Insulin-induced Early Growth Response Gene (Egr-1) Mediates a Short Term Repression of Rat Malic Enzyme Gene Transcription*

Isabel BarrosoDagger and Pilar Santisteban§

From the Instituto de Investigaciones Biomédicas "Alberto Sols," Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Arturo Duperier 4, Madrid E-28029, Spain

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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In this report we have studied insulin regulation of malic enzyme (ME) gene transcription in rat H-35 hepatoma cells and localized the insulin-responsive region of the ME promoter between positions -177 and -102. This region contains a putative insulin response element (IRE-II). When nuclear extracts from untreated or insulin-treated H-35 cells were incubated with IRE-II, transcription factors Sp1 and Sp3 were observed to bind constitutively to this element, whereas insulin induces the quick and transient binding of an insulin response factor. This induction requires de novo protein synthesis. Competition and supershift assays demonstrated that the insulin response factor is the immediate-early gene Egr-1. In vitro assays revealed that Egr-1 displaces Sp1 from its binding site in IRE-II. Insulin induces Egr-1 mRNA, with a time course pattern that corresponds perfectly to the Egr-1 binding to IRE-II. This induction depends on the activation of mitogen-activated protein (MAP) kinase, and it is phosphatidylinositol 3-kinase-independent, as demonstrated with specific inhibitors for both pathways. By cotransfecting the wild-type or a dominant negative Ras, an upstream regulator of MAP kinase, we show that Ras inhibits ME promoter activity. Furthermore, overexpression of Egr-1 in H-35 cells represses the ME gene promoter in a dose-dependent manner. These results suggest that insulin induces a quick, transient, and Ras/MAP kinase-dependent activation of Egr-1 which leads to a transient repression of ME gene transcription. On a late phase, insulin would activate a different, Egr-1-independent pathway, which would result in activation of the ME gene.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Apart from a well reported role in metabolism, insulin behaves as a mitogen (1, 2) and differentiation factor (3, 4) for a great number of cells and tissues. Insulin binds to and activates high affinity receptors located in the plasma membrane (5). The signal transduction pathways activated from these receptors include two major mediators: Ras/MAP1 kinase and phosphatidylinositol (PI) 3-kinase as well as less known effectors. Ras/MAP kinase and PI 3-kinase have been involved in mitogenic and metabolic effects induced by insulin (for review, see Ref. 6). Stimulation of any of these pathways can eventually lead to the activation/inhibition of target genes, either by transcriptional or post-transcriptional mechanisms. Regulation of transcription by insulin affects more than 100 genes (7). Insulin response elements (IREs) have been identified in the promoter of different genes, and although it is now accepted that there is not a consensus IRE, the sequence T (G/A)TTT (T/G) (G/T) has been found in several promoters repressed by insulin (8-10). The transacting factors that activate these IREs are largely unknown. Serum response factor (11), Ets (12), or high mobility group (13) families of transcription factors have been involved in gene regulation by insulin, although its action is restricted to a few genes.

The malic enzyme (ME) gene is regulated by insulin. This lipogenic enzyme catalyzes the oxidative decarboxylation of malate to pyruvate and provides NADPH for de novo synthesis of fatty acids, mainly in liver and adipose tissue (for review, see Ref. 14). ME gene expression is subjected to a complex multihormonal and nutritional regulation (for review, see Ref. 15). In addition to insulin, thyroid hormones and fatty acids are also positive regulators of ME, whereas glucagon, cAMP inducers, and retinoic acid inhibit its expression. The rat ME gene promoter lacks both a TATA box and CAAT sequence and displays an extremely GC- rich content in its proximal region (16). Functional response elements for thyroid hormone receptors (17) and peroxisome proliferator-activated receptors (18) have been located within the ME promoter. In addition, putative binding sites for AP-1 (19) and Sp1 (16) have been described in light of sequence homology.

Sp1 is a ubiquitous transcription factor that binds to consensus GC boxes (20) and mediates transcription through glutamine-rich transactivation domains (21). In many promoters, Sp1 sites overlap with binding sites for the immediate-early gene Egr-1 (22). This transcription factor (also called nerve growth factor I-A, zif 268, or Krox 24) binds to the consensus GCG (C/G) GGG CG sequence (23, 24) and either activates or represses transcription (25). In overlapping Sp1/Egr-1 binding sites, constitutively bound Sp1 is usually displaced by Egr-1, which is induced by many different external stimuli including mitogens and differentiation signals (26).

In this report we have studied insulin regulation of ME gene transcription in rat H-35 hepatoma cells. In a previous work (27) we described the existence of two putative IREs within the ME promoter: IRE-I, which resembles the negative IRE described in the phosphoenolpyruvate carboxykinase promoter (10), and IRE-II, which is similar to the positive IRE-A element of the glyceraldehyde-3-phosphate dehydrogenase gene promoter (28). We demonstrate that the region between -177 and -102, containing the IRE-II element, displays the maximal insulin response. A more detailed analysis of the IRE-II sequence revealed a putative binding site for Sp1. We show that Sp1, Sp3, and an insulin response factor (IRF) bind to IRE-II in a specific manner. By competition and supershift assays, we demonstrate that IRF is Egr-1. Insulin induces a quick and transient activation of Egr-1 transcript, which is MAP kinase-dependent. Overexpression of Egr-1 inhibits ME gene promoter activity. The same results are obtained by overexpressing Ras, an upstream regulator of Egr-1. We propose that insulin initially represses ME gene expression in H-35 cells by up-regulating Egr-1. On a delayed phase, insulin would activate a positive effector that will be responsible for the late up-regulation of ME gene.

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Plasmids-- 5'-Deletions of the rat ME promoter fused in front of the chloramphenicol acetyltransferase (CAT) gene (pME882CAT, pME315CAT, and pME177CAT) have been described before (16). pME102CAT contains the first 102 base pairs of ME promoter subcloned in front of the CAT gene and was constructed by removing the XbaI (-775)-NotI (-102) fragment from pME775CAT (29). These plasmids were generously provided by Dr. V. Nikodem (NIH, Bethesda, MD) and Dr. B. Desvergne (IBA, Lausanne, Switzerland).

Plasmid pJDM8 contains the full-length rat Egr-1 cDNA (30) and was kindly supplied by Dr. J. Milbrandt (Washington University School of Medicine, St. Louis, MO). pSG-Egr-1 was constructed by subcloning the rEgr-1 cDNA from pJDM8 into the EcoRI site of pSG5 (31).

pMMTV-rasH(Asn-17) and pMMTV-ras plasmids have been described previously (32) and contain the dominant inhibitory H-ras Asn-17 mutation or the wild-type ras oncogene, respectively, under the control of the mouse mammary tumor virus long terminal repeat.

Cell Culture and Transfections-- H-35 rat hepatoma cells (or H4IIE-ATCC CRL 1548, American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 5% newborn bovine serum, 100 µg/ml streptomycin, and 100 IU/ml penicillin. Cell culture reagents were purchased from Life Technologies, Inc. The effect of insulin was studied in serum-starved (Dulbecco's modified Eagle's medium supplemented with 0.2% fetal bovine serum) confluent cells. After 3 days, 100 nM insulin (Actrapid, Novo-Nordisk, Bagsvaerd, Denmark) was added for the times indicated in each experiment. When needed, cycloheximide (Sigma) was used at a dose of 5 µg/ml 3 h prior to insulin treatment. In some experiments, MAP kinase or PI 3-kinase activities were inhibited with PD98059 (50 µM) or wortmannin (25 nM) (Calbiochem), respectively. Both inhibitors were added 30 min prior to insulin.

Transfections were performed in 60-mm plates, with 4 × 105 cells/plate, using the calcium phosphate method (33). Each plate was transfected with 10 µg of pME reporter plasmid in the presence of 2 µg of CMV-LUC plasmid to correct for transfection efficiency. In some experiments, wild-type or mutant MMTV-ras plasmids (5 µg/plate) or pSG-Egr-1 (2-10 µg/plate) was used. The total amount of DNA was normalized using empty vector. Transfections were done in duplicate and repeated at least three times. When needed, transfected cells were serum starved for 24 h and then treated with 100 nM insulin for 24 h. Cellular extracts were prepared by four freeze-thaw cycles, and CAT and luciferase activities were determined as described previously (34, 35).

Western Blot Assays-- H-35 cells transfected with expression vectors for Egr-1, wild-type, or dominant negative ras constructs were assayed in parallel in Western blot experiments to assess expression of the transfected plasmids. In the case of Ras, total proteins were obtained by four freeze-thaw cycles in a buffer containing 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 400 µM phenylmethylsulfonyl fluoride, 20 µg/ml pepstatin A, and 40 µg/ml aprotinin. For Egr-1, nuclear proteins were prepared as described previously (36). Protein concentrations were determined with the method of Bradford (37) using the Bio-Rad protein assay kit. 40 µg of total or nuclear proteins was electrophoresed in a 12% or 7% SDS-polyacrylamide gel for Ras or Egr-1, respectively, and blotted on nitrocellulose membranes (Schleicher & Schuell). After blocking the membranes with 10% low fat dried milk in Tris-buffered saline containing 0.05% Tween 20, immunodetection was performed with 5 µg/ml of a commercial antibody for Egr-1 or H-Ras (Santa Cruz Biotechnology Inc., Santa Cruz, CA). After probing with the antibodies, membranes were incubated with a streptavidin-conjugated anti-rabbit-specific antibody. Immunoreactive bands were visualized with the Western blotting Luminol reagent (Santa Cruz Biotechnology).

RNA Analysis-- Total RNA from H-35 cells was extracted by the guanidinium isothiocyanate method (38). Samples (30 µg) were electrophoresed in 1% agarose gels containing 2.2 M formaldehyde, blotted on Nytran membranes (Schleicher & Schuell) and hybridized with a rat Egr-1 cDNA. Hybridization and washing steps were performed as described (39). Egr-1 cDNA (3.1 kilobases) was obtained from pSG-Egr-1 by digestion with EcoRI. The probe was gel purified and labeled with [alpha -32P]dCTP (ICN, Irvine, CA) to a specific activity of 1 × 109 cpm/µg DNA using the "Ready To Go" kit (Amersham Pharmacia Biotech). Methylene blue staining of transferred RNA was used as loading control.

Electophoretic Mobility Shift Assays (EMSAs)-- Nuclear extracts were prepared as described (36), and protein concentrations were determined with the method of Bradford (37) using the Bio-Rad protein assay kit (Bio-Rad). Purified human Sp1 protein was obtained from Promega (Madison, WI), and rat Egr-1 protein was translated in vitro from pSG-Egr-1 using the coupled transcription/translation system (TNT) (Promega). The following oligonucleotides were used: IRE-II, 5'-CCC GCC CCC GCC TCC TCG CA-3'; Sp1, 5'-ATT CGA TCG GGG CGG GGC GAG-3'; and Egr-1, 5'-GGA TCC AGC GGG GGC GAG CGG GGG CGA-3'. Single-stranded oligonucleotides were end labeled using T4 polynucleotide kinase (Promega) and [gamma -32P]ATP (ICN), annealed, and purified using Sephadex G-25 columns (Boehringer Mannheim). EMSAs were performed essentially as described (40). 10 µg of nuclear extracts and 20,000 cpm of probe were incubated in a final volume of 20 µl with a binding mixture containing 26 mM HEPES, pH 7.9, 50 mM KCl, 5 mM MgCl2, 1.1 mM EDTA, 1 mM dithiothreitol, 20 µM ZnSO4, 8 µg of poly(dI·dC), and 5% Ficoll or 20% glycerol as carrier. The samples were incubated on ice for 30 min. The same conditions were used in the reactions containing purified Sp1 (0.5 footprinting unit/reaction in the presence of 10 µg of bovine serum albumin) or in vitro translated Egr-1 (2.5 µl/reaction). For competition experiments, a 100-fold excess of unlabeled oligonucleotide was preincubated on ice with nuclear extracts 15 min prior to the addition of the probe. For supershift assays, 1 µg of anti-Sp1, anti-Sp3, or anti-Egr-1 antibodies (Santa Cruz Biotechnology) or preimmune antisera were preincubated overnight with nuclear extracts at 4 °C before the addition of the probe. The resulting DNA complexes were electrophoresed on a 5% polyacrylamide gel (29:1, acrylamide:bisacrylamide). Gels were run at 20 mA in a cold room in 0.5 × TBE (1 × TBE is 90 mM Tris, 90 mM boric acid, and 1 mM EDTA), vacuum dried, and visualized by autoradiography.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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The IRE of the Rat ME Gene Promoter Is Located between -177 and -102-- Previous studies from our laboratory (27) demonstrated a direct role of insulin in the regulation of ME gene, both by transcriptional and post-transcriptional mechanisms. Those studies were performed in rat liver. In this report we have chosen H-35 rat hepatoma cells to study insulin-mediated regulation of ME because these cells respond to physiological doses of insulin (41, 42) and have been used extensively in gene expression studies (8, 43, 44). We have found in this cell line the same regulation of ME mRNAs as in rat liver (data not shown).

To define the region within the ME gene promoter responsible for the insulin response, we transiently transfected H-35 cells with several 5'-deletions of the rat ME promoter fused to the CAT gene and then treated the cells with insulin. As shown in Fig. 1, the pME882 construct, which contains 882 base pairs of ME promoter, responds weakly to insulin. Serial deletions of the ME promoter increment the insulin response, with a maximum effect (3-fold) achieve by pME177. The insulin response is completely lost with a subsequent deletion of 75 base pairs (pME102), indicating that a positive IRE of the rat ME gene promoter is located between positions -177 and -102. These results are in agreement with our previous data (27) that pointed out the presence of a negative IRE (referred to as IRE-I) between positions -692 and -683 of the ME promoter, and a positive IRE (referred to as IRE-II) between positions -175 and -156. As shown in Fig. 1, deletion of the putative IRE-I increments the insulin response of ME gene promoter, indicating that IRE-I could effectively behave as a negative element. The maximum positive insulin response is restricted to the region -177/-102, which contains the putative positive IRE-II, so further studies were focused on this element.


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Fig. 1.   Identification of the insulin-responsive region within the ME promoter. Panel A, schematic diagram of the full ME promoter (pME882) and the different deletions (pME315, pME177, and pME102) linked to the CAT reporter gene. Protein binding sites detected previously and the corresponding transcription factors are indicated with different symbols. Panel B, ME promoter activity derived from 10 µg of each construct transiently transfected into H-35 cells. After transfection the cells were maintained for 24 h in the absence of serum and then treated (+) or not (-) for 24 h with 100 nM insulin. Relative CAT activity is the value of percent acetylation normalizing the results to luciferase activity derived from the 2 µg of pCMV-LUC transfected to correct for transfection efficiency. The ME promoter activity is expressed as fold induction over the basal levels (= 1) of serum-depleted cells. The results are the mean ± S.D. of four independent experiments.

Sp1, Sp3, and an IRF Bind to the -175 to -156 Region of ME Promoter-- To identify nuclear proteins that could bind to the -175/-156 ME promoter region (IRE-II), we first examined IRE-II sequence and compared it with the previously described IRE-A element of glyceraldehyde-3-phosphate dehydrogenase gene promoter. As shown in Fig. 2A, both regions contain GC boxes, which are consensus binding sites for the transcription factor Sp1 (20). We next performed a band shift assay with labeled IRE-II and nuclear extracts from H-35 cells depleted of serum for 3 days and then treated with 100 nM insulin for different times. Three different complexes (C1, C2, and C3) are detected in nuclear extracts from untreated cells, and one complex more (designated IRF) is detected only in insulin-treated cells (Fig. 2B). All four of these complexes are specific as demonstrated by competition with unlabeled IRE-II but not with an unrelated oligonucleotide (see below). The kinetics of IRF binding to IRE-II was also examined and revealed a quick and transient induction, as IRF binding activity is already detected 30 min after the addition of insulin, is maximal with 1-h treatment (Fig. 2B), and is undetectable after 4 h (data not shown).


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Fig. 2.   Characterization of protein·IRE-II complexes. Panel A, sequence of IRE-A derived from the glyceraldehyde-3-phosphate dehydrogenase gene promoter and IRE-II, located between positions -175 and -156 within the ME gene promoter. The putative Sp1 sites in both elements are indicated. Panel B, EMSA performed with the IRE-II probe, represented in panel A, and nuclear extracts from H-35 cells maintained for 3 days in the absence of serum (0) or treated with insulin for the times indicated in the figure. The protein·DNA complexes (C1, C2, C3, and IRF) are indicated with arrows.

Because IRE-II contains putative binding sites for Sp1 (Fig. 2A), we next examined if any of the complexes detected in H-35 cells corresponded to this transcription factor or a related protein. As shown in Fig. 3A, purified human Sp1 protein binds to IRE-II in a specific manner as its binding is competed by an excess of unlabeled IRE-II and is unaffected by an unrelated oligonucleotide. Furthermore, when nuclear extracts from H-35 cells treated with insulin for 1 h were incubated with a specific antibody anti-Sp1 (Fig. 3B, lane 4), a supershift of C1 complex was observed, whereas an unrelated antibody had no effect (lane 5). The mobility of IRF was not affected by incubation with anti-Sp1 antibody, indicating that IRF does not contain Sp1 protein. Taken together, these results indicate that Sp1 is present in both untreated (lane 2) and insulin-treated H-35 cells, and its binding to IRE-II is not modified by the hormone.


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Fig. 3.   Members of the Sp1 family of transcription factors bind to IRE-II. Panel A, EMSA with 0.5 footprinting unit of purified Sp1 protein (lane 2) and the IRE-II sequence. For competition, a 100-fold excess of unlabeled IRE-II (lane 3) or an unrelated oligonucleotide (lane 4) was used. Panel B, EMSA from nuclear extracts of H-35 cells maintained in a serum-depleted medium (lane 2) or treated for 1 h with 100 nM insulin (lane 3) and IRE-II probe. Supershift assays were performed in insulin-treated cells with the specific alpha -Sp-1 antibody (lane 4) or with an unrelated antibody (lane 5). The complexes found and the supershift are indicated with arrows. Panel C, EMSA from nuclear extracts of H-35 cells maintained in a serum-depleted medium (lanes 2-4) or treated for 1 h with 100 nM insulin (lanes 5-7) and the labeled IRE-II oligonucleotide. Supershift assays were performed in both serum-depleted or insulin-treated cells with the specific alpha -Sp-3 antibody (lanes 4 and 7) or with an unrelated antibody (lanes 3 and 6). The complexes found and the supershift are indicated.

Sp3 is a closely related member of the Sp1 family of proteins which has been shown to bind to Sp1 elements in a variety of gene promoters (45, 46). To investigate if any of the detected complexes bound to IRE-II corresponds to Sp3, we performed supershift assays using specific anti-Sp3 antibody. Incubation of untreated or insulin-treated H-35 cells with anti-Sp3 antibody supershifted the C2 complex (Fig. 3C, lanes 4 and 7), whereas an unrelated antibody (lanes 3 and 6) had no effect. Anti-Sp3 antibody was unable to displace IRF, indicating that this complex does not contain Sp3 protein. These results indicate that Sp3, as observed for Sp1, is constitutively bound to IRE-II in H-35 cells, and its binding is not affected by insulin.

IRF Binding to IRE-II Requires de Novo Protein Synthesis-- In an attempt to identify IRF, its requirement of ongoing protein synthesis was studied. We performed a band shift assay with labeled IRE-II and nuclear extracts from H-35 cells treated or not with insulin for 15 min or 1 h in the presence or absence of the protein synthesis inhibitor cycloheximide (Fig. 4). As shown before, IRF binding activity was undetectable 15 min after treatment of H-35 cells with insulin (lane 4), and was maximal 1 h after insulin stimulation (lane 6). Cycloheximide alone did not induce IRF, although it was able to block completely the insulin ability to induce IRF binding to IRE-II (lane 6 versus lane 7). These results demonstrate that de novo protein synthesis is essential for IRF binding.


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Fig. 4.   Cycloheximide (CHX) inhibits the insulin-responsive protein that binds to IRE-II. EMSA was performed with IRE-II probe and nuclear extracts from H-35 cells maintained for 3 days in serum-depleted medium (lane 2) or treated with insulin for 15 min (lanes 4 and 5) or 1 h (lanes 6 and 7) in the absence (lanes 4 and 6) or presence (lanes 5 and 7) of the protein inhibitor cycloheximide. One group of cells were treated with cycloheximide alone (lane 3).

IRF Is Egr-1-- The quick and transient induction of IRF binding and its dependence on protein synthesis are typical features of a subclass of genes known as immediate-early genes. Because the GC content of IRE-II is extremely high (16 base pairs out of 20) we search for immediate-early genes that bind to GC boxes. Among the candidates, Egr-1 has been described to be induced in response to insulin in a variety of cell systems including H-35 cells (47) and with the same kinetics as observed for IRF. Egr-1 has been reported to bind specifically to the sequence GCG(C/G)GGGCG (23, 24) in a zinc-dependent manner. When the IRE-II sequence was examined in more detail, we found a perfectly conserved Egr-1 binding site that overlaps the GC boxes already identified as core binding sites for Sp1. To study if IRF corresponded to Egr-1, we initially performed competition experiments. Using a radiolabeled IRE-II probe, two complexes corresponding to Sp1 and Sp3 were detected in nuclear extracts from untreated H-35 cells (Fig. 5A, lane 1). When IRE-II was incubated with nuclear extracts from cells treated with insulin for 1 h, an additional complex, IRF, was observed (lane 2). The three complexes were competed by a 100-fold excess of unlabeled IRE-II (lane 3) and were unaffected by an excess of an unrelated oligonucleotide (lane 6), indicating that all of them are specific. An oligonucleotide containing a consensus binding site for Sp1 abolished the formation of Sp1 and Sp3 complexes but did not affect IRF (lane 4). However, a 100-fold excess of an oligonucleotide containing a consensus binding site for Egr-1 completely abolished IRF binding without affecting Sp1 and Sp3 (lane 5). These data suggest that IRF could contain Egr-1 or a related protein.


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Fig. 5.   Identification of the insulin-induced protein that binds to IRE-II. Panel A, nuclear extracts from nontreated (lane 1) or 1-h insulin-treated H-35 cells (lane 2) were incubated with labeled IRE-II. Free and bound DNA were resolved in an EMSA. The bottom part of the gel was removed, so complex C3 is not shown. For competition, 100-fold excess of unlabeled, double-stranded (ds) IRE-II (lane 3), Sp1 (lane 4), Egr-1 (lane 5), or unrelated (UR) (lane 6) oligonucleotide was used. Panel B, nuclear extracts from nontreated (-) or 1-h insulin-treated (+) H-35 cells were incubated with labeled IRE-II (lanes 1 and 2) or consensus Egr-1 as probes (lanes 4-9). When Egr-1 probe was used, the competition assay was performed with a 100-fold excess of unlabeled Egr-1 (lane 6), IRE-II (lane 7), Sp1 (lane 8), or unrelated (lane 9) oligonucleotides. Panel C, EMSA with Egr-1 protein translated from programmed TNT reticulocytes (lanes 3-6) and the IRE-II sequence. As a control, nonprogrammed TNT (Np-TNT) reticulocytes were used (lane 2). For competition, a 100-fold excess of IRE-II (lane 4), Egr-1 (lane 5), or Sp1 (lane 6) oligonucleotides was used. Panel D, EMSA with nuclear extracts from 1-h insulin-treated H-35 cells and the IRE-II probe. The supershift was performed with alpha -Egr-1 (lane 3) or alpha -Sp1 (lane 4) antibody, respectively. alpha -Egr-1 antibody alone was incubated with IRE-II (lane 1) to assess for nonspecificity of the shifted band.

To study whether IRF was Egr-1 or a related protein, a similar approach using IRE-II or a consensus binding site for Egr-1 as probes was performed. Sp1 binding is detected with IRE-II probe, both in untreated (Fig. 5B, lane 1) and insulin-treated cells (lane 2). A similar complex appears with Egr-1 probe (lane 4), but its binding is almost lost when cells were treated with insulin for 1 h (lane 5). When nuclear extracts from insulin-treated cells were used, a complex (IRF) was detected for both probes (lanes 2 and 5). The specificity of this complex was demonstrated by its competition with an excess of unlabeled Egr-1 oligonucleotide. Interestingly, an excess of IRE-II competed this insulin-induced complex as efficiently as Egr-1 (compare lanes 6 and 7). Neither an excess of an oligonucleotide with a consensus binding site for Sp1 (lane 8) nor an unrelated oligonucleotide (lane 9) was able to abolish this complex. These results provide further evidence identifying IRF as either Egr-1 or a related protein.

Consistent with this hypothesis, an in vitro translated Egr-1 protein was able to bind to IRE-II in a specific manner (Fig. 5C). The identity of IRF was further confirmed with a specific antibody for Egr-1. IRF was completely supershifted with anti-Egr-1 antibody, but it was unaffected by anti-Sp1 (Fig. 5D). These data establish definitively that Egr-1 is the IRF bound to IRE-II in the rat ME promoter.

Sp1 and Egr-1 Compete for Binding to IRE-II-- As deduced from the analysis of IRE-II sequence, Sp1 and Egr-1 binding sites overlap. This is a common feature reported in several promoters (22, 48). In most of these genes, Sp1 is expressed in a constitutive manner, whereas Egr-1 expression is induced in response to external stimuli. Usually, induced Egr-1 is able to displace Sp1 from its DNA binding site (49, 50). To study if this was the case for the ME promoter, we performed a band shift assay with limiting amounts of labeled IRE-II probe, constant levels of prebound Sp1 protein and increasing amounts of in vitro translated Egr-1. In the absence of Egr-1, IRE-II was entirely bound by Sp1 (Fig. 6, lane 2). Increasing amounts of Egr-1 decreased Sp1 binding to IRE-II (lanes 3-5), indicating not only that binding of each transcription factor is mutually exclusive, but also that Egr-1 is able to release bound Sp1 from its site in IRE-II.


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Fig. 6.   Competition of Sp1 and Egr-1 proteins for binding to IRE-II. EMSA was performed with IRE-II probe and 0.5 footprinting unit of purified Sp1 protein (lane 2) or with 2.5 µl of in vitro translated Egr-1 protein (lane 6). For competition binding a constant amount of Sp1 protein (0.5 footprinting unit) and increased amounts (2.5, 5, or 10 µl) of Egr-1protein (lanes 3-5) were used.

Insulin Induction of Egr-1 in H-35 Cells Requires Activation of MAP Kinase-- The kinetics of Egr-1 induction by insulin in H-35 cells was examined next. Insulin is a well known inducer of Egr-1 in this cell line (47, 51). In agreement with these previous reports, insulin induces Egr-1 transcript in H-35 cells in a quick and transient manner (Fig. 7A). Egr-1 mRNA is absent in serum-depleted cells (0), but it is already detectable 30 min after insulin treatment, is maximum in 1 h, and disappears quickly afterward. This kinetics of Egr-1 transcript corresponds perfectly with the induction of IRF binding to IRE-II (see Fig. 2). Along with this rapid and transient induction, Egr-1 transcript has been reported to be superinduced in the presence of a protein synthesis inhibitor such as cycloheximide (52, 53). This fact was also observed in insulin-stimulated H-35 cells. As shown in Fig. 7B, cycloheximide alone was able to up-regulate Egr-1 mRNA levels and did not block insulin-induction of Egr-1 transcript.


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Fig. 7.   Characterization of Egr-1 induction by insulin in H-35 cells. Total RNA (30 µg) was extracted from H-35 cells and hybridized with Egr-1 cDNA. Panel A, cells were maintained for 3 days in a serum-depleted medium (0) or treated with 100 nM insulin for the time intervals indicated. Panel B, cells were maintained in serum-depleted medium (-) or treated with insulin for 1 h in the presence of cycloheximide (CHX, 5 µg/ml). Panel C, cells were maintained in a serum-depleted medium (-) or treated for 1 h with insulin in the presence of the MEK inhibitor PD98059 (50 µM) or with the PI 3-kinase inhibitor wortmannin (25 nM) (panel D). One group of cells was incubated with each inhibitor alone to discard possible toxic effects. Both inhibitors were added 30 min prior to insulin. For loading control, the 18 S ribosomal RNA after methylene blue staining is shown.

We next analyzed the transduction pathways involved in Egr-1 up-regulation by insulin. The Ras/MAP kinase and PI 3-kinase pathways have been reported to mediate many insulin actions (6). Insulin-mediated induction of Egr-1 in several systems depends on the activation of the Ras/MAP kinase pathway (54, 55). To analyze the participation of this cascade on Egr-1 induction in H-35 cells, serum-depleted cells were treated with the hormone for 1 h in the presence or absence of PD98059, a specific inhibitor of MEK (the upstream regulator of MAP kinase). Results showed (Fig. 7C) a complete inhibition of Egr-1 transcript by the inhibitor. The same set of experiments was performed with wortmannin, an inhibitor of PI 3-kinase activity. In this case (Fig. 7D), the addition of the inhibitor did not modify Egr-1 mRNA levels. In both cases, the inhibitors were added alone to discard possible toxic effects. Taken together, the results presented clearly establish that stimulation of H-35 cells by insulin leads to a rapid and transient activation of the immediate-early gene Egr-1 and that this up-regulation takes place through activation of the Ras/MAP kinase signaling pathway.

Overexpression of Ras Down-regulates ME Promoter Activity-- Having demonstrated that insulin stimulates Egr-1 in H-35 cells through the Ras/MAP kinase pathway, our next aim was to correlate this observation with the insulin effect on the ME gene. To do so, we cotransfected H-35 cells with pME177 reporter plasmid and wild-type or dominant negative ras constructs. As shown in Fig. 8A, overexpression of ras does not up-regulate, but inhibits ME promoter activity, whereas a dominant negative ras induces it approximately 2-fold. pME102CAT, which does not respond to insulin, was unaffected by overexpression of Ras. Expression of transfected Ras proteins was assessed by Western blot using specific anti-Ras antibodies (Fig. 8B). These results strongly suggest that the insulin-mediated induction of ME does not take place through activation of the Ras pathway, but rather through an alternative cascade.


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Fig. 8.   Role of Ras in ME promoter activity. Panel A, plasmids containing or not the IRE-II element of the ME promoter (pME177 or pME102, respectively) were cotransfected into H-35 cells with empty vector or wild type or dominant negative ras constructs (5 µg/plate) as described under "Experimental Procedures." CAT activity was determined as relative units normalized to luciferase activity derived from the pCMV-LUC transfected to correct for transfection efficiency. The ME promoter activity is expressed as fold induction over cells transfected with empty vector (=1). The results are the means ± S.D. of three independent experiments. Panel B, representative Western blot performed with 40 µg of total proteins derived from cells transfected with pME177 reporter plasmid and vector, wild type (pMMTV-ras), or dominant negative (pMMTV-ras(Asn-17)) ras constructs is shown. A similar Western blot was obtained when pME102 was used as reporter plasmid (not shown).

Egr-1 Is a Repressor of the ME Gene Promoter-- In light of the results obtained, we evaluated whether Egr-1 directly affects ME gene promoter. We overexpressed Egr-1 in H-35 cells in the presence of pME177 as reporter plasmid. As shown in Fig. 9A, Egr-1 inhibits pME177 activity in a dose-dependent manner. The expression of transfected Egr-1 protein was assessed by Western blot (Fig. 9B). These results demonstrate that insulin-induced Egr-1 represses ME gene expression. Moreover, the transient binding of Egr-1 to IRE-II in ME promoter probably indicates that this repression does not last long. It seems that insulin activates different cascades in H-35 cells which lead in a first step to the inhibition of ME gene transcription and in a final step to its activation.


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Fig. 9.   Egr-1 represses the ME gene promoter. Panel A, the construct containing the IRE-II element of the ME promoter (pME177) was cotransfected into H-35 cells with empty vector (0) or pSG-Egr-1 (2-10 µg) as described under "Experimental Procedures." CAT activity was determined as relative units normalizing to luciferase activity derived from the pCMV-LUC transfected to correct for transfection efficiency. The ME promoter activity is considered 100 for pME177 in the absence of Egr-1. The results are the means ± S.D. of three independent experiments. Panel B, representative Western blot using 40 µg of nuclear proteins obtained from overexpression experiments of Egr-1 and hybridized with specific anti-Egr-1 antibody.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this report we have studied the insulin regulation of the rat ME gene in the hepatoma cell line H-35. In agreement with our previous results (27), we delineate two different insulin response regions within ME promoter: -882/-315, which contains the IRE-I element, and the region between -177 and -102, which contains IRE-II. Both IRE-I and IRE-II have been described in light of their sequence homology with functional IREs and for the insulin ability to modify their binding patterns in rat liver (27). Regarding IRE-I, transient transfection experiments with serial 5'-deletions of ME promoter in H-35 cells indicate that it could effectively behave as a negative IRE, because its deletion increments the insulin response of the promoter (Fig. 1). In contrast with these results, a different insulin response of this region has been reported recently (56) and could be explained by technical differences regarding the ME promoter sensitivity to the insulin dose used.

Concerning IRE-II, our results demonstrate that two members of the Sp1 family of proteins (Sp1 and Sp3) bind constitutively to this element. Sp1 is a ubiquitous transcription factor that plays an essential role in the assembly of the basal transcription machinery in TATA-less promoters (57). Furthermore, Sp1 can interact and synergize with different transcription factors (58, 59) to obtain maximal promoter activity. Sp3 is a close related member of the Sp1 family that also recognizes and binds to GC boxes (60). Binding of both Sp1 and Sp3 to the same site in DNA is a common feature reported in several promoters (62, 63). In these sites Sp3 usually modulates Sp1-driven transcription (45). Although Sp1 usually participates in basal transcription, it has also been involved in the regulation of some metabolic pathways such as the glucose response of the acetyl-CoA-carboxylase gene (61). However, and to our knowledge, Sp1 has not been related to the regulation of gene transcription by insulin. In the case of the ME gene, our results demonstrate that neither Sp1 nor Sp3 binding activities are affected by insulin. However, the particular features of the ME gene promoter (it lacks a TATA box and displays a GC-rich content in its proximal region), which are typical of Sp1-regulated genes, and the observed interplay between Sp1 and the insulin-induced Egr-1 (see below) suggest a role for these transcription factors either in the basal or the insulin-mediated response of the ME gene promoter.

Immediate-early gene Egr-1 was first described as a nerve growth factor-induced gene in differentiating PC12 cells (30). In addition to nerve growth factor, Egr-1 has been reported to be induced by many mitogens and differentiation stimuli (6), including insulin (47). In this report we demonstrate that insulin induces Egr-1 binding to IRE-II in the ME gene. This up-regulation is quick and transient and requires activation of the Ras/MAP kinase pathway. The same kinetics of Egr-1 induction has been reported previously in H-35 cells (47) and other cell systems (55). Furthermore, insulin activation of Egr-1 in 32 D myeloid cells (55) also takes place through the Ras/MAP kinase pathway, suggesting that this cascade could be a common mediator of Egr-1 induction by insulin in different cell systems.

Analysis of IRE-II sequence suggests that Egr-1 and Sp1 binding sites overlap. These overlapping sites are present in other promoters (49, 50). In most of these genes, binding of each transcription factor is mutually exclusive (22), and because of the extreme induction of Egr-1, these overlapping sites are believed to confer responsiveness to external stimuli to promoters whose transcription is controlled by Sp1. This is the case for the phorbol 12-myristate 13-acetate-induced interleukin-2 receptor beta -chain promoter (64) or the vascular injury-induced platelet-derived growth factor B-chain promoter (49). Once bound to DNA, Egr-1 can either activate or inhibit transcription, as it has both transactivation and repression domains (25). In the case of ME gene promoter, our results with recombinant Egr-1 and Sp1 proteins demonstrate that in vitro, Egr-1 displaces Sp1 from its binding site in IRE-II. This observation strongly suggests that insulin-induced Egr-1 affects the activity of the ME gene promoter. This is in fact the case, as overexpression of Egr-1 leads to a dose-dependent repression of ME promoter (Fig. 9). Egr-1 has been described to inhibit the expression of other genes (65). Moreover, cotransfection of wild-type ras, an upstream regulator of Egr-1, inhibits ME gene promoter, whereas a dominant negative ras construct activates it (Fig. 8).

Insulin induction of Egr-1 takes place in a few minutes (Fig. 7A). This quick response could indicate that activation of this immediate-early gene is one of the first events that occur after insulin stimulation of this cell type. Even more, this activation of Egr-1 is transient, at least as measured by its binding to the IRE-II sequence in ME gene promoter (Fig. 2B), and could reflect that removal of Egr-1 is followed by activation of a different cascade of events. We propose that, as a first step, insulin activates the Ras/MAP kinase pathway, which in turn stimulates Egr-1 translation. The newly synthesized Egr-1 protein would almost immediately translocate to the nucleus and bind to IRE-II in the ME gene promoter, repressing its transcription. The significance of this repression is unknown. One possible explanation could be that this initial stimulation of insulin confers a mitogenic signal to the cell; it is possible that ME gene expression is not needed for this response. Two pieces of evidence support this mitogenic effect. First, in contrast to what happens with primary hepatocytes, insulin alone is a full mitogen for H-35 cells (42); second, in this initial phase insulin induces the Ras/MAP kinase pathway, and Egr-1, whose role in promoting entry of quiescent cells into G1 phase, is well known (6, 47, 51). After this initial step, insulin could activate different signals, such as the specific Egr-1 corepressor nerve growth factor IA-binding protein 2; (66), so the Egr-1 activity would be removed, allowing Sp1 to bind again to its binding site in IRE-II.

These results are not in disagreement with the well known activation of ME gene expression by insulin. This effect probably takes place through a delayed pathway that involves different transcription factors acting in ME promoter. In this regard, the results presented by Streeper et al. (56) demonstrate that members of the AP-1 complex bind to the AP-1 element located between positions -132 and -126 in the ME promoter. Furthermore, this element confers insulin responsiveness to a heterologous promoter, and its mutation results in loss of this response, probing its role in the insulin up-regulation of this gene.

    ACKNOWLEDGEMENTS

We are indebted to Dr. V. Nikodem (National Institutes of Health) for pME882 and pME177 plasmids and Dr. B. Desvergne (Institut de Biologie Animale, Lausanne, Switzerland) for pME315 and pME775. Dr. J. Milbrandt (Washington University School of Medicine, St. Louis, MO) kindly provided the rEgr-1 cDNA. We gratefully acknowledge Dr. J. A. Velasco for critical reading of the manuscript and D. L. Medina and P. Méndez for helpful suggestions.

    FOOTNOTES

* This work was supported in part by Grants DGICYT (PM97-0065), CAM (08.1/0025/1997), and Fundación Salud 2000 (Spain).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.

Dagger Recipient of fellowship from Comunidad Autónoma de Madrid (Spain).

§ To whom correspondence should be addressed: Instituto de Investigaciones Biomédicas, CSIC/UAM, Arturo Duperier 4, Madrid 28029, Spain. Tel.: 34-91-585-4644; Fax: 34-91-585-4587; E-mail: psantisteban{at}iib.uam.es.

    ABBREVIATIONS

The abbreviations used are: MAP, mitogen-activated protein; PI 3-kinase, phosphatidylinositol 3-kinase; IRE, insulin response element; ME, malic enzyme; Egr, early growth response; IRF, insulin response factor; CMV, cytomegalovirus; LUC, luciferase; CAT, chloramphenicol acetyltransferase; MMTV, mouse mammary tumor virus; EMSA, electrophoretic mobility shift assay; TNT, transcription/translation; MEK, MAP/ERK (extracellular signal-regulated kinase) kinase.

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