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Endocrinology Vol. 148, No. 5 2444-2452
Copyright © 2007 by The Endocrine Society

Nongenomic Estrogen Effects on Nitric Oxide Synthase Activity in Rat Adipocytes

Anne-Marie Jaubert, Nadia Mehebik-Mojaat, Danièle Lacasa, Dominique Sabourault, Yves Giudicelli and Catherine Ribière

Département de Biochimie et de Biologie Moléculaire (A.-M.J., N.M.-M., D.S., Y.G.), Faculté de Médecine Paris-Ile de France-Ouest, Université de Versailles Saint-Quentin en Yvelines, F-78000 Versailles, France; Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 755 (D.L.), Université Pierre et Marie Curie, Hôpital Hôtel-Dieu, Centre de Recherche en Nutrition Humaine, F-75004 Paris, France; and INSERM (C.R.) Unité Mixte de Recherche en Santé 747, Université Paris Descartes, Pharmacologie Toxicologie et Signalisation Cellulaire, F-75006 Paris, France

Address all correspondence and requests for reprints to: C. Ribière; Pharmacologie Toxicologie et Signalisation Cellulaire, 45 rue des Saints-Pères, 75270 Paris Cedex 06, France. E-mail: catherine.ribiere{at}paris-ouest.univ-paris5.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogens exert multiple genomic effects on adipose tissue through binding to nuclear estrogen receptors. However, there is evidence for additional nongenomic mechanisms whereby estrogens may exert their control on adipose tissue metabolism through rapid activation of various membrane-initiated kinase cascades. Here, we tested rapid effects of estrogens on nitric oxide production in white adipose tissue using 17-ß estradiol (E2) and its membrane impermeant albumin conjugated form (17-ß estradiol hemisuccinate BSA, E2-BSA). We found that both E2 and E2-BSA stimulate nitric oxide synthase (NOS) activity in adipocytes. These effects were abolished by 1) ICI 182–780, a selective estrogen receptor antagonist; 2) wortmannin, an inhibitor of phosphatidylinositol 3-kinase; and 3) N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide (H-89) an inhibitor of protein kinase A. In contrast to NOS activation by E2, E2-BSA-induced NOS activity was abolished by UO126, an inhibitor of MAPK kinase/ERK (p42/p44 MAPKs). Immunoblotting studies have shown that both estrogens phosphorylate endothelial NOS (NOS III) on Ser1179, an effect that is prevented by wortmannin and H89, suggesting that NOS III is the target for estrogen-induced NOS activity. Furthermore, only the E2-BSA-induced NOS III phosphorylation on Ser1179 was totally abolished by UO126. These results indicate that the signaling cascades involved in adipocyte NOS stimulation by estrogens are different depending on whether estrogens are free or conjugated to albumin and therefore underline the importance of estrogen receptor locations in the nongenomic actions of estrogens in these cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGENS PLAY IMPORTANT roles in the regulation of adipose tissue metabolism, accumulation, and distribution in males and females during development and adulthood (1). Adipose tissue is a steroid reservoir and a site of estrogen production. In this tissue, the biological effects of estrogens are mediated by the two {alpha} and ß isoforms of the estrogen receptor (ER), which are both expressed in human and rodent fat cells (2, 3). In these cells, like in many others, estrogens regulate key protein expression at the genomic level. For example, transcription of important adipose genes such as leptin, perilipin, peroxisome proliferator activated receptor {gamma} and lipoprotein lipase genes are controlled by estrogens (4, 5, 6). There is also increasing evidence that estrogens can rapidly modulate cell functions through nongenomic actions. These alternative mechanisms involve short-term activation of signals generated from cytoplasmic and/or cell membrane receptors, inducing downstream regulatory cascades such as the MAPKs, the phosphatidylinositol 3-kinase (PI3-kinase), and various tyrosine kinase cascades through nongenomic mechanisms (7). Some of these nongenomic effects seem to be ascribed to ERs located in the plasma membrane because they can be reproduced by estrogen forms that do not pass across the plasma membrane, such as the estrogen-BSA conjugates (8, 9). Estrogens exert cardioprotective effects linked at least in part to increased nitric oxide (NO) production by endothelial nitric oxide synthase (NOS III) (10). In vitro, exposure to estrogens increases rapidly the release of NO from cultured endothelial cells (11, 12, 13, 14, 15, 16). Both ER{alpha} and ERß appear to mediate this nongenomic activation of NOS III at the plasma membrane level (12, 17) because in caveolae a subpopulation of ER{alpha} and ERß are coupled to NOS III in a functional signaling module (11, 12, 17). NOS III activity is regulated by reversible phosphorylations through multiple protein kinases [AMP-activated protein kinase, Akt (protein kinase B), protein kinase A (PKA), protein kinase C] and protein phosphatases (PP1 and PP-2A) acting on Ser1179 and Thr497, which are considered to be the most important sites implicated in the enzyme activity (18, 19, 20). Ser1179 is the main regulatory site, and its phosphorylation results in the activation of NOS III (21), whereas phosphorylation of Thr497 seems to reduce NOS III activity. Adipose tissue is also a site of NO production; we have shown that this tissue expresses inducible NOS (NOS II) and NOS III (22). Because estrogens activate rapidly several kinase cascades, we postulated that nongenomic estrogen action may modulate NOS activity by phosphorylation of NOS III in adipocytes. In the present study we compared the acute effects of estrogen on NOS activity in adipose tissue of ovariectomized female rats and of male rats using 17-ß estradiol (E2) and the membrane-impermeable conjugate to BSA (17-ß estradiol hemisuccinate BSA, E2- BSA).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
L-[2,3,4,5-3H]Arginine (58 Ci /mmol) and the ECL Detection kit are products of Amersham Biosciences (Little Chalfont, UK). AG Dowex 50W-X8, Bradford protein dye reagent, and electrophoretic chemicals were from Bio-Rad Laboratories (Hercules, CA). Anti dual-phospho Thr183 and Tyr185 p42/p44 MAPKs (V8031) and anti-phospho Ser473 Akt (G7441) antibodies were obtained from Promega (Madison, WI). UO126, anti-phospho Ser1179 NOS III, anti-Akt, and anti-phospho Thr308 antibodies were from Cell Signaling Technology (Beverly, MA). Polyclonal anti-NOS II, polyclonal anti-NOS III, and anti-ERK (pan ERK) antibodies were obtained from Transduction Laboratories (San Diego, CA), and Akt inhibitor I was from Calbiochem (San Diego, CA). 17ß-Estradiol, E2-BSA, and other reagents were obtained from Sigma (St. Louis, MO).

Animals
Male and female Sprague Dawley rats (180–200 g), obtained from Centre d’Elevage de Rats Janvier (Le Genest St Isle, France) at 8 wk of age, were maintained at constant room temperature (24 C) on a 12-h light/12-h dark cycle. Female rats were either ovariectomized (OVX) or sham-operated (control) and killed 2 wk after the operation. Fed rats were killed by decapitation, and white adipose tissue from epididymal or parametrial fat depots was carefully removed and rapidly used for adipocyte preparation. All experimental protocols were approved by the University Animal Use and Care Committee.

Adipocyte incubation and NOS activity
Isolated epididymal adipocytes were prepared as previously described (22). NOS activity was measured after L-[3H]arginine conversion into L-[3H]citrulline by intact adipocytes (23). Briefly, adipocytes (3–5 x 105 cells/ml) were incubated at 37 C in Krebs-Ringer buffer (pH 7.4) containing 2% (wt/vol) BSA, 5 mM glucose, 1.5 µCi/ml L-[3H]arginine, and 50 mM valine (to inhibit arginase), in the absence or presence of various concentrations of estrogens and/or the effectors to be tested. Each incubation was performed without and with a specific NOS inhibitor, diphenyl-iodonium. Incubations were stopped by adding 250 µl ethanol followed by 5 ml of 1:1 (vol/vol) H2O/Dowex 50W-X8 (Na+ form) resin to retain arginine and were then left to settle for 10 min at 4 C. Supernatant was removed, and the main product detected by HPLC was citrulline. The citrulline production was linear between 10 and 60 min. One milliliter of supernatant was added to the liquid scintillation cocktail for counting. Values obtained in the presence of diphenyl-iodonium were subtracted from each sample. Dimethylsulfoxide, which was used as vehicle for the tested inhibitors, was added to controls, and separate experiments revealed no effect of this compound on the adipocyte NOS activity.

Cell lysates
Adipocytes (3–5 x 105 cells /ml) were incubated during 20 min at 37 C in Krebs-Ringer buffer (pH 7.4) containing 5 mM glucose in the absence or in the presence of the effectors to be tested. Then adipocytes were harvested and disrupted in buffer containing 20 mM Tris (pH 7.5), 5 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 2 mM Na3VO4, 1% Nonidet P-40, and protease inhibitors (24). Cell lysates were solubilized by continuous stirring for 30 min at 4 C and centrifuged for 10 min at 14,000 x g. Supernatants were used for protein determination according to Bradford (25) and, after Laemmli buffer addition, for electrophoresis and immunoblotting studies.

Western blotting
Cell lysates (10–20 µg protein /lane) were subjected to SDS-PAGE and then blotted onto polyvinylidene difluoride membranes. The blots were incubated with the primary antibody at 4 C overnight and incubated with the secondary antibody linked to peroxidase. Immunoreactive proteins were visualized on x-ray film by an enhanced chemiluminescence ECL reagents. Immunoblotting was performed using specific anti-NOS II, anti-NOS III, anti-phospho NOS III (Ser1179), anti-phospho Akt (Ser473), anti-phospho Akt (Thr308), anti-active MAPKs (phospho-Thr184 and phospho-Tyr185), and anti-ERK (pan ERK) antibodies.

Statistical analysis
Data are presented as means ± SEM. Statistical analyses were performed using unpaired Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Estrogens stimulate NOS activity
Because acute estrogen effects are generally attributed to membranous ER, we first tested E2-BSA. A 30-min exposure to E2-BSA resulted in a dose-dependent increase of NOS activity in adipocytes from both OVX female and male rats (Fig. 1Go, A and B). As shown in Fig. 2Go, A and B, stimulation of NOS activity was observed with E2 in a gender-independent manner. In contrast, when testing the inactive stereoisomer of estradiol, 17{alpha}-E2 (10–6 M), NOS activity remained unchanged, independently of gender (data not shown). Because the NOS responses to estrogens were similar in OVX and male rats, the following experiments were conducted in adipocytes from male rats. Estrogen’s effects on NOS activities were rapid and sustained; radioactive counts corresponding to [3H]citrulline that were measured after a 10- or 60-min estrogen exposure were, respectively, 600 ± 50 and 3500 ± 160 cpm/105 cells for E2-BSA, 530 ± 52 and 3200 ± 200 cpm/105 cells for E2, vs. 300 ± 25 and 1750 ± 160 cpm/105 cells for control. As shown in Fig. 3Go, addition of the selective ER antagonist, ICI 182–780 (1 µM), alone did not alter NOS activity but prevented both the E2-BSA- and E2-stimulated NOS activities.


Figure 1
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  		FIG. 1. E2-BSA increases NOS activity in rat adipocytes. A, Isolated adipocytes from female OVX rats were incubated with increasing concentrations of E2-BSA (10–10–10–6 M) for 30 min. B, Isolated adipocytes from male rats were incubated with increasing concentrations of E2-BSA (10–10–10–6 M) for 30 min. NOS activity was measured by the conversion of L-[3H]arginine into L-[3H]citrulline as described in Materials and Methods. Results are means ± SEM of independent experiments performed in duplicate with five separate adipocyte preparations and are expressed as the percentage of NOS activity in control adipocytes (C). *, P < 0.02; **, P < 0.01 vs. control.

 

Figure 2
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  		FIG. 2. E2 increases NOS activity in rat adipocytes. A, Isolated adipocytes from female OVX rats were incubated with increasing concentrations of E2 (10–8, 10–6 M) for 30 min. B, Isolated adipocytes from male rats were incubated with increasing concentrations of E2 (10–8, 10–6 M) for 30 min. NOS activity was measured by the conversion of L-[3H]arginine into L-[3H]citrulline as described in Materials and Methods. Results are means ± SEM of independent experiments performed in duplicate with five separate adipocyte preparations and are expressed as the percentage of NOS activity in control adipocytes (C). *, P < 0.02; **, P < 0.01 vs. control.

 

Figure 3
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  		FIG. 3. An antagonist of ERs (ICI 182–780) prevents estrogen-induced NOS activity. Adipocytes were pretreated with vehicle alone or ICI 182–780 (1 µM) for 15 min and then exposed to vehicle or estrogen (10–6 M) for another 30 min. NOS activity was assessed as described in Materials and Methods. Results are means ± SEM of independent experiments performed in duplicate with four separate adipocyte preparations and are expressed as the percentage of NOS activity in control adipocytes (C). **, P < 0.01 vs. control.

 
Estrogens phosphorylate NOS III on Ser1179
Adipocytes express NOS II (22), so we evaluated the regulation of expression of this protein isoform under E2-BSA and E2 treatments. As shown in Fig. 4AGo, estrogen treatment did not modify NOS II protein expression; the relative ratio of densitometric units of NOS II bands/constitutive NOS III bands were identical. Because NOS III (but not NOS II) is a target for protein kinases, we felt it was important to determine whether estrogen stimulation of rat adipocyte NOS activity implied phosphorylation of NOS III at Ser1179, which appeared to be crucial for the regulation of this enzyme catalytic activity (26). As shown in Fig. 4BGo, adipocyte exposure to E2-BSA or E2 resulted in a clear increase in NOS III phosphorylation at Ser1179.


Figure 4
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  		FIG. 4. E2-BSA and E2 induce phosphorylation of NOS III at Ser1179. A, Adipocytes were exposed to vehicle or estrogen (10–6 M) for 20 min. Cell lysates, prepared as described in Materials and Methods, were separated on sodium dodecyl sulfate (SDS)-7% polyacrylamide gels and then transferred to nitrocellulose membranes and analyzed with polyclonal anti-NOS II and polyclonal anti-NOS III antibodies. Densitometry was performed to quantify the ratio of NOS II/NOS III bands. Data represent means ± SEM of independent experiments with five separate adipocyte preparations. B, Adipocytes were exposed to vehicle or 10–6 M E2-BSA or 10–6 M E2 for 20 min. Cell lysates, prepared as described in Materials and Methods, were separated on SDS-7% polyacrylamide gels and then transferred to nitrocellulose membranes and analyzed with antibody specific for phosphorylated form of NOS III at Ser1179 and with polyclonal anti-NOS III antibody to ensure equal loading of the samples. Densitometry was performed to quantify phosphorylated bands. Data represent means ± SEM of independent experiments with five separate adipocyte preparations. ***, P < 0.001 vs. control.

 
Estrogen-induced NOS activity is PI3-kinase dependent
Because recent studies have demonstrated that, in endothelial cells, NOS III response to estrogens occurred via the PI3-kinase pathway (13, 15), we tested a specific PI3-kinase inhibitor, wortmannin, on adipocyte NOS activity. As shown in Fig. 5AGo, 10–6 M wortmannin failed to influence basal NOS activity but abolished NOS stimulation due to either E2-BSA or E2. LY 294002 (40 µM), another PI3-kinase inhibitor, also blocked the effect of the two estrogens (results not shown). Furthermore, both E2-BSA and E2 induced-NOS III phosphorylations at Ser1179 were severely reduced by wortmannin (Fig 5BGo). Akt activation after adipocyte exposure to estrogens was next examined by Western blot analysis. Data in Fig. 6AGo clearly show the failure of E2-BSA and E2 to phosphorylate Akt both at Ser473 or Thr308, which contrasts with the Akt phosphorylations induced by 1 nM insulin, used as positive control. However, to test a precocious or transient Akt activation implicated in NOS III Ser1179 phosphorylation induced by estrogens, we used a newly developed Akt inhibitor, Akt inhibitor I [1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate]. Akt inhibitor I was able to prevent Akt activation induced by insulin (results not shown). As shown Fig. 6BGo, Akt inhibitor I (10 µM) did not prevent NOS III Ser1179 phosphorylation induced by estrogens in contrast to that induced by insulin. These results indicate that activation of the PI3-kinase pathway without Akt activation is involved in the mechanisms whereby estrogens stimulate NOS activity in adipocytes.


Figure 5
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  		FIG. 5. PI3-kinase is involved in estrogen-induced NOS activity. A, Adipocytes were pretreated with vehicle alone or PI3-kinase inhibitor, 1 µM wortmannin (W), for 20 min and then exposed to vehicle or estrogen (10–6 M) for another 30 min. NOS activity was assessed as described in Materials and Methods. Results are means ± SEM of independent experiments performed in duplicate with four separate adipocyte preparations and are expressed as the percentage of NOS activity in control adipocytes (C). **, P < 0.01 vs. control. B, Adipocytes were pretreated with vehicle alone or 1 µM wortmannin (W) for 15 min and then exposed to vehicle or estrogen (10–6 M) for another 20 min. Cell lysates, prepared as described in Materials and Methods, were separated on SDS-7% polyacrylamide gels and then transferred to nitrocellulose membranes and analyzed with antibody specific for phosphorylated form of NOS III at Ser1179 and with polyclonal anti-NOS III antibody to ensure equal loading of the samples. Densitometry was performed to quantify phosphorylated bands. Data represent means ± SEM of independent experiments with five separate adipocyte preparations. **, P < 0.01 vs. control.

 

Figure 6
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  		FIG. 6. Akt is not involved in estrogen-induced NOS III phosphorylation. A, Adipocytes were incubated with vehicle, E2-BSA and E2 (10–6 M), or insulin (I; 1 nM), used as positive control, for 15 min. Cells lysates, prepared as described in Materials and Methods, were separated on SDS-12% polyacrylamide gels and then transferred to nitrocellulose membranes and analyzed with antibodies specific for phosphorylated form of Akt at Ser473 or at Thr308 and with polyclonal anti-Akt antibody to ensure equal loading of the samples. Densitometry was performed to quantify phosphorylated bands. Data represent means ± SEM of independent experiments with five separate adipocyte preparations. ***, P < 0.01 vs. control. B, Adipocytes were pretreated with vehicle alone or 10 µM Akt inhibitor I for 15 min and then exposed to vehicle or estrogen (10–6 M) or insulin (I; 1 nM), used as positive control, for another 20 min. Cell lysates, prepared as described in Materials and Methods, were separated on SDS-7% polyacrylamide gels and then transferred to nitrocellulose membranes and analyzed with antibody specific for phosphorylated form of NOS III at Ser1179 and with polyclonal anti-NOS III antibody to ensure equal loading of the samples. Densitometry was performed to quantify phosphorylated bands. Data represent means ± SEM of independent experiments with five separate adipocyte preparations. ***, P < 0.001 vs. control; §§§, P < 0.001 vs. insulin.

 
Estrogen stimulation of NOS activity involves PKA activation
Because estrogens were shown to increase cAMP in various cells (27, 28, 29), the possibility that PKA activation might play a role in the estrogen-stimulated NOS activity was next tested with H89, a PKA inhibitor. Interestingly, 10 µM H89, which had no effect per se on NOS activity, completely abolished both the E2-BSA- and E2-stimulated NOS activity (Fig. 7AGo). The same results (not shown) were also observed with another PKA inhibitor, the Rp-diastereomer of adenosine 3',5'-cyclic phosphorothionate. In parallel, both the E2-BSA- and E2-induced NOS III phosphorylations at Ser1179 were completely abolished by H89 (Fig. 7BGo). These results suggest an important role of PKA in the adipocyte NOS activation by estrogens.


Figure 7
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  		FIG. 7. PKA inhibitor blocks estrogen-induced NOS activity. A, Adipocytes were pretreated with vehicle alone or with PKA inhibitor, 10 µM H89, for 15 min and then exposed to vehicle or E2-BSA and E2 (10–6 M) for another 30 min. NOS activity was measured as described in Materials and Methods. Results are means ± SEM of independent experiments performed in duplicate with five separate adipocyte preparations and are expressed as the percentage of NOS activity in control adipocytes (C). **, P < 0.01 vs. control. B, Adipocytes were pretreated with vehicle alone or with PKA inhibitor, 10 µM H89, for 15 min and then exposed to vehicle or E2-BSA and E2 (10–6 M) for another 20 min. Cell lysates, prepared as described in Materials and Methods, were separated on SDS-7% polyacrylamide gels and then transferred to nitrocellulose membranes and analyzed with antibody specific for phosphorylated form of NOS III at Ser1179 and with polyclonal anti-NOS III antibody to ensure equal loading of the samples. Densitometry was performed to quantify phosphorylated bands. Data represent means ± SEM of independent experiments with five separate adipocyte preparations. **, P < 0.01 vs. control.

 
Only the E2-BSA stimulation of NOS activity involves MAPK activation
Because estrogens activate p42/p44 MAPKs in fat cells (30) and because the estrogen-induced p42/p44 MAPK activation was shown to activate NOS III in endothelial cells (12, 31), we next studied the eventual participation of p42/p44 MAPK in estrogen effect. As previously reported in adipocytes (30), E2-BSA and E2 induced p42/p44 MAPK activation, which was prevented by wortmannin and by UO126, a selective inhibitor of MAPK kinase/ERK (MEK), the immediate upstream activator of p42/p44 MAPKs (Fig. 8Go, A and B). We also tested the eventual role of PKA in the estrogen-mediated MAPK activation. Although PKA inhibition with H89 was without any significant effect on the p42/p44 MAPK activation induced by E2-BSA (Fig. 8AGo), in contrast H89 prevented the p42/p44 MAPK activation induced by E2 (Fig. 8BGo), suggesting that E2 binding to ER might at first induce PKA activation followed by p42/p44 MAPK activation. Then we tested UO126 effect on NOS activity and phosphorylation induced by estrogens. As shown in Fig. 9AGo, whereas UO126 (10–5 M) failed to alter basal NOS activity, this inhibitor drastically reduced the E2-BSA-stimulated NOS activity but did not significantly affect the E2-induced NOS activity. Although MAPKs could not directly phosphorylate NOS III at Ser1179 (32), UO126 prevented the phosphorylation induced by E2-BSA and not by E2 (Fig. 9BGo).


Figure 8
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  		FIG. 8. E2-BSA and E2 activate p42/p44 MAPKs. Adipocytes were pretreated for 15 min without or with 1 µM wortmannin (W), 1 µM H89, or 1 µM UO126 and then exposed to vehicle or 10–6 M E2-BSA (A) and 10–6 M E2 (B) for another 20 min. Cell lysates, prepared as described in Materials and Methods, were separated on SDS-12% polyacrylamide gels and then transferred to nitrocellulose membranes and analyzed with antibody specific for the dual-phosphorylated form of p42/p44 MAPKs or with anti-total p42/p44 MAPKs (pan ERK) antibody to ensure equal loading of the samples. Densitometry was performed to quantify phosphorylated bands. Data represent means ± SEM of independent experiments with five separate adipocyte preparations. *, P < 0.05; **, P < 0.01 vs. control.

 

Figure 9
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  		FIG. 9. Only E2-BSA activation of NOS requires MAPK stimulation. A, Adipocytes were pretreated with vehicle alone or a MEK inhibitor, UO126 (1 µM), for 15 min and then exposed to vehicle or E2-BSA and E2 (10–6 M) for another 30 min. NOS activity was assessed as described in Materials and Methods. Results are means ± SEM of independent experiments performed in duplicate with four separate adipocyte preparations and are expressed as the percentage of NOS activity in control adipocytes (C). *, P < 0.02 vs. control. B, Adipocytes were pretreated with vehicle alone or a MEK inhibitor, UO126 (1 µM), for 15 min and then exposed to vehicle or E2-BSA and E2 (10–6 M) for another 20 min. Cell lysates, prepared as described in Materials and Methods, were separated on SDS-7% polyacrylamide gels and then transferred to nitrocellulose membranes and analyzed with antibody specific for phosphorylated form of NOS III at Ser1179 and with polyclonal anti-NOS III antibody to ensure equal loading of the samples. Densitometry was performed to quantify phosphorylated bands. Data represent means ± SEM of independent experiments with five separate adipocyte preparations. **, P < 0.01 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major finding of this study is that estrogens increase NOS activity in adipocytes through different nongenomic signaling mechanisms depending on the nature of the estrogen form investigated (Fig. 10Go). Both E2 and E2-BSA increase phosphorylation of NOS III on Ser1179. Phosphorylation of NOS III on Ser1179 plays an important role in NOS III activation, especially in the response of this enzyme to various physiological stimuli (26). Mechanistically, NOS III phosphorylation on Ser1179 increases NO release by enhancing electron flux through the reductase domain of NOS III and by improving calcium sensitivity of the enzyme (21). Ser1179 is the target for multiple protein kinases including Akt, AMP kinase (33), PKA, protein kinase G (34), and calmodulin-dependent kinase II (18). Our results using wortmannin and H89 indicate that both E2 and E2-BSA activate a PI3-kinase/PKA cascade leading to phosphorylation of NOS III on Ser1179. Because both inhibitors also abolished the estrogen-induced NOS activity, it appears that one mechanism whereby estrogens stimulate NOS in adipocytes is an increase in NOS III phosphorylation on Ser1179. Furthermore, because NOS III and NOS II are the only NOS isoforms expressed in adipocytes (22) and because PKA is unable to phosphorylate NOS II (34), it becomes clear that NOS III is the target for the nongenomic estrogen-induced NOS stimulation in adipocytes. To our knowledge, this study is the first to report involvement of PI3-kinase/PKA activation in the mechanisms whereby estrogens stimulate NOS III activity. In endothelial cells, rapid effects of estrogens were related to activation of the PI3-kinase/Akt cascade associated with NOS III phosphorylation (13, 14, 15, 35, 36). In contrast with these previous studies, we failed to observe any Akt phosphorylation in estrogen-treated adipocytes. This lack of effect on Akt is in accordance with previous studies investigating estrogen action on NOS in uterine artery endothelial cells and in cerebellar granule cells (16, 37). Therefore, the signal transduction mechanisms mediating NOS modulation by estrogens appear different according to cell environment and/or cell phenotype. If PI3-kinase/PKA seems to play a major role in the estrogen (whether free or albumin bound)-induced NOS activation and NOS III phosphorylation on Ser1179, our results also indicate that p42/p44 MAPKs are implicated in the E2-BSA-induced NOS stimulation. In fact, although a selective inhibitor of MEK prevents both NOS activation and Ser1179 phosphorylation induced by E2-BSA, this compound does not affect NOS activation and Ser1179 phosphorylation caused by E2. Our results show for the first time that, in adipocytes, signaling involved in E2-induced NOS activity is different from that implicating E2-BSA. Although, E2-BSA binding to the plasma membrane was directly visualized using fluorescein-labeled E2-BSA (28, 38), there may be some reticence against the experimental use of this compound because there is a possibility of contamination by free steroid or partial penetration inside the cell. However, because the transduction mechanisms of NOS activation by E2-BSA and E2 appear different, such unaccounted E2-BSA effect, if any, should be minor. The mechanistic difference between E2 and E2-BSA observed by use of UO126 in the present study could be linked to different subcellular locations of ER and/or NOS III. Although a majority of ER is located in the nucleus, several biochemical and cytochemical analyses suggest that a portion of ER is also located in cytoplasm and plasma membrane (39). Most of the nongenomic estrogen signaling mechanisms provided so far occur through membrane localization or cytoplasmic retention of ER via receptor modifications, adaptor proteins, binding to membrane-associated growth factor receptors, or kinases (39). Because E2-BSA is a membrane-impermeable estrogen form, its action is mediated by membranous ER. Moreover, many reports have shown that the ability of estrogens to rapidly activate the ERK member of the MAPK family occurs only through membranous ER (8, 40, 41). p42/p44 MAPK activation induced by E2-BSA in adipocytes appears, therefore, linked to plasma membrane ER and is indispensable for NOS activation. Because p42/p44 MAPKs did not directly phosphorylate NOS III on Ser1179 (19, 34), MAPK effects are indirect but favor this phosphorylation. Prominent compartments for NOS III are the plasma membrane and the Golgi apparatus, where NOS III can be phosphorylated and activated (42, 43). At plasma membrane, NOS III resides in caveolae and directly binds caveolin-1, which prevents NOS activation (44). Moreover, as in caveolae, NOS III is colocated with membranous ER (45); therefore, it appears that p42/p44 MAPK activation induced by E2-BSA modulates NOS III activation in plasmalemmal caveolae. Thus, p42/p44 MAPKs could therefore act on NOS III protein-protein interactions (NOS III-caveolin-1/NOS III-heat shock protein 90) or on phosphorylation of another site on NOS III such as the recent identified Thr97 (46). Concerning E2, the situation is different because MAPK activation due to E2 requires PKA. One could therefore suggest that PKA activation is the primary event linked principally to a cytosolic ER, which could induce phosphorylation of NOS III at the Golgi complex. This cytosolic receptor can then be translocated to the cell membrane where it leads to p42/p44 MAPK activation, but this last activation has no effect on NOS activity. If nongenomic effects induced by estrogens involve generally resident and/or associated plasma membrane ER, other nongenomic effects could well result from E2 binding to intracellular ER because, as shown here in and in other studies, those effects are not mimicked by E2-BSA (37, 47). If p42/p44 MAPK activation is essential for NOS III stimulation by several agonists in various cells (16, 12, 48, 49), our results in adipocytes show clearly that p42/p44 MAPK activation is required when agonists such as insulin (50), leptin (51), or in the present study E2-BSA use a membranous receptor. The fact that p42/p44 MAPKs do not play a role with E2, the physiological estrogen form, is linked to easy E2 access inside the cell and suggests that membranous ER concentration is weak in adipocytes.


Figure 10
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  		FIG. 10. Schematic diagram of E2-BSA and E2 effects on the rapid activation of NOS III. E2-BSA and E2 acutely stimulate NOS activity via a PI3-kinase/PKA-dependent phosphorylation of NOS III at serine1179. Dependently of the estrogen forms, the NOS III phosphorylation and NOS activity required or not p42/p44 MAPK activation, which could be linked to different locations of ERs.

 
In conclusion, this study shows that estrogens stimulate NOS activity in white adipocytes through mechanisms including PI3-kinase/PKA, which are dependent or independent of p42/p44 MAPK activation. Location of the involved ER plays a fundamental role because the signal transduction pathways responsible for NOS activation appear to be different for E2 and E2-BSA. In contrast to previous data showing that some genomic estrogen effects such as induction of activator protein-1 complex expression were related to gender (52), in this study we did not observe sex differences in the nongenomic effect of estrogen leading to increased NOS activities. This difference between genomic and nongenomic estrogen effects underlines further the importance of estrogen-regulated expression of ER coactivators rather than ER expression. NO is a signaling molecule involved in a critical range of processes. Therefore estrogen-stimulated NOS activity could be of physiological importance in adipocyte homeostasis regulation. Previous studies have revealed that after treatment with the receptor ligand (E2), a significant increase in the concentration of S-nitrosylated cysteine residues in ER was observed. This E2-dependent S-nitrosocysteine generation could inhibit selectively the estrogen-induced gene expression without affecting nongenomic events (53, 54). Thus, estrogen-mediated production of NO could modulate the bioactivity of ER, possibly by shifting the receptor from its major role as a regulator of gene transcription toward more rapid (nongenomic) functions.


   Footnotes
 
This work was supported by Ministry of Research and Technology.

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 15, 2007

Abbreviations: Akt, Protein kinase B; E2, 17-ß estradiol; E2-BSA, 17-ß estradiol hemisuccinate BSA; ER, estrogen receptor; MEK, MAPK kinase/ERK; NO, nitric oxide; NOS, NO synthase; NOS II, inducible NOS; NOS III, endothelial NOS; OVX, ovariectomized; PI3-kinase, phosphatidylinositol-3-kinase; PKA, protein kinase A; SDS, sodium dodecyl sulfate.

Received September 28, 2006.

Accepted for publication February 5, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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