Genistein activates endothelial nitric oxide synthase in broiler pulmonary arterial endothelial cells by an akt-dependent mechanism

ABSTRACT Deregulation of endothelial nitric oxide synthase (eNOS) plays an important role in the development of multiple cardiovascular diseases. Our recent study demonstrated that genistein

supplementation attenuates pulmonary arterial hypertension in broilers by restoration of endothelial function. In this study, we investigated the molecular mechanism by using broiler

pulmonary arterial endothelial cells (PAECs). Our results showed that genistein stimulated a rapid phosphorylation of eNOS at Ser1179 which was associated with activation of eNOS/NO axis.

Further study indicated that the activation of eNOS was not mediated through estrogen receptors or tyrosine kinase inhibition, but _via_ a phosphatidylinositol 3-kinase (PI3K)/Akt-dependent

signaling pathway, as the eNOS activity and related NO release were largely abolished by pharmacological inhibitors of PI3K or Akt. Thus, our findings revealed a critical function of Akt in

mediating genistein-stimulated eNOS activity in PAECs, partially accounting for the beneficial effects of genistein on the development of cardiovascular diseases observed in animal models.

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PROMOTING ENOS EXPRESSION AND PREVENTING ENOS UNCOUPLING Article Open access 22 July 2022 INTRODUCTION Cardiovascular diseases, such as coronary heart disease, hypertension and

atherosclerosis are the leading causes of death among women in western countries. The observation that the incidence of cardiovascular diseases substantially increased in post-menopause

women supports the viewpoint that the loss of protection attributed to estrogen deficiency (Grodstein et al., 1996). Further supportive evidence for the beneficial effects comes from the

pre-clinical studies demonstrating that estrogen therapy reduces cardiovascular risk even though it increases breast cancer incidence after a prolonged treatment (Barrett-Connor and Bush,

1991; Stampfer and Colditz, 1991). The multiple benefits of estrogen have been reported to be mediated by several mechanism, including an enhanced expression of nitric oxide synthase (NOS),

relaxation of pre-contracted vessels, inhibition of calcium entry, stimulation of the production of prostacyclin or an inhibitory effect on the development of myointimal hyperplasia (Farhat

et al., 1996). Moreover, estrogen has also been shown to enhance endothelium-dependent vasodilation of coronary arteries in both postmenopausal women and primates (Farhat et al., 1996;

Lissin and Cooke, 2000). Given the demonstrated risks to conventional estrogen therapy, phytoestrogens, a class of natural occurring estrogen, have received increasing attention because of

their potentially protective roles against several types of chronic diseases, includeing cardiovascular diseases, osteoporosis and hormone-related cancer (Cassidy and Griffin, 1999; Lissin

and Cooke, 2000). Among phytoestrogens, genistein and daidzein, two major isoflavones in soybeans are the well-studied due to their wide distribution. _In vitro_ and _in vivo_ studies

indicated that genistein supplementation has abilities to improve endothelial dysfunction in postmenopausal women, ovariectomized or chronically hypoxic rats (Squadrito et al., 2000;

Karamsetty et al., 2001; Catania et al., 2002; Cuevas et al., 2003). Further studies shown that the beneficial effects of genistein were associated with the regulation on endothelial nitric

oxide synthase (eNOS), because N-nitro-L-arginine methyl ester (L-NAME), the inhibitor of eNOS, abolished the restoration of endothelial function in healthy postmenopausal women (Mishra et

al., 2000; Colacurci et al., 2005) and rats (Vera et al., 2007). Upon activation, eNOS produces and release endothelial nitric oxide, a crucial vasoactive molecule in maintaining vascular

homeostasis because of its potent activity to relax vascular arteries, prevent intra-vasal coagulation and platelet aggregation and antagonize the proliferation of smooth muscle cells (Li

and Forstermann, 2000). Deregulation of eNOS is an essential factor that correlated with the development of cardiovascular diseases and points to the possibility that genistein might

directly or indirectly regulate eNOS activity in endothelial cells. However, the underlying mechanisms are not fully known. Our recent study demonstrated that supplementation of genistein

attenuated pulmonary hypertension by restoration of endothelial function in broilers (Yang et al., 2010). Therefore the aim of the present study was to investigate the effect of genistein on

the activation of eNOS and the underlying mechanisms in broiler pulmonary arterial endothelial cells (PAECs). RESULTS GENISTEIN ENHANCES ENOS ACTIVITY IN BROILER PAECS THROUGH A NON-GENOMIC

MECHANISM INVOLVING PHOSPHORYLATION OF ENOS AT SER1179 To determine the effect of genistein on the eNOS activity, cultured cells were treated with vehicle or indicated concentrations of

genistien for 15 min (Figure 1A). In the range of 10-2 to 102 µM used in this experiment, genistein enhanced eNOS activity in a dose dependent manner, with genistein of 10 µM inducing

maximal eNOS activation, which was observed as early as 5 min post-treatment and reach maximum after 15 min of incubation and then rapidly declined thereafter but was still significant

higher than control after 1 h of genistein treatment (Figure 1B). The rapid activation of eNOS by genistein is by a non-genomic mechanism, because pre-treatment of the cells with

cycloheximide, a protein synthesis inhibitor, or the mRNA transcription inhibitor actinomycin D had no significant effect on genistein-induced eNOS activity (Figure 2). eNOS has been

reported to be activated by phosphorylation, we therefore investigated whether the rapid activation of eNOS induced by genistein is associated with phosphorylation. Western blot results

showed that genistein treatment elicited a rapid increase of phosphorylation of eNOS at Ser1179, which was maximal at the concentration of 10 µM (Figure 3); consistent with the enhanced eNOS

activity induced by genistein (Figure 1A). In contrast, genistein treatment did not affect the phosphorylation of eNOS at Thr 495 (Figure 3), suggesting a critical role of phosphorylation

of eNOS at Ser1179 in genistein-induced non-genomic eNOS activation. GENISTEIN-INDUCED ACTIVATION OF ENOS IS NOT MEDIATED BY ESTROGEN RECEPTOR (ER) It is well known that estrogen can

regulate eNOS activity by a estrogen receptor mediated mechanism (MacRitchie et al., 1997). Genistein has weak estrogenic effect by binding to estrogen receptors due to the structural

similarity to that of estrogen (Kuiper et al., 1997). We next evaluate whether ER was involved in genistein induced rapid activation of eNOS. Cells were incubated with genistein in the

absence or presence of estrogen receptor antagonist ICI 182, 780 (100 nM). As shown, ICI 182, 780 did not affect the eNOS activity (Figure 4A), nor the phosphorylation of eNOS at Ser1179

(Figures 4B and 4C), suggesting that genistein-induced activation of eNOS is not mediated by estrogen receptor in our system. ACTIVATION OF ENOS IN PAECS IS INDEPENDENT OF TYROSINE KINASE

INHIBITION Beside the estrogenic effect, genistein is widely used as a tyrosine kinase inhibitor in various experimental systems; we next investigate whether the genistein-induced activation

of eNOS is mediated by inhibition of tyrosine kinase in PAECs. Serum-starved endothelial cells were incubated with genistein or herbinycin (10 µM), a specific inhibitor of tyrosine kinase,

followed by tyrosine kinase activity assay. Genistein, at the concentrations used for the stimulation of eNOS activity in our experiment did not inhibit the tyrosine kinase activity (Figure

5). Our result is in agreement with the previous study showing that higher concentration of genistein (100 µM) is required to inhibit tyrosine kinase activity (Liu et al., 2004). In

contrast, herbimycin A, a positive control used in our experiment, potently inhibit tyrosine kinase. ACTIVATION OF ENOS BY GENISTEIN IS MEDIATED BY A PI3K/AKT-DEPENDENT MECHANISM eNOS can be

phosphorylated at the Ser1179 and thus activated by several protein kinases, such as ERK (Bernier et al., 2000), Akt (Dimmeler et al., 1999; Fulton et al., 1999) or protein kinase A (PKA)

(Boo et al., 2002) in response to various stimulus. To elucidate the potential kinase pathways responsible for genistein-induced eNOS phosphorylation, PAECs were pre-treated with vehicle or

PKA inhibitor H89 (5 µM), ERK inhibitor PD98059 (10 µM), PI3K inhibitor LY294002 (10 µM) or Akt inhibitor A443654 (5 µM) for 30 min and then incubated with or without genistein (10 µM) for

another 15 min. As shown, H89 had no effect on genistein-induced eNOS activation (Figure 6A) and phosphorylation of eNOS at Ser1179 (Figures 6B and 6C). PD98059 did not affect

genistein-induced eNOS activity, even though it partly abrogated eNOS phosphorylation. By contrast, LY294002 and A443654 significantly abolished the phosphorylation of eNOS at Ser1179 and

related eNOS activity, suggesting that genistein enhanced eNOS activity through a PI3K/Akt-dependent mechanism. GENISTEIN STIMULATES NO RELEASE IN PULMONARY ENDOTHELIAL CELLS To further

confirm the functional role of rapid activation of eNOS by genistein in PAECs, we determine the release of NO by the measuring of the sum concentration of NO2- and NO3- in culture

supernatants using a fluoremetric assay. In consistence with the activation of eNOS, genistein (10 µM) treatment significantly increased the NO release compared with that of control (Figure

7) which was completely blocked by pretreatment with L-NAME (300 µM), an inhibitor of eNOS. In agreement with eNOS activity (Figure 6), genistein-stimulated NO release was significantly

blocked by PI3K inhibitor LY294002 (10 µM) or Akt inhibitor A443654 (5 µM), instead of PKA inhibitor H89 or ERK1/2 inhibitor PD98059. DISCUSSION The phytoestrogen genistein has drawn

increasing attention due to its potential healthy benefits in preventing cardiovascular diseases. However, the underlying mechanisms are still not well defined. In this study, we

demonstrated that genistein stimulated a rapid activation of eNOS in a dose- and time-dependent manner. This effect was not mediated by a genomic mechanism since it occurred rapidly and was

not inhibited by cycloheximide or actinomycin D. Further study showed that genistein-induced eNOS activation through a PI3K/Akt-dependent phosphorylation of eNOS at Ser1179 since the

pretreatment of cells with PI3K inhibitor LY294002 or Akt inhibitor A443654 abrogated genistein stimulated eNOS activation (Figure 6) and related NO release (Figure 7), indicating an

important role of PI3K/Akt in genistein-induced rapid activation of eNOS/NO in broiler PAECs. Vascular endothelial cells play an important role in maintaining normal vascular function by

producing several vasoconstrictors and vasodilators in response to multiple mechanical, physiological, and pharmacological stimuli (Schiffrin, 2001; Mawji and Marsden, 2003). NO produced by

NOS in endothelial cells is a potent vasodilator in pulmonary vessels that maintains normal vascular tone and homeostasis through interactions with vasoconstrictor endothelin-1 (ET-1)

(Lavallee et al., 2001). Impaired endothelial NO production contributes to the increased vascular resistance and leading to the development of pulmonary hypertension (Tan et al., 2007). In

broiler chickens, pulmonary hypertension, also known as ascites, is a metabolic disease characterized by the pulmonary artery hypertension-induced right ventricular hypertrophy and failure.

Although evidence in human and rats indicates that dramatic changes in the function of the pulmonary vascular endothelium occur in pulmonary hypertension, little is known about these changes

in broiler chicken. Our recent study demonstrated that genistein supplementation enhanced eNOS activity in broiler chicken (Yang et al., 2010). To explore the mechanisms by which genistein

enhanced eNOS activity, PAECs were isolated from broiler chicken embryos and treated with genistein. The rapid activation of eNOS (Figure 1) and NO release (Figure 7), suggested that

activation of eNOS is not mediated by a genomic mechanism and this was confirmed by the treatment of cells with genistein in the absence or presence of cycloheximide or actimomycin (Figure

2). In consistence with this observation, genistein treatment did not affect eNOS protein expression (Figure 3). Further study showed that the rapid activation of eNOS was associated with

phosphorylation of eNOS at Ser1179 (Figure 3). Although genistein has weak estrogenic effects in some tissues through binding to estrogen receptor beta with an affinity comparable with

17-estradiol, and both estrogen receptors are present in vascular cells, our data show that the rapid activation of eNOS by genistein was independent of ERs, because the specific ER

antagonist ICI182, 780 did not affect genistein-induced eNOS phosphorylation and activity (Figure 4). In addition, the activation of eNOS by genistein was unrelated to tyrosine kinase

inhibition (Figure 5), which is in agreement with a previous study showing that high concentration of genistein (100 µM) is required to inhibits tyrosine kinase activity (Peterson and

Barnes, 1996), which is much higher than the concentrations used in our study to induce maximum activation of eNOS in broiler PAECs. Since several protein kinases, such as Akt, PKA and

ERK1/2, have been proposed to phosphorylate eNOS at Ser1179 and increase eNOS activity in response to various stimuli in vascular endothelial cells (Dimmeler et al., 1999; Fulton et al.,

1999; Michell et al., 1999; Bernier et al., 2000; Igarashi and Michel, 2001; Kobayashi et al., 2003), we next determine the kinses that are responsible for the activation of eNOS following

genistein treatment. PKA and ERK do not appear to play a role in the genistein-induced phosphorylation of eNOS as selective kinase inhibitors failed to influence the genistein-induced eNOS

activity (Figure 6) and NO release (Figure 7). In contrast, the activation of eNOS was largely abolished by PI3K or Akt inhibitors suggesting that PI3K/Akt activity contributed to the

genistein-induced eNOS activation. This results are not in agreement with a recent study showing genistein induces eNOS activation through a PKA-, instead of PI3K/Akt-dependent pathway in

bovine aortic endothelial cells (Liu et al., 2004). Possible explanations for this observation might include the fact we used broiler pulmonary arterial endothelial cells rather than aortic

endothelial cells. Another factor might be the different dosage used in these two studies, in our study genistein concentration (10 µM) is determined according to our _in vivo_ study in

broilers (Yang et al., 2010), which is ten times higher than that of Liu et al (2004). Further study using broiler aortic endothelial cells is needed to elucidate whether the discrepancy is

tissue specific. Many diverse beneficial effects of genistein on cardiovascular disease have been described in publication reports. An involvement of the endothelial vasorelaxing effects of

genistein has been recently reported in human and rats (Mishra et al., 2000; Colacurci et al., 2005; Vera et al., 2007), suggesting a critical role of eNOS that contributes to the

cardio-protective effect of genistein. The data presented here, along with our _in vivo_ study (Yang et al., 2010), provided evidence that genistein activated eNOS through a non-genomic

mechanism, resulting in the release of NO, restored endothelial function and attenuated pulmonary hypertension in broilers. Supplementation of genistein might be a potential therapeutic

strategy that reduced the incidence of pulmonary hypertension, a leading causes of death in poultry industry. It should be noted that in our system, PI3K or Akt inhibitor largely, but not

completely abrogate genistein-induced eNOS activity, suggesting the existence of alternative signaling pathways that contributed to the phosphorylation of eNOS and related NO release. eNOS

was initially reported to be phosphorylated on serine residues and has been studied in most detailed (Fleming et al., 2001). However, recent studies shown that other residues, such as

threonine and tyrosine residues, can also be phosphorylated and regulates eNOS activity (Fleming et al., 1998; Harris et al., 2004). Even though the phosphorylation of eNOS at Thr495 did not

change upon genistein treatment in our system (Figure 3), we can not exclude the possibility that Akt or other kinases might regulate eNOS activity through phosphorylation at other residues

that attribute to the increased activity. The exact mechanism by which Akt phosphorylates eNOS in the pulmonary arterial endothelial cells is not clear. Further study is needed to address

this question. In summary, the present data demonstrated that acute genistein treatment led to activation of eNOS in broiler pulmonary arterial endothelial cells through a PI3K/Akt-dependent

mechanism. Pharmacological inhibitor of Akt markedly reduced the phoshphorylation of eNOS at Ser1179 and related NO production. Our data provide an alternative explanation for the

beneficial effect of genistein on vascular diseases in human and animals. METHODS MATERIALS M199 media, FBS and other cell culture supplements were obtained from Invitrogen (Carlsbad, CA).

Genistein, ICI 182, 780, inhibitors of ERK1/2 (PD98059), PKA (H89), tyrosine kinase (Herbimycin), PI3K (LY294002) and Akt (A443654) were purchased from Sigma (St. Louis, MO). Protease and

phosphatase inhibitor cocktails were obtained from Roche Molecular Systems, Inc. (Alameda, CA). CELL CULTURE Twenty day-old chick embryo pulmonary arterial endothelial cells (PAECs) were

isolated by enzymatic dispersion according to the modified method (Visner et al., 1994). The purity of PAEC culture was characterized by indirect immunofluorescent staining for factor VIII

antigen, a generally accepted marker of endothelial cells (Visner et al., 1994). Cells were cultured in M199 media supplemented with 10% FBS, 100 IU/mL penicillin, 100 µg/mL streptomycin, 8

mM HEPES, and 2 mM glutamine at 39℃ in a 5% CO2/95% air environment. PAECs were passaged with 0.05% trypsin and passages 4-6 were used in all the experiments. ENOS ACTIVITY Cells were

serum-starved overnight in phenol red-free medium before eNOS activity measurements. eNOS activity was determined by measuring the conversion of [3H]L-arginine to [3H]L-citrulline as

described (Hisamoto et al., 2001). In some experiments, cells were pretreated with estrogen receptor inhibitor ICI 182,780 (100 nM); PKA inhibitor H89 (5 µM), ERK inhibitor PD98059 (10 µM),

PI3K inhibitor LY294002 (10 µM) or Akt inhibitor A443654 (5 µM) for 30 min before incubated with genistein. The reaction was terminated by aspirating the buffer and washing with ice-cold PBS

containing EGTA (5 mM) and EDTA (5 mM), followed by the addition of 1 ml ice-cold trichoroacetic acid (0.5 N). Subsequent sample handling and then radioactivity was measured with a liquid

scintillation counter. eNOS activity was normalized as picomoles of [3H] L-citrulline produced per milligram protein and expressed as a percentage of control. NITRIC OXIDE MEASUREMENT To

determine the effect of genistin on NO release _in vitro_, serum-starved PAECs were treated with genistein in the presence or absence of kinase inhibitors, PD98059 (10 µM), H89 (5 µM),

LY294002 (10 µM), A443654 (5 µM), nitric oxide was determined by measuring the sum concentration of NO2- and NO3- (NOx) in culture supernatants using a fluoremetric assay kit following the

manufacturer's instructions (Stratagene, La Jolla). Briefly, culture supernatants were collected and treated with NO3- for 30 min at room temperature to reduce NO3- to NO2-, which then

reacted with 2, 3-diaminomaphthalene for 10 min to yield the fluorescent product 1(H)-naphthotriazole. Fluorescence was measured with excitation and emission wavelengths of 365 and 450 nm,

respectively. Fluorescence readings were converted into concentration based on the standard curves and then represent as folds change compared to that of control. TYROSINE KINASE ACTIVITY

ASSAY Cells cultured in serum-free, phenol red-free M199 media for 24 h were treated with genistein as indicated in the figure legend or herbimycin A (10 µM) for 30 min. Cells were harvested

and lysed, and the supernatants were used for the tyrosine kinase activity assay according to method described (Liu et al., 2004). The relative fluorescence intensity were normalized to

corresponding protein levels and expressed as percentage of the controls. WESTERN BLOTTING Cells were lysed in ice-cold buffer containing 20mM Tris-HCl (pH 7.4), 2.5 mM EDTA, 1% Triton

X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM NaF, 1 mM Na3VO4 and 1 mM protease inhibitor cocktail from Roche (Alameda, CA). Cell lysates were centrifuged for 15 min at 12,000 _g_ to

remove cellular debris. Equal amounts of protein were separated on SDS-page gels and transfer to PVDF membranes (Millpore, Bedford, MA.). The membranes were blocked in a 5% skimmed milk

solution at room temperature for 1 h, and then incubated with anti-phospho-eNOS Ser1179, anti-phospho-eNOS Thr497 from upstate (Upstate Biotechnologies, Lake Placid, NY), or anti-eNOS

antibody from Transduction Laboratories (Lexington, KY). Blots were stripped and re-probed with anti-actin antibody (Sigma) to demonstrated equal loading. STATISTICAL ANALYSIS Comparisons

between groups were performed using one-way ANOVA followed by the Duncan test. Differences were considered statistically significant at the level of _P_ < 0.05 and values are represented

as means ± S.D. The statistical analysis was performed with the software of SPSS 11.0 for Windows. ABBREVIATIONS * Akt: protien kinase B * eNOS: endothelial nitric oxide synthase * ER:

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ACKNOWLEDGEMENTS This work was supported by Yangtz River Scholar and Innovation Research Team Development Program (Project No. IRT0945), and by the grants from the National Natural Science

Foundation of China (NO. 30700576) and State Key Laboratory of Animal Nutrition (Project No. 2004DA125184-0807). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * State key Lab of Animal

Nutrition, College of Animal Science and Technology, China Agricultural University (CAU), Beijing 100193, China., Ying Yang, Wei Nie, Jianmin Yuan, Bingkun Zhang, Zhong Wang & Yuming Guo

* College of Animal Science and Technology, CAU, Beijing 100193, China., Zhenlong Wu Authors * Ying Yang View author publications You can also search for this author inPubMed Google Scholar

* Wei Nie View author publications You can also search for this author inPubMed Google Scholar * Jianmin Yuan View author publications You can also search for this author inPubMed Google

Scholar * Bingkun Zhang View author publications You can also search for this author inPubMed Google Scholar * Zhong Wang View author publications You can also search for this author

inPubMed Google Scholar * Zhenlong Wu View author publications You can also search for this author inPubMed Google Scholar * Yuming Guo View author publications You can also search for this

author inPubMed Google Scholar CORRESPONDING AUTHORS Correspondence to Zhenlong Wu or Yuming Guo. RIGHTS AND PERMISSIONS This is an Open Access article distributed under the terms of the

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medium, provided the original work is properly cited. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Yang, Y., Nie, W., Yuan, J. _et al._ Genistein activates endothelial

nitric oxide synthase in broiler pulmonary arterial endothelial cells by an Akt-dependent mechanism. _Exp Mol Med_ 42, 768–776 (2010). https://doi.org/10.3858/emm.2010.42.11.078 Download

citation * Accepted: 30 September 2010 * Published: 06 October 2010 * Issue Date: November 2010 * DOI: https://doi.org/10.3858/emm.2010.42.11.078 SHARE THIS ARTICLE Anyone you share the

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Nature SharedIt content-sharing initiative KEYWORDS * endothelial cells * genistein * nitric oxide synthase type III * proto-oncogene proteins c-akt * pulmonary artery