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• W1989133036 abstract "We have assessed the potential role of sterol regulatory element-binding protein-1c (SREBP-1c) on the transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK-C). SREBP-1c introduced into primary hepatocytes with an adenovirus vector caused a total loss of PEPCK-C mRNA and a marked induction of fatty acid synthase mRNA that directly coincided with the appearance of SREBP-1c in the hepatocytes. It also blocked the induction of PEPCK-C mRNA by cAMP and dexamethasone in these cells. In contrast, a dominant negative form of SREBP-1c (dnSREBP-1c) stimulated the accumulation of PEPCK-C mRNA in these cells. SREBP-1c completely blocked the induction of PEPCK-C gene transcription by the catalytic subunit of protein kinase A (PKA), and increasing concentrations of dnSREBP-1c reversed the negative effect of insulin on transcription from the PEPCK-C gene promoter in WT-IR cells. The more than 10-fold induction of PKA-stimulated PEPCK-C gene transcription caused by the co-activator CBP, was also blocked by SREBP-1c. In addition, dnSREBP-1c reversed the strong negative effect of E1A and NF1 on PKA-stimulated transcription from the PEPCK-C gene promoter. An analysis of the possible site of action of SREBP-1c using stepwise truncations of the PEPCK-C gene promoter indicated that the negative effect of SREBP-1c on transcription is exerted at a site between −355 and −277. We conclude that SREBP-1c is an intermediate in the action of insulin on PEPCK-C gene transcription in the liver and acts by blocking the stimulatory effect cAMP that is mediated via an interaction with cAMP-binding protein. We have assessed the potential role of sterol regulatory element-binding protein-1c (SREBP-1c) on the transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK-C). SREBP-1c introduced into primary hepatocytes with an adenovirus vector caused a total loss of PEPCK-C mRNA and a marked induction of fatty acid synthase mRNA that directly coincided with the appearance of SREBP-1c in the hepatocytes. It also blocked the induction of PEPCK-C mRNA by cAMP and dexamethasone in these cells. In contrast, a dominant negative form of SREBP-1c (dnSREBP-1c) stimulated the accumulation of PEPCK-C mRNA in these cells. SREBP-1c completely blocked the induction of PEPCK-C gene transcription by the catalytic subunit of protein kinase A (PKA), and increasing concentrations of dnSREBP-1c reversed the negative effect of insulin on transcription from the PEPCK-C gene promoter in WT-IR cells. The more than 10-fold induction of PKA-stimulated PEPCK-C gene transcription caused by the co-activator CBP, was also blocked by SREBP-1c. In addition, dnSREBP-1c reversed the strong negative effect of E1A and NF1 on PKA-stimulated transcription from the PEPCK-C gene promoter. An analysis of the possible site of action of SREBP-1c using stepwise truncations of the PEPCK-C gene promoter indicated that the negative effect of SREBP-1c on transcription is exerted at a site between −355 and −277. We conclude that SREBP-1c is an intermediate in the action of insulin on PEPCK-C gene transcription in the liver and acts by blocking the stimulatory effect cAMP that is mediated via an interaction with cAMP-binding protein. phosphoenolpyruvate carboxykinase dibutyryl cyclic AMP insulin regulatory sequence sterol regulatory element-binding protein-1c dominant negative SREBP-1c CCAAT/enhancer-binding protein protein kinase A cAMP-binding protein hepatic nuclear factor The regulation of transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK-C)1 in the liver by insulin is a critical step in the control of glucose homeostasis in all mammals. Insulin markedly inhibits PEPCK-C gene transcription, even in the presence of cAMP; the half-time of this inhibition is ∼30 min (1Granner D.K. Andreone T. Sasaki K. Beale E. Nature. 1983; 305: 549-551Crossref PubMed Scopus (245) Google Scholar). Despite its profound effects, the mechanism of insulin action on transcription of the gene for PEPCK-C remains surprisingly elusive. A region of the PEPCK-C gene promoter termed an insulin regulatory sequence (IRS) (−420 to −402) has been identified. This sequence confers partial insulin sensitivity to heterologous promoters (2O'Brien R.M. Lucas P.C. Forest C.D. Magnunson M.A. Granner D.K. Science. 1990; 249: 533-537Crossref PubMed Scopus (285) Google Scholar), and several proteins that bind to the IRS have been proposed to have a role in the insulin control of PEPCK-C gene transcription (3O'Brien R.M. Noisin E.L. Suwanichkul A. Yamasaki T. Lucas P.C. Wang J.-C. Powell D.R. Granner D.K. Mol. Cell. Biol. 1995; 15: 1747-1758Crossref PubMed Google Scholar). However, to date, transcription factors or regulatory elements in the promoter that account for the strong negative effects of insulin on hepatic PEPCK-C gene transcription have not been identified. Recently, sterol regulatory element-binding protein-1c (SREBP-1c) has been proposed as a key intermediate in the action of insulin on genes coding for proteins involved in carbohydrate metabolism (4Azzout-Marniche D. Becard D. Guichard C. Foretz M. Ferre P. Foufelle F. Biochem. J. 2000; 350: 389-393Crossref PubMed Scopus (231) Google Scholar). We reported that SREBP-1c cDNA introduced into hepatocytes via an adenoviral vector induces glucokinase and other insulin-induced gene mRNAs (fatty acid synthase, acetyl-CoA carboxylase, and Spot 14) (4Azzout-Marniche D. Becard D. Guichard C. Foretz M. Ferre P. Foufelle F. Biochem. J. 2000; 350: 389-393Crossref PubMed Scopus (231) Google Scholar, 5Foretz M. Pacot C. Dugail I. Lemarchand P. Guichard C. Le Liepvre X. Berthelier-Lubrano C. Spiegelman B. Kim J.B. Ferre P. Foufelle F. Mol. Cell. Biol. 1999; 19: 3760-3768Crossref PubMed Scopus (450) Google Scholar). The dominant negative form of SREBP-1c (dnSREBP-1c) blocked the ability of insulin to induce the expression of the glucokinase gene (4Azzout-Marniche D. Becard D. Guichard C. Foretz M. Ferre P. Foufelle F. Biochem. J. 2000; 350: 389-393Crossref PubMed Scopus (231) Google Scholar,5Foretz M. Pacot C. Dugail I. Lemarchand P. Guichard C. Le Liepvre X. Berthelier-Lubrano C. Spiegelman B. Kim J.B. Ferre P. Foufelle F. Mol. Cell. Biol. 1999; 19: 3760-3768Crossref PubMed Scopus (450) Google Scholar). SREBP-1c is a member of a larger family of transcription factors, termed SREBPs, that were first discovered based on their involvement in cholesterol and fatty acid metabolism (6Yokoyama C. Wang X. Briggs M.R. Admon A. Wu J. Hua X. Goldstein J.L. Brown M.S. Cell. 1993; 75: 187-197Abstract Full Text PDF PubMed Scopus (776) Google Scholar). A precursor form of SREBP-1 is synthesized and immediately anchored in the endoplasmic reticulum and nuclear membranes (7DeBose-Boyd R.A. Brown M.S. Li W.P. Nohturfft A. Goldstein J.L. Espenshade P.J. Cell. 1999; 99: 703-712Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Proteolytic cleavage of the precursor generates a mature, active form of the transcription factor that migrates into the nucleus, where it can bind both sterol regulatory elements (5′-TCACCCCCCAC-3′) and E-boxes (5′-CANNTG-3′) (8Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2918) Google Scholar, 9Kim J.B. Spotts G.D. Halvorsen Y.D. Shih H.M. Ellenberger T. Towle H.C. Spiegelman B.M. Mol. Cell. Biol. 1995; 15: 2582-2588Crossref PubMed Scopus (294) Google Scholar) in the promoters of specific genes. For proteolytic cleavage to occur, SREBPs must be transported to a site in the Golgi complex by the action of a protein termed SREBP cleavage-activating protein (10Nohturfft A. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 17243-17250Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Cleavage of the precursor form of SREBP involves two proteolytic enzymes, the first of which is regulated by SREBP cleavage-activating protein (11Sakai J. Nohturfft A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1998; 273: 5785-5793Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar) and is sensitive to the concentration of sterols in the cell (12Yang T. Goldstein J.L. Brown M.S. J. Biol. Chem. 2000; 275: 29881-29886Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). There is no evidence that SREBP-1c is activated by sterols in a similar manner. It has been shown previously, using cultured rat hepatocytes, that SREBP-1c gene expression is transcriptionally stimulated by insulin and repressed by glucagon (5Foretz M. Pacot C. Dugail I. Lemarchand P. Guichard C. Le Liepvre X. Berthelier-Lubrano C. Spiegelman B. Kim J.B. Ferre P. Foufelle F. Mol. Cell. Biol. 1999; 19: 3760-3768Crossref PubMed Scopus (450) Google Scholar). SREBP-1c has also been implicated in the effect of insulin on the genes for the low density lipoprotein receptor (13Liu J. Ahlborn T.E. Briggs M.R. Kraemer F.B. J. Biol. Chem. 2000; 275: 5214-5221Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 14Bennett M.K. Osborne T.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6340-6344Crossref PubMed Scopus (100) Google Scholar) and fatty acid synthase (9Kim J.B. Spotts G.D. Halvorsen Y.D. Shih H.M. Ellenberger T. Towle H.C. Spiegelman B.M. Mol. Cell. Biol. 1995; 15: 2582-2588Crossref PubMed Scopus (294) Google Scholar). In addition, it has been suggested from in vivo studies in mice and in vitro studies in adipocyte cell lines (15Horton J.D. Bashmakov Y. Shimomura I. Shimano H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5987-5992Crossref PubMed Scopus (531) Google Scholar) (16Shimomura I. Bashmakov Y. Ikemoto S. Horton J.D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13656-13661Crossref PubMed Scopus (614) Google Scholar) that the nuclear concentration of SREBP-1c is enhanced by insulin. Finally, indirect evidence suggests that SREBP-1c could be a target of mitogen-activated protein kinase, a known intermediate of insulin action (17Kotzka J. Muller-Wieland D. Koponen A. Njamen D. Kremer L. Roth G. Munck M. Knebel B. Krone W. Biochem. Biophys. Res. Commun. 1998; 249: 375-379Crossref PubMed Scopus (72) Google Scholar). Hence, insulin could influence SREBP-1c in three ways; it could (a) increase transcription of the gene for SREBP-1c, (b) increase the mobilization of SREBP-1c to the nucleus, and (c) alter the transcriptional activity of SREBP-1c once the protein is in the nucleus. Despite the current interest in SREBP-1c as a potential regulatory factor in the pathway of insulin action in the liver, its involvement in the regulation of transcription of genes involved in the control of hepatic gluconeogenesis has not been well established. In this paper, we show that SREBP-1c markedly reduces the basal level of PEPCK-C mRNA in isolated hepatocytes and blocks cAMP and dexamethasone-induced transcription from the PEPCK-C gene. In addition, a dominant negative form of SREBP-1c stimulated basal PEPCK-C gene expression in hepatocytes and overcame the negative effect of insulin on basal transcription of PEPCK-C gene. Our findings suggest that SREBP-1c exerts its effect by blocking the interaction of specific transcription factors involved in the cAMP regulation of PEPCK-C gene transcription with CBP. We conclude that SREBP-1c is an intermediate in the action of insulin on PEPCK-C gene transcription in the liver. Luciferase assay reagents were purchased from Promega Corp. (Madison, WI). The QuickChangeTM site-directed mutagenesis kit was from Stratagene. The Slid-A-Lyser dialysis cassette was from Pierce. HepG2 cells were originally purchased from the ATCC (Manassas, VA), and the WT-IR cells (18Rother K.I. Imai Y. Caruso M. Beguinot F. Formisano P. Accili D. J. Biol. Chem. 1998; 273: 17491-17497Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) were a generous gift from Dr. Domenico Accili (Columbia University, New York). Dulbecco's modified Eagle's medium/Ham's F-12 cell culture medium and fetal calf serum were from Life Technologies, Inc., and the restriction enzymes, T4DNA ligase, DNA polymerase I (large fragment), and Klenow fragment were purchased from New England Biolabs Inc. (Beverly, MA). Anti-SREBP-1c antibody was a product of Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Plasmid Mini and Qiafilter Midi and Maxi Kits were purchased from Qiagen Inc. (Valencia, CA), while the poly(dI-dC) was from Amersham Pharmacia Biotech, and the [α-32P]dCTP (3000 Ci/mmol) was from PerkinElmer Life Sciences. FuGENE 6 was purchased fromRoche Molecular Biochemicals. Oligonucleotide primers were synthesized by Integrated DNA Technologies (IDT) Inc. (Coralville, IA). All other reagents used in this study were of the highest quality obtainable. The expression vectors for the full-length CBP and the fragments of CBP were generous gifts from Dr. Richard Goodman (Vollum Institute, Portland, OR), and the expression vector containing the catalytic subunit of protein kinase A was kindly provided by Dr. Masa-Aki Muramatsu. The cDNA for both normal SREBP-1c and the dominant negative form were a generous gift from Dr. Bruce Spiegelman (Harvard University, Boston, MA). The plasmid p2000 luc was generated by ligating the XbaI and BglII fragment of the PEPCK-C gene promoter into pGL2-basic (which contains the luciferase structural gene) that had been digested with NheI andBglII. The plasmids were isolated with Plasmid Mini or Qiafilter Midi Kits (Qiagen, Inc.) and checked for purity by electrophoresis in 0.8% agarose gels, followed by visualization under UV light. The deletions in the PEPCK-C gene promoter (68Luc, 109Luc, 134Luc, 174Luc, 277Luc, and 355Luc) were from Short et al. (19Short J.M. Wynshaw-Boris A. Short H.P. Hanson R.W. J. Biol. Chem. 1986; 261: 9721-9726Abstract Full Text PDF PubMed Google Scholar). These segments of the PEPCK-C gene promoter were ligated to the luciferase structural gene by digestion with BglII and KpnI. After verification of the appropriate size ligated product, the DNA was transformed in the DH5α Escherichia coli strain. A plasmid miniprep was conducted using the Qiagen Mini-Prep Kit, and the DNA was isolated. The isolated product was redigested with the same enzymes to verify the in-frame insertion of the PEPCK-C gene promoter fragments. HepG2 hepatoma cells were grown in Dulbecco's minimal essential medium, supplemented with 50% F-12 nutrient mixture (Dulbecco's modified Eagle's medium/F-12 and 10% fetal calf serum) and antibiotics (50 units of penicillin and 50 μg of streptomycin per ml) at 37 °C in a 95% air, 5% CO2 atmosphere in six-well plates for 24 h before transfection. WT-IR cells were passaged at 33 °C in α-minimal essential medium supplemented with 4% fetal calf serum, 2 mm glutamine, and 10 mm dexamethasone, and after transfection the cells were transferred to 37 °C, with similar conditions as mentioned above. The cells (1 × 105 cells in 2 ml of medium/well) were then transfected with plasmid DNA (1 μg of plasmid DNA to 4.5 μl of FuGENE-6 transfection reagent per 35-mm well) (Roche Molecular Biochemicals). After 24 h, the cells were washed with ice-cold 1× phosphate-buffered saline, pH 7.4, and lysed by the addition of 300 μl of 1× Cell Culture Lysis Reagent (Promega, Madison, WI). The lysate was collected and centrifuged in 1.5-ml Eppendorf tubes for 6 min at 12,000 × g. The cell lysate was separated from the pellet and used for measurement of protein concentration and luciferase activity. For luciferase activity, 10 μl of the cell lysate was used to measure the integrated light units over 10 s, using the luciferase assay system (Promega, Madison, WI) and a luminometer (Tropix, Inc., Bedford, MA) as recommended by the manufacturers. The protein content of the extracts was determined by the dc-Bio-Rad protein assay method, using several concentrations of bovine serum albumin in cell lysis buffer as a standard. Animal studies were conducted according to the French guidelines for the care and use of experimental animals. Female (200–300-g body weight) Wistar rats from Iffa-Credo (L'Arbresle, France) were used for isolation of hepatocytes. They were housed in plastic cages at a constant temperature (22 °C) with light from 0700 to 1900 h for at least 1 week before the experiments. The adenovirus vector containing the transcriptionally active amino-terminal fragment (amino acids 1–403) of SREBP-1c was constructed as described previously (20Foretz M. Guichard C. Ferre P. Foufelle F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12737-12742Crossref PubMed Scopus (590) Google Scholar). Briefly, the cDNA of the transcriptionally active fragment of SREBP-1c cDNA was subcloned into the shuttle vector pAd Track-CMV. The resultant plasmid was linearized by the restriction endonucleasePmeI and co-transformed with the supercoiled adenoviral vector pAd-Easy1 into E. coli strain BJ5183. Recombinants were selected by kanamycin resistance and screened by restriction endonuclease digestion. The recombinant adenoviral construct was then cleaved with PacI and transfected into the packaging cell line 293. The recombinant adenovirus containing the dominant negative form of SREBP-1c was constructed as described previously (5Foretz M. Pacot C. Dugail I. Lemarchand P. Guichard C. Le Liepvre X. Berthelier-Lubrano C. Spiegelman B. Kim J.B. Ferre P. Foufelle F. Mol. Cell. Biol. 1999; 19: 3760-3768Crossref PubMed Scopus (450) Google Scholar). The adenovirus vector containing the major late promoter with no exogenous gene (Ad null) was used as a control. The adenoviral vectors were propagated in 293 cells, purified by cesium chloride density centrifugation, and stored as previously described (5Foretz M. Pacot C. Dugail I. Lemarchand P. Guichard C. Le Liepvre X. Berthelier-Lubrano C. Spiegelman B. Kim J.B. Ferre P. Foufelle F. Mol. Cell. Biol. 1999; 19: 3760-3768Crossref PubMed Scopus (450) Google Scholar). Hepatocytes were isolated from rats by the collagenase method (21Berry M.N. Friend D.S. J. Cell Biol. 1969; 43: 506-520Crossref PubMed Scopus (3601) Google Scholar). Cell viability was assessed by the Trypan blue exclusion test and was always higher than 85%. Hepatocytes were seeded at a density of 8 × 106 cells/dish in 100-mm Petri dishes in medium M199 with Earle's salts (Life Technologies) supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, 0.1% (w/v) bovine serum albumin, 2% (v/v) Ultroser G (Life Technologies), 100 nm dexamethasone (Sigma), 1 nm insulin (Actrapid; Novo Nordisc, Copenhagen, Denmark), 100 nmtriiodothyronine (Sigma). After cell attachment (4 h), cells were cultured in various conditions as described in the legends to Figs.Figure 1, Figure 2, Figure 3, Figure 4.Figure 1Time course of the effect of SREBP-1c introduced into rat hepatocytes with an adenoviral vector on the concentration of mRNA for PEPCK-C and fatty acid synthase.Hepatocytes from rats were isolated as described under “Experimental Procedures.” The hepatocytes were maintained in culture for 16 h after cell attachment in the presence of 5 mm glucose, and where indicated, adenovirus was added at 1 plaque-forming unit (pfu)/cell. At various times after infection with the adenovirus, the cells were harvested and total RNA was extracted and hybridized with cDNAs for PEPCK-C, fatty acid synthase (FAS), and SREBP-1c (SREBP-1c-403). Details of the methods used for Northern blot analysis are presented under “Experimental Procedures.” The Northern blots shown are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) For the experiments involving adenoviruses, hepatocytes were cultured for 16 h after cell attachment and then incubated for 120 min at 37 °C in M199 either with or without adenovirus containing SREBP-1c or dnSREBP-1c at various titers (plaque-forming units). Fresh medium was then added, and the cells were maintained in culture for the times described in the legends to Figs. Figure 1, Figure 2, Figure 3, Figure 4. To determine the effects of SREBP-1c on cAMP or dexamethasone-stimulated PEPCK-C gene expression in hepatocytes, 0.1 mm Bt2cAMP or 0.1 mmdexamethasone was added to the cells after attachment and incubated in the presence of 5 mm glucose for 16 h. Total cellular RNA was extracted from cultured hepatocytes using the guanidine thiocyanate method (22Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62909) Google Scholar) and prepared for Northern blot hybridization as previously described (5Foretz M. Pacot C. Dugail I. Lemarchand P. Guichard C. Le Liepvre X. Berthelier-Lubrano C. Spiegelman B. Kim J.B. Ferre P. Foufelle F. Mol. Cell. Biol. 1999; 19: 3760-3768Crossref PubMed Scopus (450) Google Scholar). Labeling of each DNA probe with [α-32P]dCTP was performed by random priming (Rediprime labeling kit; Amersham Pharmacia Biotech). Autoradiograms of Northern blots were scanned using an image processor program. The cDNA probes used to determine the levels of mRNA for fatty acid synthase (FAS), albumin, SREBP-1c, and PEPCK-C were used as previously described (20Foretz M. Guichard C. Ferre P. Foufelle F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12737-12742Crossref PubMed Scopus (590) Google Scholar). The cDNA for angiotensinogen from the rat was purchased from ATCC. The effect of overexpressing the cDNA for SREBP-1c on the level of expression of the gene for PEPCK-C was assessed by infecting isolated primary rat hepatocytes with an adenoviral vector expressing this transcription factor and measuring the concentration of mRNA for PEPCK-C, fatty acid synthase, and SREBP-1c at varying times after infection. SREBP-1c mRNA appeared in the hepatocytes within 4 h of viral infection, and its accumulation corresponded directly to a decrease in the level of PEPCK-C mRNA; there was a reciprocal increase in the concentration of mRNA for fatty acid synthase (Fig.1). Within 10 h after the introduction of SREBP-1c, PEPCK-C mRNA was virtually undetectable in the hepatocytes. The effect of SREBP-1c on PEPCK-C gene expression did not require glucose in the incubation medium of the infected hepatocytes, since there was an equally robust decline in PEPCK-C mRNA with either 5 or 25 mm glucose or with 5 mm lactate/pyruvate as substrate (Fig.2). Interestingly, the level of PEPCK-C mRNA was lower in hepatocytes incubated with 25 mmglucose, supporting the observation that transcription of the gene for PEPCK-C is inhibited at high concentrations of glucose (23Scott D.K. O'Doherty R.M. Stafford J.M. Newgard C.B. Granner D.K. J. Biol. Chem. 1998; 273: 24145-24151Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar,24Cournarie F. Azzout-Marniche D. Foretz M. Guichard C. Ferre P. Foufelle F. FEBS Lett. 1999; 460: 527-532Crossref PubMed Scopus (31) Google Scholar). The dominant negative form of SREBP-1c dimerizes with the dominant positive form of SREBP-1c, thereby sequestering the active transcription factor. dnSREBP-1c increased the concentration of PEPCK-C mRNA in the primary hepatocytes in the absence of added cAMP; it had no effect, however, on the level of albumin mRNA (Fig.3). We conclude that SREBP-1c dramatically decreases PEPCK-C mRNA in hepatocytes in a concentration-dependent manner and that it is involved in maintaining the low basal level of PEPCK-C gene expression in the liver. Finally, SREBP-1c completely blocked the cAMP-induced increase in expression of the gene for PEPCK-C in hepatocytes after 16 h of incubation; it decreased the observed PEPCK-C mRNA to undetectable levels (Fig. 4). A similar inhibitory effect of SREBP-1c was noted on the normal induction of transcription from the PEPCK-C gene promoter by dexamethasone (data not shown). The long term effects of both cAMP and dexamethasone on the accumulation of PEPCK-C mRNA in hepatocytes suggest that these compounds act to stabilize the mRNA as well as stimulate gene transcription. The inhibitory effect of SREBP-1c on PEPCK-C gene expression in hepatocytes and the strong stimulation of the basal transcription of this gene by dnSREBP-1c suggested that SREBP-1c had a insulin-like action on transcription of the PEPCK-C gene. To test this hypothesis, we determined the effects of overexpressing a cDNA for SREBP-1c on basal and PKA-stimulated transcription from the PEPCK-C gene promoter (−2000 to +73) that was linked to the luciferase structural gene (Fig.5). SREBP-1c had no effect on the low level of basal transcription from the PEPCK-C gene promoter in HepG2 cells but blocked PKA-induced transcription from that promoter in a concentration-dependent manner (Fig. 5). At equivalent levels of DNA (0.5 mg), SREBP-1c caused a 60% inhibition of transcription, even in the presence of a strong stimulation of PEPCK-C gene transcription by PKA (Fig. 5). At higher concentrations of SREBP-1c cDNA, the inhibition of transcription from the PEPCK-C gene promoter was 90%. In contrast, the dominant negative form of SREBP-1c (dnSREBP-1c) greatly induced both basal and PKA-stimulated transcription in this system. The effect was dependent on the concentration of the dnSREBP-1c cDNA transfected with the PEPCK-C gene promoter (Fig. 5). The effect of SREBP-1c on PEPCK-C gene transcription parallels the strong negative effect of insulin (i.e. it markedly inhibits PEPCK-C gene transcription even in the presence of the major stimulatory action of PKA). SREBP-1c, like insulin, acts in a dominant negative fashion on PEPCK gene transcription. We next used WT-IR cells to test the effect of insulin in the presence and absence of SREBP-1c on transcription from the PEPCK-C gene promoter (Fig.6). WT-IR cells were used in this experiment, since, unlike HepG2 cells, they have been shown to respond to added insulin (18Rother K.I. Imai Y. Caruso M. Beguinot F. Formisano P. Accili D. J. Biol. Chem. 1998; 273: 17491-17497Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). We determined the concentration of mRNA for SREBP-1c in these cells using “real time” RT-PCR and noted that there was 5.8 pg of SREBP-1c/mg of total RNA. This compared with 1 pg of SREBP-1c in the liver of a starved mouse. We could not detect SREBP-1c mRNA in HepG2 cells. Increasing concentrations of insulin added to WT-IR cells caused the expected decrease in transcription from the PEPCK-C gene promoter, even in the presence of PKA. Co-transfection of dnSREBP-1c in the presence of 0.12 mm insulin resulted in a concentration-dependent increase in transcription from the PEPCK-C gene promoter. Thus, the negative effect of insulin on both basal and PKA-stimulated transcription from the PEPCK-C gene promoter could be completely reversed by transfecting the WT-IR cells with increasing concentrations of dnSREBP-1c. The regulation of PEPCK-C gene transcription by cAMP involves the transcriptional co-activator CBP (25Leahy P. Crawford D.R. Grossman G. Chaudhry A. Gronostajski R. Hanson R.W. J. Biol. Chem. 1999; 274: 8813-8822Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Several of the transcription factors that mediate the effect of cAMP on PEPCK-C gene expression have been shown to act by binding to specific regions of CBP (25Leahy P. Crawford D.R. Grossman G. Chaudhry A. Gronostajski R. Hanson R.W. J. Biol. Chem. 1999; 274: 8813-8822Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 26Cardinaux J.R. Notis J.C. Zhang Q. Vo N. Craig J.C. Fass D.M. Brennan R.G. Goodman R.H. Mol. Cell. Biol. 2000; 20: 1546-1552Crossref PubMed Scopus (153) Google Scholar, 27Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577PubMed Google Scholar). Since SREBP-1c inhibits PKA-induced transcription from the PEPCK-C gene promoter and also binds to CBP, it is reasonable to assume that it might interfere with the interaction of transcription factors such as C/EBPα, C/EBPβ, CREB, or CREM with CBP (28Oliner J.D. Andresen J.M. Hansen S.K. Zhou S. Tjian R. Genes Dev. 1996; 10: 2903-2911Crossref PubMed Scopus (138) Google Scholar). CBP stimulates transcription from the PEPCK-C gene promoter in the presence of PKA but does not alter basal transcription (Fig.7). SREBP-1c totally blocks this inductive effect and returns the level of transcription to that noted with PKA alone but not to basal levels. This suggests that SREBP-1c interferes with the CBP binding to transcription factor(s) involved in the cAMP stimulation of PEPCK-C gene transcription. This conclusion is further strengthened by the experiment shown in Fig.8 in which the effect of dnSREBP-1c on the inhibition of PEPCK-C gene transcription caused by E1A and NF1c was determined. E1A markedly inhibits both basal and PKA-stimulated transcription from the PEPCK-C gene promoter (25Leahy P. Crawford D.R. Grossman G. Chaudhry A. Gronostajski R. Hanson R.W. J. Biol. Chem. 1999; 274: 8813-8822Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 29Kalvakalanu D.V.R. Liu J. Hanson R.W. Harter M.L. Sen G.C. J. Biol. Chem. 1992; 267: 2530-2536Abstract Full Text PDF PubMed Google Scholar). Previous studies have shown that this effect on the PEPCK-C gene is exerted via the E1A binding domain (amino acids 1687–2241) on CBP that is required in the coordination of PEPCK-C gene transcription (25Leahy P. Crawford D.R. Grossman G. Chaudhry A. Gronostajski R. Hanson R.W. J. Biol. Chem. 1999; 274: 8813-8822Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Members of the NF1 family of transcription factors also totally block PKA-stimulated PEPCK-C gene transcription, but they act via the CREB binding domain (amino acids 451–682) in the PEPCK-C gene promoter (25Leahy P. Crawford D.R. Grossman G. Chaudhry A. Gronostaj" @default.
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