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Endocrinology Vol. 143, No. 3 930-940
Copyright © 2002 by The Endocrine Society

INTRACELLULAR SIGNAL SYSTEMS

Rapid Nongenomic E2 Effects on p42/p44 MAPK, Activator Protein-1, and cAMP Response Element Binding Protein in Rat White Adipocytes

Esther Garcia Dos Santos, Marie Noëlle Dieudonne, René Pecquery, Vincent Le Moal, Yves Giudicelli and Danièle Lacasa1

Faculté de Médecine Paris-Ouest, Université René Descartes and Laboratoire de Biochimie et Biologie Moléculaire, Centre Hospitalier de Poissy, 78303 France

Address all correspondence and requests for reprints to: Y. Giudicelli, Service de Biochimie et de Biologie Moléculaire, Centre Hospitalier Intercommunal, 78303 Poissy, France. E-mail: . biochip{at}wanadoo.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In some tissues, rapid effects of estrogens have been described at the plasma membrane level including activation of the MAPK activity. In rat adipocytes, the present study demonstrates that physiological concentrations (0.1–10 nM) of E2 rapidly activate the p42/p44 MAPK. This effect was blocked by the pure estrogen antagonist, ICI 182 780, and appeared specific for E2 because 17{alpha}-E2, T, and progesterone failed to change the MAPK activity. Pertussis toxin; PP2, a selective inhibitor of Src family kinase; and wortmannin all reduced the magnitude of MAPK activation by E2 suggesting involvement of the Gi-protein/Src family kinase/PI3K pathway. Classical PKCs and MAPK kinase were also involved in MAPK activation by E2. Interestingly, this activation was observed in late but not early differentiated rat preadipocytes, and the immunoreactive ER{alpha} protein was detected only in adipocyte membrane, suggesting that the adipocyte membrane structure is required for the nongenomic effect of E2. Moreover, E2 induced a rapid nuclear translocation of MAPK together with a fast MAPK- dependent activation of cAMP response element binding protein leading to a transcriptional activation of cAMP response element binding protein-responsive genes and reported plasmids. However, the E2 increase in adipocyte activator protein-1 DNA binding does not seem to be fully explained by the E2 activation of the MAPK pathway. This study provides clear evidence for an additional nongenomic mechanism whereby estrogens may exert their control on adipose tissue metabolism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGENS EXERT MULTIPLE effects on female reproductive organs but also on other target tissues such as the central nervous system (1), bone (2), vascular system (3), and adipose tissue (4). In rodents, for example, ovariectomy leads to an increased fat mass that can be reversed by in vivo E2 treatment (5, 6). Activity and mRNA expression of lipoprotein lipase, the rate-limiting enzyme for lipid storage, are depressed after estrogen treatment (7). More recently, estrogens were reported to repress lipoprotein lipase gene expression through a direct transcriptional mechanism (8). Nuclear-specific ERs are present in both precursor and mature rat fat cells (9, 10). Beside the classical ER{alpha}, another ER subtype, ERß, was recently characterized and cloned (11). ERß is also expressed in adipose tissues from various localizations (12). Adipose tissue being an important site for estrogen biosynthesis and storage (13), expression of ER by adipocytes suggests that estrogens could act as autocrine/paracrine factors on white adipose tissue.

Cellular actions of estrogens are initiated by their binding to the nuclear ER. Then the activated receptor regulates transcription of target genes either through binding to estrogen response elements (EREs) present in promoters or through interactions with other transcription factors like activator protein-1 [AP-1 (14, 15)]. In fact, ER action at AP-1 sites depends on ER isoforms ({alpha} or ß): E2-binding to ER{alpha} activates, whereas E2 binding to ERß inhibits, AP-1 transcriptional activity (15, 16) through protein-protein interactions between ER{alpha} or ß and AP-1 components and cofactor recruitment.

However, there is increasing evidence that estrogen, like other steroids, can also modulate cell functions through nongenomic actions at the plasma membrane level (17, 18) supporting the existence of putative membrane ER (19). In some cell types for example, estrogens can rapidly trigger a variety of signal transduction events leading to stimulation of calcium flux, cAMP production, PLC activation, and inositol phosphate generation (20).

The MAPK/ERK1/2 can also be rapidly stimulated by estrogens in various cell types such as breast cancer (21), endothelial (22), osteoblastic (23), and neuroblastoma (24) cells. The two isoforms of MAPK (p42/p44) play critical roles in the control of cell proliferation, differentiation, homeostasis, and survival. MAPK is activated by dual phosphorylation on specific threonine and tyrosine residues by MAPK kinases (MEKs). MEKs are themselves activated by several kinases such as the Raf proteins, which in turn are regulated by Ras family members. Tyrosine kinase receptors transmit extracellular signals through protein complexes including the adaptor protein Grb2, the guanine nucleotide exchange protein Sos, and the adaptor protein Shc leading to Ras activation (25, 26). Depending on the receptor, cell type, and upstream modulator of Ras, MAPK activation can also be modulated by pertussis toxin (PTX)-sensitive G proteins through a signaling pathway involving Src and PI3K (27, 28). Upon activation, MAPK phosphorylates multiple substrates of various subcellular localizations such as PLA2 in the cytoplasm and several transcription factors in the nucleus (25, 26). AP-1 is a nuclear target of MAPK through phosphorylation of preexisting AP-1 components leading to increased AP-1 transcriptional activity (29). Furthermore, activation by insulin of cAMP-response element-binding protein (CREB), another important transcriptional factor, has also been reported to require MAPK pathway (30).

In rat white adipocytes, we have recently shown that estrogens in vivo rapidly increased the mRNA and protein expressions of the two major components of AP-1, c-fos and c-jun, and also enhanced the AP-1 DNA binding activity (31). The present studies were undertaken to investigate the hypothesis that in rat adipocytes estrogens enhance AP-1 and CREB activations, at least in part, through a nongenomic activation of the MAPK cascade.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
FBS was obtained from Life Technologies, Inc. (Grand Island, NY). Genistein, E2 (cyclodextrine form and BSA conjugate), phenol red-free DMEM, DMEM-Ham’s F12 (50:50 mix), and lysophosphatidic acid (LPA) were obtained from Sigma (St. Louis, MO). The phospho-CREB (Ser 133) and phospho-Src (Tyr 416) antibodies were obtained from New England Biolabs, Inc. (Beverly, MA). The antisera specific for pan-ERK (E17120) and caveolin-1 (37120) were obtained from Transduction Laboratories (Lexington, WI). The antiserum specific for active MAPK (V 8031), MEK inhibitor UO 126, AP-1, nuclear factor {kappa}B (NF{kappa}B), and Oct-1 consensus double-stranded oligonucleotides, T4 polynucleotide kinase, ß-galactosidase (ß-gal), and luciferase assay system were from Promega Corp. (Madison, WI). The antiserum specific for ER{alpha} (MC-20) and the mutated AP-1 consensus double-stranded oligonucleotide were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The plasmid reporter, pCRE-Luc (no. 219076), was obtained from Stratagene (Amsterdam, The Netherlands). Western blotting protocols were from Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire, UK). ICI 182,780, LY 29 4002, and GF 109203X were from Tocris Laboratory (Bristol, UK). Wortmannin and PP2 were obtained from Calbiochem-Novabiochem (San Diego, CA). All other chemicals were of reagent grade.

Animals
Procedures with experimental animals were authorized and followed the guidelines of the Ministry of Agriculture (France) (authorization 006614). Female Sprague Dawley rats (125–150 g) were killed by decapitation. Parametrial fat pads were removed aseptically.

Cell culture
Cell preparation and cultures were performed as described elsewhere (32). Briefly, preadipocytes obtained from the stroma-vascular fraction of adipose tissue by collagenase digestion were plated at a density of 1–2 x 104 cells/cm2 in 8% FBS-DMEM. After 12 h, cultures were washed and fed with 8% FBS-DMEM. Medium was changed every other day. At confluence (3 d post plating), cells were allowed to differentiate in DMEM-Ham’s F12 containing 5 µg/ml insulin, 10 µg/ml transferrin, and 200 pM T3 (ITT medium) in the absence of serum as described elsewhere (32). Whatever the anatomical origin, at least 80% of the cells were fully differentiated at d 8–10 post confluence.

ITT medium remained unchanged 2 d before experiments. E2 [ß- cyclodextrine form or BSA conjugated (E2-BSA)] was dissolved in PBS and other compounds in either ethanol (17{alpha} and ß E2, ICI 182780) or DMSO [dimethylsulfoxide (GFX 109203X, U0126, PP2, wortmannin, LY 294002, and Genistein)] and added to cells in fresh DMEM-Ham’s F12 medium. In controls, vehicles were added at the same concentrations never exceeding 0.01% (vol/vol). The cellular toxicity of the various inhibitors was verified by measuring the lactate dehydrogenase activity released into the medium after cell preincubation with the compounds as described elsewhere (33).

Cellular extract preparation
Cellular extracts were prepared from adipocytes as follows. After washing, cells were scraped in 10 mM Tris buffer, pH 7.4, containing 150 mM NaCl, 2 mM EGTA, 2 mM dithiothreitol (DTT), 1 mM orthovanadate, 50 mM sodium ß-glycerophosphate, 10 mM NaF, 20 µg/ml 4-(2-aminoethyl-benzenesulfonyl fluoride hydrochloride) (AEBSF), 5 µg/ml aprotinin, and 5 µg/ml leupeptin. The extracts were sonicated and centrifuged at 25,000 x g for 15 min at 4 C. The resulting supernatants were denatured with Laemmli buffer (vol/vol) and stored at -20 C.

Preadipocyte and adipocyte membranes were prepared as described elsewhere (34). Briefly, cells were scraped in cold buffer containing 50 mM Tris buffer, pH 7.4, 0.25 M sucrose, 1 mM EDTA, 100 µM PMSF, and 2 µM leupeptin. After sonication and nuclei elimination at low speed, the extracts were centrifuged at 25,000 x g for 20 min at 4 C. The resulting pellets were washed and denatured with Laemmli buffer (vol/vol).

Nuclear extract preparation
Adipocytes were rapidly washed with cold PBS and scrapped in cold buffer A (10 mM HEPES, pH 7.9, 150 mM NaCl, 0.5 mM DTT, 0.6% Nonidet P-40, 1 mM EDTA, 1 mM orthovanadate, 30 mM ß-glycerophosphate, 10 mM NaF, 20 µg/ml AEBSF, 5 µg/ml aprotinin, and 5 µg/ml leupeptin). The homogenates were centrifuged at 7,000 x g for 10 min at 4 C. The nuclear extract was prepared as described by Digman et al. (35), with some modifications. After washing, crude nuclei were resuspended in cold buffer B [20 mM HEPES, pH 7.9, 25% (vol/vol) glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 0.05 mM DTT, 0.6% Nonidet P-40, 1 mM orthovanadate, 10 mM NaF, 30 mM ß-glycerophosphate, 20 µg/ml AEBSF, 5 µg/ml aprotinin, and 5 µg/ml leupeptin]. The suspension was vigorously shaken at 4 C for 30 min, followed by centrifugation at 25,000 x g for 10 min at 4 C. The supernatant containing the nuclear extract was stored in aliquots at -80 C until to be used for EMSA or diluted (vol/vol) in Laemmli buffer for Western blot analysis.

Western blot analysis
Equal amounts of protein (10–50 µg) and prestained molecular weight markers were subjected to SDS-PAGE (12.5% acrylamide). Proteins were transferred to polyvinylidenedifluoride membranes. The filters were subsequently stained to verify that equal protein amounts were loaded and transferred. After blocking by Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) and 2.5% gelatin for 2 h, filters were incubated overnight with the primary antibody diluted in TBS-T/2.5% gelatin (0.5 mg/ml). Membranes were washed and incubated with the secondary antiserum coupled to peroxidase (1:12,500 dilution in TBS-T) for 1 h and extensively washed with TBS-T. Filters were next incubated with the enhanced chemiluminescence detection solution and then exposed to x-ray films. Reprobing of the membranes gave identical results. Specificity of the immunoreactive proteins was verified by loss of sample immunoreactivity when incubated with the antiserum neutralized with the corresponding specific peptide. Signals were quantified by densitometry. Control experiments with various amounts of protein (5–50 µg) were performed to ensure that the densitometric signal intensity was proportional to the amount of protein loaded. To normalize the results, membranes were stripped and reprobed with antiserum specific for pan-ERK or C/EBPß when nuclear extracts were used.

EMSA
EMSA was performed as previously described (31). Briefly, protein-DNA complexes were formed by incubating 2–10 µg of nuclear protein in a buffer consisting of 10 mM HEPES, pH 7.9, 50 mM KCl, 5 mM MgCl2, and 1 mM DTT in the presence of 1 µg nonspecific competitor DNA poly(dI-dC) for 10 min at 4 C. The 32P-labeled probe was then added and the incubation further extended for 20 min at room temperature. The resulting DNA-protein complexes were separated from the unbound probe by electrophoresis on a native 6% polyacrylamide gel in 0.5x Tris/Borate/EDTA buffer. Gels were then dried and subjected to autoradiography. AP-1 double-stranded oligonucleotides (5'-CGC TTG ATG AGT CAG CCG GAA-3') or NF{kappa}B (5'-AGT TGA GGG GAC TTT CCC AGG C-3') were labeled with [{gamma}-32P]ATP (3000 Ci/mmol) using T4 polynucleotide kinase. Unincorporated nucleotides were removed by chromatography in a G25 Sephadex column. Signals were quantified by densitometry. Parallel gels were performed and stained to ensure that the amount of nuclear extracts loaded was identical whatever the experimental conditions. In competition experiments, 1-, 10-, or 100-fold molar excesses of unlabeled AP-1 double-stranded oligonucleotide or a 50-fold molar excess of unlabeled heterologue Oct-1 double-stranded oligonucleotide (5'-TGT CGA ATG CAA ATC ACT AGA A-3') were included in the binding reaction mixture. The reaction specificity was verified by adding a 1-, 10-, or 100-fold molar excesses of unlabeled mutated AP-1 double-stranded oligonucleotide (mAP-1:5'-CGT TTG ATG ACT CAG CCG GAA-3') with the labeled probe.

RNA preparation and RT-PCR
Total RNAs were extracted from parametrial adipocytes following the acid-guanidinium-isothiocyanate protocol (36). The reverse transcription and PCRs were performed as previously described (31). The oligonucleotide primer pairs specific for rat c-fos and ribosomal acidic protein PO (RP) were described elsewhere (37, 38). The oligonucleotide primer pairs specific for rat uncoupling protein-2 (UCP2) were: sense 5'-CAT CTT CTG GGA GGT AGC-3'/antisense 5'-AAG ACA GGG CAG GAA TGG-3'. Levels of c-fos and UCP2 mRNAs are semiquantitative relative to RP mRNA chosen as an internal control because its expression is sex steroid independent (38).

Transient transfection
Parametrial adipocytes were transiently transfected by electroporation using the conditions described elsewhere (39) with 2–5 µg CRE-Luc reporter and pSV ß-gal plasmids. One hour later, E2-BSA (10 nM) or forskolin (10 µM) were added to the cells. After 24 h, luciferase activity was measured on cell lysates and normalized to the ß-gal activity.

Other determinations
Protein concentrations were measured following the dye-binding procedure (40). All results are expressed as means ± SEM from at least three individual experiments. Comparisons between experimental groups were made using t test and ANOVA with Bonferroni P values.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MAPK (Erk-1 and Erk-2) activation in response to E2 in rat adipocytes
The presence of phosphorylated threonine and tyrosine residues on MAPK correlates with enzymatic activity (25, 26). To assess MAPK activity, we thus performed Western blot analysis of adipocyte cellular extracts using specific antibody recognizing the phosphorylated forms of p42/p44 MAPK as described elsewhere (41). The time course of the E2 effect on MAPK activation was studied on in vitro-differentiated parametrial adipocytes and is shown in Fig. 1Go. As can be seen, maximal MAPK activation by water-soluble E2 was achieved at 5 min and returned to basal levels by 60 min. Similar results were obtained with insulin confirming a previous report (42). Reprobing the membranes using a pan ERK antibody that recognizes nonphosphorylated forms of p42/p44 MAPK (Fig. 1Go) confirmed that E2 specifically altered MAPK activity but not its expression levels. It must be noted, however, that expression of the p42 isoform was higher than that of the p44 MAPK isoform. The membrane impermeable E2-BSA also rapidly activated p42/p44 MAPK (see Fig. 3Go). In contrast, 17{alpha}-E2, the biologically inactive stereoisomer, and other sex steroid hormones, progesterone and T, failed to elicit any phosphorylation of MAPK indicating the estrogen specificity of the E2 MAPK activation (data not shown).



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  		Figure 1. Time-dependent activation of MAPK by E2 or insulin in rat adipocytes. In vitro-differentiated parametrial adipocytes were incubated with E2 [10 nM (A)] or insulin [100 nM (B)] for the indicated times in serum-free F12 medium. Cell lysates were subjected to SDS-PAGE, immunoblotted, and quantified as described in Materials and Methods. Representative Western blots of time course MAPK activation by E2 (A) or insulin (B) are shown. Densitometric analysis of MAPK Western blots for E2 (C) or insulin (D) activation. Results are expressed as arbitrary units. Each bar represents the mean ± SEM of four separate experiments. *, P < 0.05.

 


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  		Figure 3. Effect of ICI 182,780 on E2 activation of MAPK in rat adipocytes. In vitro-differentiated parametrial adipocytes were pretreated with ICI 182,780 (10 nM) or the vehicle (ethanol 0.01%) for 15 min and then incubated for 5 min with E2 (10 nM) or its BSA conjugate E2-BSA (10 nM). Cell lysates were analyzed as described in Materials and Methods. A, Representative Western blot of MAPK activation. B, Densitometric analysis of MAPK Western blots. Results are expressed as arbitrary units. Each bar represents the mean ± SEM of three separate experiments. *, P < 0.01.

 
All the following experiments were next performed with water-soluble E2.

The dose-response studies presented in Fig. 2Go revealed that the E2 effect was already significant at 10-10 M, with a maximal effect at 10-8 M. The latter concentration was thus used throughout the following experiments.



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  		Figure 2. Dose-response activation of MAPK by E2 in rat adipocytes. In vitro-differentiated parametrial adipocytes were incubated for 5 min with increasing concentrations of E2. Cell lysates were subjected to SDS-PAGE, immunoblotted, and quantified as described in Materials and Methods. A, Representative Western blot analysis of E2 activation of MAPK. B, Densitometric analysis of MAPK Western blots. Results are expressed as arbitrary units. Each bar represents the mean ± SEM of three separate experiments. *, P < 0.05.

 
The fact that E2 acts rapidly on MAPK activity and that neither a transcriptional inhibitor (actinomycin D) nor a translation inhibitor (cycloheximide) affected the E2-induced activation of MAPK (data not shown), strongly suggest involvement of a nongenomic mechanism possibly mediated through a putative membrane ER. ICI 182,780, a pure ER antagonist, completely abolished the effect of E2 and its BSA conjugate on MAPK (Fig. 3Go), indicating that if a membranous receptor is required, it is structurally close to ER.

The preceding results were obtained on mature adipocytes. In the following experiments, the effects of E2 on MAPK activation were compared in proliferating, early, and late differentiated preadipocytes and were obviously present only in late differentiated cells (Fig. 4AGo).



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  		Figure 4. A, Comparison of MAPK activation by E2 in proliferating, early, and late differentiated rat preadipocytes. Proliferating, early, and late differentiated preadipocytes from parametrial fat depots were incubated with E2 (10 nM) or fetal serum (10%) for 5 min. Cell lysates were subjected to SDS-PAGE, immunoblotted, and quantified as described in Materials and Methods. A representative Western blot analysis from one experiment repeated two times is presented. B, Identification of immunoreactive ER{alpha} in adipocyte membrane. Membrane fractions (50 µg protein) of preadipocytes (pre-Ad) and adipocytes (Ad) from parametrial tissue were analyzed by Western blots using anti-C-terminal ER{alpha} or caveolin-1 antibodies. As control, data obtained with cytosolic extracts of uterus (50 µg protein) are presented. The results shown are representative from three separate experiments.

 
Detection of the immunoreactive ER{alpha} protein by Western blot was performed on preadipocyte and adipocyte membrane preparations. As shown in Fig. 4BGo, of the two prominent bands (66 kDa and 45 kDa) found in uterus extracts, the 66-kDa band was detected in adipocytes but not preadipocytes. The presence of this band was associated with that of caveolin-1, the major component of caveolae, which is strongly induced during adipogenesis (43).

To determine in more detail the mechanism of E2-induced MAPK activation, various inhibitors of upstream components of the MAPK pathway were investigated. As in breast cancer cells, E2 activation of MAPK was recently shown to require a PTX-sensitive G protein and Src kinase activity (44), we investigated the influence of long-term PTX treatment (100 ng/ml, 16 h) or PP2, a selective inhibitor of Src family tyrosine kinase (45), on MAPK activation by E2. As can be seen in Fig. 5Go, A and B, treatment by these inhibitors had no effect per se but resulted in a significant reduction of E2 activation of MAPK. These treatments also reversed almost completely the level of MAPK activation because of LPA, a known Gi-coupled agent (Fig. 5Go, A and B). Furthermore, E2 rapidly activated phosphorylation of Src kinase as demonstrated by Western blot analysis using specific antibody recognizing the phospho Src [Tyr416 (Fig. 5BGo, inset)], thus indicating a direct activation of the kinase by the steroid. Because PI3K pathway mediates insulin activation of MAPK in adipocytes (46), the role played by this pathway in E2 action was investigated using wortmannin, an inhibitor of the PI3K catalytic subunit (47). As shown in Fig. 5CGo, wortmannin pretreatment had no effect per se but considerably reduced the E2- and insulin-activated MAPK phosphorylation. These results were confirmed with LY 29 4002 (10 µM), another selective PI3K inhibitor (Ref. 48 and data not shown). Finally, other inhibitors of immediate upstream components of the MAPK signaling pathway such as Genistein, a general tyrosine kinase inhibitor (49); UO 126, a new selective MEK inhibitor (50); and GFX 109203X, a classical PKC inhibitor (51), all antagonized the activation of MAPK by E2 (Fig. 5DGo).



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  		Figure 5. Effect of various upstream component inhibitors of MAPK signaling pathway on E2 activation of MAPK in rat adipocytes. A, In vitro-differentiated parametrial adipocytes were pretreated with or without PTX (100 ng/ml) for 16 h and then incubated for 5 min with E2 (10 nM) or LPA (100 µM). B, In vitro-differentiated parametrial adipocytes were pretreated with the Src family kinase inhibitor PP2 (10 µM) or the vehicle (DMSO 0.1%) for 1 h and then incubated for 5 min with E2 (10 nM) or LPA (100 µM). B (inset), In vitro-differentiated parametrial adipocytes were incubated for 5 min with E2 (10 nM) or LPA (100 µM). Membrane fractions were prepared as described in Materials and Methods and were analyzed by Western blots using a specific antibody recognizing phospho-Src (Tyr 416). Representative Western blot analysis from one experiment repeated three times are presented. C, In vitro-differentiated parametrial adipocytes were pretreated with wortmannin (100 nM) or the vehicle (DMSO 0.1%) and then incubated with E2 (10 nM) or insulin (100 nM). D, In vitro-differentiated parametrial adipocytes were pretreated for 30 min with the tyrosine kinase inhibitor Genistein (50 µM), MEK inhibitor UO 186 (10 µM), PKC inhibitor GFX 109203X (0.5 µM), or the vehicle (DMSO 0.1%) and then incubated with E2 (10 nM) for 5 min. Cell lysates were analyzed as described in Materials and Methods. Results are expressed as arbitrary units. Each bar represents the mean ± SEM of five separate experiments. *, P < 0.05 by t test, prevention by inhibitor of MAPK activation.

 
Nuclear translocation of MAPK in response to E2 in rat adipocytes
MAPK nuclear localization was examined by Western blot analysis of nuclear extracts. As shown in Fig. 6AGo, MAPK translocation in adipocyte nucleus occurs in response to 15-min E2 exposure [p42/p44 activated MAPK increased by a factor of 4.6 ± 0.7 (n = 5), P < 0.01]. These results are similar to those obtained after 15-min insulin exposure [4.5 ± 0.8 increase (n = 5), P < 0.01 (Fig. 6BGo)].



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  		Figure 6. Nuclear translocation of MAPK in response to E2 or insulin in rat adipocytes. In vitro-differentiated parametrial adipocytes were incubated with E2 [10 nM (A)] or insulin [100 nM (B)] for the indicated times. Nuclear extracts were prepared and probed with active MAPK antiserum as described in Materials and Methods. As control loading, membranes were stripped and reprobed with C/EBPß antiserum. Representative Western blot analysis from one experiment repeated five times is presented.

 
AP-1 DNA-binding activity in response to E2 in rat adipocytes
We have recently demonstrated that E2 administration to rats induces an increase in the AP-1 activity in adipocytes (31). One mechanism that could explain this finding is the above described enhancement of MAPK nuclear translocation caused by E2. EMSA experiments were thus performed to verify this hypothesis. Data in Fig. 7AGo show that exposure to E2 for 15 min (time necessary for maximal translocation of MAPK, see Fig. 6Go) is sufficient to double the consensus AP-1 DNA-binding activity (P < 0.01, n = 5). Moreover, this increase remains sustained because it is still observed after 1 h of exposure to E2. The same kinetics of AP-1 DNA-binding activation was observed with insulin (Fig. 7BGo). In contrast, NF{kappa}B DNA-binding activity remained insensitive to E2 exposure (Fig. 7AGo), showing the selectivity of the estrogen effects toward the DNA-binding activity of transcription factors.



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  		Figure 7. Rapid effect of E2 on DNA binding activity of AP-1 and NF{kappa}B in rat adipocytes. In vitro-differentiated parametrial adipocytes were incubated with E2 [10 nM (A)] or insulin [100 nM (B)] for the indicated times. Nuclear extracts were prepared and incubated with the radiolabeled consensus AP-1 (A and B) or NF{kappa}B (A) probes as described in Materials and Methods. This figure is representative of three independent experiments.

 
To further confirm the importance of MAPK in the E2 effect on AP-1 DNA-binding activity, experiments with UO 126 were performed. Data in Fig. 8Go show that this inhibitor failed to alter basal AP-1 binding activity and only slightly reduced E2-activated AP-1-binding activity (-23 ± 5%, P < 0.05, n = 3). The latter finding contrasts with the insulin effects on AP-1, which were blocked when MAPK was inhibited (52). To ensure the validity of our experimental protocol, control experiments were conducted in parallel with IL1ß. As shown in Fig. 8Go, UO 126 induced an important reversion of the IL1ß-activated AP-1 binding activity (-66 ± 3%, P < 0.01, n = 2).



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  		Figure 8. Influence of MAPK inhibition on AP-1 DNA binding activity stimulated by E2 in rat adipocytes. In vitro-differentiated parametrial adipocytes were pretreated with 10 µM MEK inhibitor UO 126 or the vehicle (0.1% DMSO) for 30 min and then incubated with E2 (10 nM) for 15 min or IL1ß (10 ng/ml) for 60 min. Nuclear extracts were next prepared and incubated with the radiolabeled consensus AP-1 probe as described in Materials and Methods. The results shown are representative of two to three independent experiments.

 
CREB activation by E2 in rat adipocytes
The influence of E2 on CREB phosphorylation was also investigated and compared with that of insulin. Immunoblotting experiments using an antibody directed against phospho-Ser133 CREB were performed on adipocyte nuclear extracts. As shown in Fig. 9AGo, adipocyte exposure to E2 for 15 min resulted in a marked increase in CREB phosphorylation (4.4 ± 1.4-fold, P < 0.05, n = 4), which still remained observable after 1 h. Similar results were found with insulin (2.8 ± 0.7-fold, P < 0.05, n = 4 after 15 min). It is important to note that both the kinetics of CREB activation and MAPK nuclear translocation in response to E2 (Fig. 6AGo) are superimposable. This led us to determine the part played by the MAPK pathway in the E2-CREB activation. As shown in Fig. 9BGo, adipocyte pretreatment by the MEK inhibitor UO 126 resulted in a complete prevention of the E2- and insulin-induced CREB phosphorylation. These data strongly suggest that both the E2- and insulin-induced CREB phosphorylations are related to activation of the MAPK signaling pathway.



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  		Figure 9. Activation of CREB by E2 or insulin in rat adipocytes. A, In vitro-differentiated parametrial adipocytes were incubated with E2 (10 nM) or insulin (100 nM) for the indicated times. A representative Western blot analysis from one experiment repeated three times is presented. As control loading, membranes were stripped and reprobed with C/EBPß antiserum. B, In vitro-differentiated parametrial adipocytes were pretreated with 10 µM MEK inhibitor UO 126 or the vehicle (0.1% DMSO) for 30 min and then incubated with E2 (10 nM) or insulin (100 nM) for 15 min. Densitometric analysis of MAPK Western blots is presented. Results are expressed as arbitrary units. Each bar represents the mean ± SEM of four separate experiments. *, P < 0.01.

 
Transcriptional activation of CREB-responsive genes by E2 in rat adipocytes
Finally, we investigated whether the phosphorylation of Ser133 CREB caused by E2 could result in the transcriptional activation of CREB-responsive genes such as the UCP2 gene (53). As shown in Fig. 10AGo, expression of UCP2 was increased after E2-BSA exposure (2.0 ± 0.2 fold, P < 0.05, n = 3). For comparison, we studied c-fos expression, which is also regulated through CREB activation, in addition to other several pathways (54). Data in Fig. 10AGo show that E2-BSA rapidly increased c-fos mRNA levels (1.5 ± 0.2-fold, P < 0.05, n = 4).



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  		Figure 10. A, Effect of E2 on UCP2 and c-fos mRNA levels in rat adipocytes. In vitro-differentiated parametrial adipocytes were incubated for 30 min and 18 h with E2-BSA (10 nM), for c-fos and UCP2 mRNA analysis, respectively. RT-PCR was performed as described in Materials and Methods. As internal standard, PCR analysis was conducted with the RP. The results shown are representative from two to four different experiments. B, Transactivation of CRE-containing Luc reporter plasmid by E2-BSA and forskolin in rat adipocytes. The pCRE-Luc reporter plasmid was cotransfected into rat adipocytes with the pSV-ß-gal control plasmid. After 1 h, cells were incubated with or without E2-BSA (10 nM) or forskolin (10 µM) for 24 h. Luciferase and ß-gal activities were measured on cell lysates. Bars represent relative luciferase units normalized for ß-gal activity. The results are representative of two independent transfection experiments. *, P < 0.05.

 
To further confirm this transcriptional activation by E2, transient expression assays using a CRE-luciferase reporter gene were performed. As shown in Fig. 10BGo, transactivation of this reporter gene was significantly stimulated by E2-BSA. The validity of our experimental conditions was established using forskolin, a cAMP-producing agent that also promoted transactivation of the CRE-luciferase reporter gene (Fig. 10BGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The classical mechanism whereby estrogens mediate their cellular actions involves at first estrogen binding to intracellular receptors, the ER{alpha} and ERß, which then bind to DNA consensus sequences, the EREs to finally modulate gene transcription. Increasing evidences, however, indicate that estrogens, like other steroid hormones, elicit rapid, nongenomic cellular effects. To explain these nongenomic effects, the existence of putative membrane ERs coupled to various downstream transduction pathways has been postulated (20, 21, 22, 23, 24). In this study, we present the first demonstration that E2 causes a rapid activation of MAPK, AP-1, and CREB activities in rat adipocytes. In addition, the finding that this MAPK activation is observed at low concentrations of E2 renders these observations physiologically relevant.

The mechanisms whereby putative membrane ER activate signaling pathways are still not clearly defined. However, recent reports indicate that transfections with ER{alpha} and ERß cDNAs give rise to membrane receptors that interact directly with G proteins leading to activation of PLC and by the consequence to PKC stimulation (55). Our experiments showing that the ER antagonist ICI 182,780 blocks MAPK activation by E2 or its membrane impermeable conjugate E2-BSA suggest the involvement of an ER possibly located in the membrane and pharmacologically similar if not identical to the nuclear ER.

Importantly, the E2 activation of MAPK could not be observed in proliferating and early differentiated preadipocytes suggesting that the membrane structural organization of the adipocyte phenotype is required for this E2 effect. Our finding that an immunoreactive ER{alpha} protein is present together with caveolin-1 in membranes from adipocytes but not from preadipocytes gives strong support to this hypothesis. Caveolin-1 is a major component of caveolae. These specialized invaginations of plasma membrane are particularly abundant in adipose tissue (43) and have been implicated in signal transduction as several components of the MAPK pathway like Src, and MAPKs are concentrated in caveolae (56). In a recent report, ER{alpha} was shown to colocalize with caveolin-1 in endothelial cells (57). This suggests that caveolin-1 could serve as docking sites for the membranous form of ER{alpha} in adipocyte. Experiments are currently in progress to test this attractive hypothesis.

Very recently, E2 nongenomic activation of Src/Shc/MAPK in osteoblasts was shown to require only the ligand-binding domain of ER{alpha} (58). In white adipocytes, we have presently demonstrated that the Gi-protein/Src/PI3K pathway activating MAPK upstream of Ras (27, 28) and the PKC pathway activating MAPK downstream of Ras (25, 26) both intervene in E2 signaling to p42/p44 MAPK (Fig. 11Go). In endothelial cells, other reports have shown that E2 can rapidly activate PI3K through an interaction between ER{alpha} and the PI3K regulatory subunit (59, 60). PKC has also been reported to be rapidly activated by E2 in neurons and chondrocytes depending on G protein-coupled PLC (61, 62). In adipocytes, MAPK activation is PI3K dependent in response to insulin but PI3K independent in response to PKC activators (46). Thus, the latter finding together with our experiments provide strong support to the existence of a cross-talk between the PI3K and MAPK signaling pathways in adipocytes.



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  		Figure 11. A model for p42/p44 MAPK activation in response to E2 in white adipocytes.

 
Upon stimulation by various signals, part of activated MAPK rapidly translocates into the nucleus in which the enzyme phosphorylates transcription factors such as AP-1 and CREB (29, 63). Upon E2 stimulation, our experiments show a rapid nuclear localization of MAPK and, with the same kinetics, stimulation of CREB phosphorylation. Apart from its MAPK-dependent activation, CREB was originally described as a target of the cAMP signaling pathway (63). E2 activation of the cAMP signaling pathway has been described in some cell types (18, 19, 20). Therefore, the E2 activation of CREB could also result in part from cAMP signaling pathway activation. However, preliminary experiments failed to show rapid effects of E2 on cAMP production by adipocytes under the present experimental conditions.

Contrasting with the results observed on CREB, the rapid increase of AP-1 DNA binding activity following E2 exposure was only weakly altered when MAPK was inhibited. This indicates that in adipocytes the MAPK pathway alone is not sufficient to explain the AP-1 DNA-binding activity promotion by E2. This is not surprising considering that the MAPK control of AP-1 DNA-binding activity is variable according to the cell types (64, 65). It thus appears that the promoting action of E2 on AP-1 DNA-binding activity in adipocytes is owing, in part, to activation of kinases other than p42/p44 MAPK and/or inhibition of phosphatases. Supporting this hypothesis is the finding that in adipocytes as well insulin inhibits the nuclear protein phosphatase 2A leading to increased CREB activity (66). In addition, MAPK intervenes in E2 action on AP-1 at levels other than AP-1 DNA-binding activity. The steroid induces c-fos expression by acting at promoter level through Sp-1/ER{alpha} interaction and MAPK activation of Elk-1/serum response element (54).

As shown here, E2 activates the two transcriptional factors AP-1 and CREB leading for the latter factor to transcriptional activation of CREB-responsive genes like UCP2. Energy expenditure seems in part to be controlled by estrogens as suggested by recent studies on {alpha}ER-knockout mice (67) as well as by UCP2 (68, 69). It is thus tempting to speculate that the control of energy expenditure exerted by estrogens is at least in part related to UCP2 induction by E2 in adipose tissue. Moreover, as demonstrated by the present study, the transcriptional potential of E2 in adipocytes is not only restricted to target genes whose promoters contain classical EREs but is largely extended to those genes having in their promoters the AP-1 and CREB response elements. Pregnancy, which is characterized by a progressive increase in estrogen production, is also associated with the development of insulin resistance (70). In this context, it cannot be excluded that E2 through activation of AP-1 and CREB, two molecular targets of insulin, could mimic some actions of this metabolic hormone, thereby contributing to maintain insulin sensitivity in adipose tissue during pregnancy (71).

In conclusion, the present investigation describes nongenomic E2 activation of MAPK and the transcriptional factors AP-1 and CREB in adipocytes and suggests that this novel membranous ER{alpha}-dependent mechanism contributes to promote the transcriptional potency of estrogens and hence the modulation of the adipocyte metabolism by these hormones.


   Acknowledgments
 
We wish to thank Prof. G. Ailhaud, Dr. T. Issad, and Dr. I. Dugail for helpful discussions.


   Footnotes
 
This work was supported by the Université of Paris V and the Comité des Yvelines de la Ligue contre le Cancer. E.G. was sponsored by a doctoral fellowship from the Comité des Yvelines de la Ligue contre le Cancer.

1 Present address: Unité Propre de Recherche 415 Institut Cochin de Genetique Moleculaire, 22 rue Méchain, 75014 Paris, France. Back

Abbreviations: AEBSF, 4-(2-aminoethyl-benzenesulfonyl fluoride hydrochloride); AP-1, activator protein-1; CREB, cAMP-response element-binding protein; DMSO, dimethylsulfoxide; DTT, dithiothreitol; ERE, estrogen response element; ß-gal, ß-galactosidase; LPA, lysophosphatidic acid; MEK, MAPK kinase; NF{kappa}B, nuclear factor {kappa}B; PP2, 4-amino-5(4-chlorophenyl)-7-(t-butyl)pyrazdo[3,4-d]pyrimidine; PTX, pertussis toxin; RP, ribosomal acidic protein PO; TBS-T, TBS containing 0.1% Tween 20; UCP2, uncoupling protein-2.

Received June 18, 2001.

Accepted for publication November 7, 2001.


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