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Endocrinology Vol. 145, No. 12 5532-5539
Copyright © 2004 by The Endocrine Society

Genistein Acutely Stimulates Nitric Oxide Synthesis in Vascular Endothelial Cells by a Cyclic Adenosine 5'-Monophosphate-Dependent Mechanism

Dongmin Liu, Laurie L. Homan and Joseph S. Dillon

Division of Endocrinology, Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa and Veterans Affairs Medical Center, Iowa City, Iowa 52246

Address all correspondence and requests for reprints to: Joseph Dillon, M.D., VA Medical Center #3E10, Iowa City, Iowa 52246. E-mail: joseph-dillon{at}uiowa.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Genistein may improve vascular function, but the mechanism of this effect is unclear. We tested the hypothesis that genistein directly regulates vascular function through stimulation of endothelial nitric oxide synthesis. Genistein activated endothelial nitric oxide synthase (eNOS) in intact bovine aortic endothelial cells and human umbilical vein endothelial cells over an incubation period of 10 min. The maximal eNOS activity was at 1 µM genistein. Consistent with this activation pattern, 1 µM genistein maximally stimulated the phosphorylation of eNOS at serine 1179 at 10 min of incubation. The rapid activation of eNOS by genistein was not dependent on RNA transcription or new protein synthesis and was not blocked by a specific estrogen receptor antagonist. In addition, inhibition of MAPK or phosphatidylinositol 3-OH kinase/Akt kinase had no affect on eNOS activation by genistein. Furthermore, the genistein effect on eNOS was also independent of tyrosine kinase inhibition. However, inhibition of cAMP-dependent kinase [protein kinase A (PKA)] by H89 completely abolished the genistein-stimulated eNOS activation and phosphorylation, suggesting that genistein acted through a PKA-dependent pathway. These findings demonstrated that genistein had direct nongenomic effects on eNOS activity in vascular endothelial cells, leading to eNOS activation and nitric oxide synthesis. These effects were mediated by PKA and were unrelated to an estrogenic effect. This cellular mechanism may underlie some of the cardiovascular protective effects proposed for soy phytoestrogens.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EXPERIMENTAL AND CLINICAL data support vascular protective effects of estrogen in postmenopausal women (1, 2). However, therapeutic use of estrogen has been limited by recent clinical trial results suggesting that estrogen may increase the risk of coronary heart disease (3). The potential carcinogenic and feminizing effects of estrogen further limit its use as a cardioprotective agent. Given the demonstrated risks of conventional estrogen therapy, there is interest in finding novel alternative agents that may have beneficial effects on the vasculature but without some of the side effects of estrogen.

Phytoestrogens have received widespread attention over the past few years because of their potential for preventing some highly prevalent chronic diseases, including cardiovascular disease (4), osteoporosis (5), and hormone-related cancers (6). Genistein, the primary soy-derived phytoestrogen, has various biological actions, including a weak estrogenic effect (7) and inhibition of tyrosine kinases (8). Studies demonstrate that genistein has antiatherogenic effects and inhibits proliferation of vascular endothelial (9) and smooth muscle cells (10). Data from animal and in vitro studies suggest a protective role of genistein in the vasculature (11, 12, 13, 14, 15). Studies investigating its effect on plasma lipid profiles show either a moderate positive effect (16, 17, 18) or a neutral effect (19, 20). Some human intervention studies suggest a beneficial effect on atherosclerosis (21), markers of cardiovascular risk (22), vasomotor tone (23), vascular endothelial function (24), and systemic arterial compliance (19). Genistein also inhibits human platelet aggregation in vitro (25, 26) and decreases TNF{alpha}-induced monocyte chemoattractant protein-1 secretion in human vascular endothelial cells (26).

Although these data indicate a protective role of genistein in the vascular system, the mechanism underlying these beneficial effects is still largely unknown. Previous studies have established a role for estrogen in the regulation of vascular function. Estrogen can act directly on the vascular endothelial cells to enhance nitric oxide (NO) synthesis through genomic stimulation of endothelial NO synthase (eNOS) expression (27) and by receptor-mediated, nongenomic, eNOS activation (28). However, it is unknown whether the phytoestrogen genistein has a similar effect. Genistein ingestion can increase circulating nitrate/nitrite (29) and endothelium-dependent vasodilatation in humans (23, 29). In animal models, genistein induces NO-mediated relaxation of rat pulmonary arteries (13) and aorta (30), suggesting that genistein may directly act on vascular endothelium to regulate eNOS. Other studies suggest that genistein may induce vascular relaxation by cAMP-dependent mechanisms (31) or inhibition of tyrosine kinases (32). In vitro studies elucidating the cellular or molecular mechanisms of the genistein action on vascular cells are lacking.

NO produced is a potent vasodilator and also has antiinflammatory (33), antiatherogenic (34), antithrombotic (35), and antiapoptotic (36) properties. We therefore hypothesized that genistein directly regulates vascular function through stimulation of eNOS and NO synthesis from vascular endothelial cells. To test this hypothesis, we focused on the acute effects of genistein on eNOS and the cellular signaling related to this effect. We specifically tested the protein kinase A and tyrosine kinase pathways because these have been proposed in previous vascular studies (31, 32).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Bovine aortic endothelial cells (BAECs) were kindly provided by Dr. Robert Bar (University of Iowa, Iowa City, IA), and primary human umbilical vein endothelial cells (HUVECs) were obtained from the Cardiovascular Research Cell Culture Core at the University of Iowa. The following materials were used: vascular endothelial growth factors (Clonetics, San Diego, CA); phospho-eNOS (Ser1179), phospho-ERK1/2, and ERK1/2 antibodies (Cell Signaling Technology, Beverly, MA); eNOS antibody (Santa Cruz Biotechnology, Santa Cruz, CA); nitrocellulose membranes (Schleicher & Schuell, Keene, NH); SuperSignal chemiluminescence detection system and tyrosine kinase assay kit (Pierce Chemical, Rockford, IL); [3H]L-arginine, [3H]uridine, and [3H]proline (Amersham Pharmacia, Arlington Heights, IL); NO fluorometric assay kits (Cayman Chemical, Ann Arbor, MI); cAMP enzyme immunoassay (EIA) kits (Assay Designs, Ann Arbor, MI); ICI 182,780 (Tocris, Ballwin, MO); genistein, daidzein, 17ß-estradiol, isoproterenol, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89), actinomycin D, cycloheximide, N{omega}-nitro-L-arginine methyl ester, herbimycin A, LY290042, PD098059, protease and phosphatase inhibitors, and other laboratory chemicals (Sigma Chemical, St. Louis, MO). All reagents used were of analytical grade. Stock solutions of genistein and daidzein, at 20 mM in dimethyl sulfoxide, were stored at –80 C before use.

Cell culture
BAECs and HUVECs were maintained as previously described (37).

NO synthase activity
eNOS activity was determined by measuring the conversion of [3H]L-arginine to [3H]L-citrulline (38). Cell culture conditions were essentially as described previously (37), except that HUVECs were serum starved for 12 h before eNOS assay. Stimulation with various agonists was carried out in Hanks’ balanced salt solution (HBSS) buffer containing [3H]L-arginine (2.5 µCi/ml) and L-arginine (5 µM) for a range of times or 10 min at 37 C, as indicated in the figure legends. In some experiments, cells were pretreated with the estrogen receptor inhibitor ICI 182,780 (10 µM); MAPK kinase inhibitor, PD098059 (10 µM); phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002 (10 µM); or protein kinase A (PKA) inhibitor, H89 (10 µM), for 30 min before addition of agonists. The inhibitors were also present in the assays during incubation with test reagents. The concentrations of vehicle were equal in all treatment and control samples. The reaction was terminated by aspirating the buffer and washing with ice-cold PBS containing EGTA (5 mM) and EDTA (5 mM), followed by addition of 1 ml ice-cold trichloroacetic acid (0.5 N). Subsequent sample handling and scintillation counting of [3H]L-citrulline was performed as previously (39). Nonspecific activity was determined by the addition of N{omega}-nitro-L-arginine methyl ester (L-NAME, 2 mM), and subtracted from the samples. eNOS activity was normalized as picomoles of [3H]L-citrulline produced per milligram protein and expressed as a percentage of vehicle-treated controls.

NO assay
We determined NO production by measuring the sum concentration of NO2– and NO3– (NOx) in culture supernatants using a fluorometric assay kit, following the manufacturer’s instructions (Cayman). The experimental protocol was as described (37), with the following modifications. Cells were cultured for 24 h in complete media (M199, 20% fetal calf serum), followed by 24 h in phenol red-free media containing steroid-deprived 10% fetal calf serum and serum starvation for an additional 24 h in phenol red-free M199 media. The cells were then gently washed (37) and incubated with either vehicle or genistein over a range of concentrations and time points. In some experiments, cells were pretreated with H89 or ICI 182,780 as noted above before addition of agonists. Culture media were collected for NOx assay after 10 min of agonist exposure (37). Cellular fluorescence was measured with excitation and emission wavelengths of 365 and 450 nm, respectively. Nonspecific fluorescence was determined in the presence of 2 mM L-NAME and was subtracted from the samples. Fluorescence data were converted into concentrations based on standard curves constructed with sodium nitrate, normalized to protein concentration of the samples, and then expressed as percentages of vehicle-treated controls.

Tyrosine kinase activity assay
Near confluent BAECs were further cultured in serum-free, phenol red-free M199 media for 24 h. Cells were then washed and treated with genistein (10 nM to 100 µM), herbimycin A (10 µM), or vehicle for 30 min in HBSS buffer at 37 C. Cultures were either terminated or stimulated with vascular endothelial growth factor (VEGF, 50 ng/ml) for an additional 10 min. Cells were then harvested in lysis buffer, briefly sonicated, and centrifuged to discard the cell debris. The supernatants were used for the tyrosine kinase activity assay according to the manufacturer’s instructions. The relative fluorescence data were normalized to corresponding protein levels and expressed as percentages of the vehicle-treated controls.

Intracellular cAMP assay
The accumulation of cAMP in confluent BAECs under basal or stimulated conditions was determined by a specific EIA. After 24 h of serum starvation, cells were washed with HBSS and incubated with HBSS containing genistein (10 nM to 10 µM) or isoproterenol (10 µM) for 5 min at 37 C. The supernatant was then rapidly aspirated and the intracellular cAMP extraction and quantification by EIA were performed according to the manufacturer’s instructions (Assay Designs). Data were normalized to the protein concentrations of the samples.

Western blot analysis
To characterize the acute effects of genistein, BAECs were grown in 12-well plates and serum starved in phenol red-free M199 medium for 24 h. The cells were then rinsed and incubated with HBSS for 30 min and then with various concentrations of genistein or vehicle for 10 min or with 1 µM genistein for various times, as indicated. In some experiments, cells were preincubated with H89 (10 µM) or vehicle (0.01% dimethyl sulfoxide) for 30 min in HBSS before exposure to genistein. Reactions were terminated by aspiration and addition of cold lysis buffer, supplemented with protease and phosphatase inhibitors. Cells were sonicated and 50 µg of detergent-extracted lysates in Laemmli sample buffer were heated for 5 min at 95 C and resolved on 10% SDS-PAGE gels. Nitrocellulose gel blots were probed with antibody to phospho-eNOS (Ser1179) overnight at 4 C and incubated with secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature. The immunoreactive proteins were detected by chemiluminescence (SuperSignal, Pierce). Membranes were stripped and reprobed with eNOS antibody to monitor for equal protein loading. The protein bands were digitally imaged for densitometric quantitation (Silk Scientific, Orem, UT). Phosphorylated (activated) enzyme expression was normalized to total eNOS expression from the same sample.

Statistical analysis
All data were subjected to a one-way ANOVA analysis using GraphPad Prism software (GraphPad Inc., San Diego, CA), and treatment differences were subjected to a Duncan’s multiple comparison test at the 5% probability. Data in each study were derived from at least three independent experiments, and variation within treatments was expressed as SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Genistein rapidly activates eNOS in vascular endothelial cells
Based on the extensive data favoring an effect of genistein on the vasculature and the central role of NO in vascular function, we hypothesized that genistein would stimulate eNOS activity from intact vascular endothelial cells. To test this hypothesis, we performed dose-response and kinetic experiments in BAECs. Concentrations of genistein ranging from 1 nM to 10 µM and stimulating periods from 10 and 120 min were tested. Genistein significantly induced eNOS enzymatic activity from BAECs after 10 min of incubation (Fig. 1AGo). The effect was concentration dependent, with genistein 1 µM inducing maximal eNOS activation. Maximal genistein-induced eNOS activity was demonstrated between 10 and 30 min of incubation (Fig. 1BGo). The relative magnitude of the genistein response falls subsequently but is still significantly greater than control after 2 h of genistein incubation.



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  		FIG. 1. Genistein rapidly stimulates eNOS activity. Serum-deprived BAECs were incubated with various concentrations of genistein for 10 min at 37 C (A) or with 1 µM genistein for different times (B) and eNOS activity determined. Data were expressed as mean ± SEM of four to six separate experiments in triplicate. *, P < 0.05 vs. vehicle-treated control.

 
The rapid activation of eNOS by genistein is through nongenomic mechanisms
Next we examined whether the rapid activation of eNOS is mediated by a genomic or nongenomic mechanism. Pretreatment of the cells with the protein synthesis inhibitor cycloheximide or the mRNA transcription inhibitor actinomycin D at concentrations above their effective threshold (40) had no statistically significant effect on genistein-induced activation of eNOS. The eNOS activities, expressed as percentages of the vehicle-treated cells, were 176.6 ± 16.7 (genistein alone), 182.7 ± 10.1 (genistein plus cycloheximide), and 171.0 ± 19.6% (genistein plus actinomycin D). Both cycloheximide and actinomycin D were active because cycloheximide inhibited [3H]proline incorporation into protein by 82%, and actinomycin D inhibited [3H]uridine incorporation into RNA by 93% in BAECs, using the same inhibitor concentration and duration of incubation as in our experimental studies (see Figs. 1Go and 2Go, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). The eNOS inhibitor L-NAME significantly suppressed basal activity and completely blocked genistein-induced eNOS activity (basal 87.9 ± 3.3%, genistein-stimulated 78.6 ± 3.9%; P < 0.05 from basal or genistein-treated cells, respectively).



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  		FIG. 2. Nongenomic effect of genistein on eNOS activation. Serum-deprived BAECs were incubated with various concentrations of genistein for 10 min (A) or with 1 µM genistein for indicated times (B) and Western analysis performed to detect eNOS-Ser1179 and total eNOS. Means ± SEM of densitometry measurements from three separate experiments along with representative blots are shown (A and B). *, P < 0.05 vs. vehicle-treated or time zero controls.

 
Because eNOS can be activated by direct enzyme phosphorylation without an increase in protein expression, we therefore characterized this nongenomic mechanism of genistein-induced eNOS activation by evaluating whether genistein rapidly induces eNOS phosphorylation. Immunoblots showed that genistein stimulated a concentration-dependent increase in the phosphorylation of Ser1179-eNOS within 10 min of incubation. The maximal stimulation of 150% over control occurs at 1 µM genistein (Fig. 2AGo). The effect of genistein on eNOS phosphorylation peaked at 10 min of incubation and gradually decreased thereafter (Fig. 2BGo), consistent with the pattern of eNOS activity induced by genistein. The expression of eNOS protein was not changed in these studies.

Genistein activation of eNOS is not inhibited by estrogen receptor antagonists
Because genistein has weak estrogenic effects in some tissues by binding to estrogen receptor (7) and estradiol rapidly activates eNOS through a signaling pathway initiated from membrane estrogen receptors (28), we examined whether the genistein effect was mediated through estrogen receptors. Estradiol (10 nM) significantly stimulated eNOS activity from BAECs after 10 min of treatment (124.1 ± 2.0% of basal eNOS activity), although at a lower magnitude than genistein (169.5 ± 6.9%). The estrogen receptor antagonist ICI 182,780 caused no change in basal (108.2 ± 1.2%) or genistein-induced eNOS (161.7 ± 4.5%) but completely inhibited the acute response to estradiol (102.6 ± 0.6%, P < 0.05 vs. estradiol).

Activation of eNOS by genistein is not mediated by PI3K/Akt or ERK/MAPK pathways
Previous studies established that PI3K/Akt and ERK/MAPK-mediated pathways are two important signaling cascades mediating eNOS activation by many stimuli in vascular endothelial cells (41, 42). To elucidate the intracellular signaling involved in the nongenomic activation of eNOS by genistein, we tested whether the PI3K/Akt or ERK/MAPK pathways were involved in genistein-induced eNOS activation. Preincubation of BAECs with the PI3K inhibitor, LY294002, or ERK/MAPK blocker, PD098059, had no effect on either basal (data not shown) or genistein-stimulated eNOS activation (Fig. 3Go). Both LY294002 and PD098059 were active because LY294002 inhibited estradiol-induced eNOS activation by 82%, and PD098059 inhibited insulin-induced ERK1/2 phosphorylation by 90%, using the same inhibitor concentration and duration of incubation as in our experimental studies (see Figs. 3Go and 4Go, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).



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  		FIG. 3. Activation of eNOS by genistein is not mediated by PI3K/Akt or ERK/MAPK pathways. Serum-starved BAECs were preincubated with LY294002 (LY, 10 µM), PD098059 (PD, 10 µM), or vehicle (C) for 30 min and then incubated with genistein (G, 1 µM) or vehicle and eNOS activity determined. Data were expressed as mean ± SEM of three separate experiments, in triplicate. *, P < 0.05 vs. vehicle-treated control.

 


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  		FIG. 4. Activation of eNOS by genistein is independent of tyrosine kinases. A, Serum-starved BAECs were incubated with genistein, herbimycin A (H, 10 µM), or vehicle for 30 min. B, Cells were stimulated with VEGF (50 ng/ml) for 10 min after preincubation with genistein, herbimycin A, or vehicle for 30 min. Tyrosine kinase activity, expressed as relatively quenched fluorescence, was determined. Data were expressed as mean ± SEM of three separate experiments, in triplicate *, P < 0.05 vs. vehicle-treated control.

 
Genistein activation of eNOS is independent of tyrosine kinase
Because genistein is often used as a nonspecific tyrosine kinase inhibitor in studies of tyrosine kinase-mediated cellular events, and tyrosine kinase may play a role in some agonist-stimulated eNOS activation (43), we evaluated whether genistein activates eNOS by inhibition of tyrosine kinase in vascular endothelial cells. We first tested the effect of daidzein on eNOS activity. Daidzein is a phytoestrogen analog of genistein that is inactive for tyrosine kinase inhibition. Similar to genistein (167.3 ± 11.9%), incubation of BAECs with daidzein (1 µM) for 10 min significantly increased the eNOS activity (134.7 ± 8.1, P < 0.05 vs. basal), although to a lesser degree than genistein (P < 0.05 vs. genistein).

We then directly measured tyrosine kinase activity in cells treated with genistein. In parallel experiments, cells were treated with the specific tyrosine kinase inhibitor, herbimycin A. We incubated cells in 10 µM herbimycin for 30 min because this is an effective duration and concentration for inhibition of tyrosine kinase activity (44). Genistein, at any concentrations used for stimulation of eNOS activity (10 nM-10 µM), did not inhibit the basal tyrosine kinase activity (Fig. 4AGo). Significant inhibition of tyrosine kinase activity in this study was observed only at genistein 100 µM, confirming that tyrosine kinase inhibition requires higher concentrations of genistein (45). As expected, herbimycin A potently inhibited tyrosine kinase activity (Fig. 4AGo).

Because VEGF is known to activate tyrosine kinases (46), we next tested whether genistein had an effect on VEGF-stimulated tyrosine kinase activity. Incubation of the BAECs with VEGF (50 ng/ml) for 5 min stimulated a more than 2-fold increase in tyrosine kinase activity (Fig. 4BGo). This effect was completely blocked by preincubation of BAECs with herbimycin A (10 µM) for 30 min, whereas preincubating the cells with genistein at 10 µM or less had no effect. Consistent with our previous experiments, 100 µM genistein effectively inhibited the VEGF activation of tyrosine kinases (Fig. 4BGo).

Genistein activation of eNOS is mediated by PKA
Because the activation of eNOS by genistein was not mediated by PI3K/Akt, ERK/MAPK, or tyrosine kinases, we considered alternative mechanisms. Several other protein kinases, including PKA, and AMP kinase pathways are implicated in eNOS regulation (47). We chose to examine whether PKA regulates the eNOS activation by genistein. BAECs were preincubated with H89 (10 µM), a highly selective PKA inhibitor, for 30 min and then stimulated with genistein (1 µM) for 10 min. The stimulation of eNOS activity by genistein was completely inhibited by H89 (Fig. 5AGo). To examine the specificity of the PKA pathway, BAECs were preincubated in the presence of H89 (10 µM) or vehicle for 30 min before stimulation with 17ß-estradiol (10 nM) for 10 min. The PKA inhibitor H89 did not inhibit eNOS activity induced by estradiol (Fig 5AGo).



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  		FIG. 5. Activation of eNOS by genistein is mediated by the PKA pathway. Serum-starved BAECs were preincubated with H89 (10 µM) or vehicle (C) for 30 min, followed by genistein (G, 1 µM), 17ß-estradiol (E2, 10 nM), or vehicle for 10 min. Enzyme activity (A) and protein phosphorylation (B) were assayed. Experiment A was repeated four times in triplicate and data were expressed as mean ± SEM. *, P < 0.05 vs. vehicle-treated control. Experiment B was repeated three times, all with similar results.

 
To confirm this finding and ensure that it was not a species-specific phenomenon, a similar study was performed with HUVECs. The results were essentially unchanged from those with BAECs. Genistein induced eNOS activity in HUVECs (182.5 ± 6.6%), and H89 blocked the genistein effect (106.2 ± 7.3%). Inhibition of PKA had no effect on estradiol-induced eNOS activation (estradiol alone 130.5 ± 12.7%, estradiol plus H89 131.2 ± 8.9%, n = 4 experiments in triplicate, P > 0.05). Consistent with these data, the genistein-dependent phosphorylation of Ser1179-eNOS in BAECs was blocked by H89 (Fig. 5BGo).

Genistein rapidly stimulates intracellular cAMP accumulation and subsequently activates eNOS
To further characterize the PKA-dependent eNOS activation by genistein, we measured the effect of genistein on intracellular cAMP production because cAMP directly activates PKA. Genistein treatment (10 nM to 100 µM) significantly induced a concentration-dependent increase in intracellular cAMP level in BAECs, with a maximal increase at 10 µM genistein. Isoproterenol (10 µM), a ß-adrenergic receptor agonist and stimulator of adenylate cyclase, also stimulated intracellular cAMP accumulation to the same degree as genistein 10 µM (Fig. 6Go).



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  		FIG. 6. Genistein stimulates intracellular cAMP accumulation. Serum-starved BAECs were incubated with genistein (10 nM to 10 µM), isoproterenol (ISO, 10 µM), or vehicle for 5 min at 37 C. cAMP data, normalized to cellular protein, were expressed as mean ± SEM of three separate experiments in triplicates. *, P < 0.05 vs. vehicle-treated control.

 
Genistein rapidly stimulates NO release in vascular endothelial cells
To confirm the biological importance of this rapid activation of eNOS by genistein, we evaluated the NO production in BAECs. Consistent with the eNOS activation pattern, genistein rapidly stimulated NO release in a concentration-dependent manner, reaching a maximal level at 1 µM genistein (Fig. 7AGo). Preincubating the cells with H89 or L-NAME completely blunted the genistein-induced NO release (Fig. 7BGo).



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  		FIG. 7. Genistein rapidly stimulates NO production. Serum-deprived BAECs were incubated with genistein (10 nM to 10 µM) or vehicle (C) for 10 min (A) or pretreated with L-NAME (NE, 1 mM) or H89 (10 µM) for 30 min before addition of genistein (G, 1 µM) for 10 min (B) and determination of NOx. Data were expressed as mean ± SEM of four separate experiments in triplicate. *, P < 0.05 vs. vehicle-treated control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The cellular and molecular mechanisms underlying the vascular effects of genistein are still unclear. Our results defined a novel signaling pathway and an important cell biological effect for genistein, which may explain its beneficial vascular effects. We found that genistein rapidly stimulated cAMP accumulation and activated eNOS by a PKA-dependent, nongenomic, pathway. The activity of genistein was not inhibited by estrogen receptor blockade and was independent of PI3K/Akt and ERK/MAPK cascades.

The effect of genistein was rapid, with maximal Ser1179-eNOS phosphorylation and eNOS activation in BAECs at 10 min incubation. A similar rapid increase in eNOS activity was seen in human cells (HUVECs), showing that the effect was not species specific. Concentrations as low as 10 nM significantly stimulated eNOS phosphorylation and activity and NOx production. These effects were maximal at 1 µM genistein, a concentration close to that achieved in the plasma of individuals consuming soy products (0.3–0.6 µM) (48). Consistent with our data and the key role of NO in vascular function, genistein (1.8 µM) is an acute, NO-dependent vasodilator of human forearm vasculature (23). Furthermore, healthy postmenopausal women taking genistein for 6 months have increased plasma NOx concentration and enhanced flow-mediated vasodilation in the forearm (29). The response of eNOS activity to genistein declines at 10 µM. This pattern has been noted before in other experimental systems (49, 50) and has been related to activation of competing signaling systems at higher concentrations. Our in vitro data provide a molecular basis for some of these vascular effects of genistein observed in human and animal studies (13).

Endothelial NOS can be regulated at both nongenomic and genomic levels. The rapidity of eNOS activation, and lack of inhibition by cycloheximide or actinomycin D, suggest that genistein regulates NO production by a nongenomic mechanism. Enzyme activation is associated with the phosphorylation status of specific eNOS residues, which depend on the stimulus-specific pattern of protein kinase and phosphatase activation (51). Many protein kinases can modulate eNOS activity including PI3K/Akt, PKA, ERK/MAPK, AMP-activated kinase, and protein kinase G (42, 47, 52). Genistein is reported to affect Akt and MAPK (including ERK1/2 and p38) activities (53, 54). However, genistein-induced eNOS activation was not related to PI3K/Akt or ERK/MAPK activity, which regulates the acute eNOS activation in response to steroids (38), growth factors (42), and shear stress (55). The p38 MAPK is not reported to cause phosphorylation of serine 1179 in eNOS. Although neither Akt nor MAPKs mediate the genistein effects on eNOS, their potential role in other genistein-induced vascular effects, e.g. angiogenesis (9), deserves further study.

The rapid phosphorylation and activation of eNOS was dependent on PKA activation. Genistein also stimulated cAMP accumulation over the same concentration range as eNOS activation and phosphorylation. PKA is generally cAMP dependent, and cAMP stimulates Ser1179-eNOS phosphorylation by PKA-dependent mechanisms (47), suggesting that cAMP is upstream of PKA-mediated eNOS activation by genistein. Our results favor a central role of PKA in mediating eNOS activation by genistein and further demonstrate that genistein stimulates cAMP accumulation to activate eNOS through PKA.

cAMP is a central signaling molecule in a variety of cellular systems. Besides its effect on eNOS, cAMP maintains normal vascular function by inhibiting vascular endothelial (56) and smooth muscle cell proliferation (57), depressing leukocyte adhesion to endothelial cells (58), and maintaining normal endothelial barrier function (59). These events are implicated in various vascular pathologies including atherosclerosis and edema, suggesting that effects on cAMP/PKA may underlie many of the beneficial vascular effects of genistein. Whether vasoprotective effects of genistein in vivo are related to a cAMP/PKA-dependent mechanism warrants further investigation.

The mechanisms underlying the genistein effects on intracellular cAMP accumulation remain to be determined. Genistein may control the degradation of cAMP by inhibiting phosphodiesterases. However, in preliminary studies we found that inhibition of adenylate cyclase significantly attenuated the genistein-induced eNOS activity in isolated plasma membranes of BAECs. Therefore, it is possible that intracellular cAMP accumulation in response to genistein in endothelial cells is mediated, at least in part, by directly acting on the plasma membranes to facilitate receptor-dependent cAMP production involving GTP binding protein (G{alpha}s) and adenylate cyclase. We are currently testing this hypothesis.

Genistein is an inhibitor of tyrosine-specific protein kinases (8) and is often used to study tyrosine kinase-mediated signaling events. However, we do not believe that the rapid activation of eNOS by genistein is related to tyrosine kinase inhibition for the following reasons. First, genistein concentrations that stimulate eNOS activation (10 nM to 10 µM) had no effect on basal or agonist-stimulated tyrosine kinase activity. Genistein inhibits tyrosine kinase only at 100 µM concentration, consistent with previous findings (60). This concentration was 10,000-fold higher than the threshold for genistein-induced eNOS activity or cAMP accumulation. Second, daidzein, an analog of genistein that does not inhibit tyrosine kinases, also stimulated eNOS activity. The effect of daidzein on eNOS was less potent than that of genistein, as previously noted (61). Because tyrosine kinase was not inhibited by genistein at 1 µM, the differences in potency on eNOS must relate to other signaling differences between genistein and daidzein. These may include the greater cellular entry of genistein in comparison with daidzein (62) or the differences in phosphodiesterase subtype inhibition by the two agents (63). Our in vitro studies suggest that the human vasoprotective effects of genistein are not dependent on tyrosine kinase inhibition but are more likely due to a cAMP-dependent pathway because it is unlikely that circulating genistein concentrations larger than 10 µM are achieved in vivo. Rats receiving a daily genistein dose of 20 mg/kg body weight achieve a plasma concentration of only 2.2 µM (64), and the mean plasma concentration of genistein in humans consuming soy products is 0.3–0.6 µM (48). Additionally, these results suggest that genistein should be used with caution in studies of tyrosine kinase signal transduction because increased cAMP and NO may contribute to the observed effects.

Genistein has weak estrogenic effects in some tissues (7). It binds to estrogen receptor (ER)-ß with an affinity comparable with 17ß-estradiol (65) but has a considerably lower affinity for ER{alpha}. Both ERs are present in vascular endothelial cells, and estradiol stimulates acute eNOS activation in an ER-dependent manner in these cells (28). However, our data indicate that the acute activation of eNOS by genistein was independent of ERs. First, the specific ER antagonist ICI 182,780 did not inhibit the effect of genistein but completely blocked estrogen-induced eNOS activation. Second, inhibition of PI3K, which directly interacts with ER to mediate the acute effect of estrogen on eNOS activation (66), completely blocked the estrogen effect but failed to inhibit genistein-induced eNOS activity. Third, H89, a specific PKA inhibitor, inhibited the cellular response to genistein, whereas it had no effect on estrogen-stimulated eNOS activity. Thus, the genistein signaling pathway to eNOS was separate from the estrogen pathway in this study. Estrogen also stimulates acute cellular effects that are not mediated by ER{alpha} or ERß (67). The molecular identity of this alternate estrogen signaling pathway, and its possible relation to the effect of genistein, is unknown.

In summary, genistein acted directly on BAECs and HUVECs to activate eNOS and NO production through nongenomic mechanisms in whole vascular endothelial cells. The intracellular signaling pathways for activation of eNOS by genistein were independent of PI3K/Akt or ERK/MAPK but depended on the cAMP/PKA cascade. In addition, the genistein action on eNOS was not inhibited by an ER antagonist and was unrelated to tyrosine kinase inhibition. The findings suggest a molecular mechanism that may underlie some of the beneficial cardiovascular effects that have been proposed for genistein.


   Footnotes
 
This work was supported by National Institutes of Health (NIH) Grant AG55741 (to J.S.D.), Virginia Polytechnic Institute and State University (ASPIRES to D.L.), American Heart Association (postdoctoral fellowship to D.L.), and the Office of Research and Development, Department of Veterans Affairs.

Current address for D.L.: Department of Human Nutrition, Food, and Exercise, Virginia Polytechnic Institute and State University, Blacksburg, Virginia.

The contents of this paper are solely the responsibility of the authors and do not necessarily represent the official views of the NIH or the Department of Veterans Affairs.

Abbreviations: BAEC, Bovine aortic endothelial cell; EIA, enzyme immunoassay; eNOS, endothelial NO synthase; ER, estrogen receptor; H89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; HBSS, Hanks’ balanced salt solution; HUVEC, human umbilical vein endothelial cell; L-NAME, N{omega}-nitro-L-arginine methyl ester; NO, nitric oxide; NOx, NO3–; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; VEGF, vascular endothelial growth factor.

Received January 28, 2004.

Accepted for publication August 10, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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