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Originally published In Press as doi:10.1074/jbc.M602803200 on July 13, 2006

J. Biol. Chem., Vol. 281, Issue 37, 27335-27345, September 15, 2006
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The Isoflavone Equol Mediates Rapid Vascular Relaxation

Ca2+-INDEPENDENT ACTIVATION OF ENDOTHELIAL NITRIC-OXIDE SYNTHASE/Hsp90 INVOLVING ERK1/2 AND Akt PHOSPHORYLATION IN HUMAN ENDOTHELIAL CELL*

Sheeja Joy{ddagger}12, Richard C. M. Siow{ddagger}1, David J. Rowlands{ddagger}3, Marko Becker{ddagger}, Amanda W. Wyatt{ddagger}4, Philip I. Aaronson§, Clive W. Coen5, Imre Kallo§||5, Ron Jacob{ddagger}, and Giovanni E. Mann{ddagger}6

From the {ddagger}Cardiovascular Division, Reproduction and Endocrinology Division, §Asthma, Allergy, and Lung Biology Division, Schools of Biomedical and Health Sciences and Medicine, King's College London, Guy's Campus, London SE1 1UL, United Kingdom and the ||Laboratory of Endocrine and Behavioural Neurobiology, Institute of Experimental Medicine, Budapest 1083, Hungary

Received for publication, March 24, 2006 , and in revised form, May 22, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We recently reported that soy isoflavones increase gene expression of endothelial nitric-oxide synthase (eNOS) and antioxidant defense enzymes, resulting in improved endothelial function and lower blood pressure in vivo. In this study, we establish that equol (1-100 nM) causes acute endothelium- and nitric oxide (NO)-dependent relaxation of aortic rings and rapidly (2 min) activates eNOS in human aortic and umbilical vein endothelial cells. Intracellular Ca2+ and cyclic AMP levels were unaffected by treatment (100 nM, 2 min) with equol, daidzein, or genistein. Rapid phosphorylation of ERK1/2, protein kinase B/Akt, and eNOS serine 1177 by equol was paralleled by association of eNOS with heat shock protein 90 (Hsp90) and NO synthesis in human umbilical vein endothelial cells, expressing estrogen receptors (ER){alpha} and ERbeta. Inhibition of phosphatidylinositol 3-kinase and ERK1/2 inhibited eNOS activity, whereas pertussis toxin and the ER antagonists ICI 182,750 and tamoxifen had negligible effects. Our findings provide the first evidence that nutritionally relevant plasma concentrations of equol (and other soy protein isoflavones) rapidly stimulate phosphorylation of ERK1/2 and phosphatidylinositol 3-kinase/Akt, leading to the activation of NOS and increased NO production at resting cytosolic Ca2+ levels. Identification of the nongenomic mechanisms by which equol mediates vascular relaxation provides a basis for evaluating potential benefits of equol in the treatment of postmenopausal women and patients at risk of cardiovascular disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The interaction of estrogen with estrogen receptors (ER)7 leads to the transcriptional activation of estrogen-responsive genes (1-3), including eNOS (4) and key antioxidant defense genes (5). In addition to its genomic actions, estrogen rapidly stimulates eNOS activity in cultured endothelial cells. ER{alpha}, localized in plasmalemmal caveolae, has been implicated in the rapid activation of eNOS via pathways involving ERK1/2 and PI 3-kinase/Akt (6-12). In pulmonary artery endothelial cells, coupling of ER{alpha} to G{alpha}i activates downstream signaling pathways leading to NO production (13). Inhibition of the functional association of the chaperone protein Hsp90 with eNOS abolishes 17beta-estradiol-stimulated NO release (14), and studies in HUVEC have shown that fluid shear stress stimulates phosphorylation of eNOS via PI 3-kinase/Akt, leading to increased NO production independent of cytosolic Ca2+ mobilization (15). A similar Ca2+ insensitivity has been reported for eNOS activation by 17beta-estradiol (E2) in HUVEC (16), although in bovine and human aortic endothelial cells E2 appears to elevate intracellular Ca2+ (17, 18).

Genistein and daidzein are polyphenolic isoflavones contained in soy protein (19), and intestinal bacteria metabolize daidzein to equol. Isoflavones are structurally similar to estrogen and generally bind with higher affinity to estrogen receptor beta (ERbeta) compared with ER{alpha} (20). In humans consuming a soy-deficient diet, plasma concentrations of genistein are <40 nM but can reach 4 µM in Japanese consuming a soy-rich diet (19, 21). Dietary phytoestrogens have been associated with a favorable cardiovascular risk profile in postmenopausal women (22). However, the limited number of clinical trials only describe marginal benefits of isoflavones in healthy postmenopausal women, highlighting the need for further studies with postmenopausal women at risk of diabetes, cardiovascular disease, breast cancer, and osteoporosis (23).

Long term treatment with genistein improves endothelial function in humans and animal models with mild to moderate hypertension (24-27), whereas acute treatment enhances flow-mediated dilation and endothelium-dependent relaxation (28-32). Isoflavones may thus affect vascular tone by regulating either eNOS expression and/or bioavailability of NO (33). We recently reported that a soy protein diet rich in isoflavones increases the expression of eNOS and key antioxidant defense genes in aged male rats, resulting in improved NO-dependent relaxation and reduced arterial blood pressure in vivo (34).

Our present findings provide the first evidence that equol (0.1-100 nM) causes rapid activation of ERK1/2 and NO release in HUVEC and HAEC at basal cytosolic Ca2+ levels and NO-dependent relaxation. Equol-stimulated phosphorylation of Akt and eNOS serine 1177 was rapid (2 min) and accompanied by a dissociation of eNOS from caveolin-1 and association with Hsp90. Rapid activation of eNOS by equol was abrogated following inhibition of ERK1/2 and PI 3-kinase, and was not substantially affected by inhibitors of G protein-coupled receptors, Src family kinases, or the estrogen antagonists ICI 182,780 and tamoxifen.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Endothelial Cell Culture—Human umbilical vein endothelial cells (HUVEC) were isolated by collagenase digestion (1 mg ml-1) and cultured in low phenol red M199 containing 10% (v/v) fetal calf serum (FCS), 10% (v/v) newborn calf serum, 5 mM L-glutamine, and endothelial cell growth factor (20 µg ml-1) (35, 36). Human aortic endothelial cells (HAEC, Clonetics, Berkshire, UK) were initially maintained in EGM-2 medium (Clonetics) and then passaged in low phenol red M199.

Detection of Estrogen Receptors ER{alpha} and ERbeta—HUVEC were seeded into chamber slides and cultured overnight in M199. For light microscopy, cells were fixed with 4% paraformaldehyde for 10 min. Fixative was replaced with 0.01 M Kreb's phosphate-buffered saline (KPBS); one group of cells was permeabilized with KPBS containing 0.1% Triton X-100 for 10 min to facilitate penetration of reagents and another group remained nonpermeabilized. After blocking with 5% bovine serum albumin (BSA), cells were incubated with primary antibodies raised against the carboxyl terminus of human ER{alpha} (amino acids 582-595; Calbiochem, 0.5 µg ml-1) or human ERbeta (amino acids 458-477; Zymed Laboratories Inc.; D7N, 0.5 µg/ml) for 48 h (37). Biotinylated horse anti-mouse IgG (1:500) or goat anti-rabbit IgG (1:500; Vector Laboratories), respectively, were applied as secondary antibodies for 3 h, followed by avidin-biotinylated enzyme complex for 2 h. Nickel-enhanced diamino-benzidine was used to detect the immunoreactive loci in cells. After removing the chambers, HUVEC were air-dried and cover-slipped with DEPEX.

For double labeling, cells were fixed with 4% paraformaldehyde (10 min) and then placed in 0.01 M PBS followed by application of ER{alpha} and ERbeta antibodies together for 48 h. Alexa 488-conjugated goat anti-rabbit IgG and Alexa 568-conjugated goat anti-mouse IgG (1:500) were mixed and applied together for 16 h, and cells were air-dried and cover-slipped with anti-fading agent (SlowFade®, Molecular Probes).

For electron microscopy, HUVEC were treated with sucrose solutions in 0.01 M KPBS (10-30% for 10 min). After three cycles of rapid freezing and thawing, cells were transferred to 10% sucrose and washed with KPBS. After blocking with 5% BSA, cells were incubated with rabbit anti-human ER{alpha} (amino acids 458-477; Zymed Laboratories Inc.; D7N, 0.5 µg/ml) for 48 h. Cells were thoroughly rinsed with KPBS and blocked with a mixture of 0.1% cold water fish gelatin (Electron Microscopy Sciences, Hatfield, PA) and 1% BSA in KPBS. Nanogold goat anti-rabbit IgG, Fab' fragment (Molecular Probes) diluted in the same blocking solution 1:100, was used for 3 h followed by glutaraldehyde fixation (1.25%) in KPBS. Cells were washed in 0.2 M sodium citrate, pH 7.5, and gold particles were silver intensified with IntenSE kit (Amersham Biosciences). Cells were treated with 1% osmium tetroxide in 0.1 M phosphate buffer for 30 min, dehydrated in ascending series of ethanol, embedded in Durcupan ACM epoxy resin (Fluka), and polymerized at 56 °C for 2 days. Ultrathin 50-60 nm thin sections were cut using a Leica ultracut UCT ultramicrotome; ribbons were collected onto Formvar-coated single slot grids and examined with a JEOL electron microscope.

Immunocytochemical controls revealed no punctate staining after processing cells without primary antibodies (Fig. 1, B and D) or when ER antibodies were pre-absorbed with peptide antigens or recombinant proteins.

Single Cell [Ca2+]i Measurements—HUVEC were loaded with 1 µM fura 2-AM for 60 min in HEPES-buffered Dulbecco's modified Eagle's medium containing 20% FCS and then maintained in HEPES-buffered balanced salt solution (36, 38). Fura 2-AM-loaded cells were mounted on a thermostated stage (37 °C) of a Zeiss Axiovert 135 inverted microscope with a x40 oil immersion objective and excited alternately at 350 and 380 nm via a LEP dual filter wheel system (Ludl, Hawthorne, NY). Fluoresced light, >420 nm, was captured with a 14-bit cooled CCD camera (Hamamatsu C4880-80) using Openlab software (Improvision, Coventry, UK). An image pair was captured every 1-3 s, and the ratio of fluorescence at 350 and 380 nm excitation was used as a measure of intracellular calcium [Ca2+]i.

Relaxation of Isolated Aortic Rings—Thoracic aortae from male adult Wistar rats were rapidly excised, cleaned of connective tissue, and ~2 mm rings mounted onto isometric force transducers in a jacketed (37 °C) organ bath containing Krebs-Henseleit solution (mM: NaCl 118, KCl 6, NaHCO3 25, NaH2PO4 1.2, HEPES 10, D-glucose 10, CaCl2 1.6, MgSO4 1.2, pH 7.4) gassed with 95% O2 in 5% CO2 (31, 34). Resting tension (1 g) was applied to endothelium intact rings, and endothelial function was confirmed by relaxation of phenylephrine (PE, 1 µM) preconstricted rings to acetylcholine (10-6 M). After equilibration in buffer for 1 h, rings were pretreated with vehicle or the NOS inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME, 100 µM) and constricted with 1 µM PE, and relaxation to increasing doses of equol (0.01-10 µM) was monitored.

Intracellular cGMP Accumulation—Basal and stimulated cGMP levels were determined by radioimmunoassay, with inhibition of cGMP accumulation by L-NAME (100 µM, 15 min) serving as an index of NO production (36). Confluent monolayers were washed twice with warmed Krebs-Henseleit buffer and then preincubated for 15 min with buffer containing L-arginine (100 µM) and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 500 µM). Cells were then stimulated (30 s to 15 min) with 1-100 nM equol, daidzein, or genistein in the presence of IBMX and L-arginine (100 µM), and responses were compared with E2 or E2 conjugated to bovine albumin (E2-BSA).


Figure 1
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  		FIGURE 1.
Localization of ER{alpha} and ERbeta immunoreactivity in HUVEC. Cells were incubated with antibodies to ER{alpha} and ERbeta. Weak fixation together with Triton X-100 treatment facilitated penetration of immunoreagents into cell nuclei, and nuclei immunopositive for ER{alpha} (A)orERbeta (C) are shown with dotted immunoreactivity scattered in the chromatin. Using the antibody diluent (2% BSA) instead of the primary antibodies during immunocytochemical procedures (B-D) or preabsorption of primary antibodies with the corresponding peptide antigens (not shown) abolished staining of cells. Stronger fixation conditions together with omission of Triton X-100 from preincubation steps preserved IR in extranuclear sites but reduced nuclear signals for estrogen receptors; thus IR for ER{alpha} (E)orERbeta (F) appears as faint dots throughout the cell. Under these conditions, double label immunofluorescence for the two receptors (G) shows limited overlapping of immunoreactive loci (red dots represent ER{alpha}-IR and green dots ERbeta-IR); the white rectangle (G) is shown at higher magnification (H). At the EM level, ERbeta-IR was revealed by silver-intensified gold particles in the cell membrane (white arrows, I). n, nucleus. Black rectangle in I is shown at higher magnification in J. K, cytosolic Ca2+ ([Ca2+]i) levels in HUVEC are unaffected by acute exposure to 10 and 100 nM equol but elevated biphasically in response to histamine. Images represent simultaneous measurements taken from a range of 6-61 cells at any one time in two different cell cultures.

 
In other experiments, cells were pretreated with inhibitors of PI 3-kinase ([2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride], LY294,002, 10 µM, 30 min), MEK1/2 (1,4-diamino-2,3-dicyano-1,4-bis-[aminophenylthio]butadiene, U0126, 1 µM, 30 min), Src kinase (4-amino-5-(4-chlorophenyl)-7(t-butyl)pyrazolo-[3,4-d]pyrimidine (PP2, 10 µM, 30 min), pertussis toxin (100 ng ml-1, 120 min), or the estrogen receptor antagonists ICI 182,780 (10 µM, 30 min) and tamoxifen (10 µM, 60 min). Pretreated cells were then stimulated acutely with isoflavones or E2 in the presence of L-arginine (100 µM) and IBMX (0.5 mM) and vehicle or inhibitor(s). Prostacyclin (PGI2) release into the supernatant was analyzed by radioimmunoassay of 6-keto-PGF1{alpha}, the stable metabolite of PGI2 (36).

Immunoblotting Phosphorylated ERK1/2, Akt, and eNOS Ser1177— HUVEC were equilibrated in M199 containing 1% FCS for 4 h and then preincubated with Krebs-Henseleit buffer containing L-arginine (100 µM) and IBMX (0.5 mM). In other experiments, HUVEC were pretreated with vehicle or U0126 (1 µM, 30 min) or LY294,002 (10 µM, 30 min) and then stimulated for 30 s to 5 min with equol, using thrombin (1 unit/ml, 2 min) as an internal control. We reported previously (36) that the structurally distinct MEK1/2 inhibitors U0126 and PD98059 do not have inhibitory effects on p38MAPK or c-Jun NH2-terminal kinase (JNK) pathways in HUVEC. Reactions were stopped with ice-cold PBS containing 200 µM sodium orthovanadate, and cell lysates were separated by SDS-PAGE, and proteins were electrotransferred onto polyvinyldifluoride membranes and then probed with a polyclonal antibody against dually phosphorylated (threonine 183/tyrosine 185) ERK1/2 or phosphospecific antibodies against Akt or eNOS serine 1177. Protein bands were detected by ECL, and densitometric analyses were performed using Scion Image software (Scion Corp.).

Immunoprecipitation of eNOS, Association with Hsp90, and Dissociation from Caveolin-1—HUVEC in T75 culture flasks were equilibrated in M199 containing 1% FCS for 4 h prior to treatment for 2 min with equol (100 nM) or thrombin (1 unit ml-1) in Krebs-Henseleit buffer containing L-arginine (100 µM) and IBMX (0.5 mM). Cells were washed with ice-cold PBS, and cell lysates were collected in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing a protease inhibitor mixture (Sigma). Lysates were centrifuged at 13,000 x g for 10 min, and supernatants were incubated overnight at 4 °C with ExactaCruz immunoprecipitation matrix (Santa Cruz Biotechnology) precoated with an eNOS antibody. Immobilized immune complexes were washed twice with RIPA buffer, eluted from the matrix by boiling for 5 min in SDS lysis buffer, and analyzed by SDS-PAGE using antibodies against eNOS, caveolin-1, and Hsp90.

Cell Culture Reagents and Materials—All cell culture reagents, collagenase type II from Clostridium histolyticum,E2, E2-BSA, histamine, thrombin, IBMX, rolipram, L-NAME, ICI 182,780, tamoxifen, and pertussis toxin were obtained from Sigma; equol was from Apin Chemicals Ltd. (UK); daidzein and genistein were from Alexis Biochemicals; fura-2 acetoxymethyl ester (fura2-AM) was from Molecular Probes (Cambridge Bioscience, UK); U0126, LY294,002, and antibodies against ERK1/2 were from Promega (Southampton, UK); antibodies against eNOS, Hsp90, and caveolin-1 were from Santa Cruz Biotechnology, and {alpha}-tubulin was from Chemicon. ECL reagents, cyclic AMP, 125I-labeled 3',5'-cyclic GMP, and prostacyclin EIA kits were from Amersham Biosciences.

Statistical Analysis—Data are expressed as means ± S.E. of measurements in 3-7 different endothelial cell cultures. Statistical analysis was performed by use of analysis of variance followed by a Student's t test, and a level of p < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Intracellular Localization of ER{alpha} and ERbeta in HUVEC—Mild fixation and permeabilization with Triton X-100 were used to detect ER{alpha} or ERbeta immunoreactivity within the nucleus of HUVEC (Fig. 1, A and C), with staining absent when the primary antibody was omitted (Fig. 1, B and D). Stronger fixation of cells not treated with Triton X-100 showed lower signal intensity within the nucleus, but immunoreactive loci were preserved throughout the cytoplasm (Fig. 1, E and F). Doubly labeled immunofluorescence showed ER{alpha} and ERbeta reactivity in different loci of the cell (Fig. 1, G and H), and electron microscopy revealed ERbeta reactivity in the nucleus and cytoplasm of cells, with a few immunoreactive loci also detected at the plasma membrane (Fig. 1, I and J).


Figure 2
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  		FIGURE 2.
Equol mediates endothelium- and NO-dependent relaxation of rat aortic rings. Endothelium-intact aortic rings were preconstricted with PE (1 µM) and challenged with cumulative doses of vehicle or equol (0.1-10 µM). Rings were pretreated for 30 min with vehicle (A) or the NOS inhibitor L-NAME (100 µM, B) and then challenged with equol. Original contraction-relaxation response curves are shown for a single experiment. B, summary of equol-mediated relaxation expressed as a percentage of the maximal PE-induced vasoconstriction. Means ± S.E., *, p < 0.05 versus vehicle-treated rings in three different animals.

 
Acute Effects of Equol and Histamine on Mobilization of [Ca2+]i—To examine whether equol acutely alters [Ca2+]i in human endothelial cells, fura 2-AM (1 µM)-loaded HUVEC were challenged for 2 min with equol and then histamine. As shown in Fig. 1K, equol (10 or 100 nM) failed to elevate [Ca2+]i, whereas histamine (100 µM) evoked a classical biphasic increase in [Ca2+]i (36, 38). Closer examination of single cells revealed no localized [Ca2+]i responses to equol. Similar responses were observed following acute (2 min) application of 17beta-estradiol (data not shown), confirming previous findings in HUVEC (16).

Equol Causes Rapid Endothelium- and NO-dependent Relaxation—Endothelium intact aortic rings were preconstricted with PE (1 µM) and exposed to cumulative doses of equol. As shown in representative original records (Fig. 2, A and B), equol caused a dose-dependent relaxation that was inhibited by pretreatment with L-NAME (100 µM). Fig. 2C summarizes the results from three different animals.

Isoflavones Do Not Acutely Increase cAMP or PGI2 Release— Treatment of HAEC (data not shown) or HUVEC for 2 min with 100 nM equol, daidzein, or genistein had no significant effect on intracellular cAMP levels or PGI2 production, whereas forskolin and histamine increased cAMP levels and PGI2 production, respectively (Table 1).


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  		TABLE 1
Acute effects of equol, daidzein, and genistein on intracellular cAMP levels or PGI2 production in HUVEC Confluent cells were preincubated with Krebs-Henseleit buffer containing the cAMP-specific phosphodiesterase inhibitor rolipram (10 µM) and then stimulated for 2 min with 100 nM equol, daidzein, or genistein, using forskolin (1 µM, 2 min) and histamine (10 µM, 5 min) as positive controls for cAMP or PGI2 production, respectively. Data denote means ± S.E. of triplicate measurements in each of 3–6 different cell cultures, **, p < 0.01 (histamine); ***, p < 0.001 (forskolin) versus control. ND, not determined.

 
Rapid Activation of ERK1/2 and eNOS by Equol, Daidzein, and Genistein—Because ERK1/2 has been implicated in the activation of eNOS by estrogen (6, 10, 12), we compared acute effects of E2, equol, daidzein, and genistein on EKR1/2 phosphorylation in HUVEC and HAEC. Treatment with isoflavones (100 nM) led to a rapid (2 min) phosphorylation of ERK1/2 (Fig. 3, A and C) in both cell types, and densitometric analyses of different cell cultures are shown in Fig. 3, B and D.

We reported previously that activation of A2a-purinoceptors results in rapid phosphorylation of ERK1/2 and NO production in HUVEC independent of detectable increases in cytosolic Ca2+ (36). To examine whether equol, daidzein, or genistein also acutely stimulate NO synthesis in endothelial cells, we treated HUVEC and HAEC with these isoflavones (100 nM, 2 min) and measured changes in intracellular cGMP accumulation in the absence or presence of L-NAME (100 µM). Equol, daidzein, and genistein (and E2) evoked rapid increases in cGMP accumulation, which were inhibited by L-NAME (Fig. 3E).


Figure 3
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  		FIGURE 3.
Isoflavones acutely activate ERK1/2 and eNOS in HUVEC and HAEC. Serum-deprived endothelial cells were incubated in Krebs-Henseleit buffer containing L-arginine (100 µM) and then stimulated for 2 min with vehicle control (Ctrl) (0.01% Me2SO) or 100 nM equol (Eq), daidzein (Daid), genistein (Gen)or E2, using thrombin (Thr) (1 unit ml-1) as a positive control. Representative immunoblots of dually phosphorylated ERK1/2 relative to {alpha}-tubulin in HUVEC (A) and HAEC (C) cell lysates with densitometric analyses of three different HUVEC (B) and HAEC (D) cultures shown. All values for stimulated ERK1/2 phosphorylation in B and D are significantly different from control (means ± S.E., n = 4, p < 0.05). E, basal and stimulated cGMP levels in HUVEC monolayers pretreated for 15 min with vehicle or L-NAME (100 µM) and then stimulated for 2 min with 100 nM equol, daidzein, genistein, or E2 in the continued absence (filled bars) or presence (unfilled bars)of L-NAME (100 µM). cGMP accumulation is expressed as means ± S.E. or replicate measurements in each of 3-7 different cell cultures. *, p < 0.05 versus control; {diamondsuit}, p < 0.05 versus respective treatments in absence of L-NAME.

 
We then further examined concentration- and time-dependent actions of equol on ERK1/2 phosphorylation and cGMP accumulation in HUVEC. ERK1/2 was phosphorylated by equol concentrations as low as 1 nM (data not shown), with phosphorylation at higher concentrations (100 nM) detected within 30 s and maintained for up to 5 min (Fig. 4, A and B). Activation of ERK1/2 was paralleled by an increase in intracellular cGMP, with maximal NO release achieved at 2 min (Fig. 4C). All subsequent experiments were performed using 100 nM isoflavones or E2.

ERK1/2 Inhibition Abrogates Equol-mediated NO Synthesis
Preincubation of HUVEC with the MEK1/2 inhibitor U0126 (1 µM, 30min) abolished ERK1/2 phosphorylation in response to acute treatment with 100 nM equol, daidzein, genistein, or E2 (Fig. 5A), with similar findings observed in HAEC (data not shown). Under the same experimental conditions, U0126 (1 µM) abrogated equol-, daidzein-, genistein-, and E2-stimulated increases in cGMP accumulation (Fig. 5B).


Figure 4
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  		FIGURE 4.
Equol stimulates acute ERK1/2 phosphorylation and NO production in HUVEC. A, serum-deprived cells were stimulated for 2 min with vehicle or equol (0.1 and 100 nM), and lysates were immunoblotted for phosphorylated ERK1/2 relative to {alpha}-tubulin. A, time course of ERK1/2 activation by 100 nM equol with densitometric analysis of three different cell cultures as shown in B. C, time course of cGMP accumulation induced by 100 nM equol. Data are expressed as means ± S.E. of measurements in each of four (ERK1/2) or seven (cGMP) different cell cultures. Stimulated cGMP levels (1-5 min) are significantly different versus control (Ctrl) at t = 0 min, p < 0.05.

 
Equol Acutely Phosphorylates Akt and eNOS Serine 1177— Previous studies with human endothelial cells reported that E2 rapidly activates eNOS via a PI 3-kinase/Akt-dependent mechanism (8-11), involving ERK1/2 activation (6, 10, 12). To determine whether equol-stimulated NO synthesis was mediated via PI 3-kinase/Akt, HUVEC were treated with equol (100 nM) or thrombin (1 unit ml-1), and lysates were probed with phosphospecific antibodies. Equol-induced phosphorylation of Akt and eNOS was detected within 2 min (Fig. 6, A and B) and maintained for up to 10 min (data not shown). Densitometric analyses of immunoblots from different HUVEC cultures are shown in Fig. 6, C and D. To establish whether cross-talk exists between ERK1/2 and PI 3-kinase/Akt signaling pathways, HUVEC were pretreated with U0126 or LY294,002, and phosphorylation of Akt and ERK1/2 was determined. Inhibition of ERK1/2 resulted in a significant attenuation of equol-stimulated Akt phosphorylation (Fig. 6E), and similarly inhibition of PI 3-kinase attenuated ERK1/2 phosphorylation (Fig. 6F). Moreover, pretreatment with U0126 and LY294,002 (alone or in combination) markedly inhibited thrombin (data not shown), and equol mediated phosphorylation of eNOS (Fig. 6G).


Figure 5
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  		FIGURE 5.
Effects of MEK1/2 inhibition on acute activation of ERK1/2 and eNOS by equol and other isoflavones. Serum-deprived HUVEC were pretreated with U0126 (U) (1 µM. 30 min) and then stimulated for 2 min with vehicle or 100 nM equol (Eq), daidzein (Daid), genistein (Gen), or E2 in the absence or presence of U0126 (1 µM). A, cell lysates were immunoblotted for dually phosphorylated ERK1/2 and {alpha}-tubulin. Blot is representative of experiments in two different cell cultures. B, HUVEC were treated for 2 min with 100 nM isoflavones or E2 in the absence (filled bars) or presence (unfilled bars) of U0126 (1 µM, 30 min), and changes in cGMP accumulation are expressed as percentage change over control (basal cGMP levels ranged from 0.6 to 12.3 pmol (106 cells)-1). Data are expressed as means ± S.E. of duplicate measurements in each of four different cell cultures. *, p < 0.01 versus control (Ctrl); {diamondsuit}, p < 0.05 versus stimulation.

 
Equol Acutely Modulates Interactions of eNOS with Hsp90 and Caveolin-1—As eNOS activity is regulated by post-translational modifications, involving phosphorylation by Akt and interactions with regulatory proteins such as Hsp90 (14, 39, 40), we hypothesized that equol would acutely modulate eNOS interactions with caveolin-1 and Hsp90. Treatment of HUVEC for 2 min with equol (100 nM) or thrombin (1 unit ml-1) resulted in a rapid dissociation of eNOS from caveolin-1 and association with Hsp90 (Fig. 6H).

Effect of Pertussis Toxin on Isoflavone-stimulated ERK1/2 Phosphorylation and cGMP Accumulation—Previous studies have shown that activation of eNOS by E2 requires plasma membrane ER{alpha} coupling to G{alpha}i, leading to activation of downstream signaling events (13). To evaluate the potential role of G proteins in equol-stimulated eNOS activity, HUVEC were pretreated for 120 min with vehicle or pertussis toxin (PTX) and exposed to equol (100 nM) for 2 min. Pertussis toxin treatment inhibited ERK1/2 phosphorylation in response to equol, daidzein, E2, and E2-BSA, whereas stimulated cGMP accumulation was decreased only marginally (Fig. 7B).

Effect of Src Inhibition on ERK1/2 and eNOS Activation—Src kinase has been implicated in the activation of Akt and eNOS in HUVEC in response to E2 (41). To examine the potential involvement of Src kinase in the actions of equol and other isoflavones, HUVEC were pretreated for 30 min with an Src kinase inhibitor PP2 (10 µM). PP2 abrogated acute phosphorylation of ERK1/2 in response to equol, daidzein, and genistein (100 nM, 2 min) but had negligible effects on stimulated NO release (data not shown).


Figure 6
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  		FIGURE 6.
Equol acutely stimulates phosphorylation of Akt and eNOS serine 1177 and association of Hsp90 with eNOS in HUVEC. Serum-deprived cells were incubated in Krebs-Henseleit buffer containing L-arginine (100 µM) and IBMX (0.5 mM) and treated for 2 min with vehicle or 100 nM equol (Eq) or thrombin (Thr) (1 unit ml-1, positive control). A and B, representative immunoblots of Akt and eNOS serine 1177 phosphorylation relative to {alpha}-tubulin. C and D, densitometric analyses of Akt and eNOS serine 1177 phosphorylation in three different cell cultures. E, equol (2 min, 100 nM) stimulated Akt phosphorylation in cells pretreated with vehicle (Ctrl) or the PI 3-kinase inhibitor LY294,002 (LY) (10 µM, 30 min) and/or MEK1/2 inhibitor (U) (U0126, 1 µM, 30 min). F, equol stimulated ERK1/2 phosphorylation in cells pretreated with vehicle or LY294,002 and/or U0126. G, inhibition of equol stimulated eNOS phosphorylation following pretreatment of cells with vehicle or LY294,002 and/or U0126. H, immunoprecipitation (IP) of eNOS followed by Western blotting for Hsp90 and caveolin-1 reveals that equol induces a rapid dissociation of eNOS from caveolin-1 and association with Hsp90. Immunoblots in E-H are representative of similar blots obtained in 3-4 different cell cultures. Means ± S.E. *, p < 0.05 versus control (Ctrl).

 
Estrogen Receptor Antagonists Do Not Inhibit Acute Activation of ERK1/2 and eNOS by Equol and Other Isoflavones—In view of previous reports for E2 (11), we hypothesized that estrogen receptor antagonists would inhibit isoflavone-stimulated activation of ERK1/2 and eNOS. Pretreatment of HUVEC with ICI 182,780 (10 µM) or tamoxifen (10 µM) had no effect on acute (2 min) phosphorylation of ERK1/2 or cGMP accumulation in response to 100 nM equol, daidzein, genistein, or E2 (Fig. 8, A and B).

To further evaluate whether estrogen receptor antagonists affected equol and E2-stimulated cGMP accumulation over a longer time interval, we pretreated HUVEC with ICI 182,780 (10 µM) and then measured intracellular cGMP after 15 min of treatment with either equol or E2. As shown in Fig. 8C, ICI 182,780 inhibited E2 but not equol-stimulated cGMP production, confirming earlier reports of inhibition of E2-stimulated eNOS activity (after 15-30 min) in human endothelial cells by ER antagonists (9, 11, 14).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have demonstrated that nutritionally relevant plasma concentrations of equol cause rapid activation of ERK1/2 and Akt in human endothelial cells, leading to phosphorylation of eNOS, NO release, and endothelium-dependent relaxation of aortic rings. Although HUVEC express both ER{alpha} and ERbeta, acute stimulation of NO synthesis was insensitive to inhibition by the ER antagonists ICI 182,780 and tamoxifen. Rapid activation of eNOS in HUVEC by equol at basal cytosolic Ca2+ levels is consistent with previous reports of shear stress and E2-stimulated NO production at low intracellular Ca2+ (15, 16). Moreover, as reported for E2 (14, 39, 40), equol causes a rapid dissociation of eNOS from caveolin-1 and association with the chaperone protein Hsp90. To our knowledge, our findings provide the first evidence that equol, a metabolite of the isoflavone daidzein, enhances association of Hsp90 with eNOS in human endothelium.

We have shown, for the first time, a similar distribution of ER{alpha} and ERbeta immunoreactivity in HUVEC, confirming earlier reports of immunocytochemical staining (42) and detection of mRNA and protein for ER{alpha} in HUVEC (14). After mild fixation, immunoreactivity for both receptors appears in the nuclei of HUVEC, implicating a genomic function. Fixation of cells without permeabilization revealed punctate immunoreactivity over both extranuclear and nuclear sites. Such strategy has been used to detect estrogen receptors at extranuclear sites in other cell types (43). Because ER{alpha} and ERbeta can form heterodimers (44-46), we further examined whether receptors were co-localized at immunoreactive sites. Negligible co-localization at nuclear or extranuclear sites suggests that formation of ER complexes may not required for acute activation of eNOS in HUVEC in response to equol or E2. A study of en face arterial endothelium has also revealed negligible co-localization of ER{alpha} and ERbeta at extranuclear or nuclear sites (45), and dimerization of ER{alpha} is not required for rapid ER{alpha} coupling to eNOS in ovine endothelial cells (46).


Figure 7
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  		FIGURE 7.
Effects of pertussis toxin on isoflavone-stimulated activation of ERK1/2 and eNOS in HUVEC. Serum-deprived cells were pretreated with PTX (100 ng ml-1, 120 min) and then stimulated for 2 min with vehicle or 100 nM equol or E2-conjugated to bovine serum albumin (E2-BSA). A, immunoblot for phosphorylated ERK1/2 relative to {alpha}-tubulin is representative of two different cell cultures. B, PTX (100 ng ml-1, 120 min) pretreatment attenuates, but does not abolish, isoflavone- or E2-BSA (100 nM, 2 min)-stimulated cGMP accumulation. Data are expressed as means ± S.E. of replicate measurements in each of four different cell cultures. All stimulated cGMP levels were significantly different from control (Ctrl) (p < 0.05).*, p < 0.01 versus stimulation with Daid in absence of PTX.

 


Figure 8
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  		FIGURE 8.
Acute stimulation of NO production in HUVEC by equol and other isoflavones is unaffected by estrogen receptor antagonists. Cells were pretreated with the estrogen receptor antagonists ICI 182,780 (10 µM, 30 min) or tamoxifen (10 µM, 30 min), incubated in Krebs-Henseleit buffer containing L-arginine (100 µM) and IBMX (0.5 mM), and then stimulated for 2 min with 100 nM equol (Eq), daidzein (Daid), genistein (Gen), or E2 in the continued presence of the estrogen receptor antagonists. A, cell lysates were immunoblotted for dually phosphorylated ERK1/2 (not shown), and bar graph summarizes densitometric analysis of data from three different cell cultures. B, pretreatment of cells with ICI 182,780 (10 µM) or tamoxifen (10 µM) does not inhibit acute stimulation of cGMP accumulation by Eq, Daid, or E2 (100 nM, 2 min). Control basal cGMP values ranged from 2 to 6.6 pmol (106 cells)-1. C, effects of prolonged treatment with equol or E2 (100 nM, 15 min) on cGMP accumulation in cells pretreated with vehicle or ICI 182,780 (10 µM). Changes in cGMP accumulation are expressed as a percentage of control and denote means ± S.E. of duplicate measurements in each of four different cell cultures. All stimulated values in A and B were significantly different from control (Ctrl)(p < 0.05). C, *, {diamondsuit}, p < 0.05 versus control (Ctrl). Control cGMP values ranged from 2 to 6.6 pmol (106 cells)-1.

 
The distribution of ERbeta immunoreactivity in HUVEC was independent of the markers used (nickel-enhanced diamino-benzidine, fluorochromes, or ultrasmall gold particles intensified with silver), and the latter marker allowed an accurate determination of the subcellular location of ERbeta. Silver-intensified gold grains representing ERbeta were detected in the nucleus and cytoplasm of cells, with some labeling at the cell membrane. Previous studies have identified and implicated membrane ERs in nongenomic activation of signaling pathways (46-50). Further studies are required to establish whether ERbeta or ER{alpha} associate with caveolar protein complexes in HUVEC, particularly in view of reports that subpopulations of ERbeta, ER{alpha}, and alternatively spliced and truncated ER{alpha} isoforms appear to be coupled to eNOS in ovine and EAhy.926 endothelial cells (8, 46-48).

We have shown previously in HUVEC that vasoactive agonists such as histamine activate eNOS via an increase in cytosolic Ca2+ (38), whereas A2a-purinoceptor stimulation elicits rapid Ca2+-independent and ERK1/2-dependent NO release (36). We report here that acute treatment of HUVEC with equol does not alter cytosolic Ca2+ (Fig. 1K), reminiscent of E2-mediated activation of eNOS independent of Ca2+ mobilization (16). However, acute regulation of NO synthesis varies in endothelial cells from different vascular beds or species, because E2 causes a transient rise in cytosolic Ca2+ in bovine aortic and human arterial endothelial cells (17, 18). Ca2+-independent activation of eNOS in HUVEC in response to equol (Fig. 1K), E2 (14, 16), adenosine (36), or shear stress (15) may reflect the ability of phosphorylation to activate eNOS at resting cytosolic Ca2+ levels by enhancing electron flux through the reductase domain of the enzyme and reducing calmodulin dissociation (51).

We have also demonstrated that nanomolar concentrations of equol, daidzein, and genistein acutely (30 s to 2 min) stimulate phosphorylation of ERK1/2 and NO synthesis in HUVEC and HAEC. Increases in NO synthesis were inhibited by the NOS inhibitor L-NAME. As eNOS inhibition had no effect on equol-stimulated ERK1/2 phosphorylation (data not shown), this suggests that activation of ERK1/2 lies upstream of eNOS. Inhibition of ERK1/2 with U0126 significantly attenuated equol-stimulated eNOS serine 1177 phosphorylation and NO production. Previous studies established that E2 causes a rapidphosphorylation of ERK1/2 and eNOS (6-12), but to our knowledge this is the first report that the isoflavones equol, daidzein, and genistein stimulate ERK1/2 and eNOS phosphorylation in human endothelium within 2 min.


Figure 9
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  		FIGURE 9.
Signaling pathways involved in the rapid activation of eNOS and NO production by equol, daidzein, and genistein in human endothelial cells. Equol and other isoflavones acutely stimulate eNOS activity at basal cytosolic Ca2+ levels via ERK1/2 and PI 3-kinase/Akt-dependent pathways. Equol-stimulated release of NO is paralleled by a rapid dissociation of eNOS from caveolin-1 and association with the scaffolding protein Hsp90. Although ER{alpha} and ERbeta are expressed in HUVEC, the rapid actions (≤2 min) of equol, daidzein, and genistein appear not to be mediated via the coupling of ER receptors to G{alpha}i in membrane caveolae or dependent on activation of Src family kinases.

 
Activation of the serine/threonine kinase Akt, a downstream target of PI 3-kinase, leads to rapid phosphorylation of eNOS and NO release (6-12, 16, 52, 53). When we examined the effect of a PI 3-kinase inhibitor in HUVEC, we found that LY294,002 markedly inhibited equol-stimulated Akt (Fig. 6E) and eNOS serine 1177 phosphorylation. Pretreatment of HUVEC with inhibitors of both PI 3-kinase and ERK1/2 completely abrogated eNOS phosphorylation (Fig. 6G), highlighting the importance of PI 3-kinase/Akt signaling in eNOS phosphorylation (9, 11, 52, 53). E2 rapidly induces Src kinase activity in the EAhy.926 endothelial cell line, with an ER/c-Src complex associating with p85, the regulatory subunit of PI 3-kinase, to increase PI 3-kinase activity (41). However, although the Src inhibitor PP2 abolished equol-, daidzein-, and genistein-stimulated ERK1/2 phosphorylation, NO production was unaffected.

Association of Hsp90 with eNOS is critically important for eNOS-mediated NO production (14, 39), and post-translational regulation of eNOS by tyrosine kinases and Hsp90 inhibits uncoupling of the enzyme and eNOS-dependent Formula generation (54). We have shown that rapid (2 min) association of Hsp90 with eNOS induced by equol is accompanied by NO release. Studies in porcine aortic endothelial cells have implicated AMP-activated protein kinase (AMPK) in eNOS activation by 100 nM E2 and suggest that catechol conversion of E2 leads to AMPK activation and association of Hsp90 with eNOS (40). Interestingly, E2-mediated AMPK activation is unaffected by inhibition of Src family kinases, PI 3-kinase, ERK1/2, protein kinase C, and the ER antagonist ICI 182,780 (40). Thus, in porcine aortic endothelial cells co-activation of AMPK and PI 3-kinase by E2 appears to mediate eNOS activation.

Isoflavones may act as inhibitors of cAMP phosphodiesterase (55). However, as cAMP levels in HUVEC were unaffected by acute treatment with equol and other isoflavones, effects of cAMP-dependent protein kinase inhibitors on eNOS activation by isoflavones were not examined. Previous studies with bovine aortic endothelial cells (BAEC) reported that longer term treatment with genistein (100 nM, 10-120 min) stimulates activation of eNOS independent of ERK1/2, PI 3-kinase/Akt, or the involvement of estrogen receptors (56). The lack of inhibition of genistein-stimulated eNOS activity by ICI 182,780 is consistent with our present results. The discrepancies between our findings and those of Liu et al. (56), based primarily on experiments with BAEC, may reflect differences in cell type, species, and/or the time interval of genistein treatment (30 s versus 10 min). Moreover, Liu et al. (56) only reported increases in cAMP levels in BAEC in response to 10 nM to 100 µM genistein. In our study, treatment of HUVEC with isoflavone concentrations of >100 nM actually inhibited ERK1/2 phosphorylation and did not lead to enhanced NO synthesis (data not shown). The inhibition of tyrosine kinase activity by genistein (10-50 µM) is well known (57), and our present and previous results in HUVEC (36) highlight the concentration-dependent actions of genistein and other isoflavones on ERK1/2 activity.

Natural polyphenolic compounds in black and green tea and red wine also activate eNOS (58-60). Epigallocatechin-3-gallate (40-100 µM) stimulates NO release in bovine endothelial cells only after 15 min, with moderate activation of Akt and eNOS serine 1177 (58). Epigallocatechin-3-gallate-stimulated phosphorylation of eNOS was abolished by Akt and cAMP-dependent protein kinase inhibitors but notably unaffected by ERK1/2 inhibition. In porcine endothelial cells, black tea polyphenols stimulate Akt and eNOS phosphorylation within 5 min, involving a Ca2+- and p38MAPK-dependent activation of PI 3-kinase/Akt (59). Red wine polyphenols rapidly activate ERK1/2 and NO production in several endothelial cell types (60) and stimulate PI 3-kinase/Akt-dependent phosphorylation of eNOS in porcine coronary arteries (61). Although actions of trans-resveratrol on ERK1/2 and NO production in cultured endothelial cells are inhibited by ICI 182,780 and tamoxifen (60), studies with tea polyphenols have not assessed the potential inhibitory actions of ER antagonists.

Although pertussis toxin inhibited isoflavone-stimulated ERK1/2 phosphorylation, cGMP levels were only reduced marginally, suggesting activation of eNOS via PI 3-kinase/Akt. The lack of significant inhibition of equol-stimulated NO production by pertussis toxin contrasts with previous studies with E2 in ovine endothelial cells, and suggests that rapid stimulation of eNOS by isoflavones in HUVEC is not mediated via G protein-coupled receptors.

We found that isoflavone-stimulated eNOS activation was independent of ERs, because the classical ER antagonists ICI 182,780 and tamoxifen failed to inhibit equol-, daidzein-, and genistein-stimulated cGMP production. The concentration and preincubation times used in our study are similar to those in previous studies examining the actions of E2. The only difference is the much shorter treatment (2 min versus 10-30 min) used in our experiments. When we compared the inhibitory action of ICI 182,780 on E2- and equol-stimulated cGMP accumulation over 15 min, the ER antagonist selectively inhibited the actions of E2, confirming that equol-stimulated NO production was independent of ER activation. As longer term responses to 17beta-estradiol were inhibited by ICI 182,750, this implicates interactions of 17beta-estradiol with the classical ERs detected in HUVEC in this study (Fig. 1).

In summary, this study in human umbilical vein endothelial cells establishes that rapid activation of ERK1/2 and PI 3-kinase/Akt signaling pathways by equol, daidzein, and genistein is paralleled by phosphorylation of eNOS serine 1177, Hsp90 association with eNOS, and NO release (Fig. 9). Furthermore, rapid phosphorylation of eNOS serine 1177 in response to equol is abrogated by inhibitors of ERK1/2 and PI 3-kinase, and we have demonstrated cross-talk between these two signaling pathways (Fig. 6, E and F). Based on our present findings, we cannot exclude the possibility that equol also modulates NO synthesis via other NOS isoforms expressed at much lower levels in subcultured HUVEC (62, 63). Activation of tyrosine kinases and Hsp90 may act in concert to regulate the balance of NO· and Formula generation by eNOS (54). In this context, prolonged treatment of J774 cells with equol increases the bioavailability of NO by inhibiting Formula production (64). We have also documented that equol induces endothelium- and NO-dependent relaxation of isolated aortic rings. Similar studies have reported that dilatory responses to dehydroequol in isolated rat aortic rings are insensitive to inhibition of eNOS (65), whereas dilation of human forearm resistance arteries is largely NO-dependent (66).

Equol is derived from the soy isoflavone daidzein, and humans have acquired the ability to exclusively synthesize (S)-equol (67). Equol and other isoflavones may activate similar intracellular signaling pathways as a result of their phenolic ring structure. In a recent study assessing bioavailability and metabolism of soy isoflavones, it was noted that only 30% of the subjects were equol producers with negligible differences in equol production observed with age or gender (68). Thus, use of dietary sources of isoflavones as alternative treatment for postmenopausal women and patients at risk of cardiovascular disease will require evaluation of the ability of subjects to metabolize daidzein to equol. Our findings provide insight into the signaling pathways regulating eNOS activity in human endothelium and identify equol as a potent activator of acute NO production.


   FOOTNOTES
 
* This work was supported in part by British Heart Foundation Grant FS/99075, Biotechnology and Biological Sciences Research Council Grant BBS/S/K/2004/11207, Medical Research Council Grant G78/5887, and Heart Research UK Grant RG22489/04/08. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to this work. Back

2 Recipient of a British Heart Foundation studentship. Back

3 Recipient of a Biotechnology and Biological Sciences Research Council studentship. Back

4 Recipient of a Medical Research Council studentship. Back

5 Recipient of Wellcome Trust Grant 060202. Back

6 To whom correspondence should be addressed. Tel.: 44-20-7848-6209; Fax: 44-20-7848-6220; E-mail: giovanni.mann{at}kcl.ac.uk.

7 The abbreviations used are: ER, estrogen receptor; eNOS, endothelial nitric-oxide synthase; NO, nitric oxide; HAEC, human aortic endothelial cells; HUVEC, human umbilical vein endothelial cells; ERK, extracellular signal-regulated kinase; PI, phosphatidylinositol; PG, prostaglandin; NOS, nitric-oxide synthase; E2,17beta-estradiol; KPBS, Kreb's phosphate-buffered saline; BSA, bovine serum albumin; FCS, fetal calf serum; PE, phenylephrine; IBMX, 3-isobutyl-1-methylxanthine; AMPK, AMP-activated protein kinase; BAEC, bovine aortic endothelial cells; PTX, pertussis toxin; L-NAME, N{omega}-nitro-L- arginine methyl ester. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Rachel Tribe and the midwives of St. Thomas' Hospital Labor Ward for their expert assistance in the collection of human umbilical cords with informed patient consent.



   REFERENCES
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 RESULTS
 DISCUSSION
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