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Endocrinology, doi:10.1210/en.2005-1655
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Endocrinology Vol. 147, No. 9 4496-4504
Copyright © 2006 by The Endocrine Society

17ß-Estradiol Stimulates Resistin Gene Expression in 3T3-L1 Adipocytes via the Estrogen Receptor, Extracellularly Regulated Kinase, and CCAAT/Enhancer Binding Protein-{alpha} Pathways

Yen-Hang Chen1, Meng-Jung Lee1, Hsin-Huei Chang, Pei-Fang Hung and Yung-Hsi Kao

Department of Life Science, College of Science, National Central University, Chung-Li City, Taoyuan, Taiwan 32054

Address all correspondence and requests for reprints to: Dr. Yung-Hsi Kao, Department of Life Science, College of Science, National Central University, Chung-Li City, Taoyuan, Taiwan 32054. E-mail: ykao{at}cc.ncu.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Resistin is known as an adipocyte-specific secretory hormone that can cause insulin resistance and decrease adipocyte differentiation. It can be regulated by sexual hormones, but the mechanism of estrogen’s actions is still not clear. Using 3T3-L1 adipocytes, we found that 17ß-estradiol (E2) up-regulated resistin mRNA expression in a dose- and time-dependent manner. The concentration of E2 that increased resistin mRNA levels by 100–250% was approximately 1 nM for a range of 1–24 h of treatment. Treatment with either actinomycin D or cycloheximide prevented E2-stimulated resistin mRNA expression, suggesting that the effect of E2 requires new mRNA and protein synthesis. Although E2 was shown to increase activities of the estrogen receptor (ER) and MAPK kinase 1 and the association of nuclear ER{alpha} and CCAAT/enhancer binding protein-{alpha} with the resistin gene promoter, signaling was demonstrated to be blocked by pretreatment with either ICI182780 or PD98059. Neither SB203580 nor LY294002 changed the E2-increased levels of resistin mRNA, but they respectively inhibited E2-stimulated phosphorylation of p38 MAPK and Akt. These results imply the ER{alpha}, ERK, and CCAAT/enhancer binding protein-{alpha} are necessary for the E2 stimulation of transcription from the resistin promoter. Moreover, PD98059, but not SB203580 or LY294002, antagonized E2-increased resistin protein release. These data suggest that E2 likely modifies the distribution of the resistin protein between the intracellular and extracellular compartments via an ERK-dependent pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RESISTIN IS A cysteine-rich hormone that was first isolated from adipose tissues and found to link obesity to type II diabetes in rodents (1). In particular, administration of exogenous resistin into normal mice causes glucose intolerance and hyperinsulinemia, whereas an antiresistin antibody decreases blood glucose and improves insulin sensitivity in obese mice (1). In addition, resistin suppresses insulin-stimulated glucose uptake in adipocytes (1) and muscle cells (2). Moreover, transgenic mice overexpressing a dominant-negative form of resistin showed increased adipogenesis and improved insulin sensitivity (3). However, the involvement of resistin in obesity and insulin resistance in humans is still controversial. Some studies have shown no relationship of resistin gene expression with body weight or insulin resistance (4). Others found that resistin mRNA expression in adipose tissues of obese humans is higher than that in normal subjects (5), that a single-nucleotide polymorphism in the resistin gene promoter was associated with obesity (6) and diabetes (7), and that the plasma resistin levels were elevated in patients with obesity (8) and type II diabetes (9). One possible explanation for these disparate findings is the presence of the various isoforms (10, 11) or dimers (12) of resistin. This contention may also explain the functional diversity of resistin in different species or systems. For example, resistin regulates fasted blood glucose levels, lipid metabolism, catecholamine release, inflammation, hepatic insulin resistance, and proliferation and activation of endothelial cells and smooth muscle cells (4, 13, 14, 15, 16). The mechanisms of actions of resistin can stimulate muscle cell proliferation through activation of ERK1/2 and phosphatidylinositol 3- kinase (PI3K) (16) as well as inhibit insulin signaling of 3T3-L1 adipocytes through the induction of the gene expression of suppressor of cytokine signaling 3 (17).

Despite the importance of resistin, relatively little is known about the control of production of resistin by sexual hormones (18). Although sexual differences of resistin levels were found in mice (male < female) (19), rats (male > female) (20), and humans (male < female) (21), suggesting the possible involvement of sex steroids in regulating resistin production, the results did not demonstrate a direct effect of estrogens on resistin gene expression or protein secretion by adipocytes. In a murine study, an ovariectomy increased resistin mRNA abundance of adipose tissues without changing plasma resistin levels, whereas estrogen replacement reduced resistin mRNA (19). In contrast, an ovariectomy did not change any level of resistin mRNA of rats (20). Whether estrogens affect resistin gene expression and protein secretion is still controversial based on those studies. The fact that the signal element responsible for transducing the action of estrogens on resistin gene expression and secretion has not been identified has caused much controversy surrounding the possible role of estrogens in regulating resistin expression. Additional in vitro cell lines that are free from interfering influences present in whole animals and that allow precise estrogen concentrations to be achieved should be excellent systems for studying the signal element(s) through which estrogens regulate resistin level.

In this study, we used 3T3-L1 adipocytes to examine the influence and the signaling of 17ß-estradiol (E2) on resistin gene expression and protein secretion. We investigated whether E2-regulated resistin gene expression and protein secretion are dependent on the CCAAT/enhancer binding protein (C/EBP), MAPK, and/or PI3K pathways. C/EBP{alpha}, MAPK, and PI3K were chosen because they have been reported to be essential signal transducers of E2 in the regulation of other genes in 3T3-L1 adipocytes (22, 23), and because C/EBP{alpha} has been reported to stimulate resistin expression (24, 25, 26).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemical reagents
All materials (e.g. E2, estriol, estrone, and so forth) were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated. DMEM, penicillin-streptomycin, fetal bovine serum (FBS), trypsin, agarose, the 1-kb plus DNA ladder marker, and the protein marker were purchased from GibcoBRL (New York, NY). Except for the resistin antibody, which was obtained from Linco Research (St. Charles, MO), all other antibodies [phospho-ERKs, ERK1, ERK2, estrogen receptor (ER), C/EBP{alpha}, goat anti-guinea pig IgG-horseradish peroxidase, etc.) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The 3'-RACE system, Trizol, and Taq polymerase were purchased from Invitrogen Life Science Technologies (Carlsbad, CA).

Cell culture
3T3-L1 adipocytes (American Type Culture Collection, Manassas, VA) were obtained according to a published method (27), in which 2-d postconfluent 3T3-L1 preadipocytes (3 x 106 cells in a 10-cm plate) were treated with DMEM containing a final concentration of 10 µM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 10% FBS for 48 h. The medium was then changed to DMEM containing 10% FBS for an additional 6–10 d. With this protocol, greater than 90% adipocyte differentiation was achieved, as indicated by phenotypical appearance and triglyceride accumulation (27). Differentiated adipocytes expressed 3.4-fold more resistin mRNA than did preadipocytes or differentiating preadipocytes.

For all experiments, adipocytes were serum-starved for 12 h in DMEM containing 0.1% (fatty acid free) BSA and then, unless noted otherwise, incubated with or without hormones at various concentrations for the indicated time periods. Estrogens (E2, estriol, and estrone), dexamethasone, diethylstilbestrol, and genistein were dissolved in 0.1% ethanol and sterile medium for cell treatment. Actinomycin D (Acti-D; 5 µg/ml), cycloheximide (5 µg/ml), PD98059 [a MAPK kinase 1 (MEK1) inhibitor (28); 50 µM], SB203580 [a p38 MAPK inhibitor (29); 10 µM], LY294002 [a PI3K inhibitor (30); 50 µM], and ICI182780 [an ER inhibitor (31); 1 µM] were used to inhibit the transcriptional, translational, MEK1, p38 MAPK, PI3K, and ER activities, respectively (27, 32). In the experiments, serum-starved 3T3-L1 adipocytes were pretreated with or without either Acti-D for 30 min or other inhibitors for 90 min. Then, adipocytes were stimulated with or without E2 (1 nM) for the indicated time period. After treatment, resistin mRNA and protein levels were measured. Despite the high dose of some inhibitors used in the study, no adverse effects on cell viability of adipocytes for 24 h were noted (27).

ELISA for resistin mRNA and extracellular resistin protein
Resistin mRNA levels were measured using a commercial PCR ELISA kit with digoxigenin labeling and detection (Roche Applied Science, Mannheim, Germany) (27). The forward and reverse primers were 5'-GTACCCACGGGATGAAGAACC-3' and 5'-GCAGAGCCACAGGAGCAG-3' for mouse resistin (accession no. AF323080) and 5'-CCAGGGTGTGATGGTGGGAATG-3' and 5'-CGCACGATTTCCCTCTCAGCTG-3' for actin (accession no. X03672), respectively. Sample resistin mRNA levels were determined by relation to a standard curve of resistin cDNAs, ranging from 3–200 ng/well (OD405nm = 0.1141 + 0.0031 x ng DNA/well; r2 = 0.998). An almost-linear range in the number of PCR amplifications for resistin was observed between 20 and 40 cycles when compared with the ß-actin standard. Thus, 30 cycles of PCR amplification were subsequently used for all experiments. After normalization to ß-actin mRNA, resistin levels were expressed as a percentage of the control. To analyze the secreted resistin protein, a homologous ELISA procedure (27) was used. The interassay and intraassay coefficients of variation in the ELISA were 7–9 and 3–4%, respectively. The reproducible results were obtained in the range of resistin of from 5–80 ng per well (OD405nm = 0.1269 + 0.0044 x ng/well; r2 = 0.979).

Immunoprecipitation and Western blot analysis
ER{alpha} and C/EBP{alpha} were immunoprecipitated according to the method described by Chen et al. (27). After experimental treatment, adipocytes were washed twice in PBS and then lysed in 1 ml buffer containing 20 mM Tris-HCl (pH 7.6), 1 mM EDTA, 1 mM Na3VO4, 0.2% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride. Lysate was agitated for 15 min at 4 C and then centrifuged for 10 min to collect the supernatant. Nuclear protein was gained by swelling cells on ice for 10 min in a buffer containing 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and 0.5% Nonidet P-40. The nuclear lysate was centrifuged at 500 x g, and the nuclear pellet was resuspended in lysis buffer. The protein content of the lysates was determined in duplicate by the dye-binding method (33) using a Bio-Rad (Richmond, CA) microplate reader and BSA (Sigma) as a standard. An aliquot of the supernatant (1 mg protein) was preincubated for 1 h at 4 C with either rabbit polyclonal ER{alpha} or C/EBP{alpha} antibody or with preimmunized normal rabbit serum (NRS) as the control. The mixture was incubated with 20 µl protein A-agarose (Santa Cruz Biotechnology) overnight at 4 C. Total amounts of ER{alpha} and C/EBP{alpha} in the immunoprecipitates were measured by Western blot analysis with ER{alpha} and C/EBP{alpha} antibodies, respectively. The amounts of phospho-ERK1/2 proteins indicative of ER{alpha} activation (22) in the immunoprecipitates were measured by Western blot analysis using a phospho-ERK1/2 antibody. The Western blot method for analyzing phospho-ERK1/2 and other proteins was performed on supernatant fractions of adipocytes (50 µg of protein) that were separated by 12.5% SDS-PAGE with a loading buffer [100 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.2% bromophenol blue, and 10% ß-mercaptoethanol) and then blotted onto a membrane (27). The immunoblots were analyzed with the primary (~0.2 µg/ml) and secondary (~0.2 µg/ml) antibodies. After normalization to ß-actin protein, levels of the intracellular resistin protein and kinases were expressed as a percentage of the control, unless noted otherwise.

Chromatin immunoprecipitation (ChIP) assays
The ChIP method was adapted from Hartman et al. (24) to analyze the association of ER{alpha} and C/EBP{alpha} with the resistin gene promoter. After adipocytes were pretreated with either ICI182780 or PD98059 for 90 min and then stimulated with or without 1 nM E2 for the indicated time periods, they were collected by washing twice with PBS and cross-linking with 1% formaldehyde in PBS at 37 C for 10 min. Cells were then rinsed twice with ice-cold PBS, centrifuged for 4 min at 700 x g, and resuspended in lysis buffer [50 mM Tris-HCl (pH 8.1), 1% SDS, and 5 mM EDTA). After a 20-min incubation on ice, samples were sonicated with 15-sec pulses three times on ice. The lysates were centrifuged at 14,000 x g for 10 min, and then the collected supernatant was diluted in buffer I (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH 8.1) with protease inhibitors (Roche Molecular Biochemicals). Samples were precleared with 2 µg sheared salmon sperm DNA and 45 µl protein A-agarose beads (Santa Cruz Biotechnology) for 2 h. They were then immunoprecipitated with either C/EBP{alpha} or ER{alpha} antibodies or with NRS. After incubation overnight, samples were then incubated with 45 µl protein A-agarose beads for 1 h followed by 10-min sequential washes in buffer II (20 mM Tris-HCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl), buffer III (20 mM Tris-HCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl), buffer IV (10 mM Tris-HCl, 0.25% M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA), and Tris-EDTA buffer. Precipitates were then extracted by incubating with elution buffer (1% SDS, 0.1 M NaHCO3) at 65 C for 6 h or overnight. DNA fragments were purified using a Geneaid PCR purification kit. An aliquot of 2–10 µl of purified sample was used in the 30 cycles of PCR. According to the method by Hartman et al. (24), primers surrounding the resistin transcription start site had sequences of 5'-GTCTTGGCTCCTAGCCTTGC-3' and 5'-GTTGACTTCTGGCCCATCC-3'.

Statistical analysis
Data are expressed as the mean ± SE. Unpaired Student’s t test was used to examine differences between the control and E2-treated groups. One-way ANOVA followed by the Student-Newman-Keuls multiple-range test were used to examine differences among multiple groups. Differences were considered significant at P < 0.05. Statistics were performed using SigmaStat (Jandel Scientific, Palo Alto, CA) and data that were transformed with the logarithm.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of E2 on resistin mRNA expression
E2 increased the steady-state levels of resistin mRNA in a concentration-dependent (Fig. 1AGo) and time-dependent (Fig. 1BGo) manner. The activation concentration of E2 to increase 100–250% of resistin mRNA levels was approximately 1 nM after 1–24 h of treatment. The possibility that the E2-induced stimulation in resistin mRNA expression resulted from the alteration in resistin mRNA stability was also examined (Fig. 1CGo). 3T3-L1 adipocytes were pretreated with the transcriptional inhibitor Acti-D and then treated with or without 1 nM E2. E2 did not alter the basal half-life of resistin mRNA induced by Acti-D alone (Fig. 1CGo). However, treatment with Acti-D prevented E2-stimulated resistin mRNA expression (Fig. 1Go, B and C).


Figure 1
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  		FIG. 1. The stimulatory effect of E2 on resistin mRNA expression in 3T3-L1 adipocytes did not result from an alteration in resistin mRNA stability. A, Dose-dependent effect of E2 was observed 6 h after treatment; B, time-dependent effect of E2 (1 nM) was observed; C, E2 did not alter resistin mRNA stability induced by Acti-D (5 µg/ml) alone. Data are expressed as the means ± SE from replicates of three experiments after the quantitative digoxigenin-PCR ELISA. SE bars are too small to be seen in B and C. In A, the control experiment was that not treated with E2, whereas in B and C, controls were set at time 0 when E2 was added in the beginning. a–d, Groups with different letters (A and B) are significantly different (P < 0.05) from each other. *, P < 0.05 vs. control (C).

 
Effects of E2 on resistin protein expression, stability, and secretion
To determine whether E2-stimulated resistin gene expression also occurred at the level of translation, changes in the intracellular resistin protein content as well as the release of resistin into culture medium were measured (Fig. 2Go). Intracellular levels of resistin protein significantly increased in the presence of 1 nM E2 after 24 h, but not 1–12 h, of treatment (Fig. 2AGo). However, E2 induced resistin protein release after 1–24 h of treatment (Fig. 2BGo).


Figure 2
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  		FIG. 2. E2 (1 nM) increased the intracellular (A) and extracellular (B) resistin protein levels in 3T3-L1 adipocytes with time-dependent differences. Also, E2 tended to affect resistin protein stability induced by cycloheximide (CHX; 5 µg/ml) alone (C and D). Data are expressed as the means ± SE from replicates of three experiments. SE bars are too small to be seen in A–D. In A, B, and D, the control was represented without E2 treatment, whereas in C, it was set at time 0 when E2 was added in the beginning. *, P < 0.05 vs. control (A–C), CHX vs. CHX+E2 at a given time (D).

 
Effect of cycloheximide on E2-induced increases in resistin protein and mRNA expression
The possibility that E2-stimulated release of resistin protein was related to changes in resistin protein stability was also examined (Fig. 2Go, C and D). 3T3-L1 adipocytes were pretreated with the translational inhibitor cycloheximide and then treated with or without 1 nM E2. Because cycloheximide was previously demonstrated to reduce the 50% amounts of the intracellular resistin protein during 20–45 h of treatment (26), the duration of the exposure of E2 was extended to 45 h. E2 had no significant effect on protein stability based on the observations of no significant changes in the intracellular resistin protein content (Fig. 2CGo). However, E2 tended to increase the amounts of resistin protein released into the extracellular medium when compared with two cycloheximide treatment groups in the presence and absence of E2 (Fig. 2DGo). In addition, treatment with cycloheximide prevented E2-stimulated resistin protein expression (Fig. 2Go, A and C).

To further examine whether E2-stimulated expression of the adipocyte resistin gene is mediated via other proteins, adipocytes were pretreated with or without cycloheximide for 90 min and then stimulated with or without 1 nM E2 for 1 h (Table 1Go), 6 h (data not shown), and 24 h (data not shown). Treatment with cycloheximide alone did not alter resistin mRNA expression of adipocytes when compared with the control. Cycloheximide, however, prevented E2-induced increases in resistin gene expression during the 24-h treatment.


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  		TABLE 1. Cycloheximide and ICI182780 prevented the E2-induced increase in resistin mRNA of 3T3-L1 adipocytes

 
Effect of E2 on ER
To further demonstrate whether E2-induced expression of the resistin gene is mediated through ER, adipocytes were pretreated with the ER antagonist ICI182780 (1 µM) for 90 min, and then they were incubated with or without 1 nM E2 for 1 h (Table 1Go). Treatment with ICI182780 alone did not alter the steady-state level of resistin mRNA but prevented E2-induced increases in resistin mRNA expression. Because the rapid effects of estrogens on white adipocytes have been described at the plasma membrane level including activation of ERK MAPK activity and because ERK MAPK has been implicated as being necessary for the E2 stimulation of adipocyte proliferation from the ER (22), the effect of E2 on ER activity was assessed by changes in the amount of the phosphorylated form of ERK MAPK. Indeed, treatment with ICI182780 prevented E2-induced increases in the amount of phospho-ERK1/2 proteins (Table 1Go). But neither E2 nor ICI182780 changed the total amounts of ERK1 and ERK2 proteins (data not shown).

Changes in the phosphorylation of kinases
Whether E2-induced up-regulation of resistin mRNA expression is related to the kinase pathways was assessed by changes in the phosphorylation of ERK MEK1, p38 MAPK, and Akt (Table 2Go). Adipocytes were pretreated with either the ERK MEK1 inhibitor PD98059, the p38 MAPK inhibitor SB203580, or the PI3K inhibitor LY294002 and then treated with 1 nM E2 for 1 h. Activities of ERK MEK1 were assessed by changes in the amounts of the phosphorylated forms of ERK1 and ERK2. E2 alone had no effect on ERK1 and ERK2 proteins (data not shown) but increased the amounts of phospho-ERK1, phospho-ERK2, phospho-p38, and phospho-Akt proteins (Table 2Go).


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  		TABLE 2. Differential effects of PD98059, SB203580, and LY294002 on 17ß-estradiol-induced alterations in resistin mRNA and protein of 3T3-L1 adipocytes

 
In contrast, PD98059 alone reduced the basal activity of ERK MEK1 and in the presence of E2, suppressed E2-increased levels of phospho-ERK proteins (Table 2Go). When compared with the E2-treated group, PD98059 prevented E2-stimulated expression of resistin mRNA. Regardless of the presence of E2, SB203580 pretreatment significantly decreased the amount of phospho-p38 protein in adipocytes relative to the control. SB203580 did not alter increases in resistin gene expression induced by E2. LY294002 significantly reduced the amount of phospho-Akt protein in adipocytes treated with or without E2 when compared with the control. Similar to SB203580, LY294002 neither changed resistin gene expression alone nor significantly affected E2-induced increases in resistin mRNA levels.

Whether E2-induced alterations in resistin protein production and secretion are dependent on these MAPK or PI3K pathways was also examined (Table 2Go). There was a trend for SB203580 to decrease the basal content of intracellular resistin protein and to increase the basal release of resistin protein after 1 h of treatment. In addition, this p38 MAPK inhibitor decreased E2-retained levels of the intracellular resistin protein and slightly further increased E2-induced increases in resistin protein release. The effects of PD98059 and LY294002 differed from those of SB203580. There was a trend of PD98059 and LY294002 to increase the basal content of the intracellular resistin protein and to decrease the basal release of the resistin protein after 1 h of treatment. Moreover, PD98059, but not LY294002, significantly affected E2-induced alterations in both the intracellular and extracellular resistin protein contents. These data indicated that PD98059 was more significant than SB203580 or LY294002 in modifying E2-induced changes in resistin protein contents between the intracellular and extracellular compartments.

Effect of E2 on resistin mRNA expression depends on the association of ER{alpha} and C/EBP{alpha}
It has been reported that adipocyte specificity of resistin mRNA expression is a result of the C/EBP{alpha} binding, thereby leading to the recruitment of transcriptional factors and coactivators (24). Accordingly, the possibility that E2-induced expression of the resistin gene is mediated through the association of ER with C/EBP{alpha} and through association of C/EBP{alpha} with the resistin promoter was also examined (Fig. 3Go). First, we examined whether E2 affected the amounts of C/EBP{alpha} and ER{alpha} proteins (Fig. 3Go, A and B). Using Western blot analysis, we found that E2 increased the amount of C/EPB{alpha} protein in a time-dependent manner (Fig. 3AGo). At 1 h, but not at 24 h, of treatment, E2 did not significantly alter the amounts of total ER{alpha} and C/EBP{alpha} proteins (Fig. 3BGo). Next, we studied whether E2 affected the association of ER{alpha} with C/EBP{alpha} (Fig. 3Go, C and D). Adipocytes were pretreated with either ICI182780 or PD98059 for 90 min and then treated with or without 1 nM E2 for 1 h. An increase in the association of nuclear ER{alpha} to C/EBP{alpha} induced by E2 was observed when nuclear protein lysates were subjected to the immunoprecipitation of ER{alpha} (Fig. 3CGo) or C/EBP{alpha} (Fig. 3DGo) with their respective antisera, and amounts of the two proteins were then determined by Western blot analysis. Treatment with either ICI182780 or PD98059 prevented the E2-induced increases in the association of nuclear ER{alpha} to C/EBP{alpha} (Fig. 3Go, C and D) but did not affect total amounts of either protein (Fig. 3BGo). Finally, we further demonstrated whether E2 affected the association of ER{alpha} and C/EBP{alpha} with the resistin gene promoter through the use of the ChIP assay (24). In this assay, chromatin was isolated and subjected to cross-linking and shearing of the DNA before immunoprecipitation with specific antibodies against ER{alpha} (Fig. 3EGo, solid circles) or C/EBP{alpha} (Fig. 3EGo, open circles) proteins. The association of ER{alpha} and C/EBP{alpha} proteins with the resistin gene promoter was examined by PCR using primers specific for the resistin promoter (as described in Materials and Methods) after the reversal of cross-linking. Using this analysis, we found that 1 nM E2 for 1 and 6 h, but not for 24 h, induced increases in the association of ER{alpha} and C/EBP{alpha} with the resistin promoter (Fig. 3EGo), but after 90-min pretreatment with either ICI182780 or PD98059, prevented the E2-induced increases in the association of ER{alpha} and C/EBP{alpha} with the resistin promoter (Fig. 3FGo).


Figure 3
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  		FIG. 3. E2 (1 nM) increased levels of the C/EBP{alpha} protein, the association of ER{alpha} with C/EBP{alpha}, and the association of both proteins with the resistin promoter in 3T3-L1 adipocytes. A, Time-dependent effect of E2 on C/EBP{alpha} protein expression was observed; B. E2, ICI182780 (1 µM), or PD98059 (50 µM) did not affect ER{alpha} and C/EBP{alpha} protein expression when total lysates were measured by Western blot analysis 1 h after treatment; C and D, ICI182780 and PD98059, respectively, prevented the E2-induced increase in the association of ER{alpha} with C/EBP{alpha}. Nuclear protein lysates were subjected to immunoprecipitation of ER{alpha} or C/EBP{alpha} with either ER{alpha} (C) or C/EBP{alpha} (D) antisera (black bars), or with NRS (the value is too small to be illustrated) and then determined by Western blot analysis. Levels of C/EBP{alpha} (C) and ER{alpha} (D) proteins are expressed as a percentage of the control after normalization to ER{alpha} and C/EBP{alpha}, respectively. E, E2 time-dependently stimulated the association of ER{alpha} and C/EBP{alpha} with the resistin promoter as determined by the ChIP assay; F, 90 min of pretreatment with either ICI182780 or PD98059 prevented the E2-induced increases in the association of ER{alpha} and C/EBP{alpha} with the resistin gene promoter. Fragmented DNA not subjected to immunoprecipitation served as the input control, and it did not change among groups (data not shown). Data are expressed as the means ± SE from replicates of three experiments. In A and E, control experiments were set at time 0 when E2 was added in the beginning, whereas in B, C, D, and F, controls were those that were not treated with E2. a and b, Groups with different letters are significantly different (P < 0.05) from each other for C/EBP{alpha} or ER{alpha}; *, P < 0.05 vs. control; #, P < 0.05, E2 vs. E2 + inhibitor (brackets).

 
Differences among several estrogens in the regulation of resistin gene expression
Differences in regulating resistin mRNA expression among E2, estriol, estrone, genistein, and diethylstilbestrol were also assessed (Fig. 4Go). E2 and genistein, but not estriol, estrone, or diethylstilbestrol, increased the resistin mRNA levels. At a given 1 nM concentration for 6 h treatment, E2 was more effective than genistein in stimulating resistin mRNA expression.


Figure 4
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  		FIG. 4. Assessment of differences among E2, estriol, estrone, diethylstilbestrol, and genistein in altering resistin mRNA expression. Starved 3T3-L1 adipocytes were incubated in the presence or absence (control group) of 1 nM E2, estriol, estrone, diethylstilbestrol, or genistein for 6 h, and then resistin mRNA levels were analyzed by the quantitative digoxigenin-PCR ELISA. Data are expressed as the means ± SE from replicates of three experiments. a–c, Groups with different letters are significantly different (P < 0.05) from each other.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that E2 stimulates resistin gene expression in 3T3-L1 adipocytes. It is likely that E2 up-regulates resistin mRNA levels by stimulating resistin gene transcription. This conclusion is supported by the findings that treatment of Acti-D prevented the E2-induced increases in resistin mRNA levels (Fig. 1Go, B and C), that E2 treatment enhanced the association of C/EBP{alpha}, a transcriptional activator of resistin (24, 25, 26), with the resistin gene promoter (Fig. 3Go), and that overexpression of dominant-negative C/EBP{alpha} prevented E2-induced resistin mRNA expression (unpublished observations). The Acti-D-dependent effect of E2 suggests that new mRNA synthesis is required for the effect of E2. However, E2 did not affect resistin mRNA stability because it did not alter the basal half-life of resistin mRNA induced by Acti-D alone (Fig. 1CGo). Moreover, pretreatment of 3T3-L1 adipocytes with cycloheximide rapidly prevented E2-induced increases in resistin mRNA expression (Table 1Go), suggesting that new protein synthesis is also required for the effect of E2.

We attempted to search the signaling proteins required for E2 induction of resistin gene expression. It is evident from these data that ER{alpha} inactivation via the antiestrogen ICI182780 (31) prevented the E2-induced increases in resistin mRNA levels and ER{alpha} activity. This demonstrates that functional ER{alpha} is necessary for the effect of E2. We also attempted to find the downstream signaling transducers of ER{alpha} involved with the activation of resistin mRNA expression. We observed herein that the specific inhibitors of ERK MEK1, but not p38 MAPK or PI3K, such as PD98059 (28), SB203580 (29), and LY294002 (30), significantly prevented the E2-increased levels of resistin mRNA, and they also respectively antagonized E2-induced increases in the amounts of phospho-ERKs, phospho-p38, and phospho-Akt proteins. These observations suggest that the stimulatory effect of E2 on resistin mRNA expression of 3T3-L1 adipocytes is mediated via a pathway that requires activation of ERK MAPK, but not p38 MAPK and PI3K, activity. The ERK-dependent effect of E2 was also supported by our observations that ICI182780 reduced E2-stimulated MEK1 activity and resistin expression and that PD98059 completely blocked the E2-induced increases in the binding of ER to C/EBP{alpha} and the association of ER{alpha} and C/EBP{alpha} with the resistin gene promoter. It was evident that E2 induced a rapid nuclear translocation of ERK MAPK together with a fast ERK MAPK-dependent activation of some transcriptional factors [i.e. cAMP response element binding protein (CREB), activator protein-1, and ER{alpha}] in rat adipocytes leading to a transcriptional activation of E2-responsive genes (i.e. c-fos) (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). Whether this mechanism explains the ERK-dependent effect of E2 on resistin gene expression was not demonstrated in this study.

The promoter activity and expression of the resistin gene are regulated by a variety of nuclear receptors and of coactivator systems (1, 24, 25, 26, 35). Because peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) agonist are major negative regulators of resistin (1, 25) and because PPAR{gamma} mRNA expression is decreased in response to estrogens during adipogenesis of bone marrow stromal cells (36), we measured the amounts of PPAR{gamma} protein in 3T3-L1 adipocytes after they were treated with 1 nM E2 for 3, 6, or 24 h. E2 slightly decreased levels of PPAR{gamma} protein by 10–16% during 24 h of treatment (unpublished observations). Whether PPAR{gamma} mRNA expression and its ER{alpha}-, C/EBP{alpha}-, and DNA-binding activities are altered by E2 was not determined in this study. We did find herein that E2 significantly increased levels of the C/EBP{alpha} protein after 24 h, but not 3 or 6 h, of treatment (Fig. 3AGo). At 1–6 h of treatment, E2 also enhanced the association of nuclear ER{alpha} with C/EBP{alpha} and the association of both proteins with the resistin gene promoter (Fig. 3Go), whereas ICI182780 antagonized these stimulatory effects of E2. Moreover, overexpression of dominant-negative C/EBP{alpha}, but not dominant-negative C/EBPß, reduced the basal levels of resistin mRNA and prevented E2-induced resistin mRNA expression (unpublished observations). These observations imply that the stimulatory effect of E2 on resistin gene expression of 3T3-L1 adipocytes may be mediated via a pathway in which ER associates with C/EBP{alpha}. This implication is also indirectly supported by the findings that a functional C/EBP{alpha}-binding site was found in the proximal resistin promoter of both murine and human adipocytes to be necessary for the stimulation of transcription from resistin promoter (24, 25, 26) and that endogenous C/EBP{alpha} was bound to the resistin promoter in adipocytes in association with p300 and CREB-binding protein (24), which are viewed as coactivators involved in ER activation (23).

Expression and secretion of resistin protein are differently regulated by certain hormones, such as insulin, IGF-I, GH, dexamethasone, endothelin-1, and vitamin A (1, 4, 27, 35, 37). We report herein that increased intracellular resistin protein content was observed after 24 h, but not 1–12 h, of E2 treatment, whereas increased resistin protein release was observed during the entire 24 h of treatment. These observations suggest that E2 transiently modifies the distribution of resistin protein between the intracellular and extracellular compartments of 3T3-L1 adipocytes. This is consistent with the observation that E2 can regulate the translocation of the ER{alpha} (38). Our previous study implied the involvement of p38 MAPK-dependent pathways in the basal and IGF-I-stimulated distribution of the resistin protein between the intracellular and extracellular compartments (27). In the present report, we found that PD98059 inhibited E2-stimulated ERK MEK1 activity and resistin protein release. Neither SB203580 nor LY294002 significantly prevented E2-stimulated resistin protein release by 3T3-L1 adipocytes. These data suggest that the way E2 signaling increases resistin protein release from 3T3-L1 cells is similar to that by which it increases resistin mRNA levels; it is likely mediated through an ERK-dependent pathway.

Cellular actions of estrogens can be genomic or nongenomic (23). The genomic mechanism through which estrogens affect transcription of estrogen-sensitive genes is by direct binding of activated ER to the estrogen response elements. By contrast, the nongenomic mechanism is that putative membrane ER{alpha} and ERß can modulate the expression of genes without directly binding to DNA but by rapidly activating the MAPK cascade. Our experiments showing that the ER{alpha} antagonist ICI182780 (31) blocked MEK1 and resistin activation by E2 suggest the possible involvement of a functional membrane ER{alpha} in regulating resistin gene expression in 3T3-L1 adipocytes. This conclusion is consistent with those observed for the nongenomic effect of E2 on the expression of adipocyte uncoupling protein-2 and c-fos genes stimulated by E2’s activation of the MAPK cascade (22). It is interesting in our findings that estrogens (i.e. E2, estriol, and estrone) and the selective ER modulators (SERMs, such as genistein and diethylstilbestrol) were not entirely consistent in stimulating resistin expression by 3T3-L1 adipocytes 6 h after 1 nM treatment. Possible explanations for this discrepancy are that the distinct types of estrogens and SERMs bind to ER{alpha} or -ß in 3T3-L1 adipocytes at varying levels and that coactivators (i.e. CREB-binding protein and p300) and corepressors (i.e. silencing mediator of retinoid and thyroid receptors) of ERs required for the actions of E2 and SERMs vary with estrogen species or ER forms (23, 34). However, our study could not exclude the possibility that the genomic mechanism of actions of estrogens and SERMs (23) may help explain their differential effects on resistin expression.

In support of this study, E2 at 10 nM for 6 h was found to induce a 163% increase in resistin mRNA levels from primary adipocytes isolated from the abdominal adipose tissues of female C57BL/6J mice according to the collagenase-digested method (Ref. 39 and Table 3Go). In addition, pretreatment of these primary cells with 1 µM ICI182780 for 90 min prevented E2-activated resistin mRNA expression. Moreover, E2 stimulated a 60% increase in the release of resistin protein from primary adipocytes, and the E2 activation of resistin release was blocked by ICI182780 pretreatment. In different experiments using C3H10T1/2 mouse adipocytes, which were obtained according to a previously published method (40), ICI182780 pretreatment prevented the E2-stimulated increases in resistin mRNA levels, and E2 significantly increased resistin protein expression and release by C3H10T1/2 cells after 24 h of treatment (Table 3Go). These effects are similar to those observed for 3T3-L1 adipocytes. We should note that the doses (1–10 nM) of E2 used in our study are close to the higher physiological circulating E2 levels (22, 41). Similarly, the levels of resistin released from the E2-stimulated primary and secondary adipocytes ranged from 32–500 ng/ml, which correspond to higher circulating resistin levels (19, 20, 21) and are compatible with the effective doses (30–20,000 ng/ml) of resistin in regulating insulin resistance, adipogenesis, and inflammation in cells or animals (13, 14, 15, 16, 17). Recent studies have shown that estrogen replacement can decrease the adipose mass and insulin resistance in aromatase-knockout mice and postmenopausal women (34), that E2 affects inflammation via altering the cytokine levels (42), and that the effect of estrogens on insulin sensitivity is influenced by the route and dose of administration (41). For example, at physiological levels, E2 has a role in maintaining normal insulin sensitivity, but at higher levels (~100 nM), it impairs insulin sensitivity by altering insulin signaling in adipose tissues (41). Firm conclusions as to whether any of these in vivo effects of E2 (34, 41, 42) can be explained by its in vitro effects on adipocyte resistin levels will require more thorough studies.


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  		TABLE 3. E2 increased resistin mRNA expression and protein release by murine primary adipocytes and C3H10T1/2 adipocytes

 
We conclude that E2 activation of resistin gene expression in 3T3-L1 adipocytes is dependent on mRNA and protein synthesis and is likely mediated through increased mRNA transcription. Although these actions were shown to be mediated via the ER{alpha}, C/EBP{alpha}, and ERK MEK1 pathways, signaling was demonstrated to be likely independent of the p38 MAPK and PI3K pathways. However, inhibitors of ERK MEK1, but not p38 MAPK, modified the E2-stimulated distribution of the resistin protein between the intracellular and extracellular compartments of 3T3-L1 adipocytes. Results of this study showing the stimulatory effect of E2 on resistin production from murine primary and secondary adipocytes appear to support the findings that resistin mRNA and protein levels are higher in female mice than in male mice. Because E2, androgen (18, 19, 20), and progesterone (43) were found to regulate resistin gene expression or protein secretion, it would be of interest to determine whether these sex steroids interact to control resistin levels in different genders in another study.


   Acknowledgments
 
We thank Dr. Sheng-Chung Lee, a research fellow at the Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, and his associate, Dr. Ching-Jin Chang, for their gifts of pCMV-Tag2 vector constructed with and without the dominant-negative forms of C/EBP{alpha} and C/EBPß. We also thank Dr. Shun-Chern Tsaur, an assistant research fellow at the Research Center for Biodiversity, Academia Sinica, Taipei, Taiwan, for his technical assistance.


   Footnotes
 
This work was supported by the National Science Council, Taiwan (NSC 93-2311-B-008-005), the University System of Taiwan, Veteran General Hospital and University System of Taiwan Joint Research Program, and Tsou’s Foundation, Taiwan to Y.-H.K.

Disclosure statement: Y.-H.C., M.-J.L., H.-H.C., P.-F.H., and Y.-H.K. have nothing to declare.

First Published Online June 1, 2006

1 Y.-H.C. and M.-J.L. contributed equally to this work. Back

Abbreviations: Acti-D, Actinomycin D; C/EBP{alpha}, CCAAT/enhancer binding protein-{alpha}; ChIP, chromatin immunoprecipitation; CREB, cAMP response element binding protein; E2, 17ß-estradiol; ER, estrogen receptor; FBS, fetal bovine serum; MEK1, MAPK kinase 1; NRS, normal rabbit serum; PI3K, phosphatidylinositol 3-kinase; PPAR{gamma}, peroxisome proliferator-activated receptor-{gamma}; SERM, selective ER modulator.

Received December 29, 2005.

Accepted for publication May 25, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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