Estrogen Induces the Akt-dependent Activation of Endothelial Nitric-oxide Synthase in Vascular Endothelial Cells*

Koji HisamotoDagger , Masahide OhmichiDagger §, Hirohisa Kurachi, Jun HayakawaDagger , Yuki KandaDagger , Yukihiro NishioDagger , Kazushige AdachiDagger , Keiichi TasakaDagger , Eiji Miyoshi||, Noriko Fujiwara||, Naoyuki Taniguchi||, and Yuji MurataDagger

From the Departments of Dagger  Obstetrics and Gynecology and || Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871 and the  Department of Obstetrics and Gynecology, Yamagata University School of Medicine, 2-2-2 Iidanishi, Yamagata, Yamagata 990-9585, Japan

Received for publication, June 12, 2000, and in revised form, September 19, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Although estrogen is known to activate endothelial nitric oxide synthase (eNOS) in the vascular endothelium, the molecular mechanism responsible for this effect remains to be elucidated. In studies of both human umbilical vein endothelial cells (HUVECs) and simian virus 40-transformed rat lung vascular endothelial cells (TRLECs), 17beta -estradiol (E2), but not 17alpha -E2, caused acute activation of eNOS that was unaffected by actinomycin D and was specifically blocked by the pure estrogen receptor antagonist ICI-182,780. Treatment of both TRLECs and HUVECs with 17beta -E2 stimulated the activation of Akt, and the PI3K inhibitor wortmannin blocked the 17beta -E2-induced activation of Akt. 17beta -E2-induced Akt activation was also inhibited by ICI-182,780, but not by actinomycin D. Either treatment with wortmannin or exogenous expression of a dominant negative Akt in TRLECs decreased the 17beta -E2-induced eNOS activation. Moreover, 17beta -E2-induced Akt activation actually enhances the phosphorylation of eNOS. 17beta -E2-induced Akt activation was dependent on both extracellular and intracellular Ca2+. We further examined the 17beta -E2-induced Akt activity in Chinese hamster ovary (CHO) cells transiently transfected with cDNAs for estrogen receptor alpha  (ERalpha ) or estrogen receptor beta  (ERbeta ). 17beta -E2 stimulated the activation of Akt in CHO cells expressing ERalpha but not in CHO cells expressing ERbeta . Our findings suggest that 17beta -E2 induced eNOS activation through an Akt-dependent mechanism, which is mediated by ERalpha via a nongenomic mechanism.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The inhibitory effect of estrogen on the development of atherosclerosis has been suggested by abundant human epidemiological and animal experimental data (1-9). The incidence of atherosclerotic diseases is lower in premenopausal women than in men, steeply rises in postmenopausal women, and is reduced to premenopausal levels in postmenopausal women who receive estrogen therapy (10-12). Until recently, the atheroprotective effects of estrogen were attributed principally to the effects on serum lipid concentrations. However, estrogen-induced alterations in serum lipids account for only approximately one-third of the observed clinical benefits of estrogen (12-14). Recent evidence suggests that the direct actions of estrogen on blood vessels contribute to the cardioprotective effects of estrogen (13, 15). There are many kinds of direct effects of estrogen on blood vessels, such as estrogen-induced increases of vasodilatation and inhibition of the response of blood vessels to injury and the development of atherosclerosis. However, the molecular mechanism underlying the estrogen-induced vasodilatation has not yet been determined. Several studies suggest that a key mediator of this vasodilator response could be the endothelium-derived relaxing factor nitric oxide (NO), and that brief treatment with estrogen increases basal NO release in endothelial cells without elevation of eNOS mRNA or protein (16). Estrogen activates endothelial nitric oxide synthase (eNOS) without altering expression of the eNOS gene in vascular endothelium (17-20). However, the details of the mechanism of the estrogen-induced eNOS activation are not yet well understood.

The serine/threonine kinase termed Akt or protein kinase B (PKB)1 is an important regulator of various cellular processes, including glucose metabolism and cell survival (21, 22). Activation of receptor tyrosine kinases and G-protein-coupled receptors, and stimulation of cells by mechanical force, can lead to the phosphorylation and activation of Akt (23-25). Akt was identified as a downstream component of survival signaling through phosphatidylinositol 3-kinase (PI3K) (26-30). Akt may be regulated by both phosphorylation and the direct binding of PI3K lipid products to the Akt pleckstrin homology domain. Akt can then phosphorylate substrates such as glycogen synthase kinase-3, 6-phosphofructo-2-kinase, and BAD. More recently, it was found that eNOS is also an Akt substrate and is activated by Akt-dependent phosphorylation to release NO in endothelial cells (31-34).

The actions of estrogen can be mediated by the classical nuclear receptors, ERalpha and ERbeta (35, 36) or through other putative membrane receptors. By definition, rapid effects of estrogen that involve nongenomic mechanisms are independent of transcriptional activation by the nuclear ERs. These rapid effects are believed to be mediated by receptors located in or close to the plasma membrane (37, 38). Estrogen-induced vasodilatation occurs 5-20 min after estrogen administration (39, 40) and is not dependent on changes in gene expression; this action of estrogen is sometimes referred to as "nongenomic." Therefore, we sought to determine whether the estrogen-induced eNOS activation is mediated by Akt activation and which type of ER is involved in this effect using both human umbilical vein endothelial cells (HUVECs) and simian virus 40-transformed rat lung vascular endothelial cells (TRLECs) (41).


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- 17beta -E2, 17alpha -E2, E2-17-BSA (17beta -estradiol (17-hemisuccinate/BSA; 38 mol E2/mol BSA), actinomycin D, and wortmannin were purchased from Sigma Chemical Co. (St. Louis, MO). ICI-182,780 was obtained from Tocris (Ballwin, MO). ECL Western blotting detection reagents were obtained from Amersham Pharmacia Biotech (Arlington Heights, IL). Rabbit polyclonal anti-Akt antibody and an Akt kinase assay kit, including GSK-3 fusion protein and a phospho-specific GSK-3alpha /beta antibody, were obtained from New England BioLabs (Beverly, MA). Rabbit polyclonal anti-hemagglutinin (HA) antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Cell Cultures-- TRLECs (41), kindly provided by Dr. K. Fukuo and Dr. S. Morimoto (Osaka University Medical School, Japan), and Chinese hamster ovary (CHO) cells, obtained from American Type Culture Collection (Rockville, MD), were cultured at 37 °C in Dulbecco's modified Eagle's medium with 10% fetal bovine serum in a water-saturated atmosphere of 95% O2 and 5% CO2. HUVECs were isolated according to the method of Jaffe et al. (42), plated in gelatin-coated tissue culture wells, and grown in M199 medium containing 20% fetal bovine serum and 50 µg/ml endothelial cell growth supplement (Clonetics Corp., San Diego, CA). HUVECs were used at passage 2 or 3.

Constructs-- The vectors encoding the various HA-tagged forms of Akt, wild-type Akt (HA-Akt), kinase-inactive Akt (HA-AktK179M), and constitutively active Akt (HA-mDelta 4-129Akt) used in this study have been described previously (26, 43, 45-47). The vectors encoding the wild-type eNOS and mutant eNOS of serine 1179 to alanine (S1179A eNOS) was a kind gift from Dr. W. C. Sessa (Yale University, New Haven, CT) (31). The human estrogen receptor alpha  (ERalpha ) expression vector, pSG5-HEGO, was a kind gift from Dr. P. Chambon (Institut de Chimie Biologique, Strasbourg, France) (48). The plasmid pSG5-mERbeta encoding nucleotides 12-1469 of ERbeta (35) was kindly provided Dr. E. R. Levin (University of California, Irvine, CA) via Dr. K. S. Korach (National Institutes of Health, Research Triangle Park, NC).

Assay of eNOS Activity-- Cells were serum-starved overnight in phenol red-free medium before eNOS activity measurements. eNOS activity was determined as the conversion of radiolabeled L-arginine to L-citrulline by a method described previously (50, 51) with a minor modification. Briefly, 10 µl of a sample was incubated for 10 min at 37 °C in a solution consisting of 50 mM HEPES, 1 mM dithiothreitol, 1 mM CaCl2, 0.1 mM tetrahydro-L-biopterin, 1 mM NADPH, 10 µg/ml calmodulin, 10 µM FAD, and 1.55 µM L-[guanidino-14C]arginine (pH 7.8), in a final volume of 100 µl. The reaction was terminated by the addition of 200 µl of buffer A (100 mM HEPES, 10 mM EDTA, pH 5.2). The whole reaction mixture was then applied to a 0.3-ml Dowex 50-WX column (Na+ form, 200-400 mesh) that had been equilibrated with buffer A. Citrulline was eluted with 0.5 ml of buffer A, and then radioactivity was measured with a liquid scintillation counter.

Assay of eNOS Activity Using a Transient Expression System-- TRLECs cultured in 100-mm dishes were transfected with 1 µg of CMV-6, 1 µg of CMV-6 containing the gene for HA-AktK179M, 1 µg of pSG5, 1 µg of ERalpha expression vector (pSG5-HEGO), or 1 µg of ERbeta expression vector (pSG5-mERbeta ) using LipofectAMINE plus (Life Technologies, Inc.) as described previously (52, 53). Seventy-two hours after transfection, serum-deprived cells were incubated with 10-7 M 17beta -E2 for 15 min, and the eNOS activity was measured as described above.

Assay of Akt Kinase Activity-- Cells were serum-starved overnight in phenol red-free medium and then treated with various materials. They were then washed twice with phosphate-buffered saline and lysed in ice-cold lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM beta -glycerolphosphate, 1 mM sodium orthovanadate, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). The extracts were centrifuged to remove cellular debris, and the protein content of the supernatants was determined using the Bio-Rad protein assay reagent. 250 µg of protein from the lysate samples was incubated with gentle rocking at 4 °C overnight with immobilized anti-Akt antibody cross-linked to agarose hydrazide beads. After Akt was selectively immunoprecipitated from the cell lysates, the immunoprecipitated products were washed twice in lysis buffer and twice in kinase assay buffer (25 mM Tris, pH 7.5, 10 mM MgCl2, 5 mM beta -glycerolphosphate, 0.1 mM sodium orthovanadate, and 2 mM dithiothreitol), and the samples were resuspended in 40 µl of kinase assay buffer containing 200 µM ATP and 1 µg of GSK-3alpha fusion protein. The kinase reaction was allowed to proceed at 30 °C for 30 min and stopped by the addition of Laemmli SDS sample buffer (54). Reaction products were resolved by 15% SDS-PAGE followed by Western blotting (55) with an anti-phospho-GSK-3alpha /beta antibody as described previously (47).

Assay of Akt Activity Using a Transient Expression System-- CHO cells cultured in 100-mm dishes were transfected with 1 µg of pSG5, 1 µg of ERalpha expression vector (pSG5-HEGO), or 1 µg of ERbeta expression vector (pSG5-mERbeta ) using LipofectAMINE plus as described previously (52, 53). Seventy-two hours after transfection, serum-deprived cells were incubated with 10-7 M 17beta -E2 for 15 min, and the Akt activity was measured as described above.

Preparation of Partially Purified eNOS-- Human eNOS was overexpressed in Sf-21 cells, which had been infected with baculovirus carrying human eNOS cDNA (56). Human eNOS was partially purified by chromatography on 2',5'-ADP-Sepharose gel, and its specificity was determined as described previously (57).

Assay of eNOS Phosphorylation-- TRLECs cultured in 100-mm dishes were treated with 10-7 M 17beta -E2 for 15 min. Cell lysates were subjected to immunoprecipitation with anti-Akt antibody. For assay using a transient expression system, TRLECs cultured in 100-mm dishes were transfected with 1 µg of HA-Akt, 1 µg of HA-AktK179M, or 1 µg of HA-mDelta 4-129Akt using LipofectAMINE plus (Life Technologies, Inc.) as described previously (52, 53). Seventy-two hours after transfection, serum-deprived cells were incubated with 10-7 M 17beta -E2 for 15 min, and lysates were immunoprecipitated with anti-HA antibody. The immunoprecipitated products were washed once in lysis buffer and twice in kinase assay buffer, and samples were resuspended in 30 µl of kinase assay buffer containing 40 µM [gamma -32P]ATP (1 µCi) and 5 µg of partially purified eNOS, or 5 µg of recombinant wild-type or S1179A eNOS purified from Escherichia coli. The kinase reaction was allowed to proceed at room temperature for 5 min and stopped by the addition of Laemmli SDS sample buffer (54). Reaction products were resolved by 8% SDS-PAGE.

Statistics-- Statistical analysis was performed using Student's t test, and p < 0.05 was considered significant. Data are expressed as the mean ± S.E.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

eNOS Activation by 17beta -E2-- To evaluate whether eNOS is activated by 17beta -E2 in TRLECs (Fig. 1A, upper panel) and HUVECs (Fig. 1A, lower panel), cultured cells were exposed to 17beta -E2 for the indicated times. The increase in eNOS activity induced by 10-7 M 17beta -E2 reached a plateau from 15 through 30 min and rapidly declined thereafter. The dose dependence of 17beta -E2-induced eNOS activation was also evaluated in TRLECs (Fig. 1B). TRLECs were treated with various concentrations of 17beta -E2 for 15 min. In the range of 10-10 to 10-7 M, 17beta -E2 induced the activation of eNOS in a dose-dependent manner. A higher concentration (10-6 M) of 17beta -E2 did not induce a stronger response (data not shown). The response was specific for 17beta -E2, because 17alpha -E2 had no effect (Fig. 2A). To determine whether this response involves rapid ER activation, the effect of concomitant treatment with the pure ER antagonist ICI-182,780 was determined (Fig. 2B). ICI-182,780 completely abolished the induction of eNOS activation by 17beta -E2. Moreover, the effects of E2-17-BSA, a membrane-impermeable conjugate of E2, and actinomycin D, an inhibitor of gene transcription, were tested to rule out the influence of genomic events mediated by nuclear ERs (Fig. 2C). E2-17-BSA stimulated an increase in eNOS activity similar to that induced by 17beta -E2, and actinomycin D did not affect the induction of eNOS activation by 17beta -E2.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1.   Activation of eNOS in endothelial cells. Cells were grown in 60-mm dishes. A, TRLECs (upper panel) and HUVECs (lower panel) were treated with 10-7 M 17beta -E2 for the indicated times. B, TRLECs were treated with the indicated concentrations of 17beta -E2 for 15 min. eNOS activity was measured by the conversion of L-[guanidino-14C]arginine to L-[guanidino-14C]citrulline, as described under "Experimental Procedures." The basal activity of eNOS was arbitrarily set at 1.0. Data are expressed as the mean -fold activation ± S.E. of six separate experiments. *p < 0.05 and **p < 0.01 as compared with the control, respectively.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2.   Specificity of the augmentation of eNOS activation by 17beta -E2. Cells were grown in 60-mm dishes. A, TRLECs were treated with 10-7 M 17beta -E2 or 10-7 M 17alpha -E2 for 15 min. B, TRLECs were pretreated with 10-6 M ICI-182,780 for 15 min, followed by treatment with 10-7 M 17beta -E2 for 15 min. C, TRLECs were pretreated with or without 25 µg/ml actinomycin D (Act-D) for 120 min, followed by treatment with 10-7 M 17beta -E2 or 10-7 M E2-17-BSA for 15 min. eNOS activity was measured by the conversion of L-[guanidino-14C]arginine to L-[guanidino-14C]citrulline, as described under "Experimental Procedures." The basal activity of eNOS was arbitrarily set at 1.0. Data are expressed as the mean -fold activation ± S.E. of six separate experiments. **p < 0.01 as compared with the control.

Activation of Akt by 17beta -E2-- To determine whether Akt is activated by 17beta -E2 in TRLECs and HUVECs, 17beta -E2 was added to cultured cells for the indicated times (Fig. 3A) and at the indicated concentrations for 15 min (Fig. 3B). Cell lysates were subjected to immunoprecipitation with immobilized anti-Akt antibody, and then supplemented with GSK-3alpha fusion protein and analyzed by Western blotting with anti-phospho-GSK-3alpha /beta antibody. Activation of Akt by 17beta -E2 in both TRLECs and HUVECs reached a plateau at 15 min, and declined thereafter (Fig. 3A). 17beta -E2 induced the activation of Akt in a dose-dependent manner in TRLECs (Fig. 3B) and HUVECs (data not shown). The response was specific for 17beta -E2, because 10-10-10-7 M 17alpha - E2 had no effect (Fig. 4A). Because Akt is an effector of survival signaling downstream of PI3K (26-30), we next examined whether stimulation of TRLECs with 17beta -E2 could increase the activity of Akt through a PI3K-dependent mechanism. TRLECs were stimulated with 17beta -E2 in the presence or absence of wortmannin, a PI3K inhibitor, and the kinase activity of Akt was assayed. The induction of Akt activity by 17beta -E2 was inhibited by wortmannin (Fig. 4B, lane 6). These results indicate that E2 activates Akt activity through a PI3K-dependent mechanism.



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 3.   Activation of Akt by 17beta -E2 in endothelial cells. Cells were grown in 100-mm dishes. A, TRLECs (upper panel) and HUVECs (lower panel) were treated with 10-7 M 17beta -E2 for the indicated times. B, TRLECs were treated with the indicated concentrations of 17beta -E2 for 15 min. Lysates were subsequently subjected to immunoprecipitation with immobilized anti-Akt antibody, and the kinase reaction was carried out in the presence of cold ATP and GSK-3alpha fusion protein, as described under "Experimental Procedures." After the reactions were stopped with Laemmli sample buffer, samples were resolved by 12% SDS-PAGE and then analyzed by Western blotting with an anti-phospho-GSK-3alpha /beta antibody. Experiments were repeated three times with essentially identical results.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4.   Specificity of the augmentation of Akt activation by 17beta -E2. Cells were grown in 100-mm dishes. A, TRLECs were treated with 10-7 M 17beta -E2 or the indicated concentrations of 17alpha -E2 for 15 min. B, TRLECs were pretreated with or without 2 × 10-7 M wortmannin for 15 min or 10-6 M ICI-182,780 for 15 min, followed by treatment with 10-7 M 17beta -E2 for 15 min. C, TRLECs were pretreated with or without 25 µg/ml actinomycin D (Act-D) for 120 min, followed by treatment with 10-7 M 17beta -E2 or 10-7 M E2-17-BSA for 15 min. Experiments were repeated three times with essentially identical results.

To determine whether this process involves rapid ER activation, the effect of concomitant treatment with the pure ER antagonist ICI-182,780 was determined (Fig. 4B, lane 4). ICI-182,780 clearly caused a decrease in 17beta -E2-induced Akt activation. Moreover, E2-17-BSA, a membrane-impermeable conjugate of E2, and actinomycin D, an inhibitor of gene transcription, were used to rule out the influence of genomic events mediated by nuclear ERs (Fig. 4C). E2-17-BSA also stimulated an increase in Akt activity, and actinomycin D did not affect the induction of Akt activity by 17beta -E2.

Akt-dependent eNOS Phosphorylation and Activation-- To determine whether 17beta -E2-induced Akt activation is involved in the phosphorylation of eNOS, 10-7 M 17beta -E2 was added to cultured cells for 15 min. Cell lysates were subjected to immunoprecipitation with anti-Akt antibody, and then assayed in an immunocomplex kinase assay using purified eNOS (57) as a substrate (Fig. 5A, left panel). 17beta -E2 directly increased the phosphorylation of eNOS in anti-Akt immunoprecipitates. Moreover, we evaluated the effect of exogenous expression of various forms of Akt on the in vitro phosphorylation of purified eNOS. TRLEC transfected with wild-type or mutant forms of hemagglutinin (HA)-tagged Akt were exposed to 10-7 M 17beta -E2 for 15 min, and extracts from these cells were immunoprecipitated with anti-HA antibody and assayed in an immunocomplex kinase assay for their ability to phosphorylate purified eNOS. Akt constructs that were expressed in TRLEC included HA-tagged wild-type Akt (HA-Akt), an Akt derivative rendered kinase-inactive by point mutation within the Akt catalytic domain (HA-AktK179M), and an Akt derivative rendered constitutively active by targeting it to the plasma membrane with a myristoyl tag (HA-mDelta 4-129Akt) (26, 43, 45-47). 17beta -E2 directly increased the phosphorylation of eNOS in anti-HA immunoprecipitates prepared from TRLEC transfected with wild-type Akt (Fig. 5A, lane 2). Anti-HA immunoprecipitates prepared from TRLEC transfected with the kinase-inactive Akt failed to phosphorylate eNOS induced by 17beta -E2 (Fig. 5A, lane 4). In addition, anti-HA immunoprecipitates from TRLEC transfected with constitutively active Akt were found to induce eNOS phosphorylation in immunocomplex kinase assays (Fig. 5A, lane 5). Because it was reported that Akt directly phosphorylated on serine 1179 (31), an immunocomplex kinase assay with anti-Akt antibody was performed using recombinant wild-type eNOS or mutant eNOS of serine 1179 to alanine (eNOS S1179A) as a substrate (Fig. 5B). Mutation of serine 1179 to alanine markedly reduced 17beta -E2-induced phosphorylation of eNOS compared with the wild-type protein. These results suggest that 17beta -E2-induced Akt activation actually increases the phosphorylation of eNOS. Next, we sought to determine whether an Akt cascade is involved in the regulation of the eNOS activation induced by 17beta -E2 in the endothelial cells. To examine whether the stimulation of the eNOS activation by 17beta -E2 is the result of activation of Akt, either wortmannin (Fig. 5C) or an expression vector, kinase-inactive HA-AktK179M, was used (Fig. 5D). Pretreatment with 2 × 10-7 M wortmannin for 15 min completely abolished the 17beta -E2-induced eNOS activation (Fig. 5C). In addition, transfection with HA-AktK179M clearly abolished the 17beta -E2-induced eNOS activation, whereas transfection with control vector had no effect on the 17beta -E2-induced eNOS activation (Fig. 5D). These results suggest that the PI3K-Akt cascade is involved in the 17beta -E2-induced eNOS activation.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5.   Akt-dependent eNOS phosphorylation and activation. A, the effect of expressed Akt on the phosphorylation of purified eNOS induced by 10-7 M 17beta -E2 for 15 min was examined. Immunocomplex kinase assays were performed using anti-Akt immunoprecipitates from TRLECs (left panel) or using anti-HA immunoprecipitates from TRLECs expressing HA-tagged Akt constructs encoding HA-Akt (Wild-type Akt), kinase-inactive HA-AktK179M (Inactive Akt), or constitutively active HA-mDelta 4-129Akt (Active Akt) expressed in TRLECs (right panel). B, the effect of 17beta -E2 on the phosphorylation of recombinant wild-type eNOS or eNOS S1179A was examined. Immunocomplex kinase assays were performed using anti-Akt immunoprecipitates from TRLECs treated with or without 10-7 M 17beta -E2 for 15 min. C and D, the effect of wortmannin and kinase-deficient Akt on 17beta -E2-induced eNOS activation was examined. TRLECs were grown in 60-mm dishes. Cells were pretreated with or without 2 × 10-7 M wortmannin for 15 min, followed by treatment with 10-7 M E2 for 15 min (C) or cells were transfected with control vector (CMV-6) or kinase-inactive HA-AktK179M (Inactive Akt) and, after 72 h, were stimulated with 10-7 M E2 for 15 min (D). eNOS activity was measured as described in the legend for Fig. 1. The basal activity of eNOS of parent cells (C) or cells transfected with CMV-6 (D) was arbitrarily set at 1.0. Data are expressed as the mean -fold activation ± S.E. of six separate experiments. **p < 0.01 as compared with the control.

Role of Extracellular and Intracellular Ca2+ in 17beta -E2-induced Akt and eNOS Activation-- eNOS is a Ca2+/calmodulin-dependent enzyme, and it has been reported that estrogen induces translocation of eNOS in a Ca2+-dependent and receptor-mediated manner (58). A23187 induces eNOS activation and produces endothelium-dependent vascular relaxation (19, 59). Thus, eNOS activity is largely regulated by Ca2+ mobilization. We therefore evaluated the role of extracellular and intracellular Ca2+ in 17beta -E2-induced Akt and eNOS activation in TRLECs (Fig. 6). Elimination of extracellular Ca2+ by treatment with 3 mM EGTA for 1 min clearly blocked the A23187-induced Akt (Fig. 6A, upper panel) and eNOS (Fig. 6A, lower panel) activation, and similarly, treatment with 3 mM EGTA for 1 min clearly inhibited the 17beta -E2-induced Akt (Fig. 6A, upper panel) and eNOS (Fig. 6A, lower panel) activation, indicating that Ca2+ influx is required for 17beta -E2-induced Akt and eNOS activation. Next, the effect of intracellular Ca2+ on 17beta -E2-induced Akt and eNOS activation was examined (Fig. 6B). Treatment with 50 µM 1,2-bis(o-amino-phenoxy)ethane-N,N,N'-tetraacetic-acetoxymethyl ester (BAPTA-AM) for 20 min to eliminate intracellular Ca2+ (52, 53) completely blocked the 17beta -E2-induced Akt (Fig. 6B, upper panel) and eNOS (Fig. 6B, lower panel) activation. Moreover, elimination of both extracellular and intracellular Ca2+ by treatment with 3 mM EGTA for 15 min (52, 53, 60) abolished the 17beta -E2-induced Akt (Fig. 6B, upper panel) and eNOS (Fig. 6B, lower panel) activation, indicating that intracellular Ca2+ is also required for 17beta -E2-induced Akt and eNOS activation. Thus, these results suggest that Ca2+ mobilization mediated by both extracellular and intracellular Ca2+ is required for the 17beta -E2-induced Akt and eNOS activation.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 6.   Role of extracellular and intracellular Ca2+ in 17beta -E2-induced Akt and eNOS activation. A, cells were pretreated with 3 mM EGTA for 1 min, and then treated with 10-6 M A23187 or 10-7 M 17beta -E2 for 15 min. B, cells were pretreated with 50 µM BAPTA-AM for 20 min or 3 mM EGTA for 15 min, and then treated with 10-7 M 17beta -E2 for 15 min. Akt activity (upper panel) and eNOS activity (lower panel) were measured as described in the legends for Figs. 3 and 1, respectively. The basal activity of eNOS was arbitrarily set at 1.0. Data are expressed as the mean -fold activation ± S.E. of six separate experiments. **p < 0.01 as compared with the control.

Effect of ERalpha or ERbeta Expression on 17beta -E2-induced Akt and eNOS Activation-- The potential role of ERalpha or ERbeta in 17beta -E2-induced Akt activation was evaluated. Transfection of ERbeta into TRLECs had no effect on 17beta -E2-induced Akt activation compared with transfection of control vector (Fig. 7A, upper panel). On the other hand, transfection of ERalpha into TRLECs caused an increase in both basal and 17beta -E2-induced Akt activation compared with transfection of control vector (Fig. 7A, upper panel). Moreover, transfection of ERalpha into TRLECs caused an increase in 17beta -E2-induced eNOS activation compared with transfection of control vector or of ERbeta (Fig. 7A, lower panel). We confirmed that both ERalpha and ERbeta were expressed in TRLECs (data not shown). Therefore, CHO cells, which do not express ERalpha or ERbeta (61), were used to examine which of these receptors is involved in 17beta -E2-induced Akt activation. In CHO cells transfected with control vector or ERbeta , 17beta -E2 had no effect on Akt activity (Fig. 7B). However, in cells transfected with ERalpha , there was an apparent increase in Akt activity upon brief stimulation with 17beta -E2 (Fig. 7B). These results indicate that 17beta -E2 induces Akt activity through ERalpha .



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of ERalpha or ERbeta expression on 17beta -E2-induced Akt and eNOS activation. Cells were grown in 60-mm dishes. A, TRLECs were transfected with control vector (pSG5), ERalpha expression vector (pSG5-HEGO), or ERbeta expression vector (pSG5-mERbeta ) and, after 72 h, were stimulated with 10-7 M 17beta -E2 for 15 min. Akt activity (upper panel) and eNOS activity (lower panel) were measured as described in the legend for Figs. 3 and 1, respectively. The basal activity of eNOS of transfected cells was arbitrarily set at 1.0. Data are expressed as the mean -fold activation ± S.E. of six separate experiments. **p < 0.01 as compared with the control. B, CHO cells were transfected with empty vector (pSG5), ERalpha expression vector (pSG5-HEGO), or ERbeta expression vector (pSG5-mERbeta ) and, after 72 h, were stimulated with 10-7 M 17beta -E2 for 15 min. Akt activity was measured as described in the legend for Fig. 3. Experiments were repeated three times with essentially identical results.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study showed that Akt is activated in HUVECs and TRLECs by 17beta -E2 and that Akt activation is required for the activation of eNOS following brief treatment with 17beta -E2: Treatment with wortmannin, a PI3K inhibitor, attenuated the 17beta -E2-induced Akt and eNOS activation, and TRLECs expressing inactive Akt showed less induction by 17beta -E2 of eNOS activity. A pure ER antagonist, ICI-182,780 completely inhibited the 17beta -E2-induced Akt and eNOS activation. Moreover, in CHO cells transfected with ERalpha , there was an apparent increase in Akt activity upon brief stimulation with 17beta -E2. These results suggest that a 17beta -E2-induced PI3K-Akt cascade stimulates the activation of eNOS through ERalpha .

Normal endothelium secretes nitric oxide, which relaxes vascular smooth muscle and inhibits platelet activation (62). In cultured endothelial cells, physiologic concentrations of estrogen cause a rapid release of nitric oxide without altering gene expression (17, 18). The rapidity of the activation of Akt (Fig. 3) and eNOS (Fig. 1) by 17beta -E2 along with the fact that the activation was not altered by the inhibition of gene transcription by actinomycin D indicate that the process may not require the classical nuclear effects of the estrogen. However, the acute response of Akt (Fig. 4B) and eNOS (Fig. 2B) to 17beta -E2 was fully inhibited by concomitant treatment with the pure ER antagonist ICI-182,780, suggesting that the response requires a rapid ER activation. Are these rapid effects of estrogen mediated by an unidentified estrogen receptor or by the known estrogen receptors acting in a novel way? The existence of rapidly acting membrane receptors for steroid hormones in both nonvascular and vascular cells has been suggested for over two decades (63, 64), but no such receptors have been isolated or cloned. Alternatively, the rapid effects of estrogen on vascular cells could be mediated by a known estrogen receptor, perhaps located in the plasma membrane (64), which is able to activate nitric oxide synthase in a nongenomic manner (19, 20). In addition, estrogen increases the expression of genes for important vasodilatory enzymes such as prostacyclin synthase and nitric oxide synthase (65, 66). Some of the effects of estrogen may therefore be due to longer-term increases in the expression of the genes for these enzymes in vascular tissues.

There are two estrogen receptors, estrogen receptor alpha  (ERalpha ) and estrogen receptor beta  (ER beta ), both of which are members of the superfamily of steroid hormone receptors (35, 67). Genetic disruption of ERalpha in mice leads to lower levels of vascular nitric oxide (69). In addition, ERalpha can directly activate endothelial nitric oxide synthase (19, 20). We found that there was an apparent increase in Akt activity upon brief stimulation with 17beta -E2 in CHO cells transfected with ERalpha , whereas 17beta -E2 had no effect on Akt activity in cells transfected with control vector or ERbeta (Fig. 7B). These findings suggest that ERalpha is capable of mediating the acute response and rapid vasodilatation caused by estrogen. What is the role of ER beta  in endothelial cells? In addition to forming homodimers, ERalpha and ERbeta can form heterodimers with each other (70), adding a further degree of complexity in the regulation of gene expression by estrogen in cells expressing both receptors. Estrogen continues to provide protection against vascular injury in mice in which ERalpha has been disrupted (71), and the expression of ERbeta , but not ERalpha , is elevated after vascular injury in male rats (72). Estrogen also provides protection against vascular injury in mice in which ERbeta has been disrupted (73), suggesting the possibility that either of the two known estrogen receptors is sufficient to protect against vascular injury or that some other unknown signaling pathway is involved.

eNOS is a Ca2+/calmodulin-dependent enzyme, and it has been reported that estrogen induces translocation of eNOS by a Ca2+-dependent and receptor-mediated mechanism (58). Because A23187 induces eNOS activation and produces endothelium-dependent vascular relaxation, Ca2+ entry from the extracellular space into endothelial cells also plays a key role (19, 59). Ca2+/calmodulin-dependent protein kinase kinase can activate and phosphorylate Akt (74). In addition, A23187 also activates Akt (75). More recently, it was also reported that plasma-lemmal caveolae-associated ERalpha mediates the acute estrogen-stimulated NO production which occurs via a novel Ca2+-dependent signaling pathway (20). We confirmed that 17beta -E2-induced eNOS activation was dependent on both extracellular and intracellular Ca2+ (Fig. 6, lower panel). Interestingly, although it was reported that phosphorylation of eNOS by Akt represents a novel Ca2+-independent regulatory mechanism for activation of eNOS (31-34, 76), the current findings indicate that 17beta -E2-induced Akt activation was dependent on both extracellular and intracellular Ca2+ (Fig. 6, upper panel).

Estrogen can cause the rapid activation of signaling pathways involving c-src-related tyrosine kinases and MAP kinases in nonendothelial cells (77, 78). It has also been reported that tyrosine kinase-MAP kinase signaling is involved in acute ERalpha -mediated eNOS activation in endothelial cells (19, 79). Although no direct evidence that eNOS is one of the substrates of MAP kinase has been reported, it has been reported very recently that eNOS is a substrate of Akt in endothelial cells (31-34), and that PI3K and Akt contribute to the production of NO stimulated by insulin in endothelial cells (80). In addition, estrogen stimulates the activation of Akt, and Akt is a downstream effector of estrogen-dependent proliferation and survival in hormone-responsive MCF-7 breast carcinoma cells (81). However, until recently there had not been any studies addressing the role of the PI3K-Akt cascade in estrogen-induced eNOS activation. This is the first report showing that estrogen stimulates the activation of the PI3K-Akt cascade in endothelial cells and that this cascade might be involved in the eNOS activation induced by estrogen. Is there any cross-talk between the MAP kinase and PI3K-Akt signaling cascades? Although we2 and other groups (44, 49, 68) reported that MAP kinase and PI3K-Akt signaling cascades converge at BAD to suppress the apoptotic effect of BAD, further investigations are necessary to examine whether MAP kinase and PI3K-Akt signaling cascades converge at eNOS to stimulate the release of NO. We are currently investigating this possibility.


    ACKNOWLEDGEMENTS

We thank Drs. Michael E. Greenberg and Sandeep Robert Datta for the gift of the vectors encoding the various HA-tagged forms of Akt, Dr. P. Chambon for the gift of the human ERalpha expression vector (pSG5-HEGO), Drs. E. R. Levin and K. S. Korach for the gift of the mouse ERbeta expression vector (pSG5-mERbeta ), and Dr. W. C. Sessa for wild-type and S1179A eNOS.


    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 81-6-879-3354; Fax: 81-6-879-3359; E-mail: masa@gyne.med.osaka-u.ac.jp.

Published, JBC Papers in Press, October 23, 2000, DOI 10.1074/jbc.M005036200

2 Hayakawa, J., Ohmichi, M., Kurachi, H., Kanda, Y., Hisamoto, K., Nishio, Y., Adachi, K., Tasaka, K., Kanzaki, T., and Murata, Y. (2000) Cancer Res. 60, 5988-5994.


    ABBREVIATIONS

The abbreviations used are: Akt/PKB, protein kinase B; E2, estradiol; NO, nitric oxide; eNOS, endothelial nitric oxide synthase; ER, estrogen receptor; ERalpha , estrogen receptor alpha ; ERbeta , estrogen receptor beta ; TRLECs, simian virus 40-transformed rat lung vascular endothelial cells; HUVECs, human umbilical vein endothelial cells; CHO, Chinese hamster ovary; MAP kinase, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; PAGE, polyacrylamide gel electrophoresis; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester; CMV, cytomegalovirus; HA, hemagglutinin; BSA, bovine serum albumin; BAD, Bcl-2-associated death promoter.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Levy, H., and Boas, E. P. (1936) JAMA 107, 97-102
2. Matthews, K. A., Meilahan, E., Kuller, L. H., Kelsey, S. F., Caggiula, A., and Wing, R. R. (1989) N. Engl. J. Med. 321, 641-646[Abstract]
3. Gruchow, H. W., Anderson, A. J., Barboriak, J. J., and Sobocinski, K. A. (1988) Am. Heart. J. 115, 954-963[Medline] [Order article via Infotrieve]
4. Nabulsi, A. A., Folsom, A. R., White, A., Patsch, W., Heiss, G., Wu, K. K., and Szklo, M. (1993) N. Engl. J. Med. 328, 1069-1075[Abstract/Free Full Text]
5. Williams, J. K., Adams, M. R., and Klopfenstein, H. S. (1990) Circulation 81, 1680-1687[Abstract]
6. Chen, S. J., Li, H., Durand, J., Oparil, S., and Chen, Y. F. (1996) Circulation 93, 577-584[Abstract/Free Full Text]
7. Jacobsson, J., Cheng, L., Lyke, K., Kuwahara, M., Kagan, E., Ramwell, P. W., and Foegh, M. L. (1992) J. Heart Lung Transplant. 11, 1188-1193[Medline] [Order article via Infotrieve]
8. Adams, M. R., Kaplan, J. R., Koritnik, D. R., and Clarkson, T. B. (1987) Arteriosclerosis 7, 378-383[Abstract]
9. Adams, M. R., Kaplan, J. R., Manuck, S. B., Koritnik, D. R., Parks, J. S., Wolfe, M. S., and Clarkson, T. B. (1990) Arteriosclerosis 10, 1050-1057
10. Barrett-Connor, E. (1997) Circulation 95, 252-264[Free Full Text]
11. Stampfer, M. J., Colditz, G. A., Willett, W. C., Manson, J. E., Rosner, B., Speizer, F. E., and Hennekens, C. H. (1991) N. Engl. J. Med. 325, 756-762[Abstract]
12. Grady, D., Rubin, S. M., Petitti, D. B., Fox, C. S., Black, D., Ettinger, B., Ernster, V. L., and Cummings, S. R. (1992) Ann. Intern. Med. 117, 1016-1037[Medline] [Order article via Infotrieve]
13. Mendelsohn, M. E., and Karas, R. H. (1994) Curr. Opin. Cardiol. 9, 619-626[Medline] [Order article via Infotrieve]
14. Bush, T. L., Barrett-Connor, E., Cowan, L. D., Criqui, M. H., Wallace, R. B., Suchindran, C. M., Tyroler, H. A., and Rifkind, B. M. (1987) Circulation 75, 1102-1109[Abstract]
15. Farhat, M. Y., Lavigne, M. C., and Ramwell, P. W. (1996) FASEB J . 10, 615-624[Abstract/Free Full Text]
16. Lantin-Hermoso, R. L., Rosenfeld, C. R., Yuhanna, I. S., Germian, Z., Chen, Z., and Shaul, P. W. (1997) Am. J. Physiol. 273, 119-126
17. Lantin-Hermoso, R. L., Rosenfeld, C. R., Yuhanna, I. S., German, Z., Chen, Z., and Shaul, P. W. (1997) Am. J. Physiol. 273, L119-L126[Abstract/Free Full Text]
18. Caulin-Glaser, T., Garcia-Cardena, G., Sarrel, P., Sessa, W. C., and Bender, J. R. (1997) Circ. Res. 81, 885-892[Abstract/Free Full Text]
19. Chen, Z., Yuhanna, I. S., Galcheva-Gargova, Z., Karas, R. H., Mendelsohn, M. E., and Shaul, P. W. (1999) J. Clin. Invest. 103, 401-406[Abstract/Free Full Text]
20. Kim, H. P., Lee, J. Y., Jeong, J. K., Bae, S. W., Lee, H. K., and Jo, I. (1999) Biochem. Cell Biol. Commun. 263, 257-262
21. Coffer, P. J., Jin, J., and Woodgett, J. R. (1998) Biochem. J. 335, 1-13[Medline] [Order article via Infotrieve]
22. Franke, T. F., Kaplan, D. R., and Cantley, L. C. (1997) Cell 88, 435-437[Medline] [Order article via Infotrieve]
23. Downward, J. (1998) Curr. Opin. Cell Biol. 10, 262-267[CrossRef][Medline] [Order article via Infotrieve]
24. Murga, C., Laguinge, L., Wetzker, R., Cuadrado, A., and Gutkind, J. S. (1998) J. Biol. Chem. 273, 19080-19085[Abstract/Free Full Text]
25. Dimmeler, S., Assmus, B., Hermann, C., Hermann, C., Haendeler, J., and Zeiher, A. M. (1998) Circ. Res. 83, 334-341[Abstract/Free Full Text]
26. Dudek, H., Datta, S. R., Franke, T. F., Birnbaum, M. J., Yao, R., Cooper, G. M., Segal, R. A., Kaplan, D. R., and Greenberg, M. E. (1997) Science 275, 661-665[Abstract/Free Full Text]
27. Kauffmann-Zeh, A., Rodriguez-Viciana, P., Ulrich, E., Gilbert, C., Coffer, P., Downward, J., and Evan, G. (1997) Nature 385, 544-548[CrossRef][Medline] [Order article via Infotrieve]
28. Kennedy, S. G., Wagner, A. J., Conzen, S. D., Jordan, J., Bellacosa, A., Tsichlis, P. N., and Hay, N. (1997) Genes Dev. 11, 701-713[Abstract]
29. Khwaja, A., Rodriguez-Viciana, P., Wennstrom, S., Warne, P. H., and Downward, J. (1997) EMBO J. 16, 2783-2793[Abstract/Free Full Text]
30. Kulik, G., Klippel, A., and Weber, M. J. (1997) Mol. Cell. Biol. 17, 1595-1606[Abstract]
31. Fulton, D., Gratton, J. P., McCabe, T. J., Fontana, J., Fujio, Y., Walsh, K., Franke, T. F., Papapetropoulos, A., and Sessa, W. C. (1999) Nature 399, 597-601[CrossRef][Medline] [Order article via Infotrieve]
32. Dimmeler, S., Fleming, I., Fisslthaler, B., Hermann, C., Busse, R., and Zeiher, A. M. (1999) Nature 399, 601-605[CrossRef][Medline] [Order article via Infotrieve]
33. Michell, B. J., Griffiths, J. E., Mitchelhill, K. I., Rodriguez-Crespo, I., Tiganis, T., Bozinovski, S., de Montellano, P. O., Kemp, B. E., and Pearson, R. B. (1999) Curr. Biol. 9, 845-848[CrossRef][Medline] [Order article via Infotrieve]
34. Gallis, B., Corthals, G. L., Goodlett, D. R., Ueba, H., Kim, F., Presnell, S. R., Figeys, D., Harrison, D. G., Berk, B. C., Aebersold, R., and Corson, M. A. (1999) J. Biol. Chem. 274, 30101-30108[Abstract/Free Full Text]
35. Kuiper, G. G., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J-A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5925-5930[Abstract/Free Full Text]
36. Homma, H., Kurachi, H., Nishio, Y., Takeda, T., Yamamoto, T., Adachi, K., Morishige, K., Ohmichi, M., Matsuzawa, Y., and Murata, Y. (2000) J. Biol. Chem. 275, 11404-11411[Abstract/Free Full Text]
37. Pietras, R. J., and Szego, C. M. (1977) Nature 265, 69-72[Medline] [Order article via Infotrieve]
38. Pietras, R. J., and Szego, C. M. (1979) J. Steroid. Biochem. 11, 1471-1483[CrossRef][Medline] [Order article via Infotrieve]
39. Reis, S. E., Gloth, S. T., Blumenthal, R. S., Resar, J. R., Zacur, H. A., Gerstenblith, G., and Brinker, J. A. (1994) Circulation 89, 52-60[Abstract]
40. Guetta, V., Quyyumi, A. A., Prasad, A., Panza, J., Waclawiw, M., and Cannon, R. O. (1997) Circulation 96, 2795-2801[Abstract/Free Full Text]
41. Jiang, B., Morimoto, S., Yang, J., Niinoabu, T., Fukuo, K., and Ogihara, T. (1998) J. Cardiovasc. Pharmacol. 31, S142-S144[CrossRef][Medline] [Order article via Infotrieve]
42. Jaffe, E. A., Nachman, R. L., Becker, C. G., and Minick, R. C. (1973) J. Clin. Invest. 52, 2745-2756[Medline] [Order article via Infotrieve]
43. Datta, S. R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., and Greenberg, M. E. (1997) Cell 91, 231-241[Medline] [Order article via Infotrieve]
44. Bonni, A., Brunet, A., West, A. E., Datta, S. R., Takasu, M. A., and Greenberg, M. E. (1999) Science 286, 1358-1362[Abstract/Free Full Text]
45. Franke, T. F., Yang, S. I., Chan, T. O., Datta, K., Kazlauskas, A., Morison, D. K., Kaplan, D. R., and Tsichlis, P. N. (1995) Cell 81, 727-736[Medline] [Order article via Infotrieve]
46. Kohn, A. D., Takeuchi, F., and Roth, R. A. (1926) (1996) J. Biol. Chem. 271, 21920-2[Abstract/Free Full Text]
47. Hayakawa, J., Ohmichi, M., Kurachi, H., Kanda, Y., Hisamoto, K., Nishio, Y., Adachi, K., Tasaka, K., Kanzaki, T., and Murata, Y. (2000) Cancer Res. 60, 5988-5994[Abstract/Free Full Text]
48. Green, S., and Chambon, P. (1986) J. Steroid. Biochem. 24, 77-83[CrossRef][Medline] [Order article via Infotrieve]
49. Fang, X., Yu, S., Eder, A., Mao, M., Bast, R. C., Jr., Boyd, D., and Mills, G. B. (1999) Oncogene 18, 6635-6640[CrossRef][Medline] [Order article via Infotrieve]
50. Seo, H. G., Tatsumi, H., Fujii, J., Nishikawa, A., Suzuki, K., Kangawa, K., and Taniguchi, N. (1994) J. Biochem. 115, 602-607[Abstract]
51. Bredt, D. S., and Snyder, S. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 682-685[Abstract]
52. Kimura, A., Ohmichi, M., Tasaka, K., Kanda, Y., Ikegami, H., Hayakawa, J., Hisamoto, K., Morishige, K., Hinuma, S., Kurachi, H., and Murata, Y. (2000) J. Biol. Chem. 275, 3667-3674[Abstract/Free Full Text]
53. Yokoi, T., Ohmichi, M., Tsaka, K., Kimura, A., Kanda, Y., Hayakawa, J., Tahara, M., Hisamoto, K., Kurachi, H., and Murata, Y. (2000) J. Biol. Chem. 275, 21639-21647[Abstract/Free Full Text]
54. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
55. Hayakawa, J., Ohmichi, M., Kurachi, H., Ikegami, H., Kimura, A., Matsuoka, T., Jikihara, H., Mercola, D., and Murata, Y. (1999) J. Biol. Chem. 274, 31648-31654[Abstract/Free Full Text]
56. Seo, H. G., Fujii, J., Soejima, H., Niikawa, N., and Taniguchi, N. (1995) Biochem. Cell Biol. Commun. 208, 10-18
57. Naka, M., Nanbu, T., Kobayashi, K., Kamanaka, Y., Komeno, M., Yanase, R., Fukutomi, T., Fujimura, S., Seo, H. G., Fujiwara, N., Ohuchida, S., Suzuki, K., Kondo, K., and Taniguchi, N. (2000) Biochem. Cell Biol. Commun. 270, 663-667
58. Goetz, R., Thatte, H. S., Prabhakar, P., Cho, M. R., Michel, T., and Golan, D. E. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2788-2793[Abstract/Free Full Text]
59. Taniguchi, H., Tanaka, Y., Hirano, H., Tanaka, H., and Shigenobu, K. (1999) Naunyn Schmiedebergs Arch Pharmacol. 360, 69-79[CrossRef][Medline] [Order article via Infotrieve]
60. Chao, T. S. O., Byron, K. L., Lee, K. M., Villereal, M., and Rosner, M. R. (1992) J. Biol. Chem. 267, 19876-19883[Abstract/Free Full Text]
61. Razandi, M., Pedram, A., Greene, G. L., and Levin, E. R. (1999) Mol. Endocrinol. 13, 307-319[Abstract/Free Full Text]
62. Moncada, S., and Higgs, A. (1993) N. Engl. J. Med. 329, 2002-2012[Free Full Text]
63. Wehling, M. (1997) Annu. Rev. Physiol. 59, 365-393[CrossRef][Medline] [Order article via Infotrieve]
64. Pappas, T. C., Gametchu, B., and Watson, C. S. (1995) FASEB J. 9, 404-410[Abstract/Free Full Text]
65. Weiner, C. P., Lizasoain, I., Baylis, S. A., Knowles, R. G., Charles, I. G., and Moncada, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5212-5216[Abstract]
66. Binko, J., and Majewski, H. (1998) Am. J. Physiol. 274, H853-H859[Medline] [Order article via Infotrieve]
67. Walter, P., Green, S., Greene, G., Krust, A., Bornert, J. M., Jeltsch, J. M., Staub, A., Jensen, E., Scrace, G., and Waterfield, M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 7889-7893[Abstract]
68. Scheid, M. P., Schubert, K. M., and Duronio, V. (1999) J. Biol. Chem. 274, 31108-31113[Abstract/Free Full Text]
69. Rubanyi, G. M., Freay, A. D., Kauser, K., Sukovich, D., Burton, G., Lubahn, D. B., Couse, J. F., Curtis, S. W., and Korach, K. S. (1997) J. Clin. Invest. 99, 2429-2437[Abstract/Free Full Text]
70. Cowley, S. M., Hoare, S., Mosselman, S., and Parker, M. G. (1997) J. Biol. Chem. 272, 19858-19862[Abstract/Free Full Text]
71. Iafrati, M. D., Karas, R. H., Aronovitz, M., Kim, S., Sullivan, T. R., Jr., Lubahn, D. B., O'Donnell, T. F., Jr., Korach, K. S., and Mendelsohn, M. E. (1997) Nat. Med. 3, 545-548[Medline] [Order article via Infotrieve]
72. Lindner, V., Kim, S. K., Karas, R. H., Kuiper, G. G., Gustafsson, J. A., and Mendelsohn, M. E. (1998) Circ. Res. 83, 224-229[Abstract/Free Full Text]
73. Karas, R. H., Hodgin, J. B., Kwoun, M., Krege, J. H., Aronovitz, M., Mackey, W., Gustafsson, J. A., Korach, K. S., Smithies, O., and Mendelsohn, M. E. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 15133-15136[Abstract/Free Full Text]
74. Yano, S., Tokumitsu, H., and Soderling, T. R. (1998) Nature 396, 584-587[CrossRef][Medline] [Order article via Infotrieve]
75. Kubohara, Y., and Hosaka, K. (1999) Biochem. Cell Biol. Commun. 263, 790-796
76. McCabe, T. J., Fulton, D., Roman, L. J., and Sessa, W. C. (2000) J. Biol. Chem. 275, 6123-6128[Abstract/Free Full Text]
77. Migliaccio, A., Di Domenico, M., Castoria, G., de Falco, A., Bontempo, P., Nola, E., and Auricchio, F. (1996) EMBO J. 15, 1292-1300[Abstract]
78. Di Domenico, M., Castoria, G., Bilancio, A., Migliacio, A., and Auricchio, F. (1996) Cancer Res. 56, 4516-4521[Abstract]
79. Russell, K. S., Haynes, M. P., Sinha, D., Clerisme, E., and Bender, J. R. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5930-5935[Abstract/Free Full Text]
80. Zeng, G., Nystrom, F. H., Ravichandran, L. V., Cong, Li-Na., Kirby, M., Mostowski, H., and Quon, M. J. (2000) Circulation 101, 1539-1545[Abstract/Free Full Text]
81. Ahmad, S., Singh, N., and Glazer, R. I. (1999) Biochem. Pharmacol. 58, 425-430[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.