Service de Biochimie et de Biologie Moléculaire, UPRES EA 2493, Faculté Paris-Ile de France-Ouest, Université Versailles St Quentin, Centre Hospitalier de Poissy, 78303 Poissy Cedex, France
Submitted 7 July 2003 ; accepted in final form 5 November 2003
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ABSTRACT |
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estrogen receptor; human adipose tissue; primary culture cells; realtime polymerase chain reaction
In general, estrogens are assumed to mediate their effects through two specific nuclear receptor isoforms called estrogen receptor (ER)- and ER-
. More recently, five ER-
mRNA isoforms have been described in humans and designated ER-
1 to ER-
5 (23). These variants differ in their COOH-terminal sequences. The apparent full-length mRNA is called
1. The ER subtypes have different tissue distribution (22) and exert, as homodimers or heterodimers, transcriptional regulation of different target genes through their binding to estrogen response elements (ERE) (10). However, there is now strong evidence that, in addition to mediating their effects through the ERE, the ER can interact with other DNA-bound transcription factors to influence gene transcription activation. For example, the ER cooperates with proteins Fos and Jun of the activating protein-1 complex (AP-1) to confer estrogen responsiveness to various promoters (47, 50). It also seems likely that ER mediate some estrogen biological effects through SP-1 recognition sites in a number of genes (36). ER-
also has been reported to inhibit the transcriptional activity of ER-
, possibly through ER-
/ER-
heterodimer formation (18), thus suggesting that the biological response to estrogens is dependent on the relative ratios of ER-
and ER-
levels in cells. Moreover, it is becoming clear that both ER subtypes are responsible for different biological functions, as indicated by their tissuespecific expression patterns and the different features characterizing ER-
and ER-
in knockout mice (19, 30).
In human adipose tissue, ER expression is still not fully clarified. Indeed, initial studies reported the presence of ER- and ER-
in mature adipocytes (11, 33), but the presence of both isoforms in human preadipocytes is controversial (21, 32). This led us to investigate ER-
and ER-
(
1 subtype) expressions, using the real-time RT-PCR and Western blot techniques, and their regulation by estrogens in preadipocytes and adipocytes from different fat localizations in men and women. Moreover, we have tested the functionality of these ER by measuring their capacity to interact with various DNA binding sites.
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MATERIALS AND METHODS |
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The adipose tissue samples (50-100 g) obtained from subcutaneous (inguinal) and intra-abdominal (omental) fat depots were collected in saline (150 mM NaCl) and immediately transferred to the laboratory. After blood vessels and connective tissue were removed, adipose tissue was rinsed in saline containing antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin). Mature adipocytes and preadipocytes were obtained by collagenase digestion as previously described (12). The floating adipocytes were washed three times with DMEM/F-12 containing 1% bovine serum albumin (BSA), 0.1 mg/ml streptomycin, and 100 U/ml penicillin before being kept in culture. Aliquots of the adipocyte suspension (1-2 x 106 cells/ml) were rapidly dispensed and incubated for 24 h at 37°C under 5% CO2-95% air atmosphere in DMEM/F-12 containing 1.5% BSA, 0.1 mg/ml streptomycin, and 100 U/ml penicillin.
Preadipocytes, obtained essentially from women donors, were plated in DMEM/F-12 supplemented with 0.1 mg/ml streptomycin, 100 U/ml penicillin, and 10% fetal calf serum (FCS) and were maintained at 37°C under 5% CO2-95% air atmosphere. After being plated at a density of 2-3 x 104 cells/cm2, cells were extensively washed and maintained in primary culture as follows: 1) for cell growth experiments, cells were maintained in DMEM/F-12 supplemented with 10% FCS until confluence (3-4 days after plating); and 2) for differentiation studies, preadipocytes were refed immediately after being plated in DMEM/F-12 supplemented with 80 nM insulin, 10 µg/ml transferrin, 0.2 nM triiodothyronine, 100 nM hydrocortisol, antibiotics (0.1 mg/ml streptomycin and 100 U/ml penicillin), and, for the first 3 days, 0.2 mM IBMX and 1 µg/ml troglitazone as described in Ref. 48. Fully differentiated cells were obtained after 10-11 days of culture.
Isolation of RNA. At defined time intervals the cultured cells were harvested in guanidinium isothiocyanate buffer. Total RNA was isolated from the cell material according to the method of Chomczynski and Sacchi (8). RNA recovery and quality were checked by measuring the optical density ratio (260/280 nm) and by electrophoresis under denaturing conditions on 2% agarose gel.
Real-time RT-PCR. Total RNA (0.5 µg) was reverse transcribed as previously described (25). Quantitative PCR was performed by using a LightCycler instrument (Roche Diagnostics) with QuantiTect SYBR Green PCR Master Mix (Qiagen). Primer sets used are indicated in Table 1. cDNA calibrators were prepared by PCR amplification run to saturation (35 cycles) with the appropriate primers. The resulting cDNAs were purified by using the QIAquick PCR purification kit (Qiagen). The samples showed a unique band in agarose electrophoresis. Numbers of cDNA copies were calculated from the absorbance at 260 nm. Calibrators were defined to contain arbitrary units of ER and glycerol-3-phosphate dehydrogenase (GAPDH) mRNA, and all calculated ER concentrations are relative to GAPDH concentrations. In accordance with Gorzelniak et al. (17), the choice of GAPDH as housekeeping gene was based on the observation that GAPDH mRNA expression is not sensitive to estrogens in human adipose cells (11,843 ± 1,328 and 15,257 ± 3,282 copies of GAPDH cDNA in control and cells exposed to 100 nM E2, respectively). Separate calibration curves for human ER and GAPDH were constructed from serial dilutions from 108 copies to 100 copies of cDNA calibrators. Calibration curves were log-linear over the quantification range with a correlation coefficient (r2) 0.99 and slopes ranging from -3.5 to -3.8. The intra-assay variability of duplicate crossing point (Cp) values never exceeded 0.2 cycles, and the interassay variation (CV value) ranged from 1 to 5% CV values for the three or four runs of each transcript.
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Real-time PCR was performed in a total reaction volume of 20 µl per capillary for the LightCycler format. Each provided cDNA preparation (50 ng/µl) was diluted 1:10 in water. The reaction buffer contained 10 µl of 2x QuantiTect SYBR Green PCR Master Mix (including HotStar Taq DNA polymerase, reaction buffer, deoxynucleotide triphosphate mixture, and SYBR Green I), 0.5 µM of each primer, and 4 µl of diluted cDNA or calibrator. The cycling conditions were as follows: initial denaturation at 94°C for 15 min, followed by 50 cycles of denaturation at 94°C for 15 s, annealing at 56°C for human ER-, 51°C for ER-
(primer set 1), or 55°C for GAPDH for 20 s, and finally extension at 72°C for 10 s. The temperature transition rate was set at 20°C/s.
After PCR, a melting curve was constructed by increasing the temperature from 65 to 95°C with a transition rate of 0.1°C/s to verify the specificity of the desired PCR products and the absence of primer dimers. To validate the melting curve results, representative samples of PCR products were separated by 2% agarose gel electrophoresis.
The second-derivative maximum method was used to automatically determine the Cp for the individual samples. The LightCycler software (version 3.1) constructed the calibration curve by plotting the Cp vs. the logarithm of the number of copies for each calibrator.
Cellular extracts. Preadipose cells were scraped on ice into lysis buffer containing 10 mM HEPES, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.5 mM dithiothreitol (DTT), 0.6% Nonidet P-40, 1 mM sodium orthovanadate, 20 mM -glycerophosphate, 10 mM NaF, 0.57 mM PMSF, 5 µg/ml leupeptin, and 5 µg/ml aprotinin, incubated on ice for 5 min, and centrifuged at 2,500 g at 4°C for 5 min. Crude nuclei were resuspended in cold buffer containing 10 mM HEPES, pH 7.9, 0.42 M NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.5 mM MgCl2, 25% (vol/vol) glycerol, 0.5 mM DTT, 0.6% Nonidet P-40, 1 mM sodium orthovanadate, 20 mM
-glycerophosphate, 10 mM NaF, 0.57 mM PMSF, 5 µg/ml leupeptin, and 5 µg/ml aprotinin. The suspension was vigorously shaken at 4°C for 30 min, followed by centrifugation (20,000 g at 4°C for 20 min). The supernatant containing the nuclear extract was stored in aliquots at -80°C until used for EMSA or diluted (vol/vol) in Laemmli buffer for Western blot analysis.
Isolated adipocytes were homogenized (vol/vol) in buffer containing 10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.05 mM DTT, 1 mM sodium orthovanadate, 20 mM -glycerophosphate, 10 mM NaF, 0.57 mM PMSF, 5 µg/ml leupeptin, and 5 µg/ml aprotinin and were centrifuged at 5,000 g at 4°C for 10 min. The resulting supernatant was centrifuged at 100,000 g at 4°C for 30 min to obtain the membranous fractions. Crude nuclei were resuspended in cold buffer containing 20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 0.42 M NaCl, 1 mM DTT, 0.2 mM EDTA, 25% glycerol, 1 mM sodium orthovanadate, 20 mM
-glycerophosphate, 10 mM NaF, 0.57 mM PMSF, 5 µg/ml leupeptin, and 5 µg/ml aprotinin. This suspension was then treated as described above to obtain the nuclear extract.
Western blotting. Equal amounts of nuclear or membranous (100 µg) proteins were subjected to SDS-PAGE (10% acrylamide). Proteins were transferred to polyvinylidene difluoride membranes. After blocking in buffer A (20 mM Tris·HCl, 137 mM NaCl, and 0.1% Tween 20) containing 2.5% gelatin for 2 h at room temperature, membranes were incubated overnight at room temperature with rabbit polyclonal anti-ER- or anti-IGF-I receptor (IGF-IR) antibody (1: 300 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). Blots were then extensively washed with buffer A, incubated with the secondary antiserum (horseradish peroxidase-labeled anti-goat IgG, 1:10,000 dilution) for 1 h at room temperature, and washed. Finally, an enhanced chemiluminescence kit from Pierce (Interchim) was used for signal detection. Control experiments with various protein amounts (20-300 µg) were performed to ensure that the densitometric signal intensity was proportional to the loaded amount of protein. Specificity of the immunoreactive ER proteins was verified by loss of the immunoreactivity in samples exposed to the antiserum neutralized by the corresponding specific peptide. To test for the purity of the adipocyte membrane fraction, blots were stripped and reprobed with an antiserum specific for a membranous component, the IGF-I receptor protein.
EMSA. Protein-DNA complexes were formed by incubating 2-10 µg of nuclear extracts in 10 µl of binding cocktail (10 mM HEPES, 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 50 mM NaCl, 4 mM spermidine, 2 mM DTT, 100 µg/ml albumin, and 35% glycerol, pH 8) in the presence of 2.5 µg of poly(dI-dC) for 15 min at 4°C. Next, 100,000-200,000 cpm of 32P-labeled double-stranded oligonucleotides containing a binding site for AP-1 (5'-CGC TTG ATG AGT CAG CCG GAA-3'), ERE (5'-GTC AGG TCA CAG TGA CCT GAT-3'), or SP-1 (5'-ATT CGA TCG GGG CGG GGC CAG C-3') were added, and the incubations were further extended for 15 min at room temperature. The resulting DNA-protein complexes were separated from the unbound probes by electrophoresis on a native 6% polyacrylamide gel in 0.5x TBE (Tris-borate-EDTA buffer). Gels were then dried and subjected to autoradiography. In competition experiments, 1-, 10-, and 100-fold molar excesses of unlabeled AP-1, ERE, or SP-1 or double-stranded oligonucleotides were included in the binding reaction mixture. The 32P-labeled double-stranded oligonucleotides were prepared by using [-32P]ATP (3,000 Ci/mmol) and T4 polynucleotide kinase. Protein concentrations were measured according to Bradford (6) with BSA as standard.
Statistical analysis. All values are expressed as means ± SE of three to four different experiments, and statistical analysis was performed by using paired or unpaired Student's t-test.
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RESULTS |
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Regulation of ER- and ER-
mRNA expression by estrogens in human adipocytes. Because estrogens were shown to influence the body fat distribution (5) and also to modulate in vivo and in vitro expression of their own receptors in various cell types (3, 16, 29, 41, 44, 46), we next investigated the direct influence of E2, in vitro, on ER-
and ER-
mRNA expression in adipocytes from subcutaneous and intra-abdominal origins. Mature adipocytes were incubated for 24 h with or without 100 nM of E2. This relatively high concentration of steroid hormone was chosen because in the culture medium, the free hormone concentration was 10 times lower because of the presence of BSA or FCS. Under these conditions, whereas in the absence of E2 (control) ER-
and ER-
mRNA levels remained stable throughout the incubation period, exposure to E2 resulted in an upregulation of ER-
mRNA expression in both subcutaneous (x1.8 ± 0.075) and intra-abdominal (x2.3 ± 0.46) adipocytes in men but only in subcutaneous adipocytes (x3.9 ± 1.06) in women (Fig. 2A). In contrast, E2 failed to alter adipocyte ER-
mRNA expression in men, whatever their anatomic origin. In women, however, ER-
mRNA expression was upregulated (4-fold) by E2, but here again only in subcutaneous adipocytes (Fig. 2B).
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Comparison of ER- and ER-
expression in human preadipocytes and adipocytes. As shown in Fig. 3A, ER-
mRNA expression in preadipocytes was weak and about 40 times lower than in adipocytes from women. The same picture emerged when the study was performed in adipose tissue from men (data not shown). ER-
protein was also reduced, although to a lesser extent (
50% less), in preadipocytes than in adipocytes (Fig. 3B).
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As shown in Fig. 3C, ER--immunoreactive protein was also present in human adipocyte membranes. The latter finding is in accordance with previous work in rat mature adipocytes (13).
In contrast with these observations and despite our efforts using different primer sets and various RT-PCR conditions, we were unable to detect any ER- mRNA signal in preadipocytes, leading us to conclude that human preadipocytes do not express the ER-
subtype.
Regulation of ER- expression by estrogens in human preadipocytes. Under the same experimental conditions as those used for adipocytes, we found that E2 had absolutely no influence on ER mRNA and protein expressions in human confluent preadipocytes (Fig. 4).
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DNA binding activity in response to E2 in human adipose tissue. To test the functionality of ER, confluent or differentiated preadipocytes were exposed for 1 or 24 h to 100 nM E2 and their nuclear extracts were then probed for their ability to interact in vitro with various consensus oligonucleotide sequences implicated in E2 responses (AP-1, SP-1, and ERE). As shown in Fig. 5, after E2 exposure there was a significant increase in AP-1 (1.9 ± 0.28), SP-1 (1.96 ± 0.11), and ERE (1.7 ± 0.05) DNA binding activity in differentiated preadipocytes but not in confluent undifferentiated preadipocytes, which remained unresponsive to E2. Finally, these various DNA-protein complexes completely disappeared when these experiments were repeated in the presence of a 100-fold excess of specific unlabeled oligonucleotides used as probes (Fig. 5) or remained unaltered when a 100-fold excess of nonspecific unlabeled oligonucleotide was added (data not shown), suggesting that the E2-induced DNA-protein binding complexes observed were specific.
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DISCUSSION |
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In mature adipocytes both ER- and ER-
subtypes are expressed, but their levels are not significantly different between subcutaneous and intra-abdominal adipose tissue localizations. No gender difference in ER-
expression was detected. However, ER-
levels are higher in women compared with men. These results suggest that differences in adipose tissue distribution between genders are not solely the consequence of intrinsic regional differences in ER levels. Nevertheless, it cannot be excluded that the molecular basis of the sexual dimorphism of body fat distribution is related to a different pattern of expression for nuclear receptor cofactors in adipose tissue between men and women. In fact, ER regulate the transcriptional activity of specific genes by recruiting an array of coactivator proteins, including steroid receptor coactivator 1, whose expression, in anterior pituitary at least, was reported to be sex specific (28).
Furthermore, gender-specific differences in local estrogen production by adipose tissue could also contribute to the sex-related specificities of body fat distribution. Expression and regulation of P-450 aromatase, the enzyme responsible for the conversion of androgens to estrogens, have been indeed reported to be different in men and women (26).
Our knowledge of the ER- distribution among various human tissues is rather limited, and the precise role played by this receptor remains unclear. In human adipocytes, two studies (2, 32) reported the presence of ER-
and ER-
mRNA and protein, but quantitative data were not available. Our present experiments confirm these findings but also reveal that ER-
mRNA levels in human adipocytes are extremely low compared with ER-
mRNA levels, thus suggesting a minor role, if any, of the ER-
subtype in these cells.
Moreover, our finding that immunoreactive ER- protein is present not only in human adipocyte nuclear extract but also in the membrane fraction suggests that E2 is able to initiate rapid nongenomic effects in human adipocytes as it does in rat fat cells (13).
Studies performed essentially in female rats have shown that ER regulation by estrogens is tissue specific. For example, estrogens in vivo upregulate ER in liver but downregulate ER in uterus, kidney, and cerebral cortex (29). In the present study we observed that estrogens in vitro positively regulated ER- and ER-
mRNA expression in subcutaneous adipocytes from women, resulting in a stable ratio of ER-
mRNA to ER-
mRNA. In subcutaneous adipocytes from men, however, estrogens upregulated the ER-
subtype only and, hence, the ER-
/ER-
ratio was increased. This change could account for the difference in adipose tissue E2 responsiveness between men and women (7, 25). In preliminary experiments performed in 3T3-L1 preadipose cells transiently transfected with the human ER-
expression vector HEGO, we have observed an induction of ER-
mRNA (unpublished data). This finding, together with the recent identification of a natural ERE sequence in the human ER-
gene promoter (24), suggests that the estrogen-induced ER-
subtype upregulation observed only in women is mediated by the ER-
subtype and by the consequence that the ER-
is an estrogen-responsive gene.
In preadipocytes in women, ER- mRNA and protein are also present, but at a much lower level than in mature adipocytes, suggesting that an increase in their expression occurs during the course of adipogenesis. However, using different sets of primers, we were unable to detect any ER-
subtype in either human confluent or differentiated preadipocytes. The latter finding, which is in agreement with two previous studies (11, 21), contrasts, however, with one report showing the presence of different ER-
subtypes (
1,
2,
4,
5), although at very low levels, in human preadipocytes (32). One possible explanation for these discrepancies could be differences between our culture conditions and those used by these authors, who furthermore observed a decrease in ER-
expression during the differentiation period, which is just opposite to our present data. Moreover, contrasting with our observation in adipocytes, we failed to find any effect of E2 on ER-
expression (mRNA and protein) in preadipocytes.
It is well established that ER- and ER-
activate gene transcription on classic ERE, but these receptors also can have opposite effects on AP-1- and SP-1-responsive elements. Indeed, in the presence of E2, ER-
works as a transcription activator, and ER-
as an inhibitor or silencer, at the AP-1 and SP-1 sites (31, 34). These findings have led us to test the capacity of ER to bind to ERE, AP-1, and SP-1 DNA sequences in nuclear extracts from confluent and differentiated preadipocytes (which express principally the ER-
subtype) after in vitro exposure to E2. Under these conditions, we observed that E2 increased the binding to AP-1, SP-1, and ERE of nuclear proteins from differentiated, but not from confluent, preadipose cells. These results strongly suggest that in undifferentiated preadipocytes, the ER-
are not functional or that their expression is too low to observe any DNA binding activity. Consistent with these hypotheses are 1) the demonstration of a direct effect of estrogens on lipoprotein lipase and creatine kinase gene expression in transfected 3T3-L1 adipose cells overexpressing ER-
(20, 45) and 2) our observation that E2 is unable to exert any regulation on ER-
expression in human confluent preadipocytes. Arguing against these hypotheses, however, is the proliferative effect exerted by estrogens in human confluent preadipocytes (1, 40). One possible explanation of the latter effect could be the rapid nongenomic and membranous ER-mediated activation of the MAPK pathway involved in cell proliferation (42), which was recently observed in a variety of cells including human breast cancer cells, endometrial cells, ovary, bone (15, 27, 43), and adipocytes (13).
With nuclear extracts from differentiated preadipocytes, E2 increases the binding to AP-1 and SP-1 as well as to the classic ERE oligonucleotides. This suggests that in human adipose tissue, estrogen responses could be mediated by AP-1 and SP-1 DNA sequences. Indeed, an AP-1-like sequence-dependent inhibition of lipoprotein lipase gene transcription by estrogens was described in murine adipocytes (20), and we have recently observed that in 3T3-L1 cells transfected with the human ER- expression vector, estrogens increase by twofold the transcriptional activity of the leptin gene, which includes several SP-1 sequences in the promoter proximal region (unpublished data).
The functional role of the ER-1 subtype, present only in mature adipocytes, is still unclear. ER-
-mediated antiproliferative and prodifferentiating effects of estrogens were previously reported in various cell types (35). If the same effects apply to human adipocytes, any estrogen response via ER-
would be limited by the presence of ER-
. Further studies are currently in progress to firmly establish the role each isoform of ER plays in adipocyte cell function and in adipogenesis in humans.
Signal cross talk between ER- or ER-
and other transcriptional factors has been recently described. For example, ER signal transduction pathway is able to interact with the peroxisome proliferator-activated receptors (PPAR-
), which are key nuclear receptors controlling adipogenesis (49). If such a mechanism also occurs in human adipose cells, the gender-specific differences in the ER-
/ER-
ratio would induce variations in the PPAR-
biological activity in these cells. This could help us to understand how estrogens modulate sexspecific adipose tissue development and distribution in humans.
In summary, the present results demonstrate that primary cultured human preadipocytes express only ER-, whereas mature adipocytes express both the ER-
and ER-
subtypes. Moreover, the predominant expression and functionality of ER-
in human adipocytes suggest that this receptor subtype plays a major role in estrogen-dependent modulation of adipospecific genes in these cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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