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• W2091254654 abstract "Glucagon, acting via cAMP, inhibits transcription of the malic enzyme gene in chick embryo hepatocytes. In transiently transfected hepatocytes, fragments from the 5′-flanking DNA of the malic enzyme gene confer cAMP responsiveness to linked reporter genes. The major inhibitory cAMP response element at −3180/−3174 base pairs (bp) is similar to the consensus binding site for AP1. DNA fragments from −3134/−3115, −1713/−944, and −413/−147 bp also contain inhibitory cAMP response elements. The negative action of cAMP is mimicked by overexpression of the catalytic subunit of protein kinase A, inhibited by overexpression of a specific inhibitor of protein kinase A, and inhibited by overexpression of the T3 receptor; these results indicate involvement of the classical eukaryotic pathway for cAMP action and suggest interaction between the T3 and cAMP pathways. Sequence-specific complexes form between nuclear proteins and a DNA fragment containing −3192/−3158 bp of 5′-flanking DNA. In nuclear extracts prepared from cells treated with chlorophenylthio-cyclic AMP and T3, the complexes have different masses than those formed with extracts from cells treated with T3 alone. Antibodies to c-Fos or ATF-2 inhibit formation of the complex formed by proteins from cells treated with chlorophenylthio-cyclic AMP and T3 but not by those from cells treated with T3 alone. These results suggest an important role for c-Fos and ATF-2 in glucagon-mediated inhibition of transcription of the malic enzyme gene. Glucagon, acting via cAMP, inhibits transcription of the malic enzyme gene in chick embryo hepatocytes. In transiently transfected hepatocytes, fragments from the 5′-flanking DNA of the malic enzyme gene confer cAMP responsiveness to linked reporter genes. The major inhibitory cAMP response element at −3180/−3174 base pairs (bp) is similar to the consensus binding site for AP1. DNA fragments from −3134/−3115, −1713/−944, and −413/−147 bp also contain inhibitory cAMP response elements. The negative action of cAMP is mimicked by overexpression of the catalytic subunit of protein kinase A, inhibited by overexpression of a specific inhibitor of protein kinase A, and inhibited by overexpression of the T3 receptor; these results indicate involvement of the classical eukaryotic pathway for cAMP action and suggest interaction between the T3 and cAMP pathways. Sequence-specific complexes form between nuclear proteins and a DNA fragment containing −3192/−3158 bp of 5′-flanking DNA. In nuclear extracts prepared from cells treated with chlorophenylthio-cyclic AMP and T3, the complexes have different masses than those formed with extracts from cells treated with T3 alone. Antibodies to c-Fos or ATF-2 inhibit formation of the complex formed by proteins from cells treated with chlorophenylthio-cyclic AMP and T3 but not by those from cells treated with T3 alone. These results suggest an important role for c-Fos and ATF-2 in glucagon-mediated inhibition of transcription of the malic enzyme gene. Malic enzyme (ME) 1The abbreviations used are: ME, malic enzyme; ATF, activating transcription factor; CAT, chloramphenicol acetyltransferase; CRE, cAMP response element; CREB, cAMP response element binding protein; CBP, CREB-binding protein; CREM, cAMP response element modulator; CPT-cAMP, chlorophenylthiocyclic AMP; ICRE, inhibitory cAMP response element; PKA, protein kinase A; PKi; inhibitor of protein kinase A; RSV, Rous sarcoma virus; TR, thyroid hormone receptor; T3, 3,5,3′-l-triiodothyronine; T3RE, triiodothyronine response element; TK, thymidine kinase; bp, base pair(s); kb, kilobase pair(s); IL-2, interleukin-2; IL-2R, IL-2 receptor. 1The abbreviations used are: ME, malic enzyme; ATF, activating transcription factor; CAT, chloramphenicol acetyltransferase; CRE, cAMP response element; CREB, cAMP response element binding protein; CBP, CREB-binding protein; CREM, cAMP response element modulator; CPT-cAMP, chlorophenylthiocyclic AMP; ICRE, inhibitory cAMP response element; PKA, protein kinase A; PKi; inhibitor of protein kinase A; RSV, Rous sarcoma virus; TR, thyroid hormone receptor; T3, 3,5,3′-l-triiodothyronine; T3RE, triiodothyronine response element; TK, thymidine kinase; bp, base pair(s); kb, kilobase pair(s); IL-2, interleukin-2; IL-2R, IL-2 receptor. (EC1.1.1.40) catalyzes the oxidative decarboxylation of malate to pyruvate and CO2, simultaneously generating NADPH from NADP+. In avian liver, most of the NADPH used in thede novo synthesis of long chain fatty acids is generated by malic enzyme (1Goodridge A.G. Biochem. J. 1968; 108: 663-666Crossref PubMed Scopus (84) Google Scholar). Malic enzyme is a typical lipogenic enzyme; its activity in avian liver increases about 70-fold when newly hatched chicks are fed a diet high in carbohydrate (1Goodridge A.G. Biochem. J. 1968; 108: 663-666Crossref PubMed Scopus (84) Google Scholar) and decreases dramatically when animals are starved (2Goodridge A.G. Biochem. J. 1968; 108: 667-673Crossref PubMed Scopus (61) Google Scholar). In chicken embryo hepatocytes in culture, insulin plus T3 causes about a 50-fold increase in malic enzyme activity and abundance of its mRNA; glucagon or cAMP blocks these effects (3Goodridge A.G. Adelman T.G. J. Biol. Chem. 1976; 251: 3027-3032Abstract Full Text PDF PubMed Google Scholar, 4Salati L.M. Ma X.-J. McCormick C.C. Stapleton S.R. Goodridge A.G. J. Biol. Chem. 1991; 266: 4010-4016Abstract Full Text PDF PubMed Google Scholar). Within 1 h after adding T3 to chick embryo hepatocytes, transcription of the malic enzyme gene increases by 30–40-fold; cAMP completely inhibits this increase (4Salati L.M. Ma X.-J. McCormick C.C. Stapleton S.R. Goodridge A.G. J. Biol. Chem. 1991; 266: 4010-4016Abstract Full Text PDF PubMed Google Scholar). The T3-dependent increase in transcription of the malic enzyme gene is mediated by several T3 response elements (T3REs), with the major one between −3883 and −3858 bp upstream of the start site for transcription (5Hodnett D.W. Fantozzi D.A. Thurmond D.C. Klautky S.A. MacPhee K.G. Estrem S.T. Xu G. Goodridge A.G. Arch. Biochem. Biophys. 1996; 334: 309-324Crossref PubMed Scopus (32) Google Scholar). Several positive acting cAMP response elements have been described (6Delmas V. Molina C.A. Lalli E. deGroot R. Foulkes N.S. Masquilier D. Sassone-Corsi P. Rev. Physiol. Biochem. Pharmacol. 1994; 124: 1-28Crossref PubMed Google Scholar). Cyclic AMP stimulates gene expression by activating protein kinase A (PKA), which, in turn, phosphorylates members of the CREB/ATF family of transcription factors, thereby increasing their transactivation potential (7Richards J.P. Bachinger H.P. Goodman R.H. Brennan R.G. J. Biol. Chem. 1996; 271: 13716-13723Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The CREB/ATF family includes polypeptides encoded by at least seven distinct genes (6Delmas V. Molina C.A. Lalli E. deGroot R. Foulkes N.S. Masquilier D. Sassone-Corsi P. Rev. Physiol. Biochem. Pharmacol. 1994; 124: 1-28Crossref PubMed Google Scholar) that share the ability to bind, with different affinities, to CREs in the 5′-flanking DNA of genes activated by cAMP. The CREB/ATF proteins also dimerize with AP1 proteins, members of another family of leucine zipper proteins (8Benbrook D.M. Jones N.C. Oncogenes. 1990; 5: 295-302PubMed Google Scholar) for which the binding site differs from a consensus CRE by only one bp (9Angel P. Karin M. Biochem. Biophys. Acta. 1991; 1072: 129-157Crossref PubMed Scopus (3237) Google Scholar). Much less is known about negative-acting cAMP-response elements. In the gene for L-type pyruvate kinase, the L4 element, −168 to −144 bp, binds major late transcription factor; it is the glucose/insulin response element and is required for inhibition by cAMP. Inhibition by cAMP also requires the contiguous L3 element, an element that binds hepatic nuclear factor 4 (10Bergot M.O. Diaz-Guerra M.J.M. Puzenat N. Raymondjean M. Khan A. Nucleic Acids Res. 1992; 20: 1871-1878Crossref PubMed Scopus (153) Google Scholar). Cyclic AMP also inhibits transcription of the genes for IL-2 and IL-2R in EL4 cells; the inhibition requires an AP1 site. In this case, cAMP increases the binding of Jun/Fos heterodimers to the AP1 site and alters the composition of Jun proteins that participate in the AP1 complex (11Tamir A. Isakov N. J. Immunol. 1994; 152: 3391-3399PubMed Google Scholar). A third example of inhibition of transcription by cAMP involves the hepatic gene for fatty acid synthase; insulin-induced transcription of this gene is inhibited by cAMP (12Paulauskis J.D. Sul H.S. J. Biol. Chem. 1989; 264: 574-577Abstract Full Text PDF PubMed Google Scholar). The cis-acting element required for the inhibitory effect is an inverted CAAT box (13Rangan V.S. Oskouian B. Smith S. J. Biol. Chem. 1996; 271: 2307-2312Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The proteins that bind to this element have not been identified. In this study, we have found that the 5′-flanking DNA of the gene for malic enzyme contains at least four cis-acting DNA sequences that are involved in responsiveness to the inhibitory action of cAMP. We have examined the function of the probable major inhibitory element and identified some of the nuclear proteins that bind to it. Restriction enzymes were obtained from New England Biolabs (Beverly, MA) or Boehringer Mannheim. Other enzymes were obtained from the indicated sources: Taq DNA polymerase (Perkin-Elmer), T4 DNA ligase (Pharmacia Biotech Inc.), Klenow fragment of Escherichia coli DNA polymerase I and calf intestinal phosphatase (Boehringer Mannheim). CPT-cAMP, 3,5,3′-l-triiodothyronine, and corticosterone were purchased from Sigma. Crystalline bovine insulin was a gift from Lilly. LipofectAceTM and Waymouth medium MD 705/1 were obtained from Life Technologies, Inc. [α-32P]dCTP (800 Ci/mmol) was purchased from Amersham Corp., andd-threo-[dichloroacetyl-1–2-14C]chloramphenicol was from NEN Life Science Products. d-Luciferin, potassium salt, was obtained from Analytical Luminescence Laboratory (San Diego, CA). Antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); all were raised against human protein except TRα, for which the antigen was chicken in origin. All other chemicals were of reagent grade or of the highest purity commercially available. Plasmid RSV-PKAc and pRSV-PKi were from R. A. Maurer (Oregon Health Sciences University) (14Maurer R.A. J. Biol. Chem. 1989; 264: 6870-6873Abstract Full Text PDF PubMed Google Scholar). Plasmid RSV-TRα was provided by H. H. Samuels (New York University) (15Samuels H.H. Forman B.M. Horowitz Z.D. Ye Z.S. Annu. Rev. Physiol. 1989; 51: 623-639Crossref PubMed Scopus (55) Google Scholar). The luciferase reporter plasmid, pXP1 (16Nordeen S.K. BioTechniques. 1988; 6: 454-457PubMed Google Scholar), was from S. K. Nordeen (University of Colorado Health Sciences Center). Plasmid RSV-LUC was constructed as described previously (5Hodnett D.W. Fantozzi D.A. Thurmond D.C. Klautky S.A. MacPhee K.G. Estrem S.T. Xu G. Goodridge A.G. Arch. Biochem. Biophys. 1996; 334: 309-324Crossref PubMed Scopus (32) Google Scholar). B. Luckow and G. Schutz (Heidelberg, Germany) provided pBLCAT2 (Ref. 17Luckow B. Schutz G. Nucleic Acids Res. 1987; 17: 2365Google Scholar; pTKCAT). Plasmid KSCAT (promoterless plasmid), p[ME−5800/+31]CAT, and the 5′-deletions thereof were constructed as described previously (5Hodnett D.W. Fantozzi D.A. Thurmond D.C. Klautky S.A. MacPhee K.G. Estrem S.T. Xu G. Goodridge A.G. Arch. Biochem. Biophys. 1996; 334: 309-324Crossref PubMed Scopus (32) Google Scholar). For the TK constructs, we inserted various fragments of ME 5′-flanking DNA into the multiple cloning site 5′ of the HSV TK promoter in pBLCAT2 (Ref. 17Luckow B. Schutz G. Nucleic Acids Res. 1987; 17: 2365Google Scholar; TKCAT). Plasmid [ME-T3RE2]TKCAT was constructed by inserting a 36-bp oligonucleotide containing T3RE2 (major T3RE) of the malic enzyme gene (5Hodnett D.W. Fantozzi D.A. Thurmond D.C. Klautky S.A. MacPhee K.G. Estrem S.T. Xu G. Goodridge A.G. Arch. Biochem. Biophys. 1996; 334: 309-324Crossref PubMed Scopus (32) Google Scholar) into the NdeI and HindIII sites of pTKCAT. To make p[ME(T3RE2)−1713/−944]TKCAT and p[ME(T3RE2)−413/−147]TKCAT, we first amplified the −1713 to −944 and −413 to −147 fragments by polymerase chain reaction using oligonucleotides containing BamHI sites and then subcloned the resulting fragments into the BamHI site of p[ME-T3RE2]TKCAT. For p[ME−3474/−2715]TKCAT, we isolated the −3474 to −2715 fragment from p[ME−5800/+31]CAT by digesting withBglII. After blunt ending with T4 DNA polymerase, the resulting mixture was digested with HindIII. The resulting 758-bp fragment was subcloned into pTKCAT that had been digested byBamHI, blunt-ended with T4 DNA polymerase, and digested by HindIII. Plasmid [ME−3474/−2715(Δ−3259/−3115)]TKCAT and p[ME−3474/−2715(Δ−3114/−2930)]TKCAT were obtained using the TransformerTM site-directed mutagenesis kit fromCLONTECH Laboratories (Palo Alto, CA) according to the manufacturer's instructions. The 30-bp mutant primers contained two 15-bp sequences corresponding to each of the flanking regions of the internal deletions. Plasmid [ME−3474/−2715]TKCAT was the target. Plasmid [ME−3474/−2715(Δ−3474/−3260)]TKCAT and p[ME−3474/−2715(Δ−2929/−2715)]TKCAT were constructed using polymerase chain reaction and p[ME−3474/−2715]TKCAT as template. For p[ME−3474/−2715(Δ−3474/−3260)]TKCAT, the 5′-primer was a 30-bp oligonucleotide that contained two 15-bp sequences corresponding to the flanking regions of the deletion and aHindIII restriction site. The 3′-primer contained the last 24 bp of the −3474 to −2715 fragment and a BamHI site. For p[ME−3474/−2715(Δ−2929/−2715)]TKCAT, the 5′-primer contained the first 24 nucleotides of the −3474 to −2715 fragment and aHindIII site. The 3′-primer was 30 bp in length and contained two 15-bp oligonucleotides that flanked the deletion and aBamHI site. After digestion with the appropriate enzymes, the polymerase chain reaction fragments were subcloned into TKCAT. Plasmid [ME(T3RE2)−3259/−3115]TKCAT and p[ME−3114/−2930]TKCAT were constructed by polymerase chain reaction amplification of malic enzyme sequences using oligonucleotides with HindIII andBamHI sites. The resulting fragments were then subcloned into p[ME-T3RE2]TKCAT or pBLCAT2, respectively, which had been digested by the appropriate enzymes. 5′-deletions of p[ME(T3RE2)−3259/−3115]TKCAT were obtained using essentially the same procedure. Wild-type and mutant forms of p[ME(T3RE2)−3192/−3158]TKCAT were constructed by subcloning 34-bp oligonucleotides, flanked with HindIII and BamHI sites, into p[ME-T3RE2]TKCAT. Sequences of all constructs were confirmed by nucleotide sequence analysis using the Sequenase DNA sequencing kit (version 2.0, U.S. Biochemical Corp.). Isolated hepatocytes were prepared from the livers of 19-day-old chick embryos (18Roncero C. Goodridge A.G. Arch. Biochem. Biophys. 1992; 295: 258-267Crossref PubMed Scopus (23) Google Scholar). The cells were incubated in 35-mm plates with Waymouth medium MD 705/1 and streptomycin (100 μg/ml), penicillin G (60 μg/ml), insulin (50 nm), and corticosterone (1 μm). After 16–24 h, the cells were transfected with p[ME−5800/+31]CAT (2.5 μg) or an equimolar amount of another reporter plasmid, pRSV-LUC (0.5 μg) and sufficient pBluescript to bring the total amount of transfected DNA to 5 μg/plate. The cells were incubated with the DNA/lipofectACETM mixture for 16–24 h; thereafter, the medium was replaced with fresh medium with or without T3 (1.6 μm) and with or without CPT-cAMP (500 μm). After an additional 48 h of incubation, the hepatocytes were harvested, and extracts were prepared (5Hodnett D.W. Fantozzi D.A. Thurmond D.C. Klautky S.A. MacPhee K.G. Estrem S.T. Xu G. Goodridge A.G. Arch. Biochem. Biophys. 1996; 334: 309-324Crossref PubMed Scopus (32) Google Scholar). A detailed description of our transfection procedures has been published (19Baillie R.B. Klautky S.A. Goodridge A.G. J. Nutr. Biochem. 1992; 4: 431-439Crossref Scopus (25) Google Scholar). Cell extracts were analyzed for protein quantity (20Sedmark J.J. Grossberg S.E. Anal. Biochem. 1977; 79: 544-552Crossref PubMed Scopus (2471) Google Scholar), luciferase activity (21DeWet J.R. Wood K.V. DeLuca M. Helinski D.R. Subramani S. Mol. Cell. Biol. 1987; 7: 725-737Crossref PubMed Scopus (2466) Google Scholar), and CAT activity (22Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5286) Google Scholar). The results were expressed initially as percentage of substrate converted to acetylated product per milligram of unheated soluble protein and then normalized as described in the figure legends. The ratio of average relative CAT activities with and without T3 or cAMP is not always the same as the corresponding average -fold changes in CAT activities or percentages of control activities, respectively, because the latter are the averages of the individual -fold changes or percentages of control activities for each experiment. The statistical significance of differences between pairs of means was determined by the Wilcoxon matched pairs, signed rank test (23Conover W. Practical Non-parametric Statistics.2nd Ed. John Wiley & Sons, Inc., New York, NY1980Google Scholar). Standard errors of the mean are provided to indicate the degree of variability in the data. Nuclear extracts were prepared (24Ausubel F.M. Brent R. Kingston R.E. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1988: 1.1-16.20Google Scholar) from chick embryo hepatocytes incubated for 48 h with insulin, corticosterone, and T3 with or without CPT-cAMP. The extraction buffer contained 0.6 m KCl and the protease inhibitors leupeptin, benzamidine, aprotinin, and phenylmethylsulfonyl fluoride. Double-stranded oligonucleotides were labeled by a fill-in reaction with [α-32P]dCTP catalyzed by the Klenow fragment of DNA polymerase. A volume of nuclear extract containing 6 μg of protein was mixed with 12 μl of binding buffer containing 20,000 cpm of 32P-labeled probe, 2 μg of poly(dI-dC), 0.01% Nonidet P-40, 0.8 μg of bovine serum albumin, 5% (v/v) glycerol, and 5 μg of salmon sperm DNA with or without a 100-fold molar excess of competitor oligonucleotide. The reaction was incubated for 15 min at room temperature. Antibody experiments used the same incubation conditions except that 1 μl of IgG (1 μg) was incubated with the reaction mixture for an additional 15 min at room temperature. The reaction mixture was then subjected to electrophoresis on 5% polyacrylamide gels at 150 V in 25 mm Tris-HCl, 0.19m glycine, 1 mm EDTA at 4 °C (24Ausubel F.M. Brent R. Kingston R.E. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1988: 1.1-16.20Google Scholar). Gels were dried and subjected to autoradiography. When chick embryo hepatocytes were transfected with 5.8 kb of 5′-flanking DNA of the malic enzyme gene linked to the CAT gene (p[ME−5800/+31]CAT), CAT activity was increased more than 40-fold by T3 and inhibited by 96% by CPT-cAMP (Fig.1). CPT-cAMP did not have a statistically significant effect on luciferase activity in cells transfected with RSV-LUC. Thus, 5.8 kb of 5′-flanking DNA contains both positive acting T3REs, as previously reported (5Hodnett D.W. Fantozzi D.A. Thurmond D.C. Klautky S.A. MacPhee K.G. Estrem S.T. Xu G. Goodridge A.G. Arch. Biochem. Biophys. 1996; 334: 309-324Crossref PubMed Scopus (32) Google Scholar), and one or more inhibitory cAMP response elements (ICREs). The magnitude of the cAMP effect is similar to that for the inhibition of transcription of this gene caused by cAMP (4Salati L.M. Ma X.-J. McCormick C.C. Stapleton S.R. Goodridge A.G. J. Biol. Chem. 1991; 266: 4010-4016Abstract Full Text PDF PubMed Google Scholar), suggesting that all of the elements necessary for the negative effect of cAMP may be located in this fragment of DNA. We next tested a series of 5′-deletions of p[ME−5800/+31]CAT to localize the ICRE(s) (Fig. 1). Deletion of the DNA from −5800 to −3845 bp caused an increase in responsiveness to cAMP. This deletion removes part of the T3 response region (5Hodnett D.W. Fantozzi D.A. Thurmond D.C. Klautky S.A. MacPhee K.G. Estrem S.T. Xu G. Goodridge A.G. Arch. Biochem. Biophys. 1996; 334: 309-324Crossref PubMed Scopus (32) Google Scholar) and may contain sequences that dampen the ability of cAMP to inhibit transcription. When the DNA from −3845 to −3474 bp was deleted, responsiveness to cAMP increased slightly, but the increase was not statistically significant. Deletion of the region from −3474 to −2715 bp decreased cAMP-mediated inhibition from 99 to 92%. This represents an 8-fold difference in sensitivity to cAMP between sets of cells transfected with these two plasmids. An additional decrease in responsiveness occurred when DNA from −2715 to −944 bp was removed (Fig. 1). Both p[ME−944/+31]CAT and p[ME−413/+31]CAT conferred statistically significant 64 and 45% inhibitions by cAMP, respectively. When cells were transfected with p[ME−147/+31]CAT, there was measurable basal activity but no inhibition by cAMP (results not shown). These results suggest that 3474 bp of 5′-flanking DNA contains three or more ICREs that are distinct from the major T3RE and several minor T3REs that are located between −3903 and −3703 bp (5Hodnett D.W. Fantozzi D.A. Thurmond D.C. Klautky S.A. MacPhee K.G. Estrem S.T. Xu G. Goodridge A.G. Arch. Biochem. Biophys. 1996; 334: 309-324Crossref PubMed Scopus (32) Google Scholar). An expression vector containing the coding sequence for the catalytic subunit of PKA (pRSV-PKAc) (14Maurer R.A. J. Biol. Chem. 1989; 264: 6870-6873Abstract Full Text PDF PubMed Google Scholar) was cotransfected with p[ME−5800/+31]CAT. Overexpression of catalytic subunit inhibited T3-induced CAT activity in a dose-dependent manner (Fig.2 A). At 0.1 μg/plate, overexpression of the catalytic subunit inhibited T3-induced CAT activity by 80%. Even in cells treated with cAMP, overexpression of the catalytic subunit decreased CAT activity. These results suggest that the catalytic subunit of PKA itself is sufficient to inhibit promoter activity of the malic enzyme gene. The basal level of CAT activity (no T3) also was inhibited by cAMP, suggesting that inhibition by cAMP may be independent of T3. We also cotransfected a construct that expresses a specific inhibitor of the catalytic subunit (PKi or Walsh inhibitor) (25Grove J.R. Price D.J. Goodman H.M. Avruch J. Science. 1987; 238: 530-533Crossref PubMed Scopus (79) Google Scholar). When 1 or 2 μg of pRSV-PKi was cotransfected with p[ME−5800/+31]CAT, inhibition by exogenously added cAMP was completely blocked; in fact, activity was higher than that in the absence of cAMP. In addition, overexpression of pRSV-PKi in the absence of cAMP, with or without T3, stimulated CAT activity. This suggests that these cells contain a significant level of free catalytic subunit of PKA in the absence of added cAMP. We conclude that the negative effect of cyclic AMP on transcription of the malic enzyme gene is mediated by the classical eukaryotic signaling pathway that involves PKA-mediated phosphorylation of target proteins. In the absence of T3, cyclic AMP caused 53 and 75% decreases in CAT activity in cells transfected by p[ME−5800/+31]CAT and p[ME−3474/+31]CAT, respectively (Fig.3). This result is consistent with the inhibition of promoter activity caused by overexpression of the free catalytic subunit of PKA and suggests that cAMP-mediated inhibition of transcription of the malic enzyme gene does not inhibit TR functionper se. The degree of inhibition by cAMP was much greater when promoter activity of the malic enzyme gene was induced by T3 than in the absence of T3 (Fig. 3); this may be due to greater sensitivity to cAMP in T3-treated cells. Alternatively, the lower cAMP responsiveness of the malic enzyme gene in cells that were not treated with T3 may be due to a combination of the lower level of promoter activity in the absence of T3 and a constitutive basal level of activity that is independent of T3 or cAMP. The conclusion that cAMP functions independently of T3 is supported by the results of a series of experiments in which different artificial and natural T3REs were linked to TKCAT and tested for responsiveness to T3 and cAMP. One construct contained five copies of a consensus T3RE (5′-AGGTCANNNAGGTCA-3′) linked to TKCAT (26Carlisle T.L. Roncero C. El Khadir-Mounier C. Thurmond D.C. Goodridge A.G. J. Lipid Res. 1996; 37: 2088-2097Abstract Full Text PDF PubMed Google Scholar); a second construct contained a palindromic T3RE linked to TKCAT (27Damm K. Thompson C.C. Evans R.M. Nature. 1989; 339: 593-597Crossref PubMed Scopus (464) Google Scholar). Hepatocytes transfected with each of these constructs gave robust responses to T3 but did not respond to cAMP (results not shown). Similarly, cells transfected with T3RE2 of the malic enzyme gene linked directly to TKCAT also failed to respond to cAMP (Fig.4). Thus, a T3 response by itself is not sufficient to make transcription of the malic enzyme gene responsive to cAMP. Plasmid [ME−5800/+31]CAT and an expression vector for chicken TRα were cotransfected into hepatocytes. At many T3REs, TR is a repressor in the absence of T3 (15Samuels H.H. Forman B.M. Horowitz Z.D. Ye Z.S. Annu. Rev. Physiol. 1989; 51: 623-639Crossref PubMed Scopus (55) Google Scholar). Overexpression of TRα caused the expected decrease in basal activity (IC-treated cells) and an increase in T3-induced activity (Fig. 5); inhibition by cAMP decreased from ∼95 to ∼40%. Three regions appear to contain ICREs (Fig. 1). DNA fragments containing the putative ICREs were subcloned upstream of the TK promoter in pTKCAT. A 35-bp oligonucleotide containing T3RE2 of the malic enzyme gene (5Hodnett D.W. Fantozzi D.A. Thurmond D.C. Klautky S.A. MacPhee K.G. Estrem S.T. Xu G. Goodridge A.G. Arch. Biochem. Biophys. 1996; 334: 309-324Crossref PubMed Scopus (32) Google Scholar) was inserted upstream of each of the ICRE-containing fragments that lacked a T3RE. This ensured a high level of promoter activity in T3-treated cells (Fig. 4). When hepatocytes were transfected with the p[ME−3474/−2715]TKCAT, cAMP inhibited T3-induced CAT activity by 98% (Fig. 4 A). In cells transfected with p[ME(T3RE2)−1713/−944]TKCAT and p[ME(T3RE2)−413/−147]TKCAT, cAMP caused 77 and 72% inhibition of CAT activity, respectively (Fig.4 B). Cyclic AMP had no effect on promoter activity in control cells transfected with p[ME-T3RE2]TKCAT or pTKCAT. These results and those in Fig. 1 are consistent with there being at least three ICREs in the 5′-flanking DNA of the malic enzyme gene. For further localization of the 5′-most ICRE(s), we constructed and tested a series of deletions of p[ME−3474/−2715]TKCAT (Fig.4 A). Deletion of the 5′-end to −3260 bp or the 3′-end to −2929 bp did not affect either T3 or cAMP responsiveness of hepatocytes transfected with these constructs. When the DNA from −3259 to −3115 bp was deleted from the parent plasmid, T3 responsiveness was essentially unchanged, but inhibition by cAMP decreased from 98 to 22%, indicating that an ICRE was located in this DNA fragment. When the DNA from −3114 to −2930 bp was deleted, T3 responsiveness decreased to 2-fold, and inhibition by cAMP decreased to 23%. This result suggests that a T3RE is located in this region. The decrease in responsiveness to cAMP could have been due to 1) the presence of a second ICRE in this region, 2) an ICRE that overlaps both of these deletions, or 3) the deletion of the T3RE localized between −3114 and −2930 bp. T3-induced activity in the absence of a T3RE is little different from basal activity and may be too low to permit detection of a larger response to cAMP. Each of the fragments containing a potential ICRE was subcloned upstream of the TK promoter in pTKCAT and transfected into hepatocytes (Fig. 6). The major T3RE of the malic enzyme gene was inserted upstream of the −3259-/−3115-bp fragment. This was not necessary for the −3114-/−2930-bp fragment, because it contains a T3RE. In cells transfected with p[ME(T3RE2)−3259/−3115]TKCAT, CAT activity was strongly suppressed by cAMP (98%). In contrast, when the cells were transfected with p[ME−3114/−2930]TKCAT, there was no change in CAT activity in response to cAMP despite the fact that CAT activity was stimulated 12-fold by T3. These results indicate that at least one ICRE is localized between −3259 and −3115 bp in the 5′-flanking DNA of the gene for malic enzyme. They also confirm the presence of a T3RE between −3114 and −2930 bp and support the suggestion that the loss of cAMP responsiveness that occurred when this fragment was deleted from a plasmid containing the −3474 to −2715 bp fragment was due to loss of this T3RE. To localize the ICRE more precisely, we prepared a series of deletions of p[ME(T3RE2)−3259/−3115]TKCAT (Fig.7). Deletion from −3259 to −3192 bp had no effect on responsiveness to cAMP. Deletion from −3192 to −3161 bp decreased inhibition by cAMP from more than 98 to 76%, a 15-fold change in responsiveness to cAMP. Further deletion to −3134 bp had no effect. These results suggest that there are at least two ICREs in this fragment, one between −3192 and −3161 bp and one between −3134 and −3115 bp. We next focused on the ICRE between −3192 and −3161 bp. This 34-bp fragment contains a sequence that is very similar (one mismatch) to those of AP1 sites that are required for cAMP-mediated inhibition of transcription the IL-2 and IL-2R genes (11Tamir A. Isakov N. J. Immunol. 1994; 152: 3391-3399PubMed Google Scholar). p[ME(T3RE2)−3192/−3158]TKCAT was constructed in a wild-type form or with a block mutation in the putative AP1 site (complementary sequence) (Fig. 8). In cells transfected with the wild-type construct, CAT activ" @default.
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