Phytoestrogen genistein up-regulates endothelial nitric oxide synthase expression via activation of cAMP response element-binding protein in human aortic endothelial cells.

We previously reported that genistein, a phytoestrogen, up-regulates endothelial nitric oxide synthase (eNOS) and prevents hypertension in rats that are independent of estrogen signaling machinery. However, how genistein regulates eNOS expression is unknown. In the present study, we show that genistein enhanced eNOS expression and NO synthesis in primary human aortic endothelial cells. Inhibition of extracellular signal regulated kinase, phosphoinositol-3 kinase, or protein kinase C did not affect genistein-enhanced eNOS expression and NO synthesis. However, chemical inhibition of protein kinase A (PKA) or adenoviral transfer of the specific endogenous PKA inhibitor gene completely abolished PKA activity and genistein-stimulated eNOS expression and NO production. Accordingly, genistein induced PKA activity and subsequent phosphorylation of cAMP response element (CRE)-binding protein (CREB) at Ser133. Suppression of CREB by small interfering RNA transfection abolished genistein-enhanced eNOS expression and NO production. Consistently, deletion of the CRE site within human eNOS promoter eliminated genistein-stimulated eNOS promoter activity. These findings provide the first evidence to our knowledge that genistein may play a beneficial role in vascular function through targeting the PKA/CREB/eNOS/NO signaling pathway.

Endothelial-derived nitric oxide (NO), synthesized by endothelial NO synthase (eNOS) from amino acid l-arginine and molecular oxygen, plays a pivotal role in maintaining vascular homeostasis. The decline of eNOS activity and/or expression is directly associated with various cardiovascular events, including hypertension (1, 2), atherosclerosis (3), and stroke (4).

Genistein, a soy-derived phytoestrogen, has received wide attention because of its potential beneficial effects on various human degenerative diseases, such as cardiovascular disease (CVD). Data from human intervention studies suggest the beneficial effects of genistein on vascular motor tone (5, 6), systemic arterial compliance (7), atherosclerosis (8), and markers of cardiovascular risk (9, 10). Several studies have shown that genistein increases circulating NO levels in postmenopausal women (11) and animals (12, 13), although the primary source of this increased NO release is not clear. We (14, 15) and others (16) recently demonstrated that genistein acts directly on vascular endothelial cells (EC) to enhance eNOS activity and expression, which consequently increases NO synthesis. Further, data from animal studies showed that genistein enhances eNOS expression in spontaneously hypertensive rats (17). Although estrogen has been shown to regulate eNOS expression both in cultured EC and in vivo (18, 19), and genistein has weak estrogenic effect, which was presumed in many previous studies as a mechanism that mediate various genistein effect, our recent studies provided evidence that the regulatory effect of genistein on human eNOS expression is not dependent on the estrogen-related signaling mechanism (15). Therefore, it is still unknown how genistein regulates eNOS expression.

Recently, we demonstrated that genistein activates the cAMP signaling pathway that is not related to its potential estrogenic effect or inhibition of protein tyrosine kinase in vascular EC (20). cAMP is a central signaling molecule in a variety of cellular systems and plays an important role in maintaining normal vascular function. Various important biological events elicited by the cAMP/protein kinase A (PKA) signaling pathway is mediated through activation of cAMP response element (CRE)-binding protein (CREB), a transcriptional factor downstream of PKA that mediates cAMP-regulated gene transcription by binding to CRE within the gene promoter region. Interestingly, recent studies showed that eNOS gene contains CRE sites within its promoter region (21), suggesting that eNOS expression may be directly regulated by CREB. Indeed, it has been found that activation of PKA improved eNOS expression in vivo (22). In the present study, we tested the hypothesis that genistein regulates eNOS expression via the PKA/CREB-mediated mechanism in EC.

Materials and Methods

Materials

Primary human aortic EC (HAEC) and endothelial growth supplements (EGM2) were purchased from Lonza Walkersville (Walkersville, MD); M199 media and fetal bovine serum (FBS) were obtained from Invitrogen (Carlsbad, CA); pGL2-eNOS promoter-luciferase plasmid (eNOS-Luc) was from Addgene, Inc. (Cambridge, MA); PKA activity and dual luciferase reporter assay kits were from Promega (Madison, WI); CREB ShortCut small interfering RNA (siRNA), scramble sequence of siRNA, and transfection reagents were from New England Biolabs (Ipswich, MA); transfection reagents were obtained from Targeting System (Santee, CA); nitrite/nitrate fluorometric assay reagents were purchased from Cayman Chemical (Ann Arbor, MI); antibodies for eNOS, phospho-CREB, CREB, and β-actin were from Cell Signaling Technology (Beverly, MA); nitrocellulose membranes and protein assay kits were from Bio-Rad (Hercules, CA); genistein, protease, and phosphatase inhibitor cocktails, H89, P3115, PD98059, LY294002, and other general chemicals were from Sigma (St. Louis, MO). Stock solutions of genistein, at 20 mm in dimethylsulfoxide (DMSO), were stored at −80 C before use.

Cell culture

Primary HAEC were cultured in M199 medium containing 2% FBS and EGM2 at 37 C in a 5% CO2 and 95% air environment. The medium was changed every other day until the cells became confluent. HAEC were passaged after 0.05% trypsin treatment, and passages 4–6 were used in all experiments.

Western blot analysis

Equal amounts of protein from cell extracts were subjected to Western blot analysis as described previously (20, 23). Membranes were probed with antibody against phospho-CREB or eNOS. The immunoreactive proteins were detected by chemiluminescence. Nitrocellulose membranes were then stripped and reprobed with CREB or β-actin antibody. The protein bands were digitally imaged for densitometric quantitation with a software program (GeneTools; Synoptics Ltd., Cambridge, UK). The expression of phospho-CREB and eNOS was normalized to that of CREB and β-actin, respectively, and expressed as fold increase over untreated control.

NO measurement

To investigate the effect of genistein on NO release from EC, confluent HAEC grown in 12-well plates were incubated with complete medium containing genistein, vehicle (DMSO), or other agents, with culture medium renewed in the third day from initial treatment. After treatment, cells were adapted into Hanks' balanced salts solution (HBSS) [135 mm NaCl, 1.2 mm CaCl2, 1.2 mm MgCl2 1.2, 5 mm KOH, 10 mm HEPES, and 10 mm glucose (pH 7.4)] supplemented with l-arginine (0.1 mm) for 30 min, followed by stimulation with 10 μm A23187 for 30 min. Culture supernatants were then collected for NO assay as determined by measuring the sum concentration of NO2− and NO3− as previously described (14). The concentration of NO2− and NO3− was normalized to that of protein in the same sample and then expressed as fold increase over control.

Adenoviral PKA inhibitor gene construct and infection

Replication-deficient adenovirus containing the complete sequence of endogenous PKA inhibitor cDNA (AdPKI) was constructed as previously described (24). For determining infection efficiency, HAEC were exposed to adenovirus at 100-1000 multiplicities of infection (MOI) per cell in 0.15 ml of serum-free M199 medium for 1 h at 37 C and then cultured in complete medium for 24 h. Heat-inactivated AdPKI served as the control. The infection efficiency was examined by measuring PKA activity as described below. For eNOS and NO analysis, HAEC were infected with AdPKI or heat-inactivated AdPKI at 1000 MOI/cell for 24 h and then treated with 1–10 μm genistein or vehicle for 5 d, followed by eNOS protein expression and NO production assays.

PKA activity assay

HAEC or AdPKI-infected HAEC treated with genistein or vehicle were collected in PKA extraction buffer [25 mm Tris-HCl, 0.5 mm EDTA, 0.5 mm EGTA, 10 mm β-mercaptoethanol, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 5 mm phenylmethylsulfonyl fluoride (pH 7.4)]. Cytoplasmic proteins were harvested by sonication and centrifugation. The enzymatic activity of PKA in cell extracts was determined by measuring the phosphorylation of kemptide as previously described (20). Phosphorylated kemptide was separated from unphosphorylated substrate on a 0.8% agarose gel by electrophoresis and visualized under UV light using an AlphaImager Imaging System (Alpha Innotech Co., San Leandro, CA). The fluorescent gels were photographed, and the amount of substrate phosphorylation was determined by quantifying the fluorescence intensity of the peptide bands.

Transfection of CREB siRNA

HAEC (60–70% confluence) grown in 12-well plates were transfected with a heterogeneous mixture of target-specific CREB siRNA (50 nm) or scramble sequences of siRNA by using siRNA transfection reagents according to the manufacturers' protocols. After 24 h of transfection, cells were incubated with or without genistein for 15 min to determine the phosphorylation of CREB or for 5 d to examine eNOS expression and NO generation as described above.

Mutation of CRE in the eNOS promoter

CRE site in the eNOS promoter cloned in eNOS-Luc (25) was mutated via PCR-based site-directed mutagenesis. Briefly, two partially overlapping fragments of the eNOS promoter were generated by PCR. Primer pairs for first fragment were 5′-CGCGGTACCATCTGATGCTGCCTGTCACC-3′ (forward) and 5′-TTCAGCGGCCGCCGCTTCCCGGGGCCG-3′ (reverse). Primer pairs for second fragment were 5′-AAGCGGCGGCCGCTGAATGACAGGGTG-3′ (forward) and 5′-CGCAAGCTTGTTACTGTGCGTCCACTCTGC-3′ (reverse). The eNOS promoter with CRE being mutated into a NotI restriction site was amplified by a third PCR from these two PCR products using primer pairs of 5′-CGCGGTACCATCTGATGCTGCCTGTCACC-3′ (forward) and 5′-CGCAAGCTTGTTACTGTG CGTCCACTCTGC-3′ (reverse). This PCR product was then digested with the restriction enzymes KpnI and HindIII and cloned into the pGL2-basic vector (Promega) at the same restriction sites to generate the eNOS gene promoter construct bearing CRE-specific mutant (mCRE-eNOS-Luc). Mutation of the CRE and sequence of the entire eNOS promoter in this plasmid were validated by DNA sequencing.

eNOS promoter activity assay

Human eNOS-Luc and mCRE-eNOS-Luc plasmids were amplified with competent cells and purified using QIAGEN Maxi kit according to the manufacturer's instructions (QIAGEN, Valencia, CA). For transient transfection of the plasmids, HAEC were grown in 24-well plates until 50–70% confluence. The cells were then cotransfected with 1.2 μg of eNOS-Luc or mCRE-eNOS-Luc and 0.5 ng of pRL reporter control plasmid per well using F-1 transfection reagent for 24 h according to the manufacturer's protocol. The transfected cells were then treated with various concentrations of genistein or vehicle in phenol red-free M199 medium containing 2% FBS for 24 h. Treated cells were harvested in reporter lysis reagent. Luciferase activity, normalized to that of pRL in the cell extracts, was determined by using the dual luciferase reporter assay system as described (23).

Statistical analysis

Data were analyzed with one-way ANOVA using SAS software and are expressed as mean ± se of three or four independent experiments. Treatment differences were subjected to Tukey's multiple comparison tests, where P < 0.05 was considered significant.

Results

Genistein increases eNOS protein expression and NO production

To initially determine the effects of exposure of HAEC to genistein on eNOS expression and NO production, confluent HAEC were incubated with various concentrations of genistein (1–10 μm) for 5 d with culture medium refreshed in third day. As shown in Fig. 1A, genistein dose dependently increased eNOS protein expression, with 10 μm genistein inducing about 60% increase over the control. We chose this genistein concentration range because it likely overlaps the genistein levels in plasma and tissues in humans and animals after dietary ingestion of genistein (26, 27). To confirm the biological importance of this increased eNOS expression by genistein, we examined NO production in HAEC treated with genistein or vehicle. Consistent with eNOS expression pattern, genistein treatment augmented A23187-induced NO release in a concentration-dependent manner, reaching a maximal level at 10 μm genistein (Fig. 1B).

Fig. 1.

Genistein (G) enhances eNOS protein expression and NO production in HAEC. Confluent HAEC were incubated with various concentrations (1–10 μm) of genistein or vehicle (DMSO) for 5 d. A, eNOS protein levels in cell extracts were measured by Western blotting and normalized to β-actin content. B, Nitrite/nitrate (NOx) production stimulated by ionophore 23187 was measured using a fluorometric assay kit and normalized to protein content. Values (mean ± se) of three separate experiments are expressed as fold over vehicle alone-treated cells. A set of representative images and bar graph (mean ± se) are shown. *, P < 0.05 vs. vehicle alone-treated cells.

Genistein-stimulated eNOS expression and NO production are independent of protein kinase C (PKC), phosphoinositol-3 kinase (PI3K), or extracellular signal regulated kinase (ERK)

Previous studies have shown that inhibition of PKC up-regulates eNOS transcription (26), and pharmacological doses of genistein were shown to inhibit PKC activity in human chronic myeloid leukemia cells (27). We therefore tested whether PKC mediates the effect of genistein on eNOS. Coincubation of the cells with p3115, a specific PKC inhibitor (28), had no effect on eNOS-derived eNOS expression (Fig. 2A) and NO production (Fig. 2B) induced by chronic exposure of HAEC to genistein. It has been established that PI3K- and ERK-mediated pathways are two important signaling cascades mediating eNOS activation by various stimuli in EC (29–32). To elucidate the intracellular signaling involved in the genomic regulation of eNOS by genistein, we then examined whether the PI3K or ERK pathways were involved in genistein-induced eNOS expression and NO synthesis. Preincubation of HAEC with the PI3K inhibitor, LY294002, or the ERK blocker, PD098059, had no effect on either basal or genistein-induced eNOS expression (Fig. 2, C and E) and NO production (Fig. 2, D and F). Both LY294002 and PD098059 were active, because recent studies from us (14, 20, 23) and others (33–35) consistently show that LY294002 and PD098059, respectively, blocked various agonist-induced serine/threonine-specific protein kinase and ERK1/2 phosphorylation and subsequent biological events in EC, using the same inhibitor concentration as in this experiment.

Fig. 2.

The stimulatory effect of genistein (G) on NO production is not dependent on the PKC, PI3K, or ERK pathway in HAEC. Confluent HAEC were preincubated with either P3115 (100 μm), a PKC inhibitor (A and B), LY294002 (LY) (10 μm), the PI3K inhibitor (C and D), or PD 98059 (PD) (10 μm), a ERK inhibitor (E and F), for 30 min followed by addition of G (10 μm) or vehicle (V) for 5 d. Protein expression of eNOS in cell extracts was analyzed with Western blotting and normalized to β-actin content. Values (mean ± se) of three separate experiments are expressed as fold of control, and a set of representative images and bar graph (mean ± se) are shown (A, C, and E). Nitrite/nitrate (NOx) production stimulated by ionophore 23187 was measured and normalized to protein content (B, D, and F). Values (mean ± se) of three independent experiments are expressed as fold over control; *, P < 0.05 vs. vehicle alone-treated cells.

Genistein-enhanced eNOS expression and NO production are mediated via PKA

Our recent study showed that genistein activates the cAMP/PKA signaling pathway in EC (20). We then examined whether genistein stimulates eNOS expression via activation of PKA. To that end, HAEC were infected with AdPKI, an adenovirus construct containing the specific endogenous PKA inhibitor gene. As shown in Fig. 3A, treatment of HAEC with 500-1000 MOI of AdPKI resulted in up to 95% reduction in PKA activity compared with that of untreated EC, whereas infection with heat-inactivated AdPKI had no significant effect on PKA activation. Accordingly, infection of HAEC with AdPKI significantly attenuated genistein-induced eNOS expression and NO production (Fig. 3, B and C), but heat-inactivated AdPKI did not significantly alter these genistein actions in EC (data not shown). Taken together, these results suggest that PKA plays a central role in mediating genistein-induced eNOS expression. To confirm whether genistein induces cellular PKA activity, HAEC were incubated with various concentrations of genistein for 15 min. As shown in Fig. 3D, genistein significantly increased PKA activity in HAEC.

Fig. 3.

Genistein (G)-enhanced eNOS protein expression and NO production are mediated via activation of the PKA/CREB cascade in HAEC. A, Different concentrations of AdPKI or heat-inactivated AdPKI virus (H) were transfected into HAEC for 24 h. Cell lysates were used to measure PKA activity using a nonradioactive PKA assay kit with negative (−) and positive (+) controls included. B and C, HAEC transfected with (+) or without (−) AdPKI were treated in the presence or absence of G (10 μm) for 5 d. Protein expression of eNOS in cell extracts was analyzed with Western blotting and normalized to β-actin content. Values (mean ± se) of three separate experiments are expressed as fold of control, and a set of representative images and bar graph (mean ± se) are shown (B). Nitrite/nitrate (NOx) production stimulated with ionophore 23187 was measured using a fluorometric assay kit and normalized to protein content. Values (mean ± se) of three separate experiments were expressed as fold of control (C). D, Confluent HAEC were serum starved in HBSS buffer for 30 min and followed by stimulation of various concentrations of G (0, 1, and 10 μm) for 15 min. PKA activity was measured by an assay kit. Values (mean ± se) of three separate experiments are expressed as fold of control. *, P < 0.05 vs. vehicle alone-treated cells.

Genistein-stimulated eNOS expression and NO production are mediated via CREB

There is evidence that the eNOS promoter contains CRE sites, and activation of CREB phosphorylation stimulates eNOS expression in EC (36). To determine the role of CREB in mediating genistein effect on eNOS expression, we first investigated whether increased activation of PKA by genistein yields higher CREB phosphorylation. To that end, we performed Western blottings using a phospho-CREB antibody that only recognizes CREB when phosphorylated at Ser133. As shown in Fig. 4A, genistein increased CREB phosphorylation at Ser133 in HAEC in a dose-dependent manner, a pattern that is consistent with the increased eNOS protein expression induced by genistein. Pretreatment of cells with PKA inhibitor H89 completely abolished the CREB phosphorylation stimulated in the presence of genistein (Fig. 4B), suggesting that PKA mediates the genistein-stimulated phosphorylation of CREB. Next, we examined whether genistein induction of eNOS expression and NO production is mediated by CREB. To that end, we used siRNA to abolish the expression of CREB in HAEC. Transfection of HAEC with CREB siRNA reduced CREB protein expression by 97% compared with that in control cells (Fig. 4C). As a result, ablation of CREB expression blocked the effects of genistein on CREB activation in HAEC (Fig. 4C), whereas the scramble sequence of siRNA did not affect CREB expression and activation (Fig. 4C). Accordingly, disruption of CREB expression with siRNA blunted the effects of genistein on eNOS expression (Fig. 4D) and NO production (Fig. 4E), suggesting that genistein-induced eNOS expression and NO production are mediated by activation of CREB.

Fig. 4.

Genistein (G)-stimulated eNOS expression and NO production are mediated via CREB. A and B, Confluent HAEC were incubated in HBSS buffer (A) or preincubated with H89 in HBSS buffer (B) for 30 min followed by the addition of various concentrations of G (0.01–10 μm) for 15 min. CREB phosphorylation was measured by Western blotting. C–E, CREB siRNA or scramble sequence of siRNA was transfected into HAEC for 24 h. Cells were then treated with G (0, 1, and 10 μm) for 15 min in HBSS buffer for determining CREB phosphorylation and expression (C) or 5 d in EGM2 medium for determining eNOS protein expression (D) and NO production (E) as described in Materials and Methods. Values (mean ± se) of three separate experiments are expressed as fold over control; *, P < 0.05 vs. vehicle alone-treated control.

Genistein induction of eNOS expression is mediated through CRE in its promoter

To further investigate whether the CRE sites within the eNOS promoter were required for genistein-induced eNOS expression in HAEC, we mutated CRE in the eNOS promoter and then performed transient transfection assays in HAEC. As shown in Fig. 5, genistein-induced eNOS promoter activity was abolished by mutation of CRE. This result suggests that CRE plays a critical role in genistein-stimulated increases in eNOS expression.

Fig. 5.

Genistein (G) induction of eNOS expression is dependent on CRE site in the eNOS promoter. 50–70% confluent HAEC were transfected with mCRE-eNOS or wild-type eNOS promoter plasmids. Twenty-four hours later, cells were treated in the presence or absence of G (1 and 10 μm) for 1 d. The promoter activity of eNOS was measured by a dual luciferase reporter assay system. Values (mean ± se) of three separate experiments are expressed as fold over control; *, P < 0.05 vs. vehicle alone-treated cells.

Discussion

CVD are the leading cause of morbidity and mortality in the United States (37), and the critical role of eNOS in maintenance of cardiovascular health has been well established (1–4). However, eNOS expression was significantly decreased in ovariectomized and fertile rats (38), consistent with the finding that premenopausal women have lower incidence rate of CVD than that of age-matched men, but this reduced CVD rate diminishes with the onset of menopause and becomes even higher in postmenopausal women than that in men (37), suggesting a vascular protective effect of estrogen. Indeed, estrogen replacement therapy appears to reduce the risk of CVD (39) and increase the circulating NO (40) in postmenopausal women, confirming that estrogen is cardioprotective, and this beneficial effect is at least partially mediated through promoting endothelium-derived NO synthesis (41). However, administration of estrogen has potential carcinogenic effects in women and feminizing effects in men (42). These side effects limit its use as a cardio-protective agent. Therefore, search for safe, alternative eNOS-promoting agents for prevention of CVD is of major importance in the effort to decrease the burden of CVD morbidity and mortality. Genistein, a well-known phytoestrogen with much higher affinity to estrogen receptor-β (43), may be a novel candidate as an alternative to estrogen-based vascular protective agent. We (14, 15) and others (16) have previously shown that genistein up-regulates eNOS expression and NO production. However, how genistein regulates eNOS expression is still unknown.

Previous studies showed that estrogen can act directly on vascular EC to enhance NO production through both genomic stimulation of eNOS expression (44) and membrane receptor-mediated, nongenomic activation of the enzymatic activity (45). Although we previously found that genistein can also stimulate NO synthesis through rapid nongenomic activation of eNOS and genomic stimulation of its expression in EC (14, 15), these effects are independent of the estrogen receptor-dependent mechanisms, which promoted us to investigate other potential pathways that mediate genistein actions in vascular EC. It was showed that PKC phosphorylates eNOS at Thr497, which subsequently suppresses eNOS activity in bovine aortic EC (46). In addition, inhibition of PKC increase eNOS mRNA and protein expression and subsequently elevates NO synthesis in EC (26), although the mechanism of this action is not clear. However, we found that inhibition of PKC had no effect on basal or genistein-stimulated NO release, suggesting that the regulatory effect of genistein on eNOS is not related to PKC in HAEC. Genistein in high doses was reported to affect PI3K and ERK activities, which can modulate eNOS activity and NO production (30). In the present study, however, we found that genistein-induced eNOS expression and NO release were not mediated by the PI3K or ERK pathway.

It has been shown that the cAMP/PKA signaling pathway is involved in regulating eNOS gene transcription (47). We recently found that genistein activates the cAMP signaling system in EC (14), suggesting that cAMP/PKA signaling may be involved in genistein-enhanced eNOS expression. We confirmed this hypothesis by demonstrating that genistein increases PKA activity and that genistein-increased eNOS protein expression and NO production in HAEC are completely blocked by adenoviral transfer of endogenous PKA inhibitor gene, which is highly specific and efficient, because it completely blocked PKA activity, whereas heat-inactivated AdPKI had no significant effect on PKA activity. Thus, the present results provide direct evidence that genistein up-regulation of eNOS expression is through a PKA-mediated mechanism.

eNOS promoter contains several regulatory elements, including shear stress-responsive element, estrogen-responsive element, and activator protein-1 binding site, which modulate eNOS gene transcription in response to respective stimuli (48–50). In addition to these mechanisms that regulate eNOS expression, a recent study found that eNOS gene contains functional CRE that also positively regulates eNOS gene transcription (21). In the classic cAMP/PKA/CREB-mediated gene transcription process, cAMP-activated PKA translocates from cytoplasm into nuclear, where it phosphorylates CREB on Ser133. Phosphrylated CREB then binds to CRE of specific gene and therefore initiates gene transcription (51). Our further studies demonstrate that genistein rapidly activates PKA-dependent phosphorylation of CREB at Ser133 in HAEC. This result suggests that genistein may regulate eNOS through activation of PKA-dependent CREB, given that the phosphorylation of CREB at Ser133 is necessary for its binding to CRE to regulate gene transcription (52), and that eNOS promoter contains CRE sites (21). Indeed, we found that siRNA disruption of CREB expression abolished genistein-induced eNOS expression and NO production. Furthermore, genistein-increased eNOS promoter activity was eliminated after mutation of CRE within the eNOS promoter, suggesting that CREB mediates genistein effect on eNOS expression through its binding CRE site within the eNOS promoter. Although data from these complementary experiments provide evidence that genistein regulation of eNOS is mediated by CREB, it should be noted that near depletion of CREB only reduced basal eNOS expression and NO production by less than 50%. In addition to CREB, there are other transcriptional factors, such as activator protein-1, that also positively regulate eNOS expression (48–50, 53). In addition, estrogen can up-regulate eNOS transcription through estrogen-responsive element in the eNOS promoter (54). In the present study, we did not use estrogen-stripped FBS, which typically contains 192 pg/ml estrogen (55), a dose that is capable of stimulating eNOS expression in EC. Therefore, it is conceivable that knockdown of CREB cannot completely abolish basal eNOS transcription, given those alternative regulatory mechanisms.

We recently discovered that genistein activated cAMP/PKA signaling by stimulating adenylate cyclase (AC) activity EC (14). However, how AC is activated by genistein is presently unknown. AC is commonly linked to G protein-coupled receptor (GPR) (56, 57). Interestingly, it was shown that genistein can bind to an orphan plasma membrane receptor GPR30 in cancer cells (58). Although the physiological role of GPR30 is still unclear, it was shown that GPR30 is coupled to Gαs to stimulate AC in cancer cells (56). It has been shown that GPR30 is also expressed in vascular EC (59). Therefore, there is the possibility that genistein activates cAMP signaling system via GPR30-mediated mechanisms, an aspect that is currently under investigation in our laboratory.

In summary, we show in this study, for the first time to our knowledge, that genistein promotes eNOS expression and NO production through the cAMP/PKA/CREB/CRE pathway in primary human vascular EC. These findings add new information to the functional repertoire of this food-derived small molecule and form the basis for further evaluating its potential in preventing or treating CVD. Future studies therefore will be aimed at determining genistein if PKA/CREB/eNOS/NO signaling elicited by genistein in vitro is physiologically relevant in vivo.