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• W2078220726 abstract "Liver carnitine palmitoyltransferase I catalyzes the transfer of long-chain fatty acids into mitochondria. L-CPT I is considered the rate-controlling enzyme in fatty acid oxidation. Expression of the L-CPT I gene is induced by starvation in response to glucagon secretion from the pancreas, an effect mediated by cAMP. Here, the molecular mechanisms underlying the induction of L-CPT I gene expression by cAMP were characterized. We demonstrate that the cAMP response unit of the L-CPT I gene is composed of a cAMP-response element motif and a DR1 sequence located 3 kb upstream of the transcription start site. Our data strongly suggest that the coactivator PGC-1 is involved in the regulation of this gene expression by cAMP in combination with HNF4α and cAMP-response element-binding protein (CREB). Indeed, (i) cotransfection of CREB or HNF4α dominant negative mutants completely abolishes the effect of cAMP on the L-CPT I promoter, and (ii) the cAMP-responsive unit binds HNF4α and CREB through the DR1 and the cAMP-response element sequences, respectively. Moreover, cotransfection of PGC-1 strongly activates the L-CPT I promoter through HNF4α bound at the DR1 element. Finally, we show that the transcriptional induction of the PGC-1 gene by glucagon through cAMP in hepatocytes precedes that of L-CPT-1. In addition to the key role that PGC-1 plays in glucose homeostasis, it may also be critical for lipid homeostasis. Taken together these observations suggest that PGC-1 acts to coordinate the process of metabolic adaptation in the liver. Liver carnitine palmitoyltransferase I catalyzes the transfer of long-chain fatty acids into mitochondria. L-CPT I is considered the rate-controlling enzyme in fatty acid oxidation. Expression of the L-CPT I gene is induced by starvation in response to glucagon secretion from the pancreas, an effect mediated by cAMP. Here, the molecular mechanisms underlying the induction of L-CPT I gene expression by cAMP were characterized. We demonstrate that the cAMP response unit of the L-CPT I gene is composed of a cAMP-response element motif and a DR1 sequence located 3 kb upstream of the transcription start site. Our data strongly suggest that the coactivator PGC-1 is involved in the regulation of this gene expression by cAMP in combination with HNF4α and cAMP-response element-binding protein (CREB). Indeed, (i) cotransfection of CREB or HNF4α dominant negative mutants completely abolishes the effect of cAMP on the L-CPT I promoter, and (ii) the cAMP-responsive unit binds HNF4α and CREB through the DR1 and the cAMP-response element sequences, respectively. Moreover, cotransfection of PGC-1 strongly activates the L-CPT I promoter through HNF4α bound at the DR1 element. Finally, we show that the transcriptional induction of the PGC-1 gene by glucagon through cAMP in hepatocytes precedes that of L-CPT-1. In addition to the key role that PGC-1 plays in glucose homeostasis, it may also be critical for lipid homeostasis. Taken together these observations suggest that PGC-1 acts to coordinate the process of metabolic adaptation in the liver. carnitine palmitoyltransferase liver CPT cAMP response element cAMP-response element binding protein cAMP-response element modulator CAAT/enhancer binding protein protein kinase A CREB-binding protein tyrosine aminotransferase phosphoenolpyruvate carboxykinase cAMP response unit chloramphenicol acetyltransferase electrophoretic mobility shift assays hypersensitive site direct repeat hepatocyte nuclear factor Liver mitochondrial long-chain fatty acid oxidation and ketogenesis are closely related to the appearance of the carnitine palmitoyltransferase (CPT)1system. The CPT system, which controls the transfer of long-chain fatty acids inside the mitochondria, is composed of three distinct entities: CPT I localized in the outer mitochondrial membrane, carnitine-acylcarnitine translocase and CPT II localized on the inner mitochondrial membrane (1McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1323) Google Scholar). It is generally accepted that CPT I is the rate-limiting enzyme for the oxidation of long-chain fatty acids in the liver (1McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1323) Google Scholar). Two isoforms of CPT I have been cloned (2Esser V. Britton C.H. Weis B.C. Foster D.W. McGarry J.D. J. Biol. Chem. 1993; 268: 5817-5822Abstract Full Text PDF PubMed Google Scholar, 3Esser V. Brown N.F. Cowan A.T. Foster D.W. McGarry J.D. J. Biol. Chem. 1996; 271: 6972-6977Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The “liver” isoform (L-CPT I) is expressed in most tissues including liver, kidney, and pancreatic β cells (2Esser V. Britton C.H. Weis B.C. Foster D.W. McGarry J.D. J. Biol. Chem. 1993; 268: 5817-5822Abstract Full Text PDF PubMed Google Scholar) while the “muscle” isoform (M-CPT I) is expressed in heart, skeletal muscle, and adipose tissue (3Esser V. Brown N.F. Cowan A.T. Foster D.W. McGarry J.D. J. Biol. Chem. 1996; 271: 6972-6977Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 4Yamazaki N. Shinohara Y. Shima A. Terada H. FEBS Lett. 1995; 363: 41-45Crossref PubMed Scopus (110) Google Scholar).Expression of the CPT I gene in liver is regulated during development and by several hormones and nutrients (1McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1323) Google Scholar). At birth, rat hepatic CPT I mRNA increases, remains high during the suckling period, and then decreases after weaning onto a high carbohydrate diet (5Thumelin S. Esser V. Charvy D. Kolodziej M. Zammit V. McGarry J.D. Girard J. Pégorier J.P. Biochem. J. 1994; 300: 583-587Crossref PubMed Scopus (79) Google Scholar). Few studies have addressed the direct effects of pancreatic hormones on the regulation of L-CPT I gene expression. However, it was shown that the addition of glucagon or its second messenger, cyclic AMP, induces transcription of the L-CPT I gene without modification of mRNA half-life (6Louet J.F., Le May C. Pégorier J.P. Decaux J.F. Girard J. Biochem. Soc. Trans. 2001; 29: 310-316Crossref PubMed Google Scholar). In cultured fetal rat hepatocytes, insulin antagonizes the cAMP-induced accumulation of CPT I mRNA (7Chatelain F. Kohl C. Esser V. McGarry J.D. Girard J. Pégorier J.P. Eur. J. Biochem. 1996; 235: 789-798Crossref PubMed Scopus (109) Google Scholar). These observations suggest a prominent role for pancreatic hormones in the regulation of L-CPT I gene expression.The rat L-CPT I gene has been cloned, and its promoter partially characterized (8Park E.A. Steffen M.L. Song S. Park V.M. Cook G.A. Biochem. J. 1998; 330: 217-224Crossref PubMed Scopus (38) Google Scholar). Basal expression of L-CPT I is driven by Sp1 and NF-Y transcription factors (9Steffen M.L. Harrison W.R. Elder F.F.B. Cook G.A. Park E.A. Biochem. J. 1999; 340: 425-432Crossref PubMed Scopus (32) Google Scholar). Initial characterization of the DNA sequences involved in the hormonal and nutritional response of L-CPT I gene expression allowed the identification of: (i) a functional thyroid hormone (T3)-response element located approximately −3 kb from the transcription start site of the gene (10Jansen M.S. Cook G.A. Song S. Park E.A. J. Biol. Chem. 2000; 275: 34989-34997Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), (ii) a peroxisome proliferator-response element, located at −3 kb from the start site, corresponding to a classical DR1 element (11Louet J.F. Chatelain F. Decaux J.F. Park E.A. Kohl C. Pineau T. Girard J. Pégorier J.P. Biochem. J. 2001; 354: 189-197Crossref PubMed Scopus (134) Google Scholar), and (iii) a fatty acid-response element present in the first intron of the gene (11Louet J.F. Chatelain F. Decaux J.F. Park E.A. Kohl C. Pineau T. Girard J. Pégorier J.P. Biochem. J. 2001; 354: 189-197Crossref PubMed Scopus (134) Google Scholar).Cyclic AMP activates eukaryotic gene transcription via binding of nuclear factors to cAMP-responsive elements (CRE). The classical CRE consists of a relatively well conserved 8-bp palindrome (TGACGTCA) (12Mayr B. Montminy M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 599-609Crossref PubMed Scopus (2035) Google Scholar). Nuclear factors involved in cAMP-responsive transcriptional activation include cAMP-response element binding protein (CREB), cAMP-response element modulator (CREM), activating transcription factor-1 (ATF-1), and potentially, members of the nuclear receptor superfamily (13Daniel P.B. Walker W.H. Habener J.F. Annu. Rev. Nutr. 1998; 18: 353-383Crossref PubMed Scopus (212) Google Scholar). CREB proteins belong to a family of transcription factors that contain a basic region/leucine zipper (bZIP). The bZIP family includes Jun, Fos, CAAT/enhancer binding protein (C/EBP), and yeast GCN4 (13Daniel P.B. Walker W.H. Habener J.F. Annu. Rev. Nutr. 1998; 18: 353-383Crossref PubMed Scopus (212) Google Scholar). Current models of cAMP-responsive transcriptional activation invoke phosphorylation of proteins of the CREB family on a single serine residue in response to activation of protein kinase A (PKA) by cAMP. Phosphorylation of CREB promotes the recruitment of the transcriptional coactivator CREB-binding protein (CBP) and increases cAMP-induced transcription. However, one major limitation of this model is that CREB, ATF-1, and CBP genes are all ubiquitously expressed and are not transcriptionally activated by cAMP (13Daniel P.B. Walker W.H. Habener J.F. Annu. Rev. Nutr. 1998; 18: 353-383Crossref PubMed Scopus (212) Google Scholar). Thus, this model cannot explain the sensitivity of liver-specific genes such as tyrosine aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK) to cAMP. In this regard, the cAMP-dependent enhancement of TAT expression is dependent upon the cooperative action of CREB and HNF4α, allowing tissue-specific control of this gene (14Nitsch D. Boshart M. Schutz G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5479-5483Crossref PubMed Scopus (161) Google Scholar). Similarly, while the promoter of the PEPCK gene contains a classical CRE element, maximal cAMP induction of this gene requires multiple cis-acting sites within a so-called “cAMP response unit” (CRU) (15Roesler W.J. Mol. Cell. Endocrinol. 2000; 162: 1-7Crossref PubMed Scopus (70) Google Scholar). The CRU of the PEPCK promoter contains five cis-elements including the CRE and four binding sites for a C/EBP (15Roesler W.J. Mol. Cell. Endocrinol. 2000; 162: 1-7Crossref PubMed Scopus (70) Google Scholar). Therefore, the molecular mechanisms involved in the cAMP-mediated induction of target genes seem to be more complex than the simple presence of a CRE motif.Recently, PPARγ coactivator-1 (PGC-1) was shown to be involved in the regulation of gluconeogenic genes by cAMP and glucocorticoids (16Yoon J.C. Puigserver P. Chen G. Donovan J., Wu, Z. Rhee J. Adelmant G. Stafford J. Kahn C.R. Granner D.K. Newgard C.B. Spiegelman B.M. Nature. 2001; 413: 131-138Crossref PubMed Scopus (1496) Google Scholar). PGC-1 is strongly induced in mouse liver by fasting, insulin deficiency (e.g. streptozotocin-induced diabetes, ob/ob genotype, and liver insulin receptor knockout), and, in primary cultures of hepatocytes, by cAMP (16Yoon J.C. Puigserver P. Chen G. Donovan J., Wu, Z. Rhee J. Adelmant G. Stafford J. Kahn C.R. Granner D.K. Newgard C.B. Spiegelman B.M. Nature. 2001; 413: 131-138Crossref PubMed Scopus (1496) Google Scholar). Furthermore, both PEPCK and glucose-6-phosphatase genes are activated by overexpression of PGC-1 (16Yoon J.C. Puigserver P. Chen G. Donovan J., Wu, Z. Rhee J. Adelmant G. Stafford J. Kahn C.R. Granner D.K. Newgard C.B. Spiegelman B.M. Nature. 2001; 413: 131-138Crossref PubMed Scopus (1496) Google Scholar). Full transcriptional activation of the PEPCK promoter requires a coactivation of the glucocorticoid receptor and HNF4α (16Yoon J.C. Puigserver P. Chen G. Donovan J., Wu, Z. Rhee J. Adelmant G. Stafford J. Kahn C.R. Granner D.K. Newgard C.B. Spiegelman B.M. Nature. 2001; 413: 131-138Crossref PubMed Scopus (1496) Google Scholar). These data strongly suggest that PGC-1 could be a key modulator of the metabolic adaptation of the liver.Here, we have characterized a cAMP-responsive unit in the L-CPT I gene. The CRU contains two cis-acting DNA sequences: a CRE and a DR1 motif. We demonstrate that CREB and HNF4α bind to the L-CPT I promoter via the CRE and DR1, respectively. Moreover, we show that the nuclear receptor coactivator PGC-1 strongly stimulates the L-CPT I promoter. In addition, PGC-1 expression is transcriptionally activated by cAMP and glucagon in primary cultures of rat hepatocytes. As this induction precedes that of L-CPT I, we suggest that PGC-1 is involved in the activation of the L-CPT I gene by cAMP. Taken together, our results suggest that PGC-1, in addition to controlling gluconeogenesis, also plays a role in the adaptation of hepatic lipid metabolism in response to nutritional stimuli.EXPERIMENTAL PROCEDURESAnimalsFemale Wistar rats bred in our laboratory were housed in plastic cages at a constant temperature, 24 °C, with light between 6.00–20.00 h. They had free access to water and chow pellets (65% carbohydrate, 11% fat, 24% protein by energy content). Livers from 21-day-old fetuses delivered by cesarean section and 1-day-old suckling newborn rats were used for nuclear extract isolation.Isolation and Culture of Suckling Rat Hepatocytes12-day-old suckling pups were anesthetized with pentobarbital (50 mg/kg). Hepatocytes were isolated by liver perfusion as described previously (17Narkewicz M.R. Iynedjian P.B. Ferré P. Girard J. Biochem. J. 1990; 271: 585-589Crossref PubMed Scopus (44) Google Scholar). After measurement of cell viability by trypan blue exclusion, hepatocytes were suspended in M199 medium (Invitrogen) containing 5.5 mm glucose, 0,1% (mass/v) bovine serum albumin, and 2% (v/v) Ultroser G (Invitrogen). Cells (2 × 106) were plated in 2 ml of M199 medium in 12-well plates. Cultures were maintained at 37 °C and 5% CO2. After plating for 4 h, the medium containing unattached cells was removed and replaced by 1 ml of fresh M199 without Ultroser G or bovine serum albumin for the transfection experiments.Plasmids ConstructsReporter ConstructsThe reporter plasmids CPT20, CPT18, CPT14, and the CPT15 contain 6870, 4495, 1653, and 193 bp, respectively, of the 5′ flanking sequence from the transcription start site of the L-CPT I gene (8Park E.A. Steffen M.L. Song S. Park V.M. Cook G.A. Biochem. J. 1998; 330: 217-224Crossref PubMed Scopus (38) Google Scholar). A PstI-EcoRI fragment (−3000 to −1653) from the original P1 clone (8Park E.A. Steffen M.L. Song S. Park V.M. Cook G.A. Biochem. J. 1998; 330: 217-224Crossref PubMed Scopus (38) Google Scholar) was ligated into the PstI and EcoRI sites of pBS II K/S+ (Stratagene, La Jolla, CA). The SmaI-XhoI fragment from this vector was ligated intoSmaI-XhoI of CPT15 to yield CPT16.L-CPT I (−2967 to −2773) was described previously (11Louet J.F. Chatelain F. Decaux J.F. Park E.A. Kohl C. Pineau T. Girard J. Pégorier J.P. Biochem. J. 2001; 354: 189-197Crossref PubMed Scopus (134) Google Scholar). Serial deletions of this construct were created by PCR. The 3′ end deletion including L-CPT I (−2967 to −2831) and the L-CPT I (−2967 to −2871) constructs were made with the same forward primer including aHindIII restriction site: (−2967) 5′-TGAAGCTTGGGGTTTGTTATCCTTG-3′ and two different reverse primers containing XbaI restriction sites: (−2831) 5′-CCTCTAGACCTCAGTTCCCCCTGA and (−2871) 5′-CCTCTAGACGTTTTGAGTCAATA-3′. L-CPT I (−2938 to −2831) was generated with the same reverse primer (−2831) and the forward primer (−2938): 5′-CGAAGCTTTCCTCATGGAACCT-3′. These HindIII-XbaI PCR fragments were ligated into the pBLCAT5 vector (18Boshart M. Klüppel M. Schmidt A. Schütz G. Luckow B. Gene. 1992; 110: 129-130Crossref PubMed Scopus (230) Google Scholar). Automated DNA sequencing confirmed that the nucleotide sequence of the PCR product was 100% identical to the template sequence. The heterologous construct containing the CRE sequence of the rat somatostatin promoter was described previously (19de Groot R.P. Sassone-Corsi P. Oncogene. 1992; 7: 2281-2286PubMed Google Scholar).The CRE2x, DR1, CRE, and CRE/DR1 constructs were prepared by ligation of double-stranded oligonucleotides into the HindIII andXbaI sites of the pBLCAT5 vector. Oligonucleotides used for each construct were as follows: CRE2x-upper-5′-AGCTCCTGGTGACGCTGGCCCTGGTGACGCTGGC-3′, CRE2x-lower-5′-CTAGGCCAGCGTCACCAGGGCCAGCGTCACCAGG-3′, DR1-upper-5′-AGCTCTCAAAACGTGTACAGGAGCTCAAAGTTCAAGTTCAAGTTCAGG-3′, DR1-lower-5′-CTAGCCTGAACTTGAACTTTGAGCTCCTGTACACGTTTTGAG-3′, CRE-upper-5′-AGCTGCTGTCCTCATGGAACCTGGTGACGCTGGCTGAACAA-3′, CRE-lower-5′-CTAGTTGTTCAGCCAGCGTCACCAGGTTCCATGAGGACAGC-3′, CRE/DR1-upper-5′-AGCTGGTGACGCTGGCACAGGAGCTCAAAGTTCAAGTTCAGG-3′, and CRE/DR1-lower-5′-CTAGCCTGAACTTGAACTTGAACTTTGAGCTCCTGTGCCAGCGTCACC-3′. Site-directed mutagenesis was conducted using the DpnI QuikChange method (Stratagene, La Jolla, CA). Oligonucleotides used to disrupt each DNA motif were: CREm-upper-5′-CATGGAACCTGGTaAgcCTGGCTGAAC-3′, AP1m-upper-ACCTCCGCTATTCtCgtAAAACGTGTACAGG-3′, and DR1m-upper-CGTGTACAGGAGCTCAgcGaaCAAGTTCAGGG-3′. Mutation in each construct (CREm, DR1m, AP1m, and CREm-DR1m) was confirmed by sequence analysis.Expression VectorThe expression vector pSV-PKA-Cα codes for the catalytic Cα subunit of PKA (20Gourdon L. Lou D.Q. Raymondjean M. Vasseur-Cognet M. Kahn A. FEBS Lett. 1999; 459: 9-14Crossref PubMed Scopus (17) Google Scholar). The vector coding for Dn-HNF4α is a selective dominant negative mutant that forms defective heterodimers with wild-type HNF4α, preventing DNA binding and transcriptional activation by HNF4α. It was a gift from T. Leff (21Taylor D.G. Haubenwallner S. Leff T. Nucleic Acids Res. 1996; 24: 2930-2935Crossref PubMed Scopus (25) Google Scholar). The dominant negative mutant Dn-CREB that blocks binding of CREB and closely related transcription factors (such as ATF1) to DNA was a gift from D. Ginty (22Ahn S. Olive M. Aggarwal S. Krylov D. Ginty D.D. Vinson C. Mol. Cell. Biol. 1998; 18: 967-977Crossref PubMed Scopus (445) Google Scholar). The expression vectors pSVSport-PGC-1 and VP16-CREB were described previously (16Yoon J.C. Puigserver P. Chen G. Donovan J., Wu, Z. Rhee J. Adelmant G. Stafford J. Kahn C.R. Granner D.K. Newgard C.B. Spiegelman B.M. Nature. 2001; 413: 131-138Crossref PubMed Scopus (1496) Google Scholar).Transient Transfection ProtocolPrimary cultures of rat hepatocytes were transfected as described by Boussif et al. (23Boussif O. Lezoualc'h F. Zanta M.A. Mergny M.D. Scherman D. Demeneix B. Behr J.P. Proc. Natl. Acad. Sci. 1995; 92: 7297-7301Crossref PubMed Scopus (5575) Google Scholar). To increase transfection efficiency, 200 plaque-forming units/cell of Ad-RSV-nlsLacZ (Rous sarcoma virus promoter driving the nlsLacZ gene) were added simultaneously (24Meunier-Durmort C. Grimal H. Sachs L. Demeneix B. Forest C. Gene Therapy. 1997; 4: 808-814Crossref PubMed Scopus (56) Google Scholar). Briefly, cells were transfected with 2.5 μg of DNA plasmid per plate and polyethylenimine (0.56 μl of polyethylenimine/μg of DNA) in serum free medium. In some experiments, cells were cotransfected with 1 μg of expression vectors. Transfection was accomplished by dilution of polyethylenimine and plasmid in 25 μl of 150 mm NaCl. They were mixed together, and the polycation/DNA complex was diluted into 1 ml of serum-free medium in 12-well culture dishes. After 7 h of transfection, the medium was changed and Bt2cAMP at 10−4m, glucagon at 10−8m, or vehicle was added for 24 h (luciferase assay) or 48 h (CAT assay). Cells were lysed in reporter lysis buffer (Promega), harvested by scraping, and centrifuged at 8000 g at 4 °C for 5 min. Protein concentration, luciferase, and CAT activities were determined in the supernatant. Luciferase activity was assayed on 20 μl of supernatant using the Promega kit (Promega). CAT activity was assayed as described by Seed and Sheen (25Seed B. Sheen J.Y. Gene. 1988; 67: 271-277Crossref PubMed Scopus (830) Google Scholar).Chromatin Structure of the L-CPT I Promoter and Mapping of DNase I Hypersensitive SitesNuclei for chromatin studies were isolated from fresh tissues as described by Schibler et al. (26Schibler U. Hagenbuchle O. Wellauer P.K. Pittet A.C. Cell. 1983; 33: 501-508Abstract Full Text PDF PubMed Scopus (268) Google Scholar). Isolated nuclei were suspended in nuclear storage buffer: 0.3 m sucrose, 60 mm KCl, 15 mm NaCl, 0.1 mm EGTA, 0.2 mm EDTA, 0.15 mm spermine, 0.5 mm spermidine, 15 mm Tris/HCl, pH 7.5, 5% (v/v) glycerol, 0.5 mm dithiothreitol, and 0.1 mm phenylmethylsulfonyl fluoride. Frozen nuclei were thawed and resuspended at a DNA concentration of 2 mg/ml in digestion buffer containing 60 mm KCl, 0.1 mmEGTA, 5% (v/v) glycerol, 15 mm Tris/HCl, pH 7.5, and 0.5 mm dithiothreitol. Nuclei were prewarmed to 37 °C, and digestions initiated by adding nuclei into tubes containing 5 mm MgCl2 and DNase I (Invitrogen) ranging over 1–8 μg/ml. Control digestion tubes contain MgCl2 but no DNase I. After 15 min of digestion at 37 °C, the reaction was stopped by addition of the same volume of a buffer containing 150 mm NaCl, 15 mm EDTA, 0.3% (mass/v) sodium dodecyl sulfate, and 50 mm Tris/HCl, pH 7.5. Nuclei were digested by RNase A (50 μg/ml) for 1 h at 37 °C and then incubated overnight at 37 °C with proteinase K (100 μg/ml). DNA was extracted and quantified by spectrophotometry.For studies of DNase I hypersensitive sites, DNA from undigested and DNase I from digested nuclei was cut with EcoRI orHindIII (4 units/μg DNA) for 16 h at 37 °C, subjected to electrophoresis, and transferred onto Hybond N+ filter (Amersham Biosciences). For hybridization, two genomic probes were used: a 744-bp EcoRI-SphI fragment localized at −1653 bp from the transcription start site named S1 and an 855-bpHindIII-BamHI fragment located in the first intron named S2. Probes were labeled with [α-32P]dATP using the multiprime labeling system kit (Amersham Biosciences).Total RNA Extraction and AnalysisTotal RNA was extracted from cultured rat hepatocytes according to Chomczynski and Sacchi (27Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62986) Google Scholar). Total RNA (15 μg) was separated on 1% agarose gels containing 2.2 m formaldehyde and transferred to Hybond N membranes (Amersham Biosciences). Filters were hybridized overnight with 32P-labeled cDNA probes. The PEPCK probe was previously described (28Yoo-Warren H.Y. Cimbala M.A. Felz K. Monahan J.E. Leis J.P. Hanson R.W. J. Biol. Chem. 1981; 256: 10224-10227Abstract Full Text PDF PubMed Google Scholar). L-CPT I and PGC-1 probes were amplified by PCR with the following primers: CPT I-upper-5′-ACCAAGCTGTGGCCTTCCAGTTCACCGTCAC-3′; CPT I-lower-5′-GCGTGGCAGAGACGTCTGGAAGCTGTACAA-3′; PGC-1-upper-5′-TGTATGGAGTGACATAGAGT-3′; and PGC-1-lower-5′-GCTAAGACCGCTGCATTCAT-3′.Electrophoretic Mobility Shift Assays (EMSA)In vitro translated HNF4α was generated using the TnT T7-coupled reticulocyte lysate system (Promega). Recombinant HNF4α and CREB proteins were prepared as described previously (29Viollet B. Kahn A. Raymondjean M. Mol. Cell. Biol. 1997; 17: 4208-4219Crossref PubMed Scopus (147) Google Scholar,30Groussin L. Massias J.F. Bertagna X. Bertherat J. J. Clin. Endocrinol. Metab. 2000; 85: 345-354Crossref PubMed Scopus (56) Google Scholar). Nuclear protein extracts from liver and hepatocytes of rat were prepared with NE-PER kit (Pierce). In vitro translated proteins, recombinant HNF4α, recombinant CREB, or nuclear protein extracts, were incubated at 4 °C for 30 min with 30,000 cpm of labeled double-stranded oligonucleotides in a buffer containing 20 mm Tris/HCl, pH 7.5, 100 mm KCl, 14% (v/v) glycerol, 2 mm dithiothreitol, and 1 μg of poly(dI-dC) in a final volume of 20 μl. In the supershift assay antibodies to HNF4α or CREB were added to the binding reaction. Complexes were separated on a 6% (v/v) native polyacrylamide gel in 0.25× TBE running buffer at 200 V for 2 h. For DNA binding competition experiments, excess unlabeled double-stranded competitor oligonucleotide was added to the incubation reaction as detailed in the figure legends. Following electrophoresis gels were dried and exposed to x-ray films. The sense oligonucleotides used were: 5′-AGTCAAAAGTGTACAGGAGCTCAAAGTTCAAGTTCAG-3′ for L-CPT I-DR1; 5′-AGCTGCTGTCCTCATGGAACCTGGTGACGCTGGCTGAACAA-3′ for L-CPT I-CRE; and 5′-GATCGCCTCCTTGGCTGACGTCAGAGA-3′ for somatostatin-CRE.L-CPT I expression in Liver-specific HNF4α-null Mice (H4LivKO)Liver-specific HNF4α-null mice, in which disruption of a loxP-targeted allele of HNF4α is driven by an albumin-Cre transgene, have been described (31Hayhurst G.P. Lee Y.H. Lambert G. Ward J.M. Gonzalez F.J. Mol. Cell. Biol. 2001; 21: 1393-1403Crossref PubMed Scopus (849) Google Scholar). 45-day-old mice were anesthetized with 2.5% avertin and killed by decapitation, and livers were collected and frozen immediately in liquid nitrogen. Total liver RNA was prepared as previously described (31Hayhurst G.P. Lee Y.H. Lambert G. Ward J.M. Gonzalez F.J. Mol. Cell. Biol. 2001; 21: 1393-1403Crossref PubMed Scopus (849) Google Scholar). Poly A+ RNAs were prepared from total liver RNA using the Poly A Tract mRNA kit (Promega). Poly(A)+ RNAs (1 μg) were separated on 1% agarose, 2.2 m formaldehyde gels prior to blotting to Hybond-N+ membranes (Amersham Biosciences). Blots were hybridized by using successively specific probes of L-CPT I and glyceraldehyde-3-phosphate dehydrogenase (31Hayhurst G.P. Lee Y.H. Lambert G. Ward J.M. Gonzalez F.J. Mol. Cell. Biol. 2001; 21: 1393-1403Crossref PubMed Scopus (849) Google Scholar).DISCUSSIONHormones and neurotransmitters change patterns of gene expression in target cells by activating protein kinases that phosphorylate and modify the activity of different transcription factors (32Montminy M. Annu. Rev. Biochem. 1997; 66: 807-822Crossref PubMed Scopus (854) Google Scholar). CREB, the first transcription factor whose activity was shown to be regulated by phosphorylation, mediates the transcriptional effect of cAMP by binding the CRE element in the upstream region of a number of genes (33Rudolph D. Tafuri A. Gass P. Hammerling G.J. Arnold B. Schutz G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4481-4486Crossref PubMed Scopus (265) Google Scholar). This transcription factor is phosphorylated by PKA after an increase of intracellular cAMP concentration. More recently, it was demonstrated that CREB is an in vivo substrate for a variety of other kinases like the calmodulin kinase, the mitogen-activated protein kinase/p90rsk, the protein kinase C and the protein kinase B (PKB/Akt) (34Mayr B.M. Canettieri G. Montminy M.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10936-10941Crossref PubMed Scopus (154) Google Scholar). This suggests that CREB could activate transcription of genes in response to various other extracellular signals such as Ca2+ or growth factors (35Gonzalez G.A. Montminy M.R. Cell. 1989; 59: 675-680Abstract Full Text PDF PubMed Scopus (2041) Google Scholar, 36Sheng M. Thompson M.A. Greenberg M.E. Science. 1991; 252: 1427-1430Crossref PubMed Scopus (1274) Google Scholar, 37Ginty D.D. Bonni A. Greenberg M.E. Cell. 1994; 77: 713-725Abstract Full Text PDF PubMed Scopus (674) Google Scholar). Thus, the molecular mechanisms mediating the cAMP signal and its specificity are more complex than previously thought.In the present study, we have examined the regulation of L-CPT I gene expression by cAMP in hepatocytes. Our results show that two cis-acting elements, a DR1 motif and a CRE-like element (TGACG), are required for the full stimulatory effect of cAMP on L-CPT I expression. This type of “multi-responsive element” structure, termed CRU, is not a surprising observation. Indeed, it is now generally accepted that transcriptional regulation of genes by cAMP seldom occurs through a unique CRE motif as described for the somatostatin gene (38Montminy M. Brindle P. Arias J. Ferreri K. Armstrong R. Adv. Pharmacol. 1996; 36: 1-13Crossref PubMed Scopus (12) Google Scholar). For example, the full cAMP induction of glucose-6-phosphatase gene expression involves two CRE elements in HepG2 cell lines (39Lin B. Morris D.W. Chou J.Y. Biochemistry. 1997; 36: 14096-14106Crossref PubMed Scopus (81) Google Scholar, 40Schmoll D. Wasner C. Hinds C.J. Allan B.B. Walther R. Burchell A. Biochem. J. 1999; 338: 457-463Crossref PubMed Scopus (71) Google Scholar), while in kidney cell lines, the same gene is regulated by cAMP through these two CRE sequences and an HNF1 motif (41Streeper R.S. Hornbuckle L.A. Svitek C.A. Goldman J.K. Oeser J.K. O'Brien R.M. J. Biol. Chem. 2001; 276: 19111-19118Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Furthermore, the CRU of the TAT gene, which like L-CPT I, is induced by glucagon and repressed by insulin, is composed of a CRE and a DR1 motif (14Nitsch D. Boshart M. Schutz G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5479-5483Crossref PubMed Scopus (161) Google Scholar). The L-CPT I gene contains four consensus CRE sites, from CRE1 to CRE4 (6Louet J.F., Le May C. Pégorier J.P. Decaux J.F. Girard J. Biochem. Soc. Trans. 2001; 29: 310-316Crossref PubMed Google Scholar). Interestingly, the classical, symmetric CRE1 (TGACGTAA), which differs from the well characterized CRE sequence of the rat somatostatin gene (TGACGTCA) by only one nucleotide, is not sufficient for the response to cAMP. These data strongly suggest that the presence of CRE consensus sequences within the promoter of genes is not sufficient to fully explain cAMP gene activation. Our study shows that the functionality of a CRE sequence is more dependent on its promoter context (proximity to other cis-acting element(s), localization in an open chromatin structure" @default.
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• W2078220726 title "The Coactivator PGC-1 Is Involved in the Regulation of the Liver Carnitine Palmitoyltransferase I Gene Expression by cAMP in Combination with HNF4α and cAMP-response Element-binding Protein (CREB)" @default.
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