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Endocrinology Vol. 141, No. 8 2837-2846
Copyright © 2000 by The Endocrine Society

ARTICLES

Estradiol Stimulation of c-fos and c-jun Expressions and Activator Protein-1 Deoxyribonucleic Acid Binding Activity in Rat White Adipocyte1

Esther Garcia2, Danièle Lacasa and Yves Giudicelli

Department of Biochemistry and Molecular Biology, Faculty of Medicine Paris-Ouest, Université Paris V and Centre Hospitalier, Poissy 78303 France

Address all correspondence and requests for reprints to: Y. Giudicelli, Service de Biochimie et de Biologie Moléculaire 78303 Poissy, France.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In order to elucidate the molecular mechanisms whereby ovarian hormones, and particularly estrogens, modulate fat cell metabolism, we investigated the effects of estradiol administration on c-fos and c-jun expressions in fat cells from ovariectomized (OVX) rats. Estradiol treatment resulted in a rapid increase in c-fos and c-jun messenger RNA (mRNA) and protein levels (about 2-fold). These effects of estradiol on c-fos and c-jun mRNAs were blocked by actinomycin D but not by cycloheximide treatment, suggesting that estradiol modulates c-fos and c-jun transcription. Moreover, the estradiol-induction of both transcripts was partially suppressed by the estrogen-receptor antagonist ICI 182,780. In contrast, progesterone administration did not affect c-fos and c-jun mRNA levels indicating a hormonal specificity of estrogen action. However, an antagonism of estradiol-induction of both genes was observed after progesterone treatment. In addition, the estradiol-induced changes in c-fos and c-jun mRNA expressions could not be observed in castrated males suggesting a gender-specific effect of estradiol. Finally, in OVX rats, estradiol treatment stimulated the specific AP-1 DNA binding activity (about 5-fold) in adipocyte nuclear extracts as assessed by electrophoretic mobility shift assays. These results suggest that some of the estrogen effects in fat cells from female rats are mediated through induction of the AP-1 complex expression and consequently through modulation of the AP-1 dependent gene expression in adipocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OVARIAN hormones are known to influence site-specifically adipocyte development and metabolism (1). Adipose tissue is an important site for estrogen biosynthesis and sex steroid hormone storage (2). Specific receptors for ovarian hormones are present in both rodent precursor and mature fat cells (3, 4, 5). In addition, the density of estradiol receptor (ER) varies according to the anatomical origin of the fat cells (5, 6). Beside the classical ER{alpha}, another ER subtype, ERß, was recently characterized (7). This ERß is expressed in adipose tissue from various localizations (8). This suggests that adipose cells are targets for estrogens. In fact, using creatine kinase B as an estrogen induced protein marker, a site- and sex-specific direct effect of estradiol was observed in adipocytes (9, 10).

In rats, several studies have shown that ovariectomy leads to an increased body fat mass that can be reversed by injection of estradiol (11, 12, 13). The accumulation of parametrial adipose tissue induced by ovariectomy is, in part, related to increased lipogenic activity in association with reduced lipolysis due to blunted adenylate cyclase catalytic activity and cAMP production (12, 14). Conversely, estrogen treatment reduces whereas progesterone administration enhances adipose tissue lipoprotein lipase (LPL) activity, the rate limiting enzyme for lipid storage (15).

Activator protein-1 complex (AP-1) consists of Fos (c-fos, Fos B, Fra-1, and Fra-2) and Jun (c-jun, Jun B and Jun D) protein homo- or heterodimers and binds to regulatory sequences in the promoter of various target genes involved in cell growth, differentiation and metabolism (for Review, see Ref. 16). Among the fos/jun family genes, c-fos and c-jun are the two major components of the AP-1 complex. Several studies have demonstrated that estrogen and progesterone regulate nuclear protooncogene expressions in target tissues (for review, see Ref. 17 ). For example, estrogen treatment rapidly increases c-fos and c-jun messenger RNA (mRNA) levels in rat uterus, an effect that seems in large part related to transcriptional activation (18, 19). Functional estrogen-responsive elements (EREs), which bind the estrogen receptors, have been identified in the promoter of c-fos and c-jun genes (20, 21). Moreover, estrogens are also able to stimulate transcriptional activities of the AP-1 complex through a cooperative interaction of the estrogen receptors with this complex (22, 23).

Various observations have led to assign an important role to the AP-1 complex in the regulation of the adipocyte differentiation process (24, 25). As a matter of fact, this complex induces transcription of both the adipocyte intracellular lipid-binding protein P2 (aP2) gene (26) and the lipoprotein lipase (LPL) gene in Ob1771 preadipose cells (27). Furthermore, in adipocytes where insulin is the major lipogenic signal, this hormone stimulates c-fos and c-jun mRNA expressions in vivo (28) and in vitro (29) as well as the transcriptional activity through phosphorylation of the AP-1 complex (30).

The present studies were undertaken to get a better understanding on how estradiol modulates adipocyte metabolism in vivo. For this purpose, we tested the hypothesis that c-fos and c-jun expressions as well as AP-1 DNA binding activity could be possible targets for estrogens in fat cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
17ß-estradiol benzoate was purchased from Roussel (Paris, France). Progesterone, cycloheximide, actinomycin D, and phorbol 12-myristate 13-acetate (TPA) were from Sigma-Aldrich Corp. (Saint Quentin, France). Human insulin was from Novo Nordisk (Boulogne-Billancourt, France). ICI 182,780 was from Tocris (Bristol, UK). Antisera and synthetic peptides specific for c-fos (OP17) were purchased from Oncogene Science, Inc. (Cambridge, MA) and those specific for c-jun (sc-1694) and C/EBP{alpha} (sc-61) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mutated AP-1 consensus double-stranded oligonucleotides were also purchased from Santa Cruz Biotechnology, Inc.. Western blotting protocols and random sequence hexanucleotide primers DNA labeling (Megaprime kit) were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). AP-1 and Oct-1 consensus double-stranded oligonucleotides and T4 polynucleotide kinase were from Promega Corp. (Madison, WI). The PCR purification kit (QUIAquick) was obtained from QIAGEN (Santa Clarina, CA). All other chemicals were of reagent grade.

Animals and hormonal treatment
Procedures with experimental animals were authorized and followed the guidelines of the Ministry of Agriculture (France) (authorization 006614). Mature female and male Sprague Dawley rats (150 g) were ovariectomized (OVX) or castrated (CAST) at least 15 days before use. Animals received a single sc injection of benzoate-estradiol (E2) (2 µg) or vehicle solution (olive oil) and were killed 2 h later. The pure estrogen antagonist ICI 182,780 (0.2 mg/rat) was injected sc 30 min before E2. Cycloheximide (5 mg ip/rat) or progesterone (5 mg sc/rat) were given 1 h before E2. Actinomycin D (0.5 mg ip/rat) was administered 3 h before E2. Plasma estradiol concentrations were at time the rats were killed: <0.03, 0.4 ± 0.08, < 0.03 and 0.13 ± 0.01 nM in OVX, OVX- E2, CAST, and CAST- E2 rats, respectively. In other experiments, OVX rats received a single ip injection of the phorbol ester, TPA (0.06 mg/rat) and were killed 1 h later.

Preparation of isolated adipocytes
Adipocytes were isolated from sc, parametrial (para), epididymal (epi), and perirenal (peri) fat pads as originally described by Rodbell (31) with slight modifications. Rat adipose tissues were homogenized or incubated with 1 mg/ml collagenase in Krebs-Ringer bicarbonate (KRB) buffer, pH 7.4, containing 5 mM glucose and 2% BSA. The digestion was allowed to proceed with vigorous shaking at 37 C for 30–45 min. The completed digest was filtered through a 250 µm nylon membrane, and floating adipocytes were collected. The uteri were quickly excised and trimmed of connective tissue.

Northern blot analysis
Total RNAs were extracted from isolated adipocytes, adipose tissues, and from uterus following the acid-guanidium-isothiocyanate protocol (32). RNA samples (20–40 µg) were separated on denaturing gels containing 1% agarose, 12.5% formaldehyde. The electrophoresed RNAs were capillary transferred onto a Hybond N+ membrane in 0.05 N NaOH and cross-linked to this membrane (30 min at 80 C). Prehybridizations were carried out for 3 h at 68 C in Church solution (0.5 M sodium phosphate, 1 mM EDTA, and 7% SDS). Hybridizations with [32P]-complementary DNA probes labeled by random priming (2–5 108 dpm/µg) were performed overnight at 68 C. The hybridized membranes were then washed 2 times for 10 min in 2 x SSC, 0.1% SDS at room temperature, once for 5 min in 0.5 x SSC, 0.1% SDS at 65 C and finally once for 5 min in 0.1 x SSC, 0.1% SDS at 65 C before being exposed to an x-ray film at -80 C. The band intensities were measured by scanning densitometry of the autoradiograms. Membranes were next dehybridized in boiling 0.5% SDS and then hybridized with probe specific for the ribosomal acidic protein PO chosen as an internal control because its expression is sex steroid independent (33). The relative amounts of c-fos and c-jun transcripts were quantified by calculating the ratio of c-fos or c-jun-band integrated densities to those of the ribosomal acidic protein.

To verify the integrity of the RNA loaded, parallel gels were run and stained with ethidium bromide to visualize 28S and 18S ribosomal RNAs.

The probes prepared by RT-PCR methods were specific for rat c-fos, c-jun, and ribosomal acidic protein PO as described in (33, 34, 35). The PCR products were purified with a PCR purification kit (QUIAquick).

Preparation of nuclear extracts
Adipose tissues were removed rapidly and were homogenized in 3 vol (wt/vol) of cold buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.05 mM dithiothreitol (DTT), 0.57 mM PMSF, 0.5 mM sodium deoxycholate, 1 mM orthovanadate, 30 mM ß-glycerophosphate, 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 Dignam (36), with some modifications. Briefly, the supernatant was decanted and the resulting pellet was resuspended in 5 vol buffer A and centrifuged a second time at 25,000 x g for 20 min at 4 C. Crude nuclei were resuspended in cold buffer B (20 mM HEPES, pH 7.9, 25% (vol/vol) glycerol, 0.42 M NaCl, 1 mM EDTA, 0.57 mM PMSF, 0.05 mM DTT, 0.5 mM sodium deoxycholate, 1 mM orthovanadate, 30 mM ß-glycerophosphate, 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 30 min at 4 C. The supernatant containing the nuclear extracts was stored in aliquots at -80 C until use for electrophoretic mobility shift assay (EMSA) or diluted vol/vol in Laemmli buffer for Western blot analysis.

Western blot analysis
Equal amounts of protein (20–30 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 equal protein loading and transfer. 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 µg/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 (10–75 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 C/EBP{alpha}.

EMSA
Protein-DNA complexes were formed by incubating 5–10 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 of nonspecific competitor DNA poly-(deoxyinosine-deoxycytidine) for 10 min at 4 C. The 32P-labeled probe was then added and the incubation continued 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 0.5 x TBE (Tris/Borate/EDTA buffer). The gels were then dried and subjected to autoradiography. AP-1 double-stranded oligonucleotides (5'-CGC TTG ATG AGT CAG CCG GAA-3') were labeled with [{gamma}-32P] ATP (3,000 Ci/mmol) using T4 polynucleotide kinase kit (Promega Corp.). Unincorporated nucleotides were removed by chromatography in a G25 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, and 100-fold molar excesses of unlabeled AP-1 double-stranded oligonucleotides or a 50-fold molar excess of unlabeled heterologue Oct-1 double-stranded oligonucleotides (5'-TGT CGA ATG CAA ATC ACT AGA A- 3') were included in the binding reaction mixture.

The reaction specificity was verified by using a 1, 10, and 100-fold molar excesses of unlabeled mutated AP-1 double-stranded oligonucleotides (mAP-1: 5'-CGT TTG ATG ACT CAG CCG GAA-3'), which were added together with the labeled probe.

Other determinations
Protein concentrations were measured following the dye-binding procedure (37). All results are expressed as means ± SEM from at least three individual experiments. Comparisons between experimental groups were made using Student’s t test and ANOVA with Bonferroni P values.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The estradiol induction of c-fos and c-jun mRNAs has been extensively studied in the uteri of ovariectomized rats (18, 19). For this reason, we have included in all the experiments described herein parallel studies on the uteri chosen as a positive control for evaluating the steroid hormone effects under our experimental conditions.

1) Expression of c-fos and c-jun in response to estradiol
In initial studies, the time-course of the hormonal effect was determined. Data in Fig. 1Go show that low but detectable levels of c-fos and c-jun mRNAs are present in adipocytes isolated from OVX rats. After E2 administration, expression of the two protooncogene mRNAs was rapidly (2 h) and maximally induced (2.6 ± 0.2 and 2.3 ± 0.2-fold increase above control values (P < 0.001, n = 10) for c-fos and c-jun, respectively) whatever the adipocyte origins (sc, parametrial and perirenal). Furthermore and consistent with recently reported data in the pituitary gland (38), these inductions still persisted until 24 h post injection. For comparison, an approximately 10-fold increase in c-fos and c-jun mRNA expressions was found in uteri of OVX rats 2 h after the same E2 treatment (data not shown). Under the same conditions, insulin treatment (3 IU per OVX rat) also resulted in a rapid (15 min) 2- to 3-fold increase in c-fos mRNA expression (data not shown), a result that is consistent with previously reported data in male rat adipose tissue (28).



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  		Figure 1. Kinetics of estradiol treatment on c-fos and c-jun mRNA expressions in rat adipocytes. OVX rats were treated with estradiol (2 µg/rat) and then killed at the indicated times. Adipocyte total RNAs were extracted, analyzed and quantified by Northern blots as described in Materials and Methods. Results, expressed as amounts of c-fos and c-jun mRNAs after correction for the ribosomal protein mRNA signal, are given in arbitrary units. Each point represents the mean ± SEM of three independent experiments. *, Significant vs. OVX (P < 0.05).

 
Dose-response studies revealed maximal c-fos and c-jun mRNA inductions with 2 µg E2 per rat (data not shown), which is the dosage commonly used in studies devoted to the modulation of c-fos and c-jun expressions by E2 in uterus (18, 19).

To determine whether these changes were reflected at the protein level, Western blot analysis were performed on adipocyte nuclear extracts. Figure 2Go shows a rapid induction of the Fos and Jun proteins following the estradiol treatment (more than 2-fold increase over the control values; P < 0.01, n = 6). However, this effect was much lower in magnitude than that observed in the uterus (8-fold increase). A comparable effect was found after 1 h phorbol ester (TPA) injection, a treatment well known to up-regulate expression of the AP-1 members in the uterus (39). In contrast, E2 was without any significant effect on C/EBP{alpha} protein, another transcriptional factor used as an internal control (Fig. 2Go). These experiments lead to the conclusion that estradiol administration to OVX rats induces in less than 2 h c-fos and c-jun mRNA and protein expressions in adipocytes.



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  		Figure 2. Influence of estradiol administration on c-fos and c-jun protein levels in rat adipocytes. OVX rats were treated with vehicle alone (OVX) or estradiol (2 µg/rat) 2 h or with TPA (0.06 mg/rat) 1 h before they were killed. Nuclear extracts were probed with rat c-fos, c-jun, and C/EBP{alpha} antiserum as described in Materials and Methods. The data shown are from one representative experiment repeated three times.

 
2) Effects of actinomycin D and cycloheximide on estradiol-induced c-fos and c-jun mRNA expressions
To determine whether the effects of E2 described above were immediate-early events, we tested E2 induction of c-fos and c-jun transcripts in adipocytes from rats pretreated with either actinomycin D or cycloheximide. As shown in Fig. 3Go, actinomycin D abolished the E2-stimulated increase in c-fos and c-jun mRNA levels, suggesting that the effects of E2 result from de novo RNA synthesis. In contrast, cycloheximide failed to block the E2-induced expression of c-fos and c-jun mRNA (Fig. 4Go), indicating a primary response to E2 without requirement for the synthesis of intermediary proteins. Moreover, it should be noticed that pretreatment with the protein synthesis inhibitor alone caused an approximately 4-fold increase in both c-fos and c-jun mRNA levels, a phenomenon which characterizes genes having short half-live mRNAs (40). Altogether, these data strongly suggest that estrogen modulate positively c-fos and c-jun transcription in rat adipocytes.



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  		Figure 3. Influence of actinomycin D pretreatment on E2-induction of c-fos and c-jun mRNA levels in rat adipocytes. OVX rats were pretreated with vehicle (OVX) or with actinomycin D (OVX + AcD) 1 h before E2 administration (OVX + E2) (OVX + E2+ AcD). Adipocyte total RNAs were prepared, analyzed and quantified by Northern blots as described in Materials and Methods. Results, expressed as amounts of c-fos and c-jun mRNAs after correction for the ribosomal protein mRNA signal, are given in arbitrary units. Each bar represents the mean ± SEM of three separate experiments. *, Significant vs. OVX (P < 0.05). **, Significant vs. E2 (P < 0.05).

 


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  		Figure 4. Influence of cycloheximide pretreatment on E2-induction of c-fos and c-jun mRNA levels in rat adipocytes. OVX rats were pretreated with vehicle (OVX) or with cycloheximide (OVX + Cx) 1 h before E2 administration (OVX + E2) (OVX + E2+ Cx). Adipocyte total RNAs were extracted, analyzed and quantified by Northern blots as described in Materials and Methods. Results, expressed as amounts of c-fos and c-jun mRNAs after correction for the ribosomal protein mRNA signal, are given in arbitrary units. Each bar represents the mean ± SEM of three separate experiments. *, Significant vs. OVX (P < 0.05). **, Significant vs. E2 (P < 0.05).

 
3) Effects of estradiol and ICI 182,780 on c-fos and c-jun mRNA expressions
To evaluate the part played by the estrogen receptor in the effects of E2 on c-fos and c-jun mRNAs, experiments were next performed with the pure antiestrogen, ICI 182,780 (41) (Fig. 5Go). Surprisingly, pretreatment with ICI 182,780 alone caused an increase in c-fos and c-jun gene expressions (1.7 ± 0.3 and 1.7 ± 0.1-fold, respectively) in adipocytes but not in uteri from OVX rats (data not shown). However, when administered 30 min before E2, this antagonist partly reversed the positive effects of E2 on c-fos and c-jun expressions (-40 ± 7% and -34 ± 2%, respectively) in adipocytes. These results support the concept that, in rat adipocytes, the promoting action of estradiol on c-fos and c-jun protooncogene expressions requires, at least in part, estrogen receptors.



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  		Figure 5. Effects of the antiestrogen ICI 182,780 on E2-induction of c-fos and c-jun mRNA levels in rat adipocytes. OVX rats were pretreated with vehicle (OVX) or with ICI 182,780 (OVX + ICI) 30 min before the E2 injection (OVX + E2) (OVX + E2+ ICI). Adipocyte total RNAs were prepared, analyzed, and quantified by Northern blots as described in Materials and Methods. A, Representative Northern blot of c-fos and c-jun mRNAs. B, Densitometric analysis of c-fos and c-jun Northern blots. Results, expressed as amounts of c-fos and c-jun mRNA after correction for the ribosomal protein mRNA signal, are given in arbitrary units. Each bar is the mean ± SEM of three separate experiments. *, Significant vs. OVX (P < 0.05). **, Significant vs. E2 (P < 0.05).

 
4) Progesterone effect on estradiol-induced c-fos and c-jun mRNA expressions
We next examined the influence of the other ovarian steroid hormone, progesterone, on c-fos and c-jun inductions. Data presented in Fig. 6Go show that treatment with progesterone alone fails to affect c-fos and c-jun mRNA levels in adipocytes. In contrast, when administered 1 h before the E2 injection, progesterone completely prevented the E2 promoting effect on c-fos and c-jun mRNA levels.



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  		Figure 6. Effects of progesterone on E2-induction of c-fos and c-jun mRNA levels in rat adipocytes. OVX rats were pretreated with vehicle (OVX) or with progesterone (OVX + Pg) (5 mg/rat) 1 h before the E2 injection (OVX + E2) (OVX + E2+ Pg). Adipocyte total RNAs were prepared, analyzed and quantified by Northern blots as described in Materials and Methods. A, Representative Northern blot of c-fos and c-jun mRNAs. B, Densitometric analysis of c-fos and c-jun Northern blots. Results, expressed as amounts of c-fos and c-jun mRNA after correction for the ribosomal protein mRNA signal, are given in arbitrary units. Each bar is the mean ± SEM of three separate experiments. *, Significant vs. OVX (P < 0.05). **, Significant vs. E2 (P < 0.05).

 
5) Gender-specificity of estrogen effect
To determine whether the estradiol effects on c-fos and c-jun expressions are gender-specific, we treated CAST rats with benzoate estradiol for 2 h. As shown in Fig. 7Go, c-fos expression in epididymal adipocytes was totally unsensitive to estradiol and the same was true for c-jun (data not shown) demonstrating the female specificity of the estradiol regulated AP-1 gene expression in adipocytes.



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  		Figure 7. Effects of estradiol on c-fos and c-jun mRNA levels in adipocytes from OVX and CAST rats. Animals were treated with vehicle alone (OVX or CAST) or E2 (2 µg/rat) 2 h before be killed. Adipocyte total RNAs were extracted from parametrial and epipidymal adipocytes of female and male rats, respectively. Northern Blot analysis was performed as described in Materials and Methods. The data show the results of one representative experiment repeated three times.

 
6) Effect of estradiol on AP-1 DNA binding activity
Gel mobility shift assays were next performed to determine whether the estradiol modulation of c-fos and c-jun expressions is accompanied by a variation in the AP-1 DNA binding activity. First, the specificity of the AP-1 DNA binding was verified by competition experiments using consensus sequences of Oct-1 and mutated AP-1 (Fig. 8AGo). Data in Fig. 8BGo show that consensus AP-1 binding activity was increased (5 ± 1-fold; P < 0.01, n = 5) by estradiol treatment in adipose tissue of OVX rats. Interestingly, this increase in DNA binding activity remained similar in magnitude to that observed in uteri (5 ± 2-fold; P < 0.05, n = 3).



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  		Figure 8. Influence of estradiol on AP-1 DNA-binding activity in rat adipocytes. A, Adipocyte nuclear extracts were incubated with a radiolabeled consensus AP-1 probe in the presence of 1, 10, and 100-fold molar excesses of unlabeled AP-1 or mutated AP-1 or of 50-fold molar excess of unlabeled Oct-1 DNA competitor. B, Animals were treated with vehicle alone (OVX) or estradiol (2 µg/rat) 2 h before be killed. Nuclear extracts prepared from parametrial adipose tissue and uteri were incubated with the radiolabeled consensus AP-1 probe as described in Materials and Methods. This figure is representative of five independent experiments.

 
These results demonstrate that in OVX rat adipocyte estradiol treatment not only stimulates the expression of the two major AP-1 complex members but also the DNA binding activity of this complex.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we investigated the effects of E2 administration on the expression of two major members of the AP-1 complex in adipocytes from ovariectomized rat. Our results show that estradiol treatment positively affects both c-fos and c-jun mRNA and protein levels. This effect appears specific to E2 because progesterone treatment alone failed to affect these transcriptional factors. The findings that modulation of c-jun and c-fos mRNAs by E2 occurs rapidly, is abolished by actinomycin D and not blocked by cycloheximide suggest a direct primary response to E2. However, overexpression of c-fos and c-jun persisted 24 h after E2 treatment. This pattern differs markedly from that seen in the uterus where the rise in c-fos and c-jun expressions due to E2 is transient (18, 19). Similarly to our observations, a lower and sustained time-dependent induction of both protooncogenes was observed in the anterior pituitary gland after E2 administration (38). One possible explanation to these observations is that the later elevations of c-fos and c-jun expressions result from indirect effect of E2 on the induction of factors regulating the expression of AP-1 components themselves. Another explanation could be that E2 induces c-fos and c-jun mRNA stabilization. However, our recent experiments do not support this last hypothesis (our unpublished results).

The c-fos promoter has been extensively studied. Its upstream region contains several important regulatory sequences including an imperfect palindromic ERE (20), various Sp1 binding sites (42) and a serum response element (SRE) adjacent to an AP-1 binding site (43). In contrast, the c-jun promoter is less known and only a few regulatory elements have been identified such as AP-1 binding sites and an imperfect palindromic ERE (21). The presence of functional ERE in both promoter regions suggests that when bound to their receptors, estrogens will directly modulate transcription of these genes. However, some recent reports have suggested that transcriptional stimulation triggered by steroid receptors are mediated by half-palindromic recognition sequences and involve cooperativity between ER and adjacent promoter elements like the AP-1 or Sp1 sites (22, 23, 42). For example, transfection experiments in MCF-7 cells revealed that E2 inducibility of c-fos expression was dependent on the formation of a transcriptionally active ER/Sp1 complex and on its binding to an Sp1 site (42).

As shown in the experiments using the pure ER{alpha} antagonist ICI 182,780, the E2-induced c-fos and c-jun mRNA expressions appear at least in part mediated by ER{alpha}. Contrasting with the observations made in uteri (this study and Ref. 41) but in agreement with investigations realized on c-fos expression in neuroblastoma cells (44), treatment with ICI 182,780 alone enhanced like E2 c-fos and c-jun expressions in rat adipocytes. One mechanism that could explain this paradoxical agonistic activity of ICI 182,780 is that this antiestrogen acts as a transcriptional activator ligand of c-fos and c-jun genes via the novel ER-ß subtype (45), which was found in our laboratory to be expressed in female rat adipocytes as well (Dieudonné, M. N., unpublished results). Given further support to this hypothesis is our observation that in rat brain where the ER-ß subtype is abundantly expressed (46), ICI 182,780 also induces c-fos and c-jun expressions (data not shown).

In contrast to E2, progesterone in vivo failed to alter the expression of c-fos and c-jun in rat adipocytes. Moreover and in agreement with previous reports performed in the uterus (18), progesterone pretreatment antagonized the effects of E2 on c-fos and c-jun. One possible explanation to such an antagonism comes from transfection experiments demonstrating that progesterone blocks E2-stimulated AP-1 dependent transcription at the AP-1 site by forming a functional competitive interaction between ER{alpha} and progesterone receptor (PR) at this response element (23). In addition, PR have been reported to interact with different coactivators that are required for the E2-transcriptional activities (47) e.g. the CBP (CREB binding protein) and the steroid receptor coactivator-1 (SRC-1).

As shown by the present study, the E2 effects on c-fos and c-jun expressions are gender specific. Sex-specific estrogenic actions have been previously observed for E2-induced changes in AP-1 binding activity in rat hypothalamus (48). Although unestablished, the molecular basis of this sex specificity could be related to differences between male and female adipocytes in ER subtype expression but also in E2-regulated expression of ER coactivators such as SRC-1 as recently reported in the pituitary gland (49).

To further investigate the influence of E2 administration on rat adipose cells, we explored the possibility that the interaction between the AP-1 complex and its specific DNA element (AP-1 binding site) could be modulated in response to E2. Our experiments clearly show that the AP-1 DNA-binding activity increases significantly after E2 treatment. AP-1 DNA binding activity is mainly regulated by posttranslational modifications, such as phosphorylation of c-fos and c-jun proteins by various kinases including the mitogen-activated protein kinase (MAPK). Interestingly, in many tissues and cell types, estrogens have been recognized to activate several kinases (for review, see Ref. 50) through nongenomic mechanisms and particularly the MAPK pathway, which in turn is able to promote c-fos transcription in neuroblastoma cell lines (44). In vitro studies are currently in progress in our laboratory to establish the part played by such nongenomic effects of E2 on c-fos and c-jun expressions in rat adipocytes and the role played by the MAP kinase signaling pathway in these effects. Our preliminary results indicate a rapid stimulation of MAPK in adipocytes after exposure to E2 (our unpublished results). Thus, beside stimulating c-fos and c-jun expressions, E2 increases even more the AP-1 DNA binding activity through a probable enhancement of the AP-1 complex phosphorylated status.

In conclusion, the present results obtained in vivo with physiological blood estrogen levels provide information at molecular level about the way of action of estrogens in fat cells. Indeed, E2, by stimulating both the expression and the activity of AP-1 members, could regulate AP-1 dependent gene expression. However, the exact target genes modulated by the AP-1 complex remain to be identified in adipocytes. AP-1 binding elements are very pleiotropic and are probably involved in different aspects of adipocyte function such as the induction of metabolic genes. Among these targets resides the adipo-specific aP2 gene (26) whose translational product, aP2, the major lipid binding protein in adipocytes, could play an important role in adipose tissue insulin resistance (51). CHOP (C/EBP homologous protein) is another candidat gene possessing an AP-1 site in its promoter (52). This factor by sequestring C/EBP{alpha} is a dominant inhibitor of C/EBP{alpha}, a master regulator of energy homeostasis (53), which transactivates several adipospecific genes like Glut-4, SCD-1 (stearoyl CoA desaturase) (54). Thus, CHOP, by inhibiting C/EBP{alpha} transcriptional activity, could alter the activation of adipo-specific genes. In addition, a cross-talk between CHOP and AP-1 factors has been described because CHOP directly interacts with Fos and Jun proteins and then activates AP-1 target genes (55). Finally, nitric oxide synthase II, another AP-1 induced gene (56) is expressed in rat and human adipocytes (57, 58). In these cells, nitric oxide modulates lipolysis depending on its redox state (59).

Increased fat mass caused by OVX is associated with reduced lipolysis (14). Progesterone administration was found to increase fat accumulation (60). On the opposite, E2 treatment of intact and OVX rats reduces fat deposits and fat cell size (4, 11, 12, 13), a phenomenom which could be linked to the promoting effects of E2 on lipolysis (61, 62, 63). Thus, some at least of the AP-1 target genes proposed above could intervene in the effects of E2 and could help to explain the antagonism between E2 and progesterone on fat cell metabolism.


   Footnotes
 
1 Supported by the Université of Paris V and the Comité des Yvelines de la Ligue Contre le Cancer. Back

2 Sponsored by a doctoral fellowship from the Comité des Yvelines de la Ligue Contre le Cancer. Back

Received February 23, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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