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Endocrinology Vol. 143, No. 12 4718-4729
Copyright © 2002 by The Endocrine Society

ARTICLE

Transcriptional Regulation of the Epiregulin Gene in the Rat Ovary

Toshio Sekiguchi, Tetsuya Mizutani, Kazuya Yamada, Takashi Yazawa, Hiroko Kawata, Miki Yoshino, Takashi Kajitani, Takashi Kameda, Takashi Minegishi and Kaoru Miyamoto

Department of Biochemistry, Fukui Medical University (T.S., T.Miz., K.Y., T.Y., H.K., M.Y., T.Kaj., K.M.), Shimoaizuki, Matsuoka, Fukui 910-1193, Japan; Department of Obstetrics and Gynecology, Gunma University School of Medicine (T.Kam., T.Min.), Maebashi, Gunma 371-8511, Japan; and CREST, Japan Science and Technology (T.S., T.Miz., K.Y., T.Y., H.K., M.Y., T.Kaj., T.Min., K.M.), Japan

Address all correspondence and requests for reprints to: Dr. Kaoru Miyamoto, Department of Biochemistry, Fukui Medical University, and CREST, JST, Fukui 910-1193, Japan. E-mail: kmiyamot{at}fmsrsa.fukui-med.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ovarian follicular development is initiated by FSH secreted from the pituitary gland. The FSH-induced follicular development involves granulosa cell proliferation and differentiation. We demonstrated that a growth factor of epidermal growth factor (EGF) family epiregulin was rapidly induced in the primary culture of rat ovarian granulosa cells by FSH within 1 h. Epiregulin gene expression was also observed in granulosa cells of antral ovarian follicles from pregnant mare’s serum gonadotropin-primed rats in vivo. To analyze the regulation of gene expression of epiregulin, we isolated and characterized the rat epiregulin gene of 22.1 kb, including 3.8 kb of 5'-upstream region as well as all five exons and four introns. We determined the transcriptional start site of rat epiregulin gene by primer extension analysis and then characterized the upstream promoter region of the gene. By using a luciferase reporter system, deletion and mutation analyses of rat epiregulin gene promoter region revealed that 125 bp upstream of transcriptional start site was essential, and that two CT boxes and one GT box within this region were important for the gene expression. We also demonstrated by EMSAs that Sp1/Sp3 proteins were involved in the epiregulin gene expression via the upstream sequence. Involvement of Sp1/Sp3 was also demonstrated that transfection of Sp1 or Sp3 expression plasmids dramatically increased the epiregulin gene promoter activities about 90- or 7.9-fold, respectively, in Drosophila SL2 cells that lack endogenous Sp family proteins. Such an increase in the promoter activity was also observed in mammalian cells when NIH-3T3 cells were used. In conclusion, we demonstrated here for the first time that EGF-type growth factor epiregulin is rapidly and strongly induced in the ovarian granulosa cells by FSH stimulation, and that two CT boxes and one GT box present in the upstream region are essential for the promoter activity of rat epiregulin. We also demonstrated that Sp family members play crucial roles in the epiregulin promoter activity through the CT boxes. The restricted and hormonally regulated expression of epiregulin in the rat ovarian granulosa cells may correspond to the physiological relevance of this peptide growth factor to the FSH-induced ovarian follicular growth and maturation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PITUITARY LH and FSH play integral roles in the regulation of normal reproductive development and function (1, 2). During follicular development, FSH induces ovarian granulosa cell proliferation and differentiation through activation of specific ovarian genes in response to the peptide hormone (3, 4, 5). Although some of the molecular mechanisms by which FSH regulates gene expression during differentiation have been defined, less is known about genes rapidly induced by FSH and the mechanism employed to mediate this induction, particularly in undifferentiated granulosa cells. Recently, we employed subtraction cloning technology to identify several ovarian genes rapidly induced by FSH in undifferentiated granulosa cells (6). Among these rapidly induced granulosa cell genes, we found a growth factor of epidermal growth factor (EGF) family, epiregulin. Epiregulin has been identified as a growth inhibitory factor from conditioned medium of a fibroblast tumor cell line NIH-3T3/clone T7 (7, 8). Several reports have described that epiregulin has a mitotic activity for various cell types, including fibroblast, hepatocyte, and keratinocyte (7, 9). Rat epiregulin has been isolated as a major mitogenic protein present in the conditioned medium of angiotensin II-stimulated rat aortic smooth muscle cells (10). Recently, bovine epiregulin has also been identified as an ovarian gene that is associated with bovine oocyte developmental competence (11). Like other EGF-type growth factors, epiregulin exerts its proliferative effects via the tyrosine kinase pathway through autophosphorylation of EGF receptors (12). It has been shown that FSH-induced proliferation of granulosa cells can be reproduced by EGF (13). Several EGF-type growth factors, including EGF itself and TGF{alpha}, have been proposed as candidates that mediate the actions of FSH on the ovarian granulosa cells (14, 15). However, none of these growth factors proposed to date has been induced in ovarian granulosa cells by FSH. In this context, epiregulin, instead of other EGF-type growth factors, is one of the strong candidates that actually mediate FSH induced granulosa cell proliferation. From this point of view it seems extremely important to analyze the regulatory mechanisms of epiregulin gene expression in ovarian granulosa cells.

The gene expression profiles of epiregulin in human and mouse tissues have been reported (8, 16, 17). Northern blot analysis has shown that the expression of human epiregulin is detected mainly on peripheral blood macrophages and placenta. The expression is also detected in various types of tumor cell lines, highest in an epithelial tumor cell line HeLa cells (17). In mice, however, epiregulin has not been detected in several tissues, except for uterus of adult mice by Northern blot analysis. The expression was detected in 7-d-old mouse embryo and then diminished to very low or undetectable levels (8, 16). We demonstrated that epiregulin gene expression was also detected in the ovarian granulosa cells only after the cells were stimulated with FSH, although rat granulosa cells do not proliferate in serum free culture with FSH.

To clarify the regulatory mechanisms of epiregulin gene expression in the ovary, we isolated and characterized rat epiregulin gene. We determined the transcriptional start site of rat epiregulin gene and intensively characterized the upstream promoter region of the gene. In the present study we identified that the region of 125 bp upstream of transcriptional start site was essential, and that two CT boxes and one GT box in this region were important for the gene expression. We also demonstrated that Sp1/Sp3 proteins were involved in the epiregulin gene expression via the upstream sequence.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Diethylstilbestrol (DES) was purchased from Sigma (St. Louis, MO). Ovine FSH (204,453 IU/mg) was obtained from the National Hormone and Pituitary Distribution Program (Bethesda, MD). Pregnant mare’s serum gonadotropin (PMSG) was a product from Teikokuzouki, Inc. (Tokyo, Japan). The Bca BEST labeling kit and Bca BEST sequencing kit were purchased from Takara, Inc. (Shiga, Japan). The dRhodamine Terminator Cycle Sequencing FS Ready Reaction Kit was purchased fromPE Applied Biosystems (Foster City, CA). A dual luciferase reporter assay system (firefly and Renilla luciferases), pGL3-Basic, and pRL-SV vectors were purchased from Promega Corp. (Madison, WI). A plasmid kit was purchased from QIAGEN (Hilden, Germany). FuGENE-6, Oligotex dT-30 super, T7, and SP6 RNA polymerase were obtained from Roche Molecular Biochemicals (Mannheim, Germany). The QuikChange site-directed mutagenesis kit and rat liver genomic library in {lambda}DASH were purchased from Stratagene (La Jolla, CA). Superscript II reverse transcriptase, Lipofectamine Plus, Schneider’s medium, and cloning vector pSPORT1 were purchased from Invitrogen (Carlsbad, CA). [{gamma}-32P]ATP (111 TBq/mmol), [{alpha}-32P]deoxy-CTP (111 TBq/mmol) and [35S]CTP (46.2 TBq/mmol) were obtained from NEN Life Science Products (Wilmington, DE). A protein assay kit was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). Anti-Sp1 (SC-420X) and anti-Sp3 (SC-644X) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Rat granulosa cell culture
Granulosa cells were obtained from immature, kwl:Wistar female rats (21-d-old) that received an injection of 2 mg DES in 0.2 ml sesame oil once daily for 4 d. The ovaries were then excised, and granulosa cells were released by puncturing follicles with a 26-gauge needle. At all times, the animals were treated according to NIH guidelines. Granulosa cells were washed and collected by brief centrifugation, and cell viability was determined by trypan blue exclusion. The granulosa cells were then cultured in Ham’s F-12/DMEM (vol/vol) supplemented with 1.1 g/liter NaHCO3, 50 mg/liter gentamycin sulfate, and 0.1% BSA on collagen-coated plates in a humidified atmosphere containing 5% CO2 in 95% air at 37 C (18).

Northern blot analysis
Granulosa cells (5 x 106 cells) were cultured in 60-mm dishes in 5 ml medium, and ovine FSH (30 ng/ml) was added to the medium after 24 h of the cell culture. The cultures were stopped at various time intervals. Similarly, various doses of FSH were added to the culture, and cells were processed 2 h after the addition of FSH. For the in vivo study 21-d-old rats were primed with 30 IU PMSG (19), and ovaries were removed at various time intervals. Total RNA was isolated by the acid guanidium thiocyanate extraction method (20). For Northern blot analysis, 10 µg total RNA from each sample was separated by electrophoresis on denaturing agarose gels and subsequently transferred to a nylon membrane (Biodyne, ICN Biomedicals, Inc., Glen Cove, NY). A 510-bp cDNA fragment containing entire cording region of rat epiregulin was radiolabeled by the random primer labeling method and used as a probe for RNA blot hybridization. Autoradiographic bands were quantified by a fluoroimage analyzer (BAS2000, Fuji Photo Film Co., Ltd., Tokyo, Japan). The blots were striped and rehybridized with a randomly radiolabeled probe for rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to normalize for equivalent loading of RNA.

In situ hybridization
In situ hybridization was performed as described by Mori et al. (21). Rat ovaries were embedded in a matrix and frozen in dry ice. Fourteen-micron-thick sections were cut by a cryostat and mounted on 3-amino-propyl tri-ethoxy silane-coated glass slides for in situ hybridization. A 510-bp cDNA fragment containing the entire cording region of rat epiregulin was subcloned into the pSPORT1 vector. Antisense or sense [35S]CTP-labeled RNA probes were synthesized using T7 or SP6 RNA polymerase. The samples were hybridized and washed at high stringency and autoradiographed with the emulsion of NTB2 (Eastman Kodak Co., Rochester, NY). All slides were counterstained with hematoxylin, dehydrated, and mounted.

Genomic library screening
Recombinants (n = 250,000) from a rat liver genomic library constructed in {lambda}DASH were screened with a randomly labeled 510-bp fragment of rat epiregulin cDNA by a plaque-hybridization procedure. Two positive clones were plaque-purified and digested with NotI to obtain rat epiregulin gene fragments. After digestion, these fragments were subcloned into pSPORT1. The nucleotide sequences of the two clones were determined using an automated DNA sequencer (model 377, PE Applied Biosystems).

Primer extension analysis
Primer extension analysis was carried out as described by Ghosh et al. (22) using an oligonucleotide with a sequence found in exon 1 (5'-AACCGGGCTGATCCAGAGACCAGGG-3'). The primer was end-labeled with T4 polynucleotide kinase and [{gamma}-32P]ATP at 37 C for 30 min. Poly(A)+ RNA was prepared by using Oligotex dT-30 super from total RNA from immature rat ovaries primed with 30 IU PMSG for 24 h or from normal immature rat ovaries (20). The 32P-labeled primer (200,000 cpm) was mixed with or without poly(A)+ RNA from the ovaries, in 20 µl hybridization buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 250 mM KCl] and denatured at 65 C for 1 h, after which the mixture was slowly cooled to room temperature. After the addition of 80 µl RT reaction mixture [25 mM Tris-HCl (pH 8.0), 90 mM KCl, 12 mM MgCl2, 12 mM dithiothreitol, 0.6 mM each deoxy-NTP, 0.2 mM spermidine, and 400 U Superscript II reverse transcriptase], the reaction was continued at 37 C for 1 h. The extended transcripts were recovered after RNA digestion with ribonuclease A, phenol extraction, and ethanol precipitation and were resolved on an 8% denaturing PAGE. M13mp18 DNA ladders were coelectrophoresed in adjacent lanes as size markers, and the resulting gel was then dried and autoradiographed.

Plasmids
Fragments of rat epiregulin promoter region, -3858/+12, -1825/+12, -1286/+12, -947/+12, -583/+12, -268/+12, -125/+12, -110/+12, and -42/+12 were inserted into a pGL3-Basic luciferase vector to obtain reporter constructs, pEpi3858, pEpi1825, pEpi1286, pEpi947, pEpi583, pEpi268, pEpi125, pEpi110, and pEpi42, respectively. The numbering of the nucleotides is relative to the transcription start site (+1). To generate a reporter plasmid with a mutation in box A, synthesized oligonucleotides 5'-CGCCACCACTCGAATTCCC-3' and 5'-GGGAATTCGAGTGGTGGCGGTAC-3' were annealed and inserted into a wild-type reporter plasmid lacking box A. Constructs having mutations in boxes B, C, AB, AC, BC, and ABC were prepared using a QuikChange site-directed mutagenesis kit. Primer pairs of (5'-CTCCCCCCGGGGGTTTGCATTAGGAAAC-3' and 5'-GTTTCCTAATGCAAACCCCCGGGGGGAG-3'), and (5'-CGTGCCTAAGCACGAATTCAGACTTATAAAGG-3' and 5'-CCTTTATAAGTCTGAATTCGTGCTTAGGCACG-3') were used to generate mutations in boxes B and C, respectively. pGM4 vector, a Rous sarcoma virus promoter-directed expression vector, was a gift from Drs. Paolo Monaci and Alfredo Nicosia (Instituto di Richerche di Biologia Moleculare, Rome, Italy). pPac, pPac-Sp1, pPac-Sp3, and pPac-USp3 vectors were gifts from Dr. Guntram Suske (Philipps Universität, Marburg, Germany). pPac-ß-galactosidase was a gift from Dr. Timothy F. Osborne (University of California, Irvine, CA). A 2.3-kb BamHI fragment of pPac-Sp3 was isolated and subcloned into the BamHI site of pGM4 to produce an Sp3 expression vector (pRSV-Sp3). pRSV-Sp1 was a gift from Dr. Yosiaki Fujii-Kuriyama (Tohoku University, Tohoku, Japan) (23). The nucleotide sequences of all the constructs were confirmed by DNA sequencing.

Oligonucleotides
Oligonucleotides used for EMSA studies are listed in Table 2Go. These oligonucleotides were annealed with their complementary oligonucleotides to produce double-stranded DNAs.


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  		Table 2. Nucleotide sequences of oligonucleotides used in EMSAs

 
Cell culture, transient transfection, and luciferase assay
All plasmids used for transfection were prepared with the QIAGEN plasmid kit, followed by CsCl gradient ultracentrifugation. Rat granulosa cells were cultured as described previously in 48-well plates at 1.25 x 105 cells/well. Each reporter plasmid and pRL Renilla luciferase control vector (for normalization) were mixed with 0.37 µl FuGENE 6, and the resulting mixture was added to the cells. After 44 h of transfection, cells were treated with or without ovine FSH (30 ng/ml), and the culture was continued for 4 h before harvesting cells.

NIH-3T3 cells were maintained in DMEM with 10% fetal bovine serum and antibiotics. Cells were dispensed into 24-well plates at 2.5 x 104 cells/well. DNA samples that contained each reporter plasmid and pRL Renilla luciferase control vector (for normalization) with or without an expression plasmid (pRSV-Sp1 and pRSV-Sp3) were transfected using Lipofectamine Plus. The total amount of DNA (µg) was adjusted by adding the pRSV-neo plasmid if any. Cells were harvested 48 h after transfection. Luciferase activity was determined using a dual luciferase reporter assay system. Measurements were made using a Lumat LB9501 luminometer (Berthold, Wildbad, Germany) in a single tube, with the first assay from firefly luciferase, followed by the Renilla luciferase assay. Firefly luciferase activities (relative light units) were normalized by Renilla luciferase activities.

Schneider line 2 (SL2) cells, a Drosophila cell line, were a gift from Dr. Tamio Noguchi (Nagoya University, Nagoya, Japan). SL2 cells were grown in Schneider’s medium supplemented with 10% fetal bovine serum at 25 C. Transfection to SL2 cells was carried out by a calcium-phosphate method (24). Cells were plated at 1 x 106 cells/60-mm dish on d 0. On d 1 the cells were transfected with 2 µg luciferase reporter plasmid, 100 ng pPac-Sp1 or pPac-USp3, and 100 ng pPac-ß-galactosidase control vector (for normalization). The total DNA amount was adjusted by the addition of the pPac plasmid. The culture medium was not changed before or after the addition of DNA. Cells were harvested 48 h after the transfection, and firefly luciferase and ß-galactosidase activities were determined. ß-Galactosidase assays were performed using a standard colorimetric procedure with o-nitrophenyl-D-galactoside (25).

Isolation of nuclei and preparation of nuclear extracts
In this experiment we used an in vivo stimulation protocol to mimic the in vitro culture system of undifferentiated granulosa cells. Immature Wistar rats that had received an injection of DES daily for 4 d were primed with PMSG for 4 h, and then ovaries were collected. Epiregulin gene expression in the ovaries under such conditions was confirmed by RT-PCR (see Fig. 8AGo). Cell nuclei were isolated by the method of Hagenbuchle and Wellauer (26) with minor modifications (27). All operations described below were carried out at 4 C. Ovaries were homogenized in a glass Dounce homogenizer (Kontes Co., Vineland, NJ) in 5 vol buffer A [15 mM HEPES/KOH (pH 7.8), 60 mM KCl, 15 mM NaCl, 14 mM 2-mercaptoethanol, 0.15 mM spermine, 0.5 mM spermidine, 1 mM phenylmethylsulfonylfluoride, and 0.2% Nonidet P-40] containing 0.3 M sucrose. The homogenate was layered on top of a cushion of 0.9 M sucrose in buffer A and centrifuged at 2,500 x g for 10 min at 4 C. The precipitated crude nuclei were resuspended in buffer A containing 0.9 M sucrose and recentrifuged. The resulting pellet was resuspended in buffer A and centrifuged again at 2,500 x g for 10 min at 4 C. The preparation of nuclear extracts was performed by the method reported by Frain et al. (28) with minor modifications. Briefly, pure nuclei were resuspended in 5 vol buffer B [20 mM HEPES/NaOH (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride, and 10% glycerol] using a Dounce homogenizer, gently stirred with a magnetic stirrer on ice for 45 min, and then centrifuged at 27,000 x g for 15 min. Ten percent Nonidet P-40 (0.01 vol) was added to the supernatant, and the resulting suspension was gently stirred on ice for 10 min and centrifuged at 27,000 x g for 5 min. The clear supernatant was dialyzed twice for 2 h each time against 50 vol buffer C [25 mM Tris/HCl (pH 8.0), 1 mM EDTA, 5 mM MgCl2, 0.1% Nonidet P-40, 1 mM dithiothreitol, and 10% glycerol] containing 0.1 M KCl. The dialysate was centrifuged at 27,000 x g for 10 min at 4 C to remove the insoluble materials. Protein concentrations of the supernatant were determined using a protein assay kit (Bio-Rad Laboratories, Inc.).



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  		Figure 8. EMSA analysis of boxes A and C within the 125 bp upstream of the rat epiregulin gene. A, Epiregulin gene expression in ovaries treated with DES once daily for 4 d and with 30 IU PMSG for the indicated times was analyzed by RT-PCR to confirm the gene expression in the ovaries from which nuclear extracts were prepared. P3, PMSG treatment for 3 h; P6, PMSG treatment for 6 h; NC, negative control. End-labeled box A and C oligonucleotides were incubated with 10 µg nuclear extracts from DES-pretreated and PMSG-primed immature rat ovaries. B, Unlabeled oligonucleotides (200-fold excess) were used as competitor DNAs. Protein-DNA complexes were separated by a 6% PAGE and subjected to autoradiography. The arrows on the left indicate the positions of the protein-DNA complexes. C, The nuclear extracts were preincubated with antibodies directed against Sp1 and/or Sp3 for 30 min before addition of the probes. Protein-DNA complexes were separated by 6% PAGE and subjected to autoradiography. The arrows on the right represent the positions of supershifted bands (SS). D, For control experiments, the nuclear extracts were preincubated with an anti-GST antibody for 30 min before addition of the probes. E, Nuclear extracts were prepared from ovaries primed with or without 30 IU PMSG. The box C probe and a consensus E box probe were labeled and used for EMSA experiments.

 
EMSAs
A nuclear extract (10 µg proteins) was incubated for 30 min with a 32P-labeled oligonucleotide (0.1 ng) and unlabeled poly(dA-dT) or poly(dI-dC) in buffer C. In the competition experiments a 200-fold molar excess of unlabeled competitor DNAs was added. A supershift assay was carried out by preincubating the nuclear extracts for 30 min with 3.5 µl anti-Sp1 or anti-Sp3 antibody. An anti-glutathione-S-transferase (anti-GST) antibody was also used for control experiments. After the binding reaction, the mixture was subjected to 6% PAGE in 45 mM Tris-HCl, 45 mM boric acid, and 1 mM EDTA at 200 V for 1 h, and then the gel was dried and autoradiographed. Autoradiographic bands were detected by a fluoroimage analyzer (BAS2000, Fuji Photo Film Co., Ltd.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We have previously isolated hundreds of clones of FSH-inducible genes from a rat ovarian granulosa cell cDNA library by subtraction cloning (6). Among these clones we found a cDNA clone that encodes a growth factor of EGF family, epiregulin. To confirm the induction of the gene in the cultured rat granulosa cells by FSH, Northern blot analysis was performed. As shown in Fig. 1AGo, a major transcript of 4.6 kb was detected 1 h after treatment with 30 ng/ml FSH, whereas no expression of epiregulin was detected in untreated cells. Maximal induction of the gene was observed 2 h after FSH treatment, and then the levels rapidly decreased to low levels 4 h after treatment. This indicates rapid, strong, and transient induction of the epiregulin gene expression in ovarian granulosa cells. Figure 1BGo showed a dose-dependent induction of the gene by FSH. Forskolin (10 µM) also induced the expression of epiregulin gene, indicating that the induction is through the protein kinase A pathway (Fig. 1CGo).



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  		Figure 1. Northern blot analysis of rat epiregulin mRNA in cultured rat granulosa cells. A, Total RNA was isolated from the cells at the indicated times after the addition of FSH (30 ng/ml). B, Total RNA was isolated from the cells 2 h after FSH treatment at various concentrations. Each 10 µg total RNA were loaded on lanes. After electrophoresis, RNA was transferred to nylon membranes, which were hybridized with 32P-labeled cDNAs of epiregulin or GAPDH. C, Total RNA was isolated from cells at the indicated times after the addition of 10 µM forskolin.

 
As shown in Fig. 2AGo, epiregulin gene expression in vivo was also confirmed. Although epiregulin gene expression was not observed in untreated 21-d-old rat ovaries, the expression was evident 24 h after treatment of rats with PMSG. In situ hybridization study revealed that the expression of epiregulin 24 h after PMSG treatment was clearly demonstrated in the granulosa cells of large antral follicles (Fig. 2BGo).



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  		Figure 2. In situ hybridization of epiregulin in PMSG-primed immature rat ovaries. A, Induction of epiregulin gene expression in ovaries with 30 IU PMSG. Total RNA was isolated from the ovaries at the indicated times. B, In situ hybridization of epiregulin in immature rat ovaries. Ovaries from 21-d-old rats were dissected, sectioned, and hybridized with 35S-labeled antisense (a, b, e, and f) or sense (c and d) epiregulin probes. Sections of ovaries from 21-d-old immature rats were primed as follows: a, b, c, and d, with PMSG (30 IU) for 24 h (magnification, x50); and e and f, with no treatment (magnification, x50). Brightfield images (a, c, and e) and darkfield images hybridized with antisense or sense epiregulin probes on the same section (b, d, and f, respectively) are shown. The scale bar for 250 µm is indicated.

 
Next we isolated rat genomic clones encoding epiregulin gene. After screening of 250,000 recombinant clones from Wistar rat liver genomic library with a 32P-labeled rat epiregulin cDNA probe, four genomic clones were obtained. Nucleotide sequences of two independent clones were determined, and the determined sequences were compared with rat epiregulin cDNA sequence. As a result, the genomic structure of the rat epiregulin gene, including the upstream region, was determined as schematically illustrated in Fig. 3Go. The rat epiregulin gene is composed of about 22.1-kb nucleotides with five exons and four introns. All exon-intron boundaries showed conserved sequences of splice donor-acceptor site as shown in Table 1Go (29, 30). The rat epiregulin gene sequence was deposited to a DNA data bank, DDBJ (AB078739).



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  		Figure 3. The genomic structure of the rat epiregulin gene is schematically drawn. Exons are represented by boxes. and {square}, Coding and noncording regions, respectively. The restriction enzymes used are indicated as follows: B, BglII; P, PstI; X, XbaI. The scale bar for 1 kb is indicated.

 

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  		Table 1. Characteristics of the exon-intron junctions in the gene for rat epiregulin

 
To determine the transcriptional start site of rat epiregulin gene, a primer extension analysis was performed. An end-labeled oligonucleotide primer that is complementary to the 5'-untranslated region of rat epiregulin mRNA was prepared. Poly(A)+ RNA from immature rat ovaries stimulated with 30 IU PMSG for 24 h was used for the extension reaction. As shown in Fig. 4Go, a radiolabeled product of 62 bp was detected in samples containing the RNA from PMSG-stimulated ovaries, where strong epiregulin gene expression was observed, whereas no product was detected in samples containing RNA from untreated ovaries. The primer extension study indicates that a start site for the transcription is located 131 bp upstream of the translation start site.



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  		Figure 4. Determination of the transcription start site by primer extension analysis. The 32P-labeled primer (200,000 cpm) was mixed with 0.2 µg (lane 1) or 1.4 µg (lane 2) poly(A)+ RNA prepared from immature rat ovaries. Lanes 3 and 4 contain the 32P-labeled primer (200,000 cpm) and 0.2 µg (lane 3) or 1.4 µg (lane 4) poly(A)+RNA prepared from immature rat ovaries primed with 30 IU PMSG for 24 h. The primer was extended with 400 U reverse transcriptase at 37 C for 1 h. Extension products were resolved on an 8% denaturing polyacrylamide gel. A dideoxy sequence ladder is also shown next to the extension products for size determination.

 
As a first step in determining the sequence required for transcriptional activity of rat epiregulin, a series of plasmids was prepared. DNA fragments of -3858/+12 (pEpi3858), -1825/+12(pEpi1825), -1286/+12(pEpi1286), -947/+12-(pEpi947), -583/+12(pEpi583), -268/+12(pEpi268), -125/ +12(pEpi125), -110/+12(pEpi110), and -42/+12(pEpi42) were placed upstream of the luciferase reporter gene, respectively. These plasmids were transiently transfected into cultured rat ovarian granulosa cells, and reporter activities were determined. As shown in Fig. 5AGo, the pEpi125 construct had stronger promoter activity than the other longer promoter constructs. This may correspond to the presence of a weak suppressor activity between -3858 and -125. Figure 5AGo also shows that deletion of -125 to -42 completely abolished the promoter activity. These data suggest that the region of 125 bp upstream of transcription start site may be important for the positive regulation of rat epiregulin gene expression. The ratios of luciferase activities (FSH stimulated/unstimulated) were almost constant regardless of the length of the 5'-upstream of epiregulin gene in the reporter constructs.



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  		Figure 5. Analysis of the rat epiregulin promoter region. A, Deletion analysis of rat epiregulin gene promoter region. Constructs used in this experiment are schematically drawn and were prepared as described in Materials and Methods. Cultured granulosa cells were transiently transfected with 0.1 µg reporter plasmids along with 0.001 µg pRL Renilla luciferase vector. Cells were treated with () or without ({square}) 30 ng/ml FSH. Each value represents the mean and SE of three independent transfection experiments. B, A nucleotide sequence of promoter region of rat epiregulin. The transcription start site is indicated by an arrow, a potential TATA box is underlined, and one GT and two CT boxes are enclosed. The GT box is designated box B; the two CT boxes are boxes A and C, respectively. C, Effects of mutations in boxes A, B, and C within the promoter region of the rat epiregulin gene. The mutant promoter constructs used are schematically drawn. The pEpi125 construct was used as a wild-type promoter. Cultured granulosa cells were transiently transfected with 0.1 µg reporter plasmids along with 0.001 µg pRL Renilla luciferase control vector. Cells were treated with () or without ({square}) 30 ng/ml FSH. pRL Renilla luciferase activity was measured for normalization. Each value represents the mean and SE of three independent transfection experiments.

 
As shown in Fig. 5Go, there are two CT boxes and one GT box within the 125-bp upstream promoter region. Those are designated boxes A, B, and C as schematically drawn in Fig. 5BGo. Those CT and GT boxes are compatible with putative Sp1/Sp3 binding sites (31, 32). As shown in Fig. 5CGo, mutations in any one of these boxes resulted in a decrease in promoter activity (38–68% relative to the wild-type promoter pEpi125). Introduction of double mutations caused a further decrease in activity to 22–42% of the wild-type activity. A construct with mutations in all boxes showed no activity. These results suggest that these CT and GT boxes are required for rat epiregulin gene expression.

As these CT and GT boxes are putative Sp1/Sp3-binding sites, we next examined whether Sp1 and Sp3 are involved in the regulation of epiregulin gene expression. Drosophila SL2 cells, which lack endogenous Sp family proteins (33), were transiently cotransfected with the luciferase reporter plasmid pEpi125 and expression vectors for Sp1 and Sp3. As shown in Fig. 6AGo, cotransfection of the Sp1 expression vector resulted in a marked increase in epiregulin promoter activity (90-fold). The increment in promoter activity caused by Sp1 was practically negligible when the ABC mut (mutations in all boxes) reporter construct was used. An expression vector of Sp3 showed similar, but weaker, effects on epiregulin promoter activity. Figure 6BGo showed that Sp3 increased reporter activity by 7.9-fold and was also not effective on the ABCmut reporter construct. We also examined synergistic effects of Sp1 and Sp3. When a constant amount of the Sp3 vectors was expressed with increasing amount of Sp1 vector, an additive increase in luciferase activity was observed. However, when a constant amount of the Sp1 vector was expressed with an increased amount of Sp3 vector, we could not confirm the additive effects (data not shown). This may reflect the fact that Sp1 has much stronger effects than Sp3 on the regulation of epiregulin gene expression. These data indicate that Sp family proteins positively regulate epiregulin promoter activity.



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  		Figure 6. Effects of Sp1 and Sp3 on the promoter activity of the rat epiregulin gene in SL2 cells. SL2 cells that lack endogenous Sp families were transiently transfected with 2 µg reporter plasmids along with 0.1 µg pPac-ß-galactosidase control vector. A, The cells were simultaneously transfected with 0.1 µg pPac-Sp1 plasmid that can express Sp1 or with control pPac plasmid in SL2 cells. B, The cells were simultaneously transfected with 0.1 µg pPac-USp3 plasmid that can express Sp3 or with empty pPac plasmid in SL2 cells. The ß-galactosidase activity of a pPac-ß-galactosidase control vector was also measured for normalization. Each value represents the mean and SE of three independent transfection experiments.

 
We next examined the effects of Sp1 and Sp3 on epiregulin promoter activity in mammalian cells. NIH-3T3 cells were used for the experiments, because the cells also express the epiregulin gene. Epiregulin reporter plasmids were cotransfected with the Sp1 and Sp3 expression vectors in NIH-3T3 cells. As shown in Fig. 7Go, A and B, cotransfection of Sp1 and Sp3 expression vectors increased epiregulin promoter activity by about 5- and 2-fold, respectively. In contrast, neither Sp1 nor Sp3 was effective when the ABC mut reporter construct was used for cotransfection. This also indicates that Sp1 and Sp3 work positively on epiregulin promoter activity in mammalian cells.



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  		Figure 7. Effects of Sp1 and Sp3 on the promoter activity of the rat epiregulin gene in NIH-3T3 cells. NIH-3T3 cells were transiently transfected with 0.1 µg reporter plasmids along with 0.001 µg pRL Renilla luciferase control vector. A, The indicated amount of Rous sarcoma virus promoter-directed Sp1 expression vector was simultaneously transfected into the cells. B, The indicated amount of RSV promoter-directed Sp3 expression vector was simultaneously transfected into the cells. A pRSV-neo empty vector was added to some samples for adjusting the amount of DNA. The pRL Renilla luciferase activity was also measured for normalization. Each value represents the mean and SE of three independent transfection experiments.

 
To examine endogenous nuclear proteins that bind to these boxes, EMSAs were performed using nuclear extracts from immature rat ovaries primed with DES/PMSG. The expression of the epiregulin gene in the ovary under such stimulatory conditions was confirmed by RT-PCR (Fig. 8AGo). Oligonucleotides used in the experiments were shown in Table 2Go. Radiolabeled oligonucleotides of boxes A, B, and C were mixed with the nuclear extracts with or without excess unlabeled oligonucleotides. With respect to box B, we could not confirm that any specific complex was formed (data not shown). As shown in Fig. 8BGo, two major complexes were formed. The formation of the complexes was prevented by the addition of an excess of the unlabeled homologous probe as well as an unlabeled oligonucleotide with a Sp1 consensus sequence (34). On the other hand, a 200-fold excess of unlabeled probes with mutations in each box had no effect on the prevention of the complex formation. These results suggest that Sp1 or those closely related to Sp1 in the ovarian nuclear extracts bind to the CT boxes.

We further confirmed that Sp family members in the ovarian extracts are actually involved in the binding to these boxes by using specific antibodies against Sp1 and Sp3. As shown in Fig. 8CGo, incubation of the nuclear extracts with box A or C in the presence of the anti-Sp1 antibody resulted in a supershift of the complex bands. The effects of anti-Sp3 antibody on complex formation were similar, but less evident, compared with those of anti-Sp1 antibody. The simultaneous addition of anti-Sp1 and anti-Sp3 antibodies caused complete disappearance of the initial complex bands. As shown in Fig. 8DGo, anti-GST antibody had no effect on the migration of the initial bands. In addition, nuclear extracts from ovaries primed with PMSG for 4 h bound slightly more strongly to the box C probe than those from untreated ovaries. Such an increase in binding was not observed when an USF-II consensus (E box) probe was used (Fig. 8EGo).

These data clearly showed that Sp1 and Sp3 in the ovarian extracts were actually bound to these boxes.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Ovarian follicle development is mainly controlled by gonadotropins, LH and FSH, secreted from the pituitary (1, 2). The proliferation and differentiation of granulosa cells induced by FSH are essential for ovarian follicle development. Early responsive genes induced by FSH are supposed to mediate such proliferation and differentiation of granulosa cells in the ovary. In this context, we have previously isolated many rat ovarian genes that are rapidly induced in granulosa cells within 1.5 h by FSH. Epiregulin is one such gene isolated from granulosa cells by subtraction cloning. Epiregulin is a growth factor of the EGF family. Among the growth factor family, EGF and TGF{alpha} are previously reported to be associated with granulosa cell proliferation. It has been shown that TGF{alpha} is expressed in theca interna cells and stimulates DNA synthesis in bovine granulosa cells (15). EGF also increases DNA synthesis moderately in cultured rat granulosa cells (14). Roy and Greenwald (13) reported that in hamster follicle culture EGF and TGF{alpha} significantly stimulated follicular DNA synthesis. These reports all indicate participation of the EGF family in granulosa cell proliferation. However, neither EGF nor TGF{alpha} has been shown to be induced in ovarian cells by gonadotropins, whereas only epiregulin among the EGF family members we have examined, including heparin-binding-EGF, TGF{alpha}, betacellulin, EGF, and heregulin, was rapidly induced in granulosa cells in response to FSH (data not shown). This suggests the physiological importance of epiregulin in ovarian follicular development induced by FSH. Epiregulin was originally identified as a growth inhibitory factor from conditioned medium of the murine tumor cell line NIH-3T3/clone T7 (7, 8). It was subsequently demonstrated to act as a mitogen for various cell types, including fibroblasts and hepatocytes (8). More recent studies have shown that epiregulin expression in vivo is very restricted. Besides its constitutive expression in certain tumor cell lines and developing mouse embryo, significant levels of epiregulin mRNA in human tissues are limited to placenta, macrophages, and, to a lesser extent, heart (7, 17). Epiregulin expression in the ovary was also limited only after stimulation with gonadotropins, as shown in Fig. 1Go. Forskolin also induces epiregulin expression in granulosa cells, suggesting involvement of the protein kinase A pathway in the induction. A recent study has shown that epiregulin gene expression was induced in vascular smooth muscle cells by vasoactive GTP-binding protein-coupled receptor agonists that evoke intracellular second messenger molecules (10). Induction of epiregulin expression in the cardiovascular system, therefore, may represent one mechanism by which GTP-binding protein-coupled receptor agonists stimulate cell proliferation in certain vascular diseases (10). Similarly, FSH is believed to act through a second messenger cAMP, and therefore, rapid induction of epiregulin gene expression by FSH suggests that epiregulin may serve as a physiological mediator of FSH for the proliferation of ovarian granulosa cells.

In vivo expression of the epiregulin gene was also confirmed, and the expression was predominant in granulosa cells of antral follicles (Fig. 2Go).

To analyze the regulation of epiregulin gene expression, we isolated the complete rat epiregulin gene, including 3.8 kb upstream of the gene. The rat epiregulin gene has five exons and four introns, and all exon-intron boundaries have consensus splice donor-acceptor sequences (29, 30). There are three potential polyadenylation signals in the 3'-untranslated region of the fifth exon. As shown in Fig. 1Go, a 4.6-kb transcript was predominant in rat ovarian granulosa cells, indicating usage of the most downstream polyadenylation site. On the other hand, in rat aortic smooth muscle cells, two transcripts of 1.2 and 4.8 kb have been induced in response to vasoactive GTP-binding protein-coupled receptor agonists, suggesting the usage of both the downstream and upstream of these polyadenylation sites in vascular smooth muscle cell (10).

To determine the mechanisms underlining the expression of the epiregulin gene by FSH, we analyzed the functional region of rat epiregulin gene promoter activity. Deletion and mutation analyses of the epiregulin promoter showed that two CT boxes and one GT box are important for FSH activation of reporter genes. Based on EMSAs and supershift assay, our results indicate that Sp1 and Sp3 are transcription factors that contribute to the regulation of epiregulin gene expression. Sp1 has been traditionally characterized as a ubiquitous regulator of basal promoter activity, partly because of its critical role in transcription from TATA-less promoters (32, 35). Sp3 is an Sp family member previously demonstrated to antagonize Sp1 activity by competing for Sp1 binding sites (36). Sp3 has also been demonstrated to activate transcription (34, 37, 38). The results described herein and those of others indicate that Sp1 and/or Sp3 can also function as enhancers, enabling hormone-inducible transcription from TATA box-containing promoters. Bovine cholesterol side-chain cleavage cytochrome P450 (39, 40), the LH receptor (41), and the rat progesterone receptor (42) require Sp1 and/or its binding sites for protein kinase A-mediated induction. The results presented herein extend these observations and provide evidence that Sp1 and/or Sp3 binding to the CT boxes mediates the FSH induction of a specific gene, epiregulin, in granulosa cells.

The known mechanisms by which Sp family members can mediate hormone-regulated expression of genes are complex. Regulation of Sp1 binding by phosphorylation (43), Sp1 inhibitor (44), or competition for binding sites with repressors (45) can also control Sp1 activity. Tissue-specific regulation of Sp1 protein levels may also be critical. In addition, regulated interaction with other factors may be important. Some of the factors with which Sp1 is known to interact include steroidogenic factor-1, p53, signal transducer and activator of transcription-1, GATA-1, activating protein-1, nuclear factor-{kappa}B, and estrogen receptor (46, 47, 48, 49, 50, 51, 52). Although no consensus sequences for these factors known to interact with Sp1 are present in the -125-bp promoter region of rat epiregulin gene, the additional factors or cofactors may bind directly to Sp1 or Sp3 instead of to the DNA. Actually, p53 or glucocorticoid receptor can potentially interact with Sp1 and/or Sp3 in trans (53, 54). Further study is needed to clarify the relationship between the Sp family members and cAMP-dependent activation of rat epiregulin gene.

In conclusion, we demonstrated here for the first time that the EGF-type growth factor epiregulin is rapidly and strongly induced in ovarian granulosa cells by gonadotropin stimulation, and that two CT boxes and one GT box present in the upstream region are essential for the promoter activity of rat epiregulin. We also demonstrated that Sp family members play crucial roles in epiregulin promoter activity through the CT boxes. The restricted and hormonally regulated expression of epiregulin in the rat ovarian granulosa cells may represent the physiological importance of this peptide growth factor in gonadotropin-induced follicular growth and maturation.


   Acknowledgments
 
We thank the National Pituitary and Hormone Distribution Program of the NIDDK for ovine FSH. We thank Dr. Yoshifumi Yokota (Fukui Medical University, Fukui, Japan) for advice about the performance of in situ hybridization. We are grateful to Dr. Paolo Monaci, Alfredo Nicosia, Guntram Suske, Timothy F. Osborne, and Yosiaki Fujii-Kuriyama for providing plasmids; to Dr. Tamio Noguchi for the generous gift of SL2 cells; and to Ms. Yoshiko Inoue for secretarial assistance.


   Footnotes
 
This work was supported in part by a Grant-in-Aid from the Ministry of Science, Culture, Sports, and Education, and a grant from the Smoking Research Foundation. The nucleotide sequence of rat epiregulin gene was submitted to the DNA Data Bank of Japan (accession no. AB078739).

Abbreviations: DES, Diethylstilbestrol; EGF, epidermal growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione-S-transferase; PMSG, pregnant mare’s serum gonadotropin; SL2, Schneider line 2.

Received April 23, 2002.

Accepted for publication August 5, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Richards JS 1980 Maturation of ovarian follicles: actions and interactions of pituitary and ovarian hormones on follicular cell differentiation. Physiol Rev 60:51–89[Free Full Text]
  2. Hirshfield AN 1991 Development of follicles in the mammalian ovary. Int Rev Cytol 124:43–101[Medline]
  3. Zeleznik AJ, Midgley Jr AR, Reichert Jr LE 1974 Granulosa cell maturation in the rat: increased binding of human chorionic gonadotropin following treatment with follicle-stimulating hormone in vivo. Endocrinology 95:818–825[Medline]
  4. Erickson GF, Wang C, Hsueh AJ 1979 FSH induction of functional LH receptors in granulosa cells cultured in a chemically defined medium. Nature 279:336–338[CrossRef][Medline]
  5. Fitzpatrick SL, Richards JS 1991 Regulation of cytochrome P450 aromatase messenger ribonucleic acid and activity by steroids and gonadotropins in rat granulosa cells. Endocrinology 129:1452–1462[Abstract]
  6. Kameda T, Mizutani T, Minegishi T, Ibuki Y, Miyamoto K 1999 Regulation of cAMP responsive element binding modulator isoforms in cultured rat ovarian granulosa cells. Biochim Biophys Acta 1445:31–38[Medline]
  7. Toyoda H, Komurasaki T, Ikeda Y, Yoshimoto M, Morimoto S 1995 Molecular cloning of mouse epiregulin, a novel epidermal growth factor-related protein, expressed in the early stage of development. FEBS Lett 377:403–407[CrossRef][Medline]
  8. Toyoda H, Komurasaki T, Uchida D, Takayama Y, Isobe T, Okuyama T, Hanada K 1995 Epiregulin. A novel epidermal growth factor with mitogenic activity for rat primary hepatocytes. J Biol Chem 270:7495–7500[Abstract/Free Full Text]
  9. Shirakata Y, Komurasaki T, Toyoda H, Hanakawa Y, Yamasaki K, Tokumaru S, Sayama K, Hashimoto K 2000 Epiregulin, a novel member of the epidermal growth factor family, is an autocrine growth factor in normal human keratinocytes. J Biol Chem 275:5748–5753[Abstract/Free Full Text]
  10. Taylor DS, Cheng X, Pawlowski JE, Wallace AR, Ferrer P, Molloy CJ 1999 Epiregulin is a potent vascular smooth muscle cell-derived mitogen induced by angiotensin II, endothelin-1, and thrombin. Proc Natl Acad Sci USA 96:1633–1638[Abstract/Free Full Text]
  11. Robert C, Gagne D, Bousquet D, Barnes FL, Sirard MA 2001 Differential display and suppressive subtractive hybridization used to identify granulosa cell messenger RNA associated with bovine oocyte developmental competence. Biol Reprod 64:1812–1820[Abstract/Free Full Text]
  12. Sasaki E, Arakawa T, Fujiwara Y, Kawada N, Fukuda T, Higuchi K, Komurasaki T, Kobayashi K 1997 Epiregulin stimulates proliferation of rabbit gastric cells in primary culture through autophosphorylation of the epidermal growth factor receptor. Eur J Pharmacol 338:253–258[CrossRef][Medline]
  13. Roy SK, Greenwald GS 1991 Mediation of follicle-stimulating hormone action on follicular deoxyribonucleic acid synthesis by epidermal growth factor. Endocrinology 129:1903–1908[Abstract]
  14. Liang SG, Yano T, Tsutsumi O, Taketani Y 1994 Modulatory role of epidermal growth factor in follicle-stimulating hormone-induced DNA synthesis in cultured rat granulosa cells. Endocr J 41:319–323[Medline]
  15. Skinner MK, Coffey Jr RJ 1988 Regulation of ovarian cell growth through the local production of transforming growth factor-{alpha} by theca cells. Endocrinology 123:2632–2638[Abstract]
  16. Das SK, Das N, Wang J, Lim H, Schryver B, Plowman GD, Dey SK 1997 Expression of betacellulin and epiregulin genes in the mouse uterus temporally by the blastocyst solely at the site of its apposition is coincident with the "window" of implantation. Dev Biol 190:178–190[CrossRef][Medline]
  17. Toyoda H, Komurasaki T, Uchida D, Morimoto S 1997 Distribution of mRNA for human epiregulin, a differentially expressed member of the epidermal growth factor family. Biochem J 326:69–75
  18. Minegishi T, Tano M, Nakamura K, Nakamura M, Igarashi S, Ito I, Shinozaki H, Karino S, Ibuki Y, Miyamoto K 1996 Regulation of follicle-stimulating hormone receptor. Horm Res 46:37–44
  19. Szoltys M, Galas J, Jablonka A, Tabarowski Z 1994 Some morphological and hormonal aspects of ovulation and superovulation in the rat. J Endocrinol 141:91–100[Abstract]
  20. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  21. Mori S, Sugawara S, Kikuchi T, Tanji M, Narumi O, Stoykova A, Nishikawa S I, Yokota Y 1999 The leukemic oncogene tal-2 is expressed in the developing mouse brain. Brain Res Mol Brain Res 64:199–210[Medline]
  22. Ghosh PK, Reddy VB, Piatak M, Lebowitz P, Weissman SM 1980 Determination of RNA sequences by primer directed synthesis and sequencing of their cDNA transcripts. Methods Enzymol 65:580–595[Medline]
  23. Imataka H, Sogawa K, Yasumoto K, Kikuchi Y, Sasano K, Kobayashi A, Hayami M, Fujii-Kuriyama Y 1992 Two regulatory proteins that bind to the basic transcription element (BTE), a GC box sequence in the promoter region of the rat P-4501A1 gene. EMBO J 11:3663–3671[Medline]
  24. Di Nocera PP, Dawid IB 1983 Transient expression of genes introduced into cultured cells of Drosophila. Proc Natl Acad Sci USA 80:7095–7098[Abstract/Free Full Text]
  25. Kikkawa E, Hinata M, Keng VW, Myint Z, Sato A, Yamada K, Tanaka T, Noguchi T 2001 Sp family members stimulate transcription of the hex gene via interactions with GC boxes. J Biochem 130:885–891[Abstract/Free Full Text]
  26. Hagenbuchle O, Wellauer PK 1992 A rapid method for the isolation of DNA-binding proteins from purified nuclei of tissues and cells in culture. Nucleic Acids Res 20:3555–3559[Abstract/Free Full Text]
  27. Yamada K, Tanaka T, Noguchi T 1997 Members of the nuclear factor 1 family and hepatocyte nuclear factor 4 bind to overlapping sequences of the L-II element on the rat pyruvate kinase L gene promoter and regulate its expression. Biochem J 324:917–925
  28. Frain M, Swart G, Monaci P, Nicosia A, Stampfli S, Frank R, Cortese R 1989 The liver-specific transcription factor LF-B1 contains a highly diverged homeobox DNA binding domain. Cell 59:145–157[CrossRef][Medline]
  29. Sharp PA 1981 Speculations on RNA splicing. Cell 23:643–646[CrossRef][Medline]
  30. Mount SM 1982 A catalogue of splice junction sequences. Nucleic Acids Res 10:459–472[Abstract/Free Full Text]
  31. DesJardins E, Hay N 1993 Repeated CT elements bound by zinc finger proteins control the absolute and relative activities of the two principal human c-myc promoters. Mol Cell Biol 13:5710–5724[Abstract/Free Full Text]
  32. Philipsen S, Suske G 1999 A tale of three fingers: the family of mammalian Sp/XKLF transcription factors. Nucleic Acids Res 27:2991–3000[Abstract/Free Full Text]
  33. Courey AJ, Holtzman DA, Jackson SP, Tjian R 1989 Synergistic activation by the glutamine-rich domains of human transcription factor Sp1. Cell 59:827–836[CrossRef][Medline]
  34. Yamada K, Tanaka T, Miyamoto K, Noguchi T 2000 Sp family members and nuclear factor-Y cooperatively stimulate transcription from the rat pyruvate kinase M gene distal promoter region via their direct interactions. J Biol Chem 275:18129–18137[Abstract/Free Full Text]
  35. Lania L, Majello B, De Luca P 1997 Transcriptional regulation by the Sp family proteins. Int J Biochem Cell Biol 29:1313–1323[CrossRef][Medline]
  36. Kingsley C, Winoto A 1992 Cloning of GT box-binding proteins: a novel Sp1 multigene family regulating T-cell receptor gene expression. Mol Cell Biol 12:4251–4261[Abstract/Free Full Text]
  37. Majello B, De Luca P, Lania L 1997 Sp3 is a bifunctional transcription regulator with modular independent activation and repression domains. J Biol Chem 272:4021–4026[Abstract/Free Full Text]
  38. Mizutani T, Yamada K, Minegishi T, Miyamoto K 2000 Transcriptional regulation of rat scavenger receptor class B type I gene. J Biol Chem 275:22512–22519[Abstract/Free Full Text]
  39. Ahlgren R, Simpson ER, Waterman MR, Lund J 1990 Characterization of the promoter/regulatory region of the bovine CYP11A (P-450scc) gene. Basal and cAMP-dependent expression. J Biol Chem 265:3313–3319[Abstract/Free Full Text]
  40. Ahlgren R, Suske G, Waterman MR, Lund J 1999 Role of Sp1 in cAMP-dependent transcriptional regulation of the bovine CYP11A gene. J Biol Chem 274:19422–19428[Abstract/Free Full Text]
  41. Chen S, Shi H, Liu X, Segaloff DL 1999 Multiple elements and protein factors coordinate the basal and cyclic adenosine 3',5'-monophosphate-induced transcription of the lutropin receptor gene in rat granulosa cells. Endocrinology 140:2100–2109[Abstract/Free Full Text]
  42. Park-Sarge OK, Sarge KD 1995 Cis-regulatory elements conferring cyclic 3',5'-adenosine monophosphate responsiveness of the progesterone receptor gene in transfected rat granulosa cells. Endocrinology 136:5430–5437[Abstract]
  43. Leggett RW, Armstrong SA, Barry D, Mueller CR 1995 Sp1 is phosphorylated and its DNA binding activity down-regulated upon terminal differentiation of the liver. J Biol Chem 270:25879–25884[Abstract/Free Full Text]
  44. Chen LI, Nishinaka T, Kwan K, Kitabayashi I, Yokoyama K, Fu YH, Grunwald S, Chiu R 1994 The retinoblastoma gene product RB stimulates Sp1-mediated transcription by liberating Sp1 from a negative regulator. Mol Cell Biol 14:4380–4389[Abstract/Free Full Text]
  45. Hagen G, Muller S, Beato M, Suske G 1994 Sp1-mediated transcriptional activation is repressed by Sp3. EMBO J 13:3843–3851[Medline]
  46. Liu Z, Simpson ER 1997 Steroidogenic factor 1 (SF-1) and SP1 are required for regulation of bovine CYP11A gene expression in bovine luteal cells and adrenal Y1 cells. Mol Endocrinol 11:127–137[Abstract/Free Full Text]
  47. Koutsodontis G, Tentes I, Papakosta P, Moustakas A, Kardassis D 2001 Sp1 plays a critical role in the transcriptional activation of the human cyclin-dependent kinase inhibitor p21(WAF1/Cip1) gene by the p53 tumor suppressor protein. J Biol Chem 276:29116–29125[Abstract/Free Full Text]
  48. Look DC, Pelletier MR, Tidwell RM, Roswit WT, Holtzman MJ 1995 Stat1 depends on transcriptional synergy with Sp1. J Biol Chem 270:30264–30267[Abstract/Free Full Text]
  49. Merika M, Orkin SH 1995 Functional synergy and physical interactions of the erythroid transcription factor GATA-1 with the Kruppel family proteins Sp1 and EKLF. Mol Cell Biol 15:2437–2447[Abstract]
  50. Noti JD, Reinemann BC, Petrus MN 1996 Sp1 binds two sites in the CD11c promoter in vivo specifically in myeloid cells and cooperates with AP1 to activate transcription. Mol Cell Biol 16:2940–2950[Abstract]
  51. Perkins ND, Agranoff AB, Pascal E, Nabel GJ 1994 An interaction between the DNA-binding domains of RelA(p65) and Sp1 mediates human immunodeficiency virus gene activation. Mol Cell Biol 14:6570–6583[Abstract/Free Full Text]
  52. Porter W, Wang F, Wang W, Duan R, Safe S 1996 Role of estrogen receptor/Sp1 complexes in estrogen-induced heat shock protein 27 gene expression. Mol Endocrinol 10:1371–1378[Abstract]
  53. Maiyar AC, Phu PT, Huang AJ, Firestone GL 1997 Repression of glucocorticoid receptor transactivation and DNA binding of a glucocorticoid response element within the serum/glucocorticoid-inducible protein kinase (sgk) gene promoter by the p53 tumor suppressor protein. Mol Endocrinol 11:312–329[Abstract/Free Full Text]
  54. Stocklin E, Wissler M, Gouilleux F, Groner B 1996 Functional interactions between Stat5 and the glucocorticoid receptor. Nature 383:726–728[CrossRef][Medline]



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