Promoter Characterization of the Rat Gene for Ca2+-binding Protein Regucalcin
TRANSCRIPTIONAL REGULATION BY SIGNALING FACTORS*

Tomiyasu Murata and Masayoshi YamaguchiDagger

From the Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka City 422-8526, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

To understand the mechanism underlying the regulation of the Ca2+-binding protein regucalcin gene expression, we characterized the 5'-flanking region of the rat regucalcin gene. The transcriptional start site of the rat regucalcin gene was determined by the cap site hunting method with rat liver cap site cDNA. The 5'-flanking region of the rat regucalcin gene ligated to a luciferase reporter gene possessed functional promoter activity in rat H4-II-E hepatoma cells. 3'- and 5'-deletion analyses indicated the sequence required for basal functional promoter activity of the rat regucalcin gene. The promoter activity of the rat regucalcin gene was enhanced by treatment with Bay K 8644, dibutyryl cAMP, phorbol esters, insulin, and dexamethasone. Using gel mobility shift assays, we found that nuclear proteins from H4-II-E cells specifically bind to the 5'-flanking region of the rat regucalcin gene. Moreover, gel mobility shift assays revealed that Bay K 8644, dibutyryl cAMP, phorbol esters, and insulin stimulated the binding of nuclear factors to the 5'-flanking region of the rat regucalcin gene in H4-II-E cells. These results suggest that Bay K 8644-, dibutyryl cAMP-, phorbol ester-, and insulin-inducible nuclear factors mediate the stimulatory effect of each regulator on promoter activity of the rat regucalcin gene.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Ca2+ plays an important role in the regulation of many cell functions. Ca2+ signals are partly transmitted to intracellular responses, which are mediated through a family of Ca2+-binding proteins (1). We have reported previously that the novel Ca2+-binding protein regucalcin, which differs from calmodulin, is distributed in the cytoplasm of hepatocytes in rats (2). This protein has a reversible effect on the activation and inhibition of various enzymes by Ca2+ in liver cells (2-4). Regucalcin may play a regulatory role in liver cell functions related to Ca2+.

The rat regucalcin gene consists of seven exons and six introns, and several consensus regulatory elements have been found in the 5'-flanking region of the gene (5). The regucalcin gene is localized on the proximal end of rat chromosome Xqll.1-12 (6), and has been demonstrated in human, mouse, bovine, monkey, dog, rabbit, and chicken but not yeast (7). We have previously reported that rat regucalcin mRNA is mainly present in the liver and only to a small extent in the kidney, as assayed by Northern blotting analysis (8), suggesting that it is expressed in a highly tissue-specific manner. In fact, it has been demonstrated that tissue-specific nuclear factor-DNA complexes are formed on the 5'-flanking region of the rat regucalcin gene in gel mobility shift assays (9).

The expression of hepatic regucalcin mRNA is induced by various factors. We have shown that the expression of regucalcin mRNA in the liver is markedly stimulated by administration of CaCl2 to rats; the expression may be mediated through Ca2+/calmodulin (8, 10). With respect to this regulatory mechanism by which Ca2+ administration stimulates the expression of hepatic regucalcin mRNA, it has been demonstrated that Ca2+ administration stimulates the additional binding of AP-1 factor to the 5'-flanking region of the rat regucalcin gene, and that this binding may be mediated through a Ca2+/calmodulin-dependent pathway (11). The expression of hepatic regucalcin mRNA is clearly stimulated by a single subcutaneous administration of insulin to fasted rats (12). In addition, we reported that the expression of regucalcin mRNA in human HepG2 hepatoma cells is stimulated by insulin treatment (13), and that 17beta -estradiol stimulates the expression of hepatic regucalcin mRNA in rats (14). These results suggest that the regucalcin promoter contains sequence elements that are targets for regulatory transcription factors.

Regulation of gene expression at the transcription level is mediated by the interaction of a trans-acting factor with a cis-acting DNA sequence in genes (15). Accordingly, the interaction between a trans-acting regulatory factor and a cis-acting regulatory element may be important for regucalcin gene expression. However, the molecular mechanism of the transcriptional regulation of regucalcin gene has not been clarified. Therefore, identification of basal and regulatory DNA elements in the 5'-flanking region of the rat regucalcin gene will provide important insight into the molecular mechanisms underlying regulation of expression of this gene.

The aim of the present study was to examine the characteristics of the functional promoter of the rat regucalcin gene. To examine regulation of the rat regucalcin gene promoter, chimeric constructs containing serial deletions of the 5'-flanking region of the rat regucalcin gene ligated to the luciferase reporter gene were prepared and transfected into rat H4-II-E hepatoma cells. We have identified the region that plays an important role in determining basal promoter activity of the gene. Moreover, it was found that Bay K 8644, dibutyryl cAMP, phorbol ester, insulin, and dexamethasone response sequences are located within the 5'-flanking region of the rat regucalcin gene. In addition, gel mobility shift assays indicated that Bay K 8644-, dibutyryl cAMP-, phorbol ester-, and insulin-inducible nuclear proteins from H4-II-E cells bind specifically to the 5'-flanking region of the rat regucalcin gene.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- pBluescript II SK+ vector was obtained from Stratagene (La Jolla, CA). Leupeptin, aprotinin, dithiothreitol, phenylmethylsulfonyl fluoride, bovine serum albumin, N6,2'-dibutyryl cyclic adenosine 3',5'-monophosphate (dibutyryl cAMP), phorbol 12-myristate 13-acetate (PMA),1 insulin, and dexamethasone were purchased from Sigma. S(-)-Bay K 8644 was obtained from Research Biochemicals International (Natick, MA). The TA cloning vector pCRII was purchased from Invitrogen (San Diego, CA). pUC18 vector, restriction enzymes, and T4 polynucleotide kinase were obtained from Takara Shuzo Co. (Shiga, Japan). Poly(dI-dC)·(dI-dC) was purchased from Pharmacia Biotech (Uppsala, Sweden). A 100-base pair DNA ladder was obtained from New England Biolabs (Beverly, MA). Adenosine 5'-[gamma -32P]triphosphate ([gamma -32P]ATP; 111 TBq/mmol) was purchased from Amersham (Buckinghamshire, UK). Working stocks of PMA (1 mM), S(-)-Bay K 8644 (5 mM), dexamethasone (2 mM), and estradiol (2 mM) were prepared in dimethyl sulfoxide (Me2SO). Working stocks of dibutyryl cAMP (200 mM) and insulin (20 µM) were freshly prepared in water and phosphate buffer, respectively. pGL3-Basic vector, pRL-TK vector, AP-1 consensus oligonucleotide (5'-CGCTTGATGAGTCAGCCGGAA-3'), and AP-2 consensus oligonucleotide (5'-GATCGAACTGACCGCCCGCGGCCCGT-3') were obtained from Promega (Madison, WI). Fetal bovine serum, alpha -minimum essential medium (alpha -MEM), penicillin, and streptomycin were purchased from Life Technologies, Inc. Reagents (analytical grade) were obtained from Sigma and Wako Pure Chemical Co. (Osaka, Japan).

Determination of the Transcriptional Start Site-- The transcriptional start site of the rat regucalcin gene was determined by the cap site hunting method with rat liver cap site cDNA (Nippon Gene, Toyama, Japan) according to the manufacturer's instructions. This method consists of removing the cap with tobacco acid pyrophosphatase and ligating r-oligos to decapped mRNAs with T4 RNA ligase. This reaction was made cap-specific by removing 5'-phosphates of non-capped RNAs with alkaline phosphatase prior to tobacco acid pyrophosphatase treatment. Unlike the conventional methods that label the 5' end of cDNAs, this method specifically labels the capped ends of mRNAs with a synthetic r-oligo prior to first-strand cDNA synthesis. The linked mRNA was used as template to synthesize the first-strand cDNA by reverse transcriptase in the presence of random primers. The cap site region of the mRNA was identified simply by reverse transcription-polymerase chain reaction (PCR).

The first round of PCR was performed using a sense DNA primer complementary to r-oligo (1RC, 5'-CAAGGTACGCCACAGCGTATG-3', provided by the supplier) paired with the gene-specific antisense primer 3 (GSAP3, 5'-CACCAACTCGCTGCACTCGATTG-3', see Fig. 2). Samples were amplified for 35 cycles under the following conditions: denaturation for 30 s at 94 °C, annealing for 1 min at 55 °C, and extension for 1 min at 72 °C. Aliquots of the first PCR reaction were used as the template in the second round of PCR reaction (nested PCR). Nested PCR was performed using a nested sense DNA primer complementary to r-oligo (2RC, 5'-GTACGCCACAGCGTATGATGC-3', provided by the supplier) paired with the nested gene-specific antisense primer 1 or 2 (GSAP1, 5'-TTGAAGGGATGTCTACAAACAGCA-3; GSAP2, 5'-GATCGAATCCCATCGGCAGACAG-3', see Fig. 2). Samples were amplified for 20 cycles under the following conditions: denaturation for 30 s at 94 °C, annealing for 1 min at 55 °C, and extension for 1 min at 72 °C. The PCR products were excised from the 2% low melting temperature agarose gel, cloned into TA vector, and sequenced using a DNA sequencer (Applied Biosystems Inc.).

Construction of the Reporter Gene Plasmid-- The reporter gene plasmids were generated by cloning restriction fragments isolated from the 5'-flanking region of the rat regucalcin gene. PCR was performed using pBluescript SK+ containing the 5.5-kilobase pair EcoRI-XhoI fragment of genomic lambda RCB2 (5) as the substrate to obtain DNA fragments -710/+157, -710/+18, -710/-223, -710/-343, -582/+157, -462/+157, -342/+157, -222/+157, and -102/+157 using the following primer pairs: fragment -710/+157, 5'-ACAGGTACCGAATTCCTGACTGATCTTT-3' and 5'-ACACTCGAGAAGAAAGAGCTGATAAGAC-3'; fragment -710/+18, 5'-ACAGGTACCGAATTCCTGACTGATCTTT-3' and 5'-ACACTCGAGGGTTGTAATGACTCCTGGC-3'; fragment -710/-223, 5'- ACAGGTACCGAATTCCTGACTGATCTTT-3' and 5'-ACACTCGAGGTATATGGCTGAGGTTGAA-3'; fragment -710/-343, 5'-ACAGGTACCGAATTCCTGACTGATCTTT-3' and 5'-ACACTCGAGGAAGGGCAATTTCCCTGGG-3'; fragment -582/+157, 5'-ACAGGTACCCCAGTTCACTGGTCTTTGG-3' and 5'-ACACTCGAGAAGAAAGAGCTGATAAGAC-3'; fragment -462/+157, 5'-ACAGGTACCTCATCCACTGCAGTGGAGC-3' and 5'-ACACTCGAGAAGAAAGAGCTGATAAGAC-3'; fragment -342/+157, 5'-ACAGGTACCACACCTGCCATTGTCCGAA-3' and 5'-ACACTCGAGAAGAAAGAGCTGATAAGAC-3'; fragment -222/+157, 5'-ACAGGTACCCAAGCCTCTGGCTGTTAAC-3' and 5'-ACACTCGAGAAGAAAGAGCTGATAAGAC-3'; and fragment -102/+157, 5'-ACAGGTACCGGGTAACCTGCAGACACCC-3' and 5'-ACACTCGAGAAGAAAGAGCTGATAAGAC-3'. Samples were amplified for 30 cycles under the following conditions: denaturation for 1 min at 94 °C, annealing for 1 min at 55 °C, and extension for 1 min at 72 °C. A DNA fragment was then separated by electrophoresis on a 2% low melting temperature agarose gel, cloned into TA vector, and sequenced using a DNA sequencer (Applied Biosystems Inc.). The DNA fragments -710/+157, -710/+18, -710/-223, -710/-343, -582/+157, -462/+157, -342/+157, -222/+157, and -102/+157 were prepared from each vector by KpnI/XhoI restriction digestion. A series of DNA fragments with different 3' and 5' ends were cloned into the pGL3-Basic promoterless plasmid containing the firefly luciferase gene.

Cell Culture and Transfection-- Rat H4-II-E hepatoma cells were maintained in alpha -MEM supplemented with 5 mM glucose, 10% heat-inactivated fetal bovine serum, 50 units/ml penicillin, and 50 µg/ml streptomycin in humidified 5% CO2, 95% air at 37 °C. For the transfection experiments, the cells were grown on 15-mm dishes to approximately 70% confluence and washed once with serum-free alpha -MEM. Either 2 µg of pGL3-Basic plasmid or an equivalent molar amount of test plasmid was co-transfected into H4-II-E cells along with 0.5 µg of pRL-TK plasmid using the synthetic cationic lipid component, Tfx-20 reagent, according to the manufacturer's instructions (Promega). The pRL-TK vector containing the Renilla luciferase gene under control of the herpes simplex virus thymidine kinase promoter (Promega) was used as an internal control for differences in transfection efficiency and cell number. For functional analysis of the basal promoter region of the rat regucalcin gene, the transfected cells were maintained for 48 h in serum-supplemented medium before harvesting. For analysis of regulation of the regucalcin promoter by signaling factors, the transfected cells were maintained for 24 h in serum-supplemented medium and preincubated for 14 h in serum-free alpha -MEM supplemented with 0.1% bovine serum albumin, 50 units/ml penicillin, and 50 µg/ml streptomycin. After preincubation, the transfectants were incubated for 16-20 h in the same medium supplemented with or without Bay K 8644 (2.5 µM), dibutyryl cAMP (0.5 mM), PMA (1 µM), insulin (10 nM), dexamethasone (1 µM), and estradiol (1 µM) before harvesting. At the end of the culture period, the transfectants were lysed, and the luciferase activity in the cell lysates was measured by dual-luciferase reporter assay system (Promega).

Preparation of Nuclear Extracts-- All steps were carried out at 4 °C or on ice. The cells were grown on 35-mm dishes to approximately 80% confluence, washed twice with ice-cold phosphate-buffered saline, and harvested by scraping into 1 ml of ice-cold phosphate-buffered saline containing 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and 2 µg/ml aprotinin. The cells were pelleted by centrifugation at 500 × g for 5 min, gently resuspended in 1 ml of hypotonic buffer (10 mM HEPES-NaOH, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and 2 µg/ml aprotinin), and incubated on ice for 10 min. The cells were lysed by homogenization, and the nuclei were collected by centrifugation for 5 min at 1000 × g. Nuclear extracts were prepared by a modification of the method of Dignam et al. (16). The nuclei were resuspended in 150 µl of ice-cold extraction buffer (10 mM HEPES-NaOH, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EGTA, 0.5 mM dithiothreitol, 5% glycerol, and 1 mM phenylmethylsulfonyl fluoride). After slowly mixing for 30 min at 4 °C, the suspensions were centrifuged at 13,000 × g for 15 min. The supernatant was dialyzed against dialysis buffer (20 mM HEPES-NaOH, pH 7.9, 75 mM NaCl, 0.1 mM EDTA, 0.5 mM dithiothreitol, 5% glycerol, and 1 mM phenylmethylsulfonyl fluoride) for 2 h. The dialysate was then centrifuged at 13,000 × g for 15 min, divided into aliquots, and stored at -80 °C. The protein concentration was determined by the method of Bradford (17) with a kit from Bio-Rad and bovine serum albumin as a standard.

DNA Fragments for Gel Mobility Shift Assays-- The radiolabeled probes and competitor fragments used in the binding assays are shown in Fig. 5A. Fragment -710/-343 was prepared by digesting -710/-343 TA vector with KpnI and XhoI. Fragment -342/+58 was prepared by digesting -342/+157 TA vector with KpnI and DraI. The double-stranded DNA probes were end-labeled with [gamma -32P]ATP and T4 polynucleotide kinase. The labeled DNA fragments were separated by electrophoresis through 4% nondenaturing polyacrylamide gels (acrylamide/bisacrylamide ratio, 30: 1), eluted with a high salt buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 1 M NaCl) overnight at room temperature, and purified.

Gel Mobility Shift Assays-- Gel mobility shift assays were carried out according to the method of Garner and Revzin (18). Nuclear extracts (1-6 µg of protein) were preincubated in 20 µl of binding buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 150 µg/ml poly(dI-dC)·(dI-dC), and 5% glycerol) for 15 min at 24 °C. The labeled probes (0.15 ng) were then added and incubated at 24 °C for an additional 30 min. The reaction mixtures were loaded onto 4% nondenaturing polyacrylamide gels (acrylamide/bisacrylamide ratio, 30:1) and electrophoresed at 10 V/cm for 90 min in 0.5× TBE (45 mM Tris, 45 mM boric acid, and 1 mM EDTA). The gels were dried and analyzed by autoradiography on x-ray film. The density of the autoradiographic data was quantified by densitometry (Dual-wavelength Flying-spot Scanner, CS-9000, Shimadzu Co., Tokyo, Japan). For the competition experiments, preincubation was performed in the presence of unlabeled competitor DNA fragment at the indicated molar excess.

Statistical Analysis-- The significance of difference between values was estimated by Student's t-test. A P value of less than 0.05 was considered significant.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Mapping of the Transcriptional Start Site-- As the first step toward characterizing the promoter of the rat regucalcin gene, the transcriptional start site was mapped by the cap site hunting method with rat liver cap site cDNA. In the first amplification, no specific PCR products were detectable using 1RC as the sense primer complementary to r-oligo and GSAP3 as the gene-specific antisense primer (data not shown). The resulting cDNA extending to the cap site was then amplified by nested PCR in the presence of 2RC as the sense primer complementary to r-oligo and either GSAP1 or GSAP2 as the gene-specific antisense primer. The amplified products were electrophoresed in agarose gels and stained with ethidium bromide (Fig. 1). Products of approximately 220 and 250 bp were obtained using 2RC/GSAP1 and 2RC/GSAP2, respectively. Two specific PCR products were cloned and sequenced. The results of sequencing analysis demonstrated that all 17 selected clones terminated at the same nucleotide (designated as +1) at their 5' ends (Fig. 2), indicating that this nucleotide is the major transcriptional initiation site of the rat regucalcin gene. Several putative DNA-binding elements that may play roles in basal and regulated regucalcin transcriptional activity were identified (Fig. 2). The TATA-like sequence and CCAAT box were located at nucleotide -25 and -69, respectively. Putative binding sites for AP-1, GATA-1, and AP-2 were also found.


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Fig. 1.   Gel electrophoresis of PCR products of oligo-capped regucalcin mRNA. Rat liver cap site cDNA was used as a PCR template to identify the transcriptional start site of the rat regucalcin gene. The first round of PCR was performed using 1RC as the sense DNA primer complementary to r-oligo and GSAP3 as the gene-specific antisense primer. An aliquot of the initial PCR reaction served the template for nested PCR. The resulting cDNA extending to the cap site was amplified by nested PCR using 2RC as the nested sense DNA primer complementary to r-oligo and either GSAP1 or GSAP2 as the nested gene-specific antisense primer. The nested PCR products were electrophoresed in 2% agarose gels and stained with ethidium bromide. The positions of GSAP1, -2, and -3 are indicated in Fig. 2. A 100-bp DNA ladder was used as the molecular size marker. Lane 1, 100-bp ladder; lane 2, nested PCR product (approximately 220 bp) using 2RC/GSAP1; lane 3, nested PCR product (approximately 250 bp) using 2RC/GSAP2.


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Fig. 2.   Nucleotide sequence of the 5'-flanking region of the rat regucalcin gene. The transcriptional initiation site determined by the cap site hunting was assigned as +1. The positions of gene-specific antisense primers used for the cap site hunting method are underlined. The translation initiator codon ATG is marked by a double underline. The arrows indicate the deletion sites of the constructs used in the reporter assays. Potential regulatory cis-elements are indicated by boxes. The intron sequences are indicated by lowercase letters. These genomic sequences have been deposited in the GenBank data base under accession numbers D67069 and D67071.

Basal Promoter Region of the Rat Regucalcin Gene-- To determine whether the 5'-flanking sequence of the rat regucalcin gene possesses functional promoter activity, we directionally subcloned an amplified DNA fragment at KpnI/XhoI sites of the pGL3 promoterless luciferase plasmid. The -710/+157 region of the rat regucalcin gene ligated to the luciferase reporter gene showed promoter activity in H4-II-E hepatoma cells.

To identify the region regulating basal promoter activity of the rat regucalcin gene, a series of 3'- and 5'-deletion constructions were transiently transfected into H4-II-E cells and assayed for luciferase activity. 3'-Deletion construct deleted to -223 did not show promoter activity (Fig. 3), suggesting that the sequence between nucleotides -223 and +18 contributes to basal promoter activity. 5'-Deletion constructs deleted to -102 retained the promoter activity (Fig. 4), suggesting that the region between nucleotides -102 and +157 contributes to basal promoter activity. The results of 3'- and 5'-deletion analyses suggested that the region between nucleotides -102 and +18 is needed for basal activity of the regucalcin promoter in H4-II-E cells.


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Fig. 3.   Functional analysis of 3'-deletion constructs of the rat regucalcin gene. A series of DNA fragments with different 3' ends (nucleotides +157, +18, -223, and -343) and a common 5' end (nucleotide -710) were ligated into the pGL3-Basic promoterless plasmid. H4-II-E hepatoma cells were transiently co-transfected with test plasmid and pRL-TK internal control plasmid. 3'-Deletion constructs are schematically shown on the left. Luciferase activity in the cell lysates was measured 48 h after transfection. The firefly luciferase activity of the test plasmid was corrected for Renilla luciferase activity of the pRL-TK plasmid. Luciferase activity of each construct is graphically shown in the right panel. Results are expressed as means ± S.D. from four to seven independent experiments.


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Fig. 4.   Functional analysis of 5'-deletion constructs of the rat regucalcin gene. A series of DNA fragments with different 5' ends (nucleotides -710, -582, -462, -342, -222, and -102) and a common 3' end (nucleotide +157) were ligated into the pGL3-Basic promoterless plasmid. H4-II-E hepatoma cells were transiently co-transfected with test plasmid and pRL-TK internal control plasmid. 5'-Deletion constructs are schematically shown on the left. Luciferase activity in the cell lysates was measured 48 h after transfection. The firefly luciferase activity of the test plasmid was corrected for Renilla luciferase activity of the pRL-TK plasmid. Luciferase activity of each construct is graphically shown in the right panel. Results are expressed as means ±S.D. from four to seven independent experiments.

Regulation of the Regucalcin Promoter by Bay K 8644, Dibutyryl cAMP, PMA, Insulin, Dexamethasone, and Estradiol-- To identify regions involved in the physiologically regulated expression of regucalcin gene, H4-II-E cells transfected with either -710/+157 LUC construct or -342/+157 LUC construct were treated with Bay K 8644, dibutyryl cAMP, PMA, insulin, dexamethasone, and estradiol. Treatment with Bay K 8644, PMA, and insulin significantly increased luciferase activity in H4-II-E cells transfected with the -710/+157 LUC construct, but -342/+157 LUC construct was completely unresponsive (Tables I and II). These results suggested that the cis-acting DNA sequences that mediate Bay K 8644, PMA, and insulin responsiveness in H4-II-E cells are located in the region -710/-343 of the rat regucalcin gene. Treatment with dibutyryl cAMP and dexamethasone increased luciferase activity in H4-II-E cells transfected with either -710/+157 LUC construct or -342/+157 LUC construct (Tables I and II). These results suggested that regulatory elements that are essential for the stimulation of the regucalcin promoter activity by dibutyryl cAMP and dexamethasone are located between nucleotides -342 and +157. Luciferase activity in H4-II-E cells transfected with either -710/+157 LUC construct or -342/+157 LUC construct was not affected by estradiol (Tables I and II).

                              
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Table I
Effects of Bay K 8644, dibutyryl cAMP, PMA, insulin, dexamethasone, and estradiol on promoter activity from -710/+157 LUC in H4-II-E hepatoma cells
Fragment -710/+157 was ligated into the pGL3-Basic promoterless plasmid (Basic LUC). H4-II-E hepatoma cells were transiently co-transfected with test plasmid and pRL-TK internal control plasmid and maintained in serum-supplemented medium for 24 h. The cells were preincubated in serum-free medium for 14 h, and then incubated for 20 h in the same medium supplemented with or without 2.5 µM Bay K 8644, 0.5 mM dibutyryl cAMP, 1 µM PMA, 10 nM insulin, 1 µM dexamethasone, and 1 µM estradiol. Luciferase activity was measured by the dual-luciferase reporter assay system. The firefly luciferase activity of the test plasmid was corrected for Renilla luciferase activity of the pRL-TK plasmid. The results are expressed as -fold stimulation compared to the luciferase activity measured after transfection with Basic LUC, which was set as 1.0. All values represent the means ± S.D. of six independent experiments, which were all carried out in triplicate. *, p < 0.05 compared with -710/+157 LUC alone.

                              
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Table II
Effects of Bay K 8644, dibutyryl cAMP, PMA, insulin, dexamethasone, and estradiol on promoter activity from -342/+157 LUC in H4-II-E hepatoma cells
Fragment -342/+157 was ligated into the pGL3-Basic promoterless plasmid (Basic LUC). H4-II-E hepatoma cells were transiently co-transfected with test plasmid and pRL-TK internal control plasmid, and maintained in serum-supplemented medium for 24 h. The cells were preincubated in serum-free medium for 14 h, and then incubated for 20 h in the same medium supplemented with or without 2.5 µM Bay K 8644, 0.5 mM dibutyryl cAMP, 1 µM PMA, 10 nM insulin, 1 µM dexamethasone, and 1 µM estradiol. Luciferase activity was measured by the dual-luciferase reporter assay system. The firefly luciferase activity of the test plasmid was corrected for Renilla luciferase activity of the pRL-TK plasmid. The results are expressed as -fold stimulation compared to the luciferase activity measured after transfection with Basic LUC, which was set as 1.0. All values represent the means ± S.D. of six independent experiments, which were all carried out in triplicate. *, p < 0.05 compared with -342/+157 LUC alone.

Binding of Nuclear Proteins from H4-II-E Cells to the 5'-Flanking Region of the Rat Regucalcin Gene-- To investigate the contribution of nuclear factors to the transcriptional activity of the rat regucalcin gene, gel mobility shift assays using nuclear extracts obtained from H4-II-E cells were employed. Fragments -710/-343 and -342/+58 were used as radiolabeled probes in gel mobility shift assays (Fig. 5A). When radiolabeled fragment -710/-343 was incubated with nuclear extracts from H4-II-E cells, gel mobility shift assays revealed the formation of complexes I and II, which were shifted upward from the free DNA (Fig. 5B, lanes 1 and 2). The presence of unlabeled fragment -710/-343 prevented the formation of the indicated complexes when the competition reactions were performed following a 15-min preincubation with a 10-100-fold molar excess of unlabeled fragment -710/-343 (Fig. 5B, lanes 2-5). The nonspecific oligonucleotides were unable to displace any of the complexes I and II (Fig. 5B, lanes 2, 6, and 7). These results indicated that complexes I and II are specifically formed by interaction of nuclear proteins to fragment -710/-343. When radiolabeled fragment -342/+58 was incubated with nuclear extracts obtained from H4-II-E cells, gel mobility shift assays revealed the formation of complexes III and IV, which were shifted upward from the free DNA (Fig. 5C, lanes 1 and 2). The presence of unlabeled fragment -342/+58 prevented the formation of the indicated complexes when the competition reactions were performed following a 15-min preincubation with a 10-100-fold molar excess of unlabeled fragment -342/+58 (Fig. 5C, lanes 2-5). The nonspecific oligonucleotides did not diminish formation of these two complexes (Fig. 5C, lanes 2, 6, and 7). These results indicated that complexes III and IV are formed due to sequence-specific binding.


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Fig. 5.   Binding profile of nuclear proteins from H4-II-E cells to the 5'-flanking region of the rat regucalcin gene. A, fragments -710/-343 and -342/+58 of the 5'-flanking region of the rat regucalcin gene used in gel mobility shift assays. These fragments were produced from the 5'-flanking region of the rat regucalcin gene as described under "Experimental Procedures." B, gel retardation analysis of fragment -710/-343 with nuclear proteins extracted from H4-II-E cells. An end-labeled fragment -710/-343 was incubated with nuclear extracts (1 µg of protein) prepared from H4-II-E cells. Competition assays were performed in the presence of a 10-100-fold molar excess of unlabeled DNA fragment as a competitor. Lane 1, no extracts; lane 2, no competitor; lane 3, 10-fold molar excess of fragment -710/-343 as competitor; lane 4, 50-fold molar excess of fragment -710/-343 as competitor; lane 5, 100-fold molar excess of fragment -710/-343 as competitor; lane 6, 100-fold molar excess of pUC18 DNA fragment (a 322-bp PvuII restriction fragment) as nonspecific competitor; lane 7, 100-fold molar excess of regucalcin gene fragment (a 314-bp HincII-SacI restriction fragment in Ref. 11) as nonspecific competitor. C, gel retardation analysis of fragment -342/+58 with nuclear proteins extracted from H4-II-E cells. An end-labeled fragment -342/+58 was incubated with nuclear extracts (4 µg of protein) prepared from H4-II-E cells. Competition assays were performed in the presence of a 10-100-fold molar excess of unlabeled DNA fragment as a competitor. Lane 1, no extracts; lane 2, no competitor; lane 3, 10-fold molar excess of fragment -342/+58 as competitor; lane 4, 50-fold molar excess of fragment -342/+58 as competitor; lane 5, 100-fold molar excess of fragment -342/+58 as competitor; lane 6, 100-fold molar excess of pUC18 DNA fragment (a 322-bp PvuII restriction fragment) as nonspecific competitor; lane 7, 100-fold molar excess of regucalcin gene fragment (a 314-bp HincII-SacI restriction fragment in Ref. 11) as nonspecific competitor.

Effects of Bay K 8644, Dibutyryl cAMP, PMA, Insulin, Dexamethasone, and Estradiol on Nuclear Protein Binding Activity to the 5'-Flanking Region of the Rat Regucalcin Gene-- Gel mobility shift assays were performed to detect the presence of nuclear proteins responsive to various physiological mediators. When the radiolabeled fragment -710/-343 was incubated with nuclear extracts obtained from control and mediator-treated cells, the formation of complex I, which appeared as a single band, was clearly increased in Bay K 8644-, PMA-, and insulin-treated cells (Bay K 8644-, PMA-, and insulin-treated: 3.9 ± 0.3-, 3.7 ± 0.5-, and 3.2 ± 0.4-fold, respectively, compared with control cells; p < 0.05; Fig. 6, lanes 1-3, 5, and 6). Whether these inducible nuclear proteins are common or distinct trans-acting factors is unclear. A comparison of nuclear extracts obtained from control cells and dibutyryl cAMP-, dexamethasone-, and estradiol-treated cells revealed no changes in the binding pattern of complex I (dibutyryl cAMP-, dexamethasone-, and estradiol-treated: 1.1 ± 0.3-, 1.0 ± 0.4-, and 1.2 ± 0.6-fold, respectively compared with control cells; not significant; Fig. 6, lanes 1, 2, 4, 7, and 8). In contrast, the formation of complex II was not affected by any of these six mediators (Fig. 6, lanes 1-8).


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Fig. 6.   Effects of Bay K 8644, dibutyryl cAMP, PMA, insulin, dexamethasone, and estradiol on nuclear protein binding activity to region -710/-343 of the rat regucalcin gene. H4-II-E cells were deprived of serum for 14 h and then treated with control solution (water and 0.05% Me2SO), 2.5 µM Bay K 8644, 0.5 mM dibutyryl cAMP, 1 µM PMA, 10 nM insulin, 1 µM dexamethasone, or 1 µM estradiol for 16 h. In the gel mobility shift experiments, end-labeled fragment -710/-343 was incubated with nuclear extracts (1 µg of protein) obtained from control cells (lanes 1 and 2), Bay K 8644-treated cells (lane 3), dibutyryl cAMP-treated cells (lane 4), PMA-treated cells (lane 5), insulin-treated cells (lane 6), dexamethasone-treated cells (lane 7), or estradiol-treated cells (lane 8). The figure shows representative results of four separate experiments. The density of the autoradiographic data was quantified by densitometry. The band intensities of complex I in Bay K 8644-, PMA-, and insulin-treated cells were significantly increased 3.9 ± 0.3-, 3.7 ± 0.5-, and 3.2 ± 0.4-fold (mean ± S.D., p < 0.05, n = 4), respectively, as compared with control cells.

When radiolabeled fragment -342/+58 was incubated with nuclear extracts obtained from control and mediator-treated cells, the formation of complex III, which appeared as a single band, was clearly increased in dibutyryl cAMP-treated cells (dibutyryl cAMP-treated, 4.1 ± 0.6-fold compared with control cells; p < 0.05; Fig. 7, lanes 1 and 4). A comparison of nuclear extracts obtained from control cells and Bay K 8644-, PMA-, insulin-, dexamethasone-, and estradiol-treated cells revealed no changes in the binding pattern of complex III (Bay K 8644-, PMA-, insulin-, dexamethasone-, and estradiol-treated: 1.1 ± 0.3-, 1.0 ± 0.5-, and 1.0 ± 0.6-fold, respectively compared with control; not significant; Fig. 7, lanes 1-3 and 5-8). There were no differences in binding pattern or intensity of complex IV between nuclear extracts from control cells and those treated with any of these six mediators (Fig. 7, lanes 1-8).


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Fig. 7.   Effects of Bay K 8644, dibutyryl cAMP, PMA, insulin, dexamethasone, and estradiol on nuclear protein binding activity to region -342/+58 of the rat regucalcin gene. H4-II-E cells were deprived of serum for 14 h and then treated with control solution (water and 0.05% Me2SO), 2.5 µM Bay K 8644, 0.5 mM dibutyryl cAMP, 1 µM PMA, 10 nM insulin, 1 µM dexamethasone, or 1 µM estradiol for 16 h. In the gel mobility shift experiments, end-labeled fragment -342/+58 was incubated with nuclear extracts (4 µg of protein) obtained from control cells (lanes 1 and 2), Bay K 8644-treated cells (lane 3), dibutyryl cAMP-treated cells (lane 4), PMA-treated cells (lane 5), insulin-treated cells (lane 6), dexamethasone-treated cells (lane 7), or estradiol-treated cells (lane 8). The figure shows representative results of four separate experiments. The density of the autoradiographic data was quantified by densitometry. The band intensities of complex III were significantly increased 4.1 ± 0.6-fold (mean ± S.D., p < 0.05, n = 4) as compared with control cells.


    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

To characterize the 5'-flanking region of the rat regucalcin gene, we employed reporter plasmids consisting of the 3'- or 5'-deletion fragment linked to the luciferase reporter gene. As Northern blotting analysis showed detectable amounts of regucalcin mRNA in rat H4-II-E hepatoma cells,2 H4-II-E hepatoma cells were used in transfection experiments. In the present study, we demonstrated that the 5'-flanking region of the rat regucalcin gene possesses functional promoter activity when transfected into H4-II-E cells. The results of 3'- and 5'-deletion analyses indicated that the region -102/+18 is essential for basal functional promoter activity of the rat regucalcin gene. A TATA-like sequence (-30ATAAA-25) and CCAAT box (-73CCAAT-69) were found at appropriate positions relative to the start site. These results suggested that both a TATA-like sequence and a CCAAT box located between nucleotides -102 and +18 play an important role in determining basal promoter activity of the rat regucalcin gene.

A previous study suggested that the induction of hepatic regucalcin mRNA expression by Ca2+ administration is attributable to the activation of a Ca2+/calmodulin-dependent pathway (10). The elevation of intracellular Ca2+ level may lead to the formation of Ca2+/calmodulin complex and the activation of Ca2+/calmodulin-dependent protein kinases. Therefore, it was possible that an increase in intracellular-free Ca2+ concentration triggers the induction of hepatic regucalcin mRNA expression. To assess whether the DNA sequence of the 5'-flanking region of the rat regucalcin gene can functionally respond to elevation of intracellular Ca2+ level, we transiently transfected H4-II-E cells with chimeric constructs containing serial deletions of the 5'-flanking region of the regucalcin gene and examined the effects of Bay K 8644, a Ca2+-channel agonist, on promoter activity. Analysis of this deletion series suggested that the cis-acting Ca2+ response sequence(s) is located within region -710/-343 of the rat regucalcin gene. Also, gel mobility shift assays indicated that the amount of complex formed on the regucalcin gene sequence -710/-343 was increased in Bay K 8644-treated cells. The agreement between the results of functional and binding analyses suggested that the Ca2+-inducible nuclear factor(s) is involved in positive regulation of regucalcin gene transcription by Ca2+.

It is also interesting to note that PMA stimulated luciferase activity from -710/+157 LUC plasmid. The deletion of fragment -710/-343 abolished PMA responses. This suggested that the stimulatory effect of PMA on rat regucalcin gene expression is mediated through a cis-acting sequence(s) located between nucleotides -710 and -343. It has also been demonstrated in electrophoretic mobility shift assays that PMA-inducible nuclear factor(s) bound to region -710/-343 of the rat regucalcin gene is present in nuclear extracts from PMA-stimulated cells. These results suggest that PMA-sensitive nuclear factor(s) plays a crucial role in PMA-regulated expression of the regucalcin gene. PMA has been demonstrated to activate transcription by regulating AP-1 binding (19). Further evidence that AP-1 is not involved in PMA-induced formation of complexes in the region -710/-343 was obtained in mobility shift experiments using a consensus oligonucleotide for this factor (data not shown). It is generally accepted that changes in the activity of protein kinase C mediated by PMA initiate a cascade of events which ultimately affects the action of specific transcription factors (20). It is possible, therefore, that the activity of identified PMA-inducible nuclear factor(s) may be modulated by the protein kinase C signaling pathway.

The expression of hepatic regucalcin mRNA is clearly stimulated by a single subcutaneous administration of insulin to fasted rats (12). Additionally, the expression of regucalcin mRNA in human HepG2 hepatoma cells is stimulated by insulin treatment (13). In the present study, we demonstrated that the stimulatory effect of insulin on rat regucalcin promoter activity in H4-II-E cells is mediated through a cis-acting sequence(s) located between nucleotides -710 and -343 of the rat regucalcin gene. In addition, we identified the insulin-inducible nuclear protein(s) that binds to region -710/-343 of the rat regucalcin gene. These results suggest that insulin-inducible nuclear protein(s) mediates the stimulatory effect of insulin on the regucalcin promoter activity.

We have recently reported that Ca2+ administration stimulates the additional binding of AP-1 to region -710/-575 of the rat regucalcin gene, suggesting that this factor participates in the regulation of regucalcin gene expression (11). In fact, the nucleotide sequence matching a potential AP-1-binding site was found in the region -710/-575 of the gene (5). In many cells, AP-1, which consists of homo- and/or heterodimers of the c-jun and c-fos gene products (21, 22), regulates the expression of some genes that contain specific AP-1 binding sites, PMA-responsive elements. In addition, it has been reported that haloperidol stimulates the DNA binding activity of AP-1 in PC12 pheochromocytoma cells and that its effect is dependent on calcium influx (23), suggesting that the elevation of intracellular Ca2+ leads to the activation of DNA binding activity of AP-1. Furthermore, insulin has been shown to stimulate AP-1-mediated gene expression and the phosphorylation of AP-1 transcription factor and several Fos-related proteins, suggesting that the phosphorylation of AP-1 by insulin plays an important role in the hormonal regulation of gene expression (24). In view of these results, endogenous AP-1 may also mediate the effects of Bay K 8644, PMA, and insulin on the regucalcin promoter activity in intact cells.

There is evidence that treatment with dibutyryl cAMP increases luciferase activity in H4-II-E cells transfected with the -710/+157 LUC plasmid. When regucalcin sequences were deleted to nucleotide -342, no change in reporter gene activity was observed in dibutyryl cAMP-treated H4-II-E cells. These results suggest that the cis-acting element(s) that mediates cAMP responsiveness is located within the region -342/+157 of the rat regucalcin gene. Using gel mobility shift assays, we found the cAMP-sensitive nuclear protein(s) which specifically binds to the region -342/+58 of the rat regucalcin gene. Therefore, the identified trans-acting factors may be involved in the induction of regucalcin gene promoter activity by cAMP. Transcriptional responses to cAMP are most commonly mediated by cAMP response element (25) and AP-2 element (26). The region between nucleotides -342 and +58 of the rat regucalcin gene contains the potential AP-2-binding sites (consensus sequence, 5'-CCC(A/C)N(G/C)(G/C)(G/C)-3' (27); -150CCCACCCC-143, -58CCCGCCCC-51), whereas a sequence homologous to the consensus cAMP response element is not present in the region. Further evidence that AP-2 is not involved in dibutyryl cAMP-induced formation of complex in region -342/+58 was obtained from mobility shift experiments using a consensus oligonucleotide for this factor (data not shown). Presumably, binding of the identified cAMP-inducible nuclear protein(s) to the regucalcin gene is mediated through cAMP-dependent protein kinase signaling pathway.

It has been reported that dexamethasone administration causes a marked increase in regucalcin mRNA levels in the kidney cortex of rats (28). Although this report was based on data from the kidney, it is possible that the positive regulation of regucalcin mRNA by dexamethasone also occurs in hepatoma cells. Our results indicated that the promoter activity of regucalcin gene in H4-II-E cells is enhanced by dexamethasone treatment. 5'-Deletion analysis indicated that the dexamethasone response sequence(s) may be located within region -342/+157 of the rat regucalcin gene, although a potential glucocorticoid response element (29) is not present in this region. Thus, as dexamethasone-inducible nuclear protein bound to region -342/+58 was not detected by gel mobility shift experiments, the results of promoter analysis suggested that an endogenous regulatory mechanism may exist in the stimulation of regucalcin gene promoter by dexamethasone.

17beta -Estradiol has been demonstrated to stimulate the expression of hepatic regucalcin mRNA in rats (14). It was suggested that an estradiol response sequence(s) may be localized within the 5'-flanking region of the rat regucalcin gene. However, region -710/+157 of the rat regucalcin gene ligated to the luciferase reporter gene did not show estradiol responsiveness. In gel mobility shift experiments, there was no difference in the binding pattern between nuclear extracts from control or estradiol-treated cells. These results suggested that the cis-acting element for estradiol is localized in the sequence upstream from the position -710 of the regucalcin gene.

In conclusion, it has been demonstrated that the 5'-flanking region of the rat regucalcin gene ligated to the luciferase reporter gene possesses promoter activity in H4-II-E hepatoma cells. We have identified the region regulating basal promoter activity of the regucalcin gene. Moreover, it was found that Ca2+, cAMP, phorbol ester, insulin, and dexamethasone response sequences are located within the 5'-flanking region of the rat regucalcin gene. We have identified the existence of trans-acting factors responsible for Ca2+, cAMP, phorbol ester, and insulin responses of the rat regucalcin gene. These results provide a basis for examining the nature of both cis- and trans-acting factors involved in the transcriptional regulation of the rat regucalcin gene.

    FOOTNOTES

* This work was supported in part by Grant-in-aid 08672922 from the Ministry of Education, Science and Culture, Japan.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D67069 and D67071.

Dagger To whom correspondence should be addressed. Tel./Fax: 81-54-264-5580; E-mail: yamaguch{at}fns1.u-shizuoka-ken.ac.jp.

The abbreviations used are: PMA, phorbol 12-myristate 13-acetate; alpha -MEM, alpha -minimum essential medium; bp, base pair(s); PCR, polymerase chain reaction; r-oligo, oligoribonucleotide.

2 M. Yamaguchi and T. Murata, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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