPES, 1 mM EDTA, 1 mM dithiothreitol, 10 µg/ml leupeptin, 2 µg/ml aprotinin, and 100 µg/ml phenylmethylsulfonylfluoride. Rat aortas were homogenized in the same buffer, and after centrifugation to discard the cell debris, the supernatants were used for eNOS assay. eNOS activity was determined as the conversion of [3H]arginine to [3H]citrulline, incubating cell extracts for 10 min at 30 C in 50 mM potassium phosphate, 1.2 mM L-citrulline, 1.2 mM CaCl2, 120 µM NADPH, and 24 µM L-[3H]arginine. Converted citrulline was separated by unconverted arginine using the acidic ion exchange resin Dowex 50 W, 200–400 mesh (Sigma-Aldrich Corp., St. Louis, MO), as previously described (26). Extracts incubated with the eNOS inhibitor, N
-nitro-L-arginine methyl ester (L-NAME; 1 mM), served as the blank. Converted eNOS activity was obtained subtracting the blank from the samples.
Nitrite assay
NO production was determined by a modified nitrite assay using 2,3-diaminonaphtalene as described previously (27). The fluorescence of 1-(H)-naphtotriazole was measured with excitation and emission wavelengths of 365 and 450 nm, respectively. Standard curves were constructed with sodium nitrite. Nonspecific fluorescence was determined in the presence of NG-monomethyl-L-arginine (3 mM).
Immunoblottings
Cell lysates or rat aorta protein extracts were separated by SDS-PAGE. The antibodies (Ab) used were eNOS (Transduction Laboratories, Lexington, KY), wild-type or Tyr204-phosphorylated ERK1/2 and wild-type or Ser217/221-phosphorylated MEK1/2, Raf-1, antiphosphoserine Ab, antiphosphotyrosine (Calbiochem, San Diego, CA), wild-type and Thr308- or Ser473-phosphorylated Akt, phospho-myelin basic protein (phospho-MBP; Upstate Biotechnology, Inc., Lake Placid, NY). Primary Ab were incubated with the membranes overnight at 4 C. The blots were hybridized with a secondary Ab coupled to horseradish peroxidase as previously described (21). Immunodetection was accomplished using enhanced chemiluminescence.
Raf-1 kinase assay
Equal amounts of protein extracts were immunoprecipitated using an Ab vs. Raf-1, as previously described (21). The immunoprecipitates were resuspended in 39 µl kinase assay buffer (20 mM 3-morpholinopropanesulfonic acid, 25 mM ß-glycerophosphate, and 5 mM EGTA added with protease inhibitors). Ten microliters of Mg/ATP (500 µM ATP, 75 mM MgCl2, in kinase assay buffer) and 5 µg dephosphorylated human recombinant myelin basic protein (Upstate Biotechnology, Inc.) were added, and the samples were kept at 37 C for 20 min. The reaction was stopped by adding 50 µl Laemmli buffer. The samples were boiled, and after centrifugation the supernatants were separated by SDS-PAGE and immunoblotted with antiphospho-MBP Ab.
Statistical analysis
Values are expressed as the mean ± SD. Statistical differences between mean values were determined by ANOVA, followed by Fisher’s protected least significance difference test for comparison of mean values.
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). NO synthesis was significantly enhanced starting from physiological DHEA concentrations and reached a plateau at 100 nM.
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). In contrast, upon DHEA administration, significant increases in eNOS enzymatic activity were found in cell lysates, which paralleled the increases in NO release (Fig. 1A). The activation of eNOS and NO synthesis was effective as early as 10 min after DHEA treatment (Fig. 1C) and was maximal after 1 h, with a subsequent progressive decline (Fig. 1C).
DHEA rapidly activates eNOS via nongenomic mechanisms
DHEA-induced activation of NO production and eNOS was not prevented by pretreatment with effective concentrations (see Figs. I and II, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org) of inhibitors of mRNA (actinomycin D) or protein (cycloheximide) synthesis (Fig. 2A), confirming that DHEA acts via nontranscriptional mechanisms. The addition of the eNOS inhibitor, L-NAME, completely blunted basal and DHEA-induced eNOS activity (Fig. 2A), confirming that DHEA-induced NO synthesis depends on activation of the type III NOS isoform.
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). In parallel, using RIA techniques, no conversion to 17ß-estradiol, estrone, or androstenedione was found in the culture medium at any of the DHEA concentrations used (estradiol, estrone, and androstenedione levels undetectable; data not shown).
Nongenomic activation of eNOS by DHEA involves a G protein-coupled receptor (GPCR)-MAPK cascade
To study the signal transduction pathways that mediate the nontranscriptional activation of eNOS by DHEA, we pretreated HUVEC with phosphatidylinositol 3-kinase (PI3K) or MAPK inhibitors. Effective concentrations (see Fig. III published as supplemental data) of the PI3K blocker, wortmannin, had no effect on DHEA-induced NO synthesis and eNOS activation (Fig. 2B), whereas the ERK1/2 inhibitor, PD 98059, significantly reduced DHEA effect (Fig. 2B), thus linking the ERK1/2 pathway to DHEA signaling in endothelial cells. As DHEA has been shown to recruit a GPCR (23), we added the G protein inhibitor, pertussis toxin (PTX), to endothelial cells treated with DHEA. PTX effectively inhibited DHEA-induced eNOS activation (Fig. 2B), indicating that the as yet unidentified receptor for this steroid may be a membrane-bound GPCR able to activate MAPK. As estradiol activates eNOS predominantly via the estrogen receptor /PI3K/Akt pathway (21), we tested the effects of concurrent estradiol and DHEA treatment on eNOS activation. The presence of the two steroids resulted in a slightly additive effect, with a final activation of eNOS that was higher than that in the presence of the two single molecules (Fig. 2B), further confirming that estrogens and DHEA signal to eNOS via different pathways.
Molecular characterization of the MAPK-dependent eNOS activation by DHEA
To confirm the role of MAPK in DHEA signaling, we studied the activation of ERK1/2 with the use of phospho-specific Abs recognizing the active forms of the enzymes. DHEA administration resulted in rapid phosphorylation of ERK1/2 that was largely prevented by the MEK inhibitor PD 98059 (Fig. 3A). In a time-consistent fashion, phosphorylation of the ERK1/2 kinase, MEK1/2, was found (Fig. 3A). The upstream component of the three-step ERK1/2 module is Raf-1. By immunoprecipitating Raf-1 and using the immunoprecipitates in a kinase assay with MBP as a phosphorylation acceptor, we found that Raf-1 is rapidly activated after DHEA treatment (Fig. 3B), and that a specific Raf-1 inhibitor is able to prevent both DHEA-dependent Raf-1 activation (Fig. 3B) as well as eNOS activation (not shown). In parallel, no phosphorylation of the two regulatory residues (Thr308 and Ser473) of protein kinase Akt, the final PI3K effector mediating activation of eNOS by estrogens or glucocorticoids (21, 28), could be found (Fig. 4A), confirming that nongenomic activation of eNOS by DHEA occurs through an independent pathway. As eNOS activation by other stimuli is associated with enzyme phosphorylation, we checked whether DHEA affects eNOS phosphorylation. We therefore immunoblotted eNOS immunoprecipitates with Ab recognizing serine- or tyrosine-phosphorylated amino acid residues, but we could not detect any Ser or Tyr phosphorylation of eNOS protein (Fig. 4B), suggesting that the nongenomic activation of eNOS by DHEA through MAPK may instead be mediated by rapid modulation of the intracellular calcium concentration (29) or by other mechanisms, rather than by direct phosphorylation of the enzyme.
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). NO synthesis induction corresponded to consistent increases in detectable eNOS enzymatic activity, which followed a concentration-dependent pattern (Fig. 5A). When cell lysates were assayed with Western analysis, parallel increases in eNOS protein were found (Fig. 5B).
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), whereas both effects of DHEA were blocked by the eNOS inhibitor L-NAME (Fig. 5C). Even after 48 h of exposure to DHEA, no detectable conversion to 17ß-estradiol, estrone, or androstenedione by HUVEC could be found at any concentration used (estradiol, estrone, and androstenedione levels undetectable; data not shown), suggesting a direct effect of DHEA.
Posttranslational effects of DHEA on eNOS protein stability
As eNOS protein amounts are controlled at the level of both protein synthesis as well as protein turnover, we checked whether administration of DHEA to endothelial cells where protein synthesis was inhibited by pretreatment with cycloheximide resulted in modifications of eNOS protein stability. Indeed, we found that in the presence of DHEA, eNOS turnover was markedly decreased, as shown by the prolonged persistence of eNOS in HUVEC (Fig. 6, A and B), corresponding to an approximate doubling of the eNOS half-life (Fig. 6, A and B).
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). However, oral DHEA administration was associated with a dose-dependent restoration of aortic eNOS protein content, with the 1 mg/kg·d dose inducing eNOS expression similar to that in fertile rats (Fig. 7B). Additionally, when aortic eNOS enzymatic activity was assayed, the same pattern was observed (Fig. 7B), confirming that DHEA administration is associated with increases in functional eNOS.
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Discussion |
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Understanding the molecular basis of DHEA action is mandatory to answer these questions, and our results contribute several original concepts: 1) DHEA is able to directly regulate the human vascular wall by controlling NO synthesis in endothelial cells; 2) DHEA activates both transcriptional and nontranscriptional signaling pathways; 3) DHEA actions are exerted independently by other steroid hormone receptors, supporting the presence of a specific DHEA receptor; 4) the nongenomic effects of DHEA in endothelial cells are mediated by activation of a GPCR-ERK1/2 MAPK cascade; 5) DHEA regulates eNOS protein synthesis as well as stability; and 6) DHEA effects on eNOS are also present in vivo, confirming the biological relevance of the in vitro findings.
Our observation of NO synthesis induction by DHEA is in agreement with a previous report on ovariectomized rabbits, showing a reduction of cholesterol-induced atherosclerosis and higher NO plasma levels in animals receiving high dietary DHEA supplementation (22). Indeed, diabetic rats receiving DHEA show improved vessel dilatation (31). Moreover, a recent report shows that DHEA treatment of bovine endothelial cells results in Gi2 and Gi3 activation, possibly via binding to specific cell membrane binding sites (23). In agreement with our findings, Gi protein activation was associated with eNOS activation (23).
NO production by endothelial cells is regulated at multiple levels. Rapid regulation of eNOS activity is exerted by estrogens and glucocorticoids by activating MAPK and PI3K/Akt pathways (21, 28).
A primary aim of our study was to characterize the molecular mechanisms of action of DHEA in human endothelial cells, and our data provide a description of a rapid DHEA action. Indeed, DHEA increases eNOS activity in a matter of minutes, independently by the activation of gene expression or protein synthesis. The nontranscriptional effects of DHEA depend on the recruitment of a GPCR and results in activation of the ERK1/2 MAPK cascade, but not of PI3K, suggesting that DHEA may activate eNOS via a different mechanism than estrogen (21).
As the reported Kd for DHEA binding to bovine endothelial cell membranes (23) and Gi2/3 activation corresponds to the range of concentrations that activate MAPK and eNOS in our study, it may be possible that activation of Gi proteins by DHEA at the cell membrane may represent the first step in the recruitment of the ERK1/2 cascade in endothelial cells. This is supported by our finding of a complete prevention of DHEA-induced eNOS activation by the G protein inhibitor pertussis toxin. Cell membrane GPCR recruitment by DHEA may signal to MAPK via the activation of p21Ras or Src, which are well known activators of Raf-1 (29, 32).
By linking DHEA to MAPK activation in endothelial cells, the role of DHEA in vascular tissues may be considerably broadened, as MAPK are multifunctional regulators of critical cellular events such as DNA synthesis, cell proliferation and survival, and ion flux regulation and control the activity of other signaling pathways, including steroid hormone receptors (29).
In addition to nongenomic regulation of eNOS activity, we provide evidence that DHEA activates eNOS expression and decreases eNOS turnover, thereby promoting NO synthesis at multiple levels.
Our findings characterize human endothelial cells as primary targets for DHEA. Endothelial-derived NO is a vasodilator, but also has antiinflammatory and antiatherogenic functions (24). The evidence for a direct induction of NO synthesis by DHEA is therefore important, providing a molecular explanation for the antiatherogenic effects of this steroid in animals (22). Additionally, some of the antiatherogenic (10) and antiinflammatory (11) effects of DHEA in humans may ultimately be explained by DHEA induction of endothelial NO synthesis.
Previous studies, performed with high DHEA daily intake and for longer times, indicate that part of the effects of DHEA on the vascular wall are mediated by conversion to estrogens (22). However, our in vitro data indicate that DHEA genomic or nongenomic actions are not blocked by interfering with either specific estrogen (ICI 182,780) and androgen (flutamide) inhibitors or by a mixed progesterone/glucocorticoid receptor inhibitor (RU486). These results, together with the absence of detectable conversion of DHEA to estrogens or androgens by endothelial cells, consistently suggest that DHEA acts directly through an as yet unrecognized receptor. Indeed, the presence of a putative receptor for DHEA on vascular cells has been suggested by recent reports showing the presence of specific binding sites in vascular smooth muscle cells (33) or endothelial cells (23), with estimated Kd values in agreement with our concentration-effect curve on eNOS activation and NO synthesis.
Our animal data support the conclusions of the in vitro studies, suggesting that the observed effects may also be important in vivo. Indeed, DHEA administration to ovariectomized rats completely restores eNOS protein amounts and activity, suggesting that exogenous DHEA can reproduce the effects of the lost ovarian hormones. In this setting, part of the effects of DHEA is probably mediated by peripheral conversion to estrogens (22), as occurs in postmenopausal women (30). However, the evidence of a direct induction of eNOS protein expression in isolated endothelial cells may indicate that DHEA also exerts direct genomic regulation at this level, independently from estrogen receptors.
In conclusion, we show that DHEA directly regulates human endothelial cells, activating eNOS through both genomic and nongenomic mechanisms, and that these effects are also found in vivo in Wistar rats. Our results contribute to the understanding of the molecular mechanisms of DHEA action, suggesting the existence of specific signaling pathways for DHEA, not requiring conversion to other steroids. Furthermore, endothelial NO induction by DHEA provides a rationale for some of the vascular effects of this steroid.
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Acknowledgments |
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Footnotes |
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Abbreviations: Ab, Antibody; DHEA, dehydroepiandrosterone; DHEAS, DHEA sulfate; eNOS, endothelial NO synthase; GPCR, G protein-coupled receptor; HUVEC, human umbilical vein endothelial cells; L-NAME, N-nitro-L-arginine methyl ester; MBP, myelin basic protein; NO, nitric oxide; PI3K, phosphatidylinositol 3-kinase; PTX, pertussis toxin.
Received January 9, 2003.
Accepted for publication April 15, 2003.
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), and cell lysates were harvested for measurement of eNOS activity (
). B, Western analysis of eNOS protein. Experiments were repeated three times in triplicate, and the mean ± SD of the nine replicates are shown. Asterisks indicate a significant difference (P < 0.05) vs. control (experiment C, all time points significantly different from time zero). The blot is representative of three different experiments with comparable results.
, significant difference (P < 0.05) vs. DHEA alone and E2 alone.
