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Cloning and reporter analysis of human mitochondrial phosphoenolpyruvate carboxykinase gene promoter
Gene 338 (2004) 157 – 162 www.elsevier.com/locate/gene
Cloning and reporter analysis of human mitochondrial phosphoenolpyruvate carboxykinase gene promoter Miwako Suzuki a, Tomoyuki Yamasaki a,*, Ryoko Shinohata a,1, Miho Hata a, Hiromu Nakajima b, Norio Kono a a
Department of Basic Laboratory Science, School of Allied Health Science, Faculty of Medicine, Osaka University, 1-7 Yamadaoka, Suita, Osaka 565-0871, Japan b Department of Clinical Laboratory, Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-3 Nakamichi, Higashinari, Osaka, Osaka 537-8511, Japan Received 11 December 2003; received in revised form 21 May 2004; accepted 1 June 2004 Available online 23 July 2004 Received by T. Sekiya
Abstract Phosphoenolpyruvate carboxykinase (PEPCK) is one of the key regulatory enzymes in gluconeogenesis. In human liver, PEPCK is about equally distributed in both cytosol (PEPCK-1) and mitochondria (PEPCK-2). The human pepck2 gene and cDNA have been reported, but the cloning of the promoter region of the pepck2 gene has not been elucidated yet. We isolated and characterized human genomic P1-artificial chromosome (PAC) clones carrying the human pepck2 gene promoter. The oligocapping method revealed that the transcriptional start point (tsp) of the human pepck2 gene is located at 97 bp upstream of the first adenine residue of the translation start site. We also determined the nucleotide sequence to 1819 bp upstream of tsp. Sequence analysis of this region revealed that it contained several potential regulatory elements, including five GC boxes and three CCAAT boxes. Reporter analysis using transient transfection with firefly luciferase synthetic gene indicated 5Vflanking region up to 822 bp, and 317 bp upstream of tsp had transcriptional activity. These results suggest that these regions of the human pepck2 gene play an important role for its expression. D 2004 Elsevier B.V. All rights reserved. Keywords: Gluconeogenesis; Mitochondria; Metabolism; Luciferase
1. Introduction Glucose is utilized as the main energy source in human tissues, especially in erythrocytes and neurons. Thus, maintenance of glucose homeostasis is quite important. For this purpose, various metabolic pathways, such as glycolysis, gluconeogenesis, and glycogenolysis, are involved. Phosphoenolpyruvate carboxykinase [GTP: oxaloacetate car-
Abbreviations: PEPCK, phosphoenolpyruvate carboxykinase; PAC, P1artificial chromosome; PCR, polymerase chain reaction; TAP, tobacco acid phosphatase. * Corresponding author. Present address: Department of Clinical Laboratory, Osaka Medical Center for Cancer and Cardiovascular Disease, 1-3-3 Nakamichi, Higashinari, Osaka, Osaka 537-8511, Japan. E-mail address: [email protected] (T. Yamasaki). 1 Present address: Department of Medical Technology, Faculty of Health Sciences, Okayama University Medical School. 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.06.005
boxylyase (transphosphorylating); phosphoenolpyruvate carboxykinase (PEPCK); EC. 4. 1. 1. 32] plays a central role in glucose homeostasis as one of the rate-limiting enzymes in gluconeogenesis (Tilghman et al., 1976). Two isozymes with different subcellular localization were identified, known as the cytosolic form (PEPCK-1) and the mitochondrial form (PEPCK-2), which are located in the cytosol and the mitochondria, respectively. The intracellular distribution of these isozymes varies widely with species and organs (Ballard and Hanson, 1969; Brech et al., 1970; Nolte et al., 1972; Elliott and Pogson, 1977; Watford et al., 1981; Cornell et al., 1986; Hod et al., 1986). In human and chicken liver, PEPCK-1 and PEPCK-2 are about equally distributed. In rabbit and hamster, PEPCK activity is detected mainly in the mitochondria, whereas almost no activity is detected in rat liver mitochondria. It has been generally thought that the activity of PEPCK was not subject to the allosteric regulation but is controlled
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via gene expression (Pilkis and Granner, 1992; Deutschman et al., 1995). The activity of PEPCK-1 is altered by starvation, refeeding, and various hormones, such as insulin, glucocorticoid, thyroid hormone, and glucagon. Its expression was regulated through both transcriptional and posttranscriptional level. Massive analysis on rat PEPCK gene promoter identified a number of hormone-responsive cis-elements (Guder et al., 1971; Runge et al., 1991; McGrane et al., 1992; O’Brien and Granner, 1996) in the region. But regulation of pepck2 expression was not analyzed well except the chicken and bovine gene (Weldon et al., 1990; Agca et al., 2002). The excessive hepatic glucose production is a main factor contributing to the fasting hyperglycemia in the patients with both type 1 and type 2 diabetes mellitus (Consoli et al., 1989). Overexpression of PEPCK-1 in transgenic mice and rats show characteristic symptoms of type 2 diabetes mellitus, such as hyperglycemia, hyperinsulinemia, increased gluconeogenesis, insulin resistance, and impaired glucose tolerances (Valera et al., 1994; Rosella et al., 1995). These findings suggested that PEPCK plays an important role in the carbohydrate metabolism of diabetic patients. Thus, it would be a potential target in the development of antidiabetic drugs. In human species, gene and cDNA of both pepck1 and pepck2 have been already cloned (Stoffel et al., 1993; Ting et al., 1993; Modaressi et al., 1996, 1998), but the promoter region of pepck2 has not been determined yet. The mechanism of the regulation of its expression remains to be analyzed. Therefore, we report the cloning and functional characterization of human pepck2 gene promoter region in this article.
2. Materials and methods
Table 1 Nucleotide sequences of oligonucleotide primers Primer name
Sequence
hPCK-2ex-F1 hPCK-2int-R1 hPCK2-rev hPCK-2SP-A1 hPCK-2SP-A2 hPCK-2SP-A3 hPCK-2pr-F1 hPCK-2pr-R1
5V-CGCCCTCCATACCTCCCCGGCTCC 5V-TCCGCCTAGGATGGGAAACCTGGC 5V-GGGAGCTTTCGGATGA 5V-TGCTACGGCATGATGGCCAGCC 5V-TGTGGATGCCCTCTGGTTGGCA 5V-GCCCAAGGGGCTCAGCCCATGCCAG 5V-TTTGCGAGGTCGTGTCTCTCC 5V-AGCGGAGCCGGGGAGGTATGG
After thermal-cycle amplification, a step at 72 jC for 7 min was added to minimize the insufficient extension of the strand. Reaction products were analyzed by agarose gel electrophoresis. 2.3. Sequence analysis Based on the result of the Southern blotting analysis (data not shown), several restriction fragments from the obtained clones seemed to contain the hpepck2 gene promoter region. These fragments were subcloned into pUC118 (Takara, Kyoto, Japan) and analyzed. The DNA sequence was determined by the dideoxynucleotide termination procedure using ABI PRISMR Dyek Terminator, BigDyek Terminator or dGTP BigDyek Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems), and the ABI PRISMR Model 377 or Model 310 autosequencer (Applied Biosystems). Obtained sequences were analyzed by SDCGenetyx software package (Genetyx, Tokyo, Japan). TRANSFAC database (Heinemeyer et al., 1999) was used to analyze the sequence for the potential transcription factor binding motifs.
2.1. Materials 2.4. Determination of the transcription start point Biochemical and molecular biological reagents were of the commercially available highest grade, unless otherwise noted. For the polymerase chain reaction (PCR), AmpliTaq Goldk DNA Polymerase (Applied Biosystems, Foster City, USA) was used. Usual molecular biological procedures are essentially the same as described in the reference (Sambrook and Russel, 2001). 2.2. Cloning the hpepck2 gene promoter region A pair of gene-specific primers, hPCK-2ex1-F1 and hPCK-2int1-R1 (Table 1), was designed based on the sequence reported by Modaressi et al. (1996, 1998). The screening of ‘‘Down To The Well’’k human P1-artificial chromosome (PACk) DNA pools, which is consisted of 123246 human genomic clones in the PAC vector (Genome Systems, St. Louis, USA), was carried out by PCR with this pair of primers in the condition of an initial denaturation and enzyme-activation step of 10 min at 95 jC followed by 35 cycles of 30 s at 95 jC, 30 s at 60 jC, and 30 s at 72 jC.
To determine the transcription start point, we applied the rapid amplification of cDNA end (RACE) procedure with the oligocapping method (Maruyama and Sugano, 1994) using FirstChoicek RLM-RACE Kit (Ambion, St. Austin, USA). Total cellular RNA was dephosphorylated by calf intestine alkaline phosphatase. Then, the removal of the cap structure at the 5Vend of mRNA by tobacco acid phosphatase (TAP) was followed by the ligation of RNA adapter. After the cDNA was synthesized from genespecific primer hPCK2-rev (Table 1), primary PCR was performed by using the gene-specific primer hPCK-2SPA1 (Table 1) and the Outer RNA Adaptor primer in the kit. The gene-specific primer hPCK-2SP-A2 (Table 1) and the Inner RNA Adaptor primer in the kit were used in sondary PCR. The gene-specific primer hPCK-2SP-A3 (Table 1) and the Inner RNA Adaptor primer were used in the final PCR. The final amplified fragments were analyzed by agarose gel electrophoresis and were cloned into the pUC118 vector for sequencing analysis.
M. Suzuki et al. / Gene 338 (2004) 157–162
2.5. Reporter assay of cloned putative promoter region 2.5.1. Construction of plasmids In order to approve the activity of the cloned putative promoter region, we constructed two synthetic reporter gene plasmids, pPCK2(-921/-29)luc and pPCK2(416/-38)luc by inserting 824 bp Msc I restriction fragment and 378 bp PCR fragment using hPCK-2pr-F1 and hPCK-2pr-R1 (Table 1) into the Sma I site of pGL3Basic vector (Promega, Madison, USA), respectively. Each plasmid was purified twice by using equilibrium centrifugation in CsCl-ethidium bromide gradients method. 2.5.2. Cell culture and transient transfection assay The human hepatoma cell line, HuH-7, was grown in Dulbecco’s modified Eagle’s Medium (DMEM, SigmaAldrich, St. Louis, USA) supplemented with 10% fetal calf serum (FCS). Twenty-four hours before transfection, the cells were plated on the 24-well plates at a density of 5 104 cells per well. Plasmids pRL-TK and pGL3Basic (Promega) were served as an internal control to normalize the transfection efficiency and negative control, respectively. The cells were cotransfected with 0.5 Ag/well of one of the reporter genes [pPCK2(-921/-29)luc, pPCK2(416/-38)luc, or pGL3Basic) and 0.5 Ag/well pRL-TK by using Lipofect
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Aminek 2000 Reagent (Gibco BRL, Rockvill, USA) according to the manufacturer’s instructions. Then, cells were grown in DMEM without serum at 37 jC. The transfected cells were washed three times with phosphate-buffered saline and were lysed by the active lysis buffer supplied with the Dual-LuciferaseR Reporter assay System (Promega) at 24 h after the start of the transfection. Luciferase activity was determined using the same kit and the luminometer TD-20/20 (Turner Designs, Sunnyvale, USA). Firefly luciferase activity of the cell lysate was normalized to Renilla luciferase activity of the same cell lysate.
3. Result and discussion 3.1. Isolation of the genomic clones carrying hpepck2 gene promoter Using primers hPCK-2ex1-F1 and hPCK-2int1-R1 with human genomic DNA as a template, single distinct 208-bp fragment could be amplified by PCR (data not shown). Thus, we PCR-screened the human genomic library ‘‘Down To The Well’’k human PACk DNA pools by using this primer pair and obtained two positive clones (hPCK2-P1 and -P2). Both clones were confirmed to contain at least the
Fig. 1. Nucleotide sequence of the hpck2 gene promoter region. The putative tsp, guanosine residue 97 bp upstream from the first adenine residue of the translation initiation codon (ATG, enclosed in a rectangle), is indicated by a bold letter. Nucleotide number, designated the tsp as + 1, is indicated on the left margin. The potential binding sites for transcription factors are underlined. This nucleotide sequence was deposited in the EMBL/GenBank/DDBJ database under accession number AB126037.
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part of hpepck2 gene by Southern blot analysis using the 208-bp amplicon as a probe (data not shown). 3.2. Southern blots and DNA sequencing of the hpepck2 gene promoter
Fig. 2. Agarose gel electrophoresis of the final PCR products by RACE analysis with oligocapping method. RACE analysis with oligocapping was performed using the RLM-RACE kit with 1 Ag of total RNA from HuH7 cells as recommended. Final PCR products were analyzed by using 1.5% agarose gel electrophoresis. Lane (+): the final amplified product started with RNA treated with TAP. Lane ( ): the final amplified product started with RNA without TAP reaction. Lane M: DNA size marker; 100-bp ladder DNA (New England Biolabs, Bethesda, MD, USA).
According to the series of Southern blot analyses of the clones hPCK2-P1 and -P2 with the hPCK2 DNA probe, four DNA fragments, 2.5 kb with Xba I, 4.5 kb with Hind III, 4.0 kb and 7.0 Kb with Hinc II, were identified to encompass the first exon (data not shown). These fragments were subcloned and were analyzed. Using the primerwalking technique along the hpepck2 gene promoter region, we determined the nucleotide sequence to 1916 bp upstream from the translation initiation site (Fig. 2). Comparing the sequencing data derived from this study with the published cDNA and gene data (Modaressi et al., 1996, 1998), several minor discrepancies were observed. In order to identify known consensus sequences of various transcription factors, search analysis on this region was performed using the TRANSFAC database. We failed to identify the conventional TATA box. However, four CCAAT box and five GC box motifs are located between 146 and 566 nucleotides upstream of the translation start codon of the hpepck2 gene. The CCAAT box, GC box, and other elements might play a significant role to promote the basal transcriptional activity of the hpepck2 gene. As shown in Fig. 1, various consensus sequences, including CREB, SREBP, AP-2, AP-1, C/EBP, and SRY elements were observed within the promoter region of
Fig. 3. Transcriptional activity of hpck2 gene promoter region. Panel A: schematic representation of hpck2 synthetic reporter constructs. DNA fragments containing putative promoter region of the hpck2 gene were cloned into Sma I site of pGL3Basic vector. Open, closed, and hatched rectangles indicate relative position of AP-1, C/EBP, and SP1 binding sites, respectively. Panel B: relative luciferase activities of hpck2 synthetic reporter constructs in HuH-7 cells. Results are expressed as the relative luciferase (firefly luciferase) activity normalized to Renilla luciferase activity. The promoterless plasmid, pGL3Basic, served as the negative control. Transient transfection was performed in triplicate in each experiment, and the results represent data from at least five independent experiments.
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hpepck2 gene. The presence of multiple transcription factor binding sites near the tsp strongly suggests that this region contains the functional hpepck2 gene promoter. 3.3. The transcription start point (tsp) of the hpepck2 gene To identify the tsp of the hpepck2 gene, RACE analysis with oligocapping method was performed. As shown in Fig. 2, the size of the final amplified products was estimated to be about 220 bp. Because the oligocapping method specifically labels the capped end of the mRNA (Maruyama and Sugano, 1994), 5V end of the PCR amplicon was assumed to be the tsp of the gene. Then, PCR products were cloned and sequenced. Sequence analysis revealed that the adjacent region to the tsp of the RACE product was essentially identical to the corresponding position in the genomic region. Consequently, the tsp, the first base next to the RNA adaptor sequence, was located 97 bp upstream from the first adenine residue of the translation initiation codon (Fig. 1). According to the previous report (Modaressi et al., 1998), the tsp was assigned at 134 bp upstream from the translation start site by primer extension analysis. This discrepancy may be due to the cell-type-specific difference. The tsp of the hpepck2 may need more considerations. 3.4. Activity of the hpepck2 gene promoter To examine the transcriptional activity of the hpepck2 gene promoter, synthetic reporter plasmids and pRL-TK were cotransfected into HuH7 cells. The transfection efficiency was compensated by using the relative firefly luciferase activity to that of Renilla luciferase. As shown in Fig. 3, both pPCK2(-921/-29)luc and pPCK2(416/-38)luc showed significantly higher luciferase activity than that of negative controls (pGL3Basic). This indicates that this region has apparent promoter activity. The activity of pPCK2(-921/-29)luc seemed to be lesser than that of pPCK2(416/-38)luc (Fig. 3), but the difference between them was not statistically significant. These data indicate that the region from -921 to -416, containing two AP-1 binding sites and a SRY binding site, may not have significant effect on the hpepck2 basal transcription.
Acknowledgements This work was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
References Agca, C., Greenfield, R.B., Hartwell, J.R., Donkin, S.S., 2002. Cloning and characterization of bovine cytosolic and mitochondrial PEPCK during transition to lactation. Physiol. Genomics 11, 53 – 63.
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Ballard, F.J., Hanson, R.W., 1969. Purification of phosphoenolpyruvate carboxykinase from the cytosol fraction of rat liver and the immunochemical demonstration of differences between this enzyme and the mitochondrial phosphoenolpyruvate carboxykinase. J. Biol. Chem. 244, 5625 – 5630. Brech, W., Shrago, E., Wilken, D., 1970. Studies on pyruvate carboxylase in rat and human liver. Biochim. Biophys. Acta 201, 145 – 154. Consoli, A., Nurjhan, N., Capani, F., Gerich, J., 1989. Predominant role of gluconeogenesis in increased hepatic glucose production in NIDDM. Diabetes 38, 550 – 557. Cornell, N.W., Schramm, V.L., Kerich, M.J., Emig, F.A., 1986. Subcellular location of phosphoenolpyruvate carboxykinase in hepatocytes from fed and starved rats. J. Nutr. 116, 1101 – 1108. Deutschman, C.S., De Maio, A., Clemens, M.G., 1995. Sepsis-induced attenuation of glucagon and 8-BrcAMP modulation of the phosphoenolpyruvate carboxykinase gene. Am. J. Physiol. 269, R584 – R591. Elliott, K.R., Pogson, C.I., 1977. The effects of starvation and experimental diabetes on phosphoenol-pyruvate carboxykinase in the guinea pig. Biochem. J. 164, 357 – 361. Guder, W., Wiesner, W., Stukowski, B., Wieland, O., 1971. Metabolism of isolated kidney tubules. Oxygen consumption, gluconeogenesis and the effect of cyclic nucleotides in tubules from starved rats. Hoppe-Seyler Z. Physiol. Chem. 352, 1319 – 1328. Heinemeyer, T., Chen, X., Karas, H., Kel, A.E., Kel, O.V., Liebich, I., Meinhardt, T., Reuter, I., Schacherer, F., Wingender, E., 1999. Expanding the TRANSFAC database towards an expert system of regulatory molecular mechanisms. Nucleic Acids Res. 27, 318 – 322. Hod, Y., Cook, J.S., Weldon, S.L., Short, J.M., Wynshaw Boris, A., Hanson, R.W., 1986. Differential expression of the genes for the mitochondrial and cytosolic forms of phosphoenolpyruvate carboxykinase. Ann. N.Y. Acad. Sci. 478, 31 – 45. Maruyama, K., Sugano, S., 1994. Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171 – 174. McGrane, M.M., Yun, J.S., Patel, Y.M., Hanson, R.W., 1992. Metabolic control of gene expression: in vivo studies with transgenic mice. Trends Biochem. Sci. 17, 40 – 44. Modaressi, S., Christ, B., Bratke, J., Zahn, S., Heise, T., Jungermann, K., 1996. Molecular cloning, sequencing and expression of the cDNA of the mitochondrial form of phosphoenolpyruvate carboxykinase from human liver. Biochem. J. 315, 807 – 814. Modaressi, S., Brechtel, K., Christ, B., Jungermann, K., 1998. Human mitochondrial phosphoenolpyruvate carboxykinase 2 gene. Structure, chromosomal localization and tissue-specific expression. Biochem. J. 333, 359 – 366. Nolte, J., Brdiczka, D., Pette, D., 1972. Intracellular distribution of phosphoenolpyruvate carboxylase and (NADP) malate dehydrogenase in different muscle types. Biochim. Biophys. Acta 284, 497 – 507. O’Brien, R.M., Granner, D.K., 1996. Regulation of gene expression by insulin. Physiol. Rev. 76, 1109 – 1161. Pilkis, S.J., Granner, D.K., 1992. Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis. Annu. Rev. Physiol. 54, 885 – 909. Runge, D., Schmidt, H., Christ, B., Jungermann, K., 1991. Mechanism of the permissive action of dexamethasone on the glucagon-dependent activation of the phosphoenolpyruvate carboxykinase gene in cultured rat hepatocytes. Eur. J. Biochem. 198, 641 – 649. Rosella, G., Zajac, J.D., Baker, L., Kaczmarczyk, S.J., Andrikopoulos, S., Adams, T.E., Proietto, J., 1995. Impaired glucose tolerance and increased weight gain in transgenic rats overexpressing a non-insulinresponsive phosphoenolpyruvate carboxykinase gene. Mol. Endocrinol. 9, 1396 – 1404. Sambrook, J., Russel, D.W., 2001. Molecular Cloning—A Laboratory Manual, Third edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Stoffel, M., Xiang, K.S., Espinosa, III, R., Cox, N.J., Le Beau, M.M., Bell, G.I., 1993. cDNA sequence and localization of polymorphic human
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cytosolic phosphoenolpyruvate carboxykinase gene (PCK1) to chromosome 20, band q13.31: PCK1 is not tightly linked to maturity-onset diabetes of the young. Hum. Mol. Genet. 2, 1 – 4. Tilghman, S.M., Hanson, R.W., Ballard, F.J., 1976. Hormonal regulation of phosphoenolpyruvate carboxykinase (GTP) in mammalian tissues. In: Hanson, R.W., Mehlman, M.A. (Eds.), Gluconeogenesis: Its Regulation in Mammalian Species. John Wiley and Sons, New York, pp. 369 – 462. Ting, C.N., Burgess, D.L., Chamberlain, J.S., Keith, T.P., Falls, K., Meisler, M.H., 1993. Phosphoenolpyruvate carboxykinase (GTP): characterization of the human PCK1 gene and localization distal to MODY on chromosome 20. Genomics 16, 698 – 706. Valera, A., Pujol, A., Pelegrin, M., Bosch, F., 1994. Transgenic mice over-
expressing phosphoenolpyruvate carboxykinase develop non-insulindependent diabetes mellitus. Proc. Natl. Acad. Sci. U. S. A. 91, 9151 – 9154. Watford, M., Hod, Y., Chiao, Y.B., Utter, M.F., Hanson, R.W., 1981. The unique role of the kidney in gluconeogenesis in the chicken. The significance of a cytosolic form of phosphoenolpyruvate carboxykinase. J. Biol. Chem. 256, 10023 – 10027. Weldon, S.L., Rando, A., Matathias, A.S., Hod, Y., Kalonick, P.A., Savon, S., Cook, J.S., Hanson, R.W., 1990. Mitochondrial phosphoenolpyruvate carboxykinase from the chicken. Comparison of the cDNA and protein sequences with the cytosolic isozyme. J. Biol. Chem. 265, 7308 – 7317.
Cloning and reporter analysis of human mitochondrial phosphoenolpyruvate carboxykinase gene promoter Miwako Suzuki a, Tomoyuki Yamasaki a,*, Ryoko Shinohata a,1, Miho Hata a, Hiromu Nakajima b, Norio Kono a a
Department of Basic Laboratory Science, School of Allied Health Science, Faculty of Medicine, Osaka University, 1-7 Yamadaoka, Suita, Osaka 565-0871, Japan b Department of Clinical Laboratory, Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-3 Nakamichi, Higashinari, Osaka, Osaka 537-8511, Japan Received 11 December 2003; received in revised form 21 May 2004; accepted 1 June 2004 Available online 23 July 2004 Received by T. Sekiya
Abstract Phosphoenolpyruvate carboxykinase (PEPCK) is one of the key regulatory enzymes in gluconeogenesis. In human liver, PEPCK is about equally distributed in both cytosol (PEPCK-1) and mitochondria (PEPCK-2). The human pepck2 gene and cDNA have been reported, but the cloning of the promoter region of the pepck2 gene has not been elucidated yet. We isolated and characterized human genomic P1-artificial chromosome (PAC) clones carrying the human pepck2 gene promoter. The oligocapping method revealed that the transcriptional start point (tsp) of the human pepck2 gene is located at 97 bp upstream of the first adenine residue of the translation start site. We also determined the nucleotide sequence to 1819 bp upstream of tsp. Sequence analysis of this region revealed that it contained several potential regulatory elements, including five GC boxes and three CCAAT boxes. Reporter analysis using transient transfection with firefly luciferase synthetic gene indicated 5Vflanking region up to 822 bp, and 317 bp upstream of tsp had transcriptional activity. These results suggest that these regions of the human pepck2 gene play an important role for its expression. D 2004 Elsevier B.V. All rights reserved. Keywords: Gluconeogenesis; Mitochondria; Metabolism; Luciferase
1. Introduction Glucose is utilized as the main energy source in human tissues, especially in erythrocytes and neurons. Thus, maintenance of glucose homeostasis is quite important. For this purpose, various metabolic pathways, such as glycolysis, gluconeogenesis, and glycogenolysis, are involved. Phosphoenolpyruvate carboxykinase [GTP: oxaloacetate car-
Abbreviations: PEPCK, phosphoenolpyruvate carboxykinase; PAC, P1artificial chromosome; PCR, polymerase chain reaction; TAP, tobacco acid phosphatase. * Corresponding author. Present address: Department of Clinical Laboratory, Osaka Medical Center for Cancer and Cardiovascular Disease, 1-3-3 Nakamichi, Higashinari, Osaka, Osaka 537-8511, Japan. E-mail address: [email protected] (T. Yamasaki). 1 Present address: Department of Medical Technology, Faculty of Health Sciences, Okayama University Medical School. 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.06.005
boxylyase (transphosphorylating); phosphoenolpyruvate carboxykinase (PEPCK); EC. 4. 1. 1. 32] plays a central role in glucose homeostasis as one of the rate-limiting enzymes in gluconeogenesis (Tilghman et al., 1976). Two isozymes with different subcellular localization were identified, known as the cytosolic form (PEPCK-1) and the mitochondrial form (PEPCK-2), which are located in the cytosol and the mitochondria, respectively. The intracellular distribution of these isozymes varies widely with species and organs (Ballard and Hanson, 1969; Brech et al., 1970; Nolte et al., 1972; Elliott and Pogson, 1977; Watford et al., 1981; Cornell et al., 1986; Hod et al., 1986). In human and chicken liver, PEPCK-1 and PEPCK-2 are about equally distributed. In rabbit and hamster, PEPCK activity is detected mainly in the mitochondria, whereas almost no activity is detected in rat liver mitochondria. It has been generally thought that the activity of PEPCK was not subject to the allosteric regulation but is controlled
158
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via gene expression (Pilkis and Granner, 1992; Deutschman et al., 1995). The activity of PEPCK-1 is altered by starvation, refeeding, and various hormones, such as insulin, glucocorticoid, thyroid hormone, and glucagon. Its expression was regulated through both transcriptional and posttranscriptional level. Massive analysis on rat PEPCK gene promoter identified a number of hormone-responsive cis-elements (Guder et al., 1971; Runge et al., 1991; McGrane et al., 1992; O’Brien and Granner, 1996) in the region. But regulation of pepck2 expression was not analyzed well except the chicken and bovine gene (Weldon et al., 1990; Agca et al., 2002). The excessive hepatic glucose production is a main factor contributing to the fasting hyperglycemia in the patients with both type 1 and type 2 diabetes mellitus (Consoli et al., 1989). Overexpression of PEPCK-1 in transgenic mice and rats show characteristic symptoms of type 2 diabetes mellitus, such as hyperglycemia, hyperinsulinemia, increased gluconeogenesis, insulin resistance, and impaired glucose tolerances (Valera et al., 1994; Rosella et al., 1995). These findings suggested that PEPCK plays an important role in the carbohydrate metabolism of diabetic patients. Thus, it would be a potential target in the development of antidiabetic drugs. In human species, gene and cDNA of both pepck1 and pepck2 have been already cloned (Stoffel et al., 1993; Ting et al., 1993; Modaressi et al., 1996, 1998), but the promoter region of pepck2 has not been determined yet. The mechanism of the regulation of its expression remains to be analyzed. Therefore, we report the cloning and functional characterization of human pepck2 gene promoter region in this article.
2. Materials and methods
Table 1 Nucleotide sequences of oligonucleotide primers Primer name
Sequence
hPCK-2ex-F1 hPCK-2int-R1 hPCK2-rev hPCK-2SP-A1 hPCK-2SP-A2 hPCK-2SP-A3 hPCK-2pr-F1 hPCK-2pr-R1
5V-CGCCCTCCATACCTCCCCGGCTCC 5V-TCCGCCTAGGATGGGAAACCTGGC 5V-GGGAGCTTTCGGATGA 5V-TGCTACGGCATGATGGCCAGCC 5V-TGTGGATGCCCTCTGGTTGGCA 5V-GCCCAAGGGGCTCAGCCCATGCCAG 5V-TTTGCGAGGTCGTGTCTCTCC 5V-AGCGGAGCCGGGGAGGTATGG
After thermal-cycle amplification, a step at 72 jC for 7 min was added to minimize the insufficient extension of the strand. Reaction products were analyzed by agarose gel electrophoresis. 2.3. Sequence analysis Based on the result of the Southern blotting analysis (data not shown), several restriction fragments from the obtained clones seemed to contain the hpepck2 gene promoter region. These fragments were subcloned into pUC118 (Takara, Kyoto, Japan) and analyzed. The DNA sequence was determined by the dideoxynucleotide termination procedure using ABI PRISMR Dyek Terminator, BigDyek Terminator or dGTP BigDyek Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems), and the ABI PRISMR Model 377 or Model 310 autosequencer (Applied Biosystems). Obtained sequences were analyzed by SDCGenetyx software package (Genetyx, Tokyo, Japan). TRANSFAC database (Heinemeyer et al., 1999) was used to analyze the sequence for the potential transcription factor binding motifs.
2.1. Materials 2.4. Determination of the transcription start point Biochemical and molecular biological reagents were of the commercially available highest grade, unless otherwise noted. For the polymerase chain reaction (PCR), AmpliTaq Goldk DNA Polymerase (Applied Biosystems, Foster City, USA) was used. Usual molecular biological procedures are essentially the same as described in the reference (Sambrook and Russel, 2001). 2.2. Cloning the hpepck2 gene promoter region A pair of gene-specific primers, hPCK-2ex1-F1 and hPCK-2int1-R1 (Table 1), was designed based on the sequence reported by Modaressi et al. (1996, 1998). The screening of ‘‘Down To The Well’’k human P1-artificial chromosome (PACk) DNA pools, which is consisted of 123246 human genomic clones in the PAC vector (Genome Systems, St. Louis, USA), was carried out by PCR with this pair of primers in the condition of an initial denaturation and enzyme-activation step of 10 min at 95 jC followed by 35 cycles of 30 s at 95 jC, 30 s at 60 jC, and 30 s at 72 jC.
To determine the transcription start point, we applied the rapid amplification of cDNA end (RACE) procedure with the oligocapping method (Maruyama and Sugano, 1994) using FirstChoicek RLM-RACE Kit (Ambion, St. Austin, USA). Total cellular RNA was dephosphorylated by calf intestine alkaline phosphatase. Then, the removal of the cap structure at the 5Vend of mRNA by tobacco acid phosphatase (TAP) was followed by the ligation of RNA adapter. After the cDNA was synthesized from genespecific primer hPCK2-rev (Table 1), primary PCR was performed by using the gene-specific primer hPCK-2SPA1 (Table 1) and the Outer RNA Adaptor primer in the kit. The gene-specific primer hPCK-2SP-A2 (Table 1) and the Inner RNA Adaptor primer in the kit were used in sondary PCR. The gene-specific primer hPCK-2SP-A3 (Table 1) and the Inner RNA Adaptor primer were used in the final PCR. The final amplified fragments were analyzed by agarose gel electrophoresis and were cloned into the pUC118 vector for sequencing analysis.
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2.5. Reporter assay of cloned putative promoter region 2.5.1. Construction of plasmids In order to approve the activity of the cloned putative promoter region, we constructed two synthetic reporter gene plasmids, pPCK2(-921/-29)luc and pPCK2(416/-38)luc by inserting 824 bp Msc I restriction fragment and 378 bp PCR fragment using hPCK-2pr-F1 and hPCK-2pr-R1 (Table 1) into the Sma I site of pGL3Basic vector (Promega, Madison, USA), respectively. Each plasmid was purified twice by using equilibrium centrifugation in CsCl-ethidium bromide gradients method. 2.5.2. Cell culture and transient transfection assay The human hepatoma cell line, HuH-7, was grown in Dulbecco’s modified Eagle’s Medium (DMEM, SigmaAldrich, St. Louis, USA) supplemented with 10% fetal calf serum (FCS). Twenty-four hours before transfection, the cells were plated on the 24-well plates at a density of 5 104 cells per well. Plasmids pRL-TK and pGL3Basic (Promega) were served as an internal control to normalize the transfection efficiency and negative control, respectively. The cells were cotransfected with 0.5 Ag/well of one of the reporter genes [pPCK2(-921/-29)luc, pPCK2(416/-38)luc, or pGL3Basic) and 0.5 Ag/well pRL-TK by using Lipofect
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Aminek 2000 Reagent (Gibco BRL, Rockvill, USA) according to the manufacturer’s instructions. Then, cells were grown in DMEM without serum at 37 jC. The transfected cells were washed three times with phosphate-buffered saline and were lysed by the active lysis buffer supplied with the Dual-LuciferaseR Reporter assay System (Promega) at 24 h after the start of the transfection. Luciferase activity was determined using the same kit and the luminometer TD-20/20 (Turner Designs, Sunnyvale, USA). Firefly luciferase activity of the cell lysate was normalized to Renilla luciferase activity of the same cell lysate.
3. Result and discussion 3.1. Isolation of the genomic clones carrying hpepck2 gene promoter Using primers hPCK-2ex1-F1 and hPCK-2int1-R1 with human genomic DNA as a template, single distinct 208-bp fragment could be amplified by PCR (data not shown). Thus, we PCR-screened the human genomic library ‘‘Down To The Well’’k human PACk DNA pools by using this primer pair and obtained two positive clones (hPCK2-P1 and -P2). Both clones were confirmed to contain at least the
Fig. 1. Nucleotide sequence of the hpck2 gene promoter region. The putative tsp, guanosine residue 97 bp upstream from the first adenine residue of the translation initiation codon (ATG, enclosed in a rectangle), is indicated by a bold letter. Nucleotide number, designated the tsp as + 1, is indicated on the left margin. The potential binding sites for transcription factors are underlined. This nucleotide sequence was deposited in the EMBL/GenBank/DDBJ database under accession number AB126037.
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part of hpepck2 gene by Southern blot analysis using the 208-bp amplicon as a probe (data not shown). 3.2. Southern blots and DNA sequencing of the hpepck2 gene promoter
Fig. 2. Agarose gel electrophoresis of the final PCR products by RACE analysis with oligocapping method. RACE analysis with oligocapping was performed using the RLM-RACE kit with 1 Ag of total RNA from HuH7 cells as recommended. Final PCR products were analyzed by using 1.5% agarose gel electrophoresis. Lane (+): the final amplified product started with RNA treated with TAP. Lane ( ): the final amplified product started with RNA without TAP reaction. Lane M: DNA size marker; 100-bp ladder DNA (New England Biolabs, Bethesda, MD, USA).
According to the series of Southern blot analyses of the clones hPCK2-P1 and -P2 with the hPCK2 DNA probe, four DNA fragments, 2.5 kb with Xba I, 4.5 kb with Hind III, 4.0 kb and 7.0 Kb with Hinc II, were identified to encompass the first exon (data not shown). These fragments were subcloned and were analyzed. Using the primerwalking technique along the hpepck2 gene promoter region, we determined the nucleotide sequence to 1916 bp upstream from the translation initiation site (Fig. 2). Comparing the sequencing data derived from this study with the published cDNA and gene data (Modaressi et al., 1996, 1998), several minor discrepancies were observed. In order to identify known consensus sequences of various transcription factors, search analysis on this region was performed using the TRANSFAC database. We failed to identify the conventional TATA box. However, four CCAAT box and five GC box motifs are located between 146 and 566 nucleotides upstream of the translation start codon of the hpepck2 gene. The CCAAT box, GC box, and other elements might play a significant role to promote the basal transcriptional activity of the hpepck2 gene. As shown in Fig. 1, various consensus sequences, including CREB, SREBP, AP-2, AP-1, C/EBP, and SRY elements were observed within the promoter region of
Fig. 3. Transcriptional activity of hpck2 gene promoter region. Panel A: schematic representation of hpck2 synthetic reporter constructs. DNA fragments containing putative promoter region of the hpck2 gene were cloned into Sma I site of pGL3Basic vector. Open, closed, and hatched rectangles indicate relative position of AP-1, C/EBP, and SP1 binding sites, respectively. Panel B: relative luciferase activities of hpck2 synthetic reporter constructs in HuH-7 cells. Results are expressed as the relative luciferase (firefly luciferase) activity normalized to Renilla luciferase activity. The promoterless plasmid, pGL3Basic, served as the negative control. Transient transfection was performed in triplicate in each experiment, and the results represent data from at least five independent experiments.
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hpepck2 gene. The presence of multiple transcription factor binding sites near the tsp strongly suggests that this region contains the functional hpepck2 gene promoter. 3.3. The transcription start point (tsp) of the hpepck2 gene To identify the tsp of the hpepck2 gene, RACE analysis with oligocapping method was performed. As shown in Fig. 2, the size of the final amplified products was estimated to be about 220 bp. Because the oligocapping method specifically labels the capped end of the mRNA (Maruyama and Sugano, 1994), 5V end of the PCR amplicon was assumed to be the tsp of the gene. Then, PCR products were cloned and sequenced. Sequence analysis revealed that the adjacent region to the tsp of the RACE product was essentially identical to the corresponding position in the genomic region. Consequently, the tsp, the first base next to the RNA adaptor sequence, was located 97 bp upstream from the first adenine residue of the translation initiation codon (Fig. 1). According to the previous report (Modaressi et al., 1998), the tsp was assigned at 134 bp upstream from the translation start site by primer extension analysis. This discrepancy may be due to the cell-type-specific difference. The tsp of the hpepck2 may need more considerations. 3.4. Activity of the hpepck2 gene promoter To examine the transcriptional activity of the hpepck2 gene promoter, synthetic reporter plasmids and pRL-TK were cotransfected into HuH7 cells. The transfection efficiency was compensated by using the relative firefly luciferase activity to that of Renilla luciferase. As shown in Fig. 3, both pPCK2(-921/-29)luc and pPCK2(416/-38)luc showed significantly higher luciferase activity than that of negative controls (pGL3Basic). This indicates that this region has apparent promoter activity. The activity of pPCK2(-921/-29)luc seemed to be lesser than that of pPCK2(416/-38)luc (Fig. 3), but the difference between them was not statistically significant. These data indicate that the region from -921 to -416, containing two AP-1 binding sites and a SRY binding site, may not have significant effect on the hpepck2 basal transcription.
Acknowledgements This work was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
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