NF-κB Inhibits Glucocorticoid and cAMP-mediated Expression of the Phosphoenolpyruvate Carboxykinase Gene
2000; Elsevier BV; Volume: 275; Issue: 41 Linguagem: Inglês
10.1074/jbc.m003656200
ISSN1083-351X
AutoresMary Waltner‐Law, Marc C. Daniels, Calum Sutherland, Daryl K. Granner,
Tópico(s)Estrogen and related hormone effects
ResumoTranscription of the phosphoenolpyruvate carboxykinase (PEPCK) gene is regulated by a variety of agents. Glucocorticoids, retinoic acid, and glucagon (via its second messenger, cAMP) stimulate PEPCK gene transcription, whereas insulin, phorbol esters, cytokines, and oxidative stress have an opposing effect. Stimulation of PEPCK gene expression has been extensively studied, and a number of important DNA elements and binding proteins that regulate the transcription of this gene have been identified. However, the mechanisms utilized to turn off expression of this gene are not well-defined. Many of the negative regulators of PEPCK gene transcription also stimulate the nuclear localization and activation of the transcription factor NF-κB, so we hypothesized that this factor could be involved in the repression of PEPCK gene expression. We find that the p65 subunit of NF-κB represses the increase of PEPCK gene transcription mediated by glucocorticoids and cAMP in a concentration-dependent manner. The mutation of an NF-κB binding element identified in the PEPCK gene promoter fails to abrogate this repression. Further analysis suggests that p65 represses PEPCK gene transcription through a protein·protein interaction with the coactivator, CREB binding protein. Transcription of the phosphoenolpyruvate carboxykinase (PEPCK) gene is regulated by a variety of agents. Glucocorticoids, retinoic acid, and glucagon (via its second messenger, cAMP) stimulate PEPCK gene transcription, whereas insulin, phorbol esters, cytokines, and oxidative stress have an opposing effect. Stimulation of PEPCK gene expression has been extensively studied, and a number of important DNA elements and binding proteins that regulate the transcription of this gene have been identified. However, the mechanisms utilized to turn off expression of this gene are not well-defined. Many of the negative regulators of PEPCK gene transcription also stimulate the nuclear localization and activation of the transcription factor NF-κB, so we hypothesized that this factor could be involved in the repression of PEPCK gene expression. We find that the p65 subunit of NF-κB represses the increase of PEPCK gene transcription mediated by glucocorticoids and cAMP in a concentration-dependent manner. The mutation of an NF-κB binding element identified in the PEPCK gene promoter fails to abrogate this repression. Further analysis suggests that p65 represses PEPCK gene transcription through a protein·protein interaction with the coactivator, CREB binding protein. phosphoenolpyruvate carboxykinase (GTP:oxaloacetate caboxy-lyase (transphosphorylating), EC 4.1.1.32) glucocorticoid response unit glucocorticoid response element glucocorticoid receptor cAMP response element gAF2 and gAF3, accessory factor binding sites chicken ovalbumin upstream promoter transcription factor hepatic nuclear factor 3 cAMP response unit mitogen-activated protein kinase nuclear factor κB insulin response sequence CREB binding protein steroid receptor coactivator-1 cAMP-dependent protein kinase 8-(4-chlorophenylthio)-cAMP chloramphenicol acetyltransferase phorbol 12-myristate 13-acetate interleukin-6 interleukin-1 tumor necrosis factor-α dexamethasone rel homology domain transactivation domains 1 and 2 Dulbecco's modified Eagle's medium insulin response unit phosphatidylinositol 3-kinase protein kinase B electrophoretic mobility shift assay signal transducers and activators of transcription Phosphoenolpyruvate carboxykinase (PEPCK)1 catalyzes a rate-controlling step in hepatic gluconeogenesis, and the transcription of this gene is regulated by several hormones (1Granner D. Pilkis S. J. Biol. Chem. 1990; 265: 10173-10176Abstract Full Text PDF PubMed Google Scholar, 2O'Brien R.M. Granner D.K. Biochem. J. 1991; 278: 609-619Crossref PubMed Scopus (247) Google Scholar), including glucocorticoids, retinoic acid, and glucagon (via its second messenger, cAMP) (3Petersen D.D. Magnuson M.A. Granner D.K. Mol. Cell. Biol. 1988; 8: 96-104Crossref PubMed Scopus (86) Google Scholar, 4Yamada K. Duong D.T. Scott D.K. Wang J.C. Granner D.K. J. Biol. Chem. 1999; 274: 5880-5887Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 5Hall R.K. Scott D.K. Noisin E.L. Lucas P.C. Granner D.K. Mol. Cell. Biol. 1992; 12: 5527-5535Crossref PubMed Scopus (77) Google Scholar, 6Lucas P.C. Granner D.K. Annu. Rev. Biochem. 1992; 61: 1131-1173Crossref PubMed Scopus (163) Google Scholar). A detailed analysis of the PEPCK promoter has revealed that each hormone response is mediated by a set of DNA elements that comprise a complex hormone response unit. For instance, the glucocorticoid response unit (GRU), which is positioned between −455 and −86 relative to the transcription start site, is required for the stimulatory effect of glucocorticoids. The GRU includes two glucocorticoid receptor binding sites (GR1 and GR2), three accessory factor binding sites (gAF1, gAF2, and gAF3), and a cAMP response element (CRE) (7Imai E. Stromstedt P.E. Quinn P.G. Carlstedt-Duke J. Gustafsson J.A. Granner D.K. Mol. Cell. Biol. 1990; 10: 4712-4719Crossref PubMed Scopus (244) Google Scholar, 8Sugiyama T. Scott D.K. Wang J.C. Granner D.K. Mol. Endocrinol. 1998; 12: 1487-1498Crossref PubMed Scopus (55) Google Scholar, 9Scott D.K. Mitchell J.A. Granner D.K. J. Biol. Chem. 1996; 271: 31909-31914Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Transcription factors that bind to these sites have been identified (Fig. 1). Hepatic nuclear factor 4 (HNF-4) and chicken ovalbumin upstream promoter transcription factor (COUP-TF) bind to gAF1, members of the hepatic nuclear factor 3 (HNF-3) family bind to gAF2, and COUP-TF binds to gAF3 to mediate the glucocorticoid response (9Scott D.K. Mitchell J.A. Granner D.K. J. Biol. Chem. 1996; 271: 31909-31914Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 10Hall R.K. Sladek F.M. Granner D.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 412-416Crossref PubMed Scopus (199) Google Scholar, 11Wang J.C. Stromstedt P.E. O'Brien R.M. Granner D.K. Mol. Endocrinol. 1996; 10: 794-800PubMed Google Scholar). Although a number of proteins bind the CRE, we showed that C/EBP-β acts as an accessory factor for the glucocorticoid response through this element (4Yamada K. Duong D.T. Scott D.K. Wang J.C. Granner D.K. J. Biol. Chem. 1999; 274: 5880-5887Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar).Figure 1Schematic representation of the PEPCK gene promoter. The cis elements and associatedtrans factors required for the glucocorticoid-, cAMP-, or insulin-mediated responses of the PEPCK gene are shown. The central position of each element with respect to the transcription start site is shown above each site. Trans-acting factors involved in the GRU, CRU, or IRU are shown below their respective binding elements. An unidentified IRS binding protein binds to gAF2 to mediate the insulin response through this element. Because deletion of gAF2 only partially represses insulin-mediated effects on PEPCK gene expression, it is believed that another element/factor complex proximal to gAF2 is also involved in this response. This unknown factor is designated as X.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Stimulation of PEPCK gene expression by cAMP, which is mediated by cAMP-dependent protein kinase A (PKA), also involves a cAMP response unit (CRU) that consists of several cis-acting elements (12Quinn P.G. Wong T.W. Magnuson M.A. Shabb J.B. Granner D.K. Mol. Cell. Biol. 1988; 8: 3467-3475Crossref PubMed Scopus (125) Google Scholar, 13Liu J.S. Park E.A. Gurney A.L. Roesler W.J. Hanson R.W. J. Biol. Chem. 1991; 266: 19095-19102Abstract Full Text PDF PubMed Google Scholar) (Fig. 1). The CRE at −90 to −82, a C/EBP-α binding site in the P3I region of the PEPCK promoter (between −246 and −238), and an AP-1 binding site extending from −260 to −250 are essential components of this CRU (14Hanson R.W. Reshef L. Annu. Rev. Biochem. 1997; 66: 581-611Crossref PubMed Scopus (634) Google Scholar, 15Roesler W.J. Vandenbark G.R. Hanson R.W. J. Biol. Chem. 1989; 264: 9657-9664Abstract Full Text PDF PubMed Google Scholar, 16Park E.A. Song S. Vinson C. Roesler W.J. J. Biol. Chem. 1999; 274: 211-217Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 17Roesler W.J. Graham J.G. Kolen R. Klemm D.J. McFie P.J. J. Biol. Chem. 1995; 270: 8225-8332Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). A variety of agents, including insulin, phorbol esters, compounds that elicit oxidative and cellular stress (such as H2O2 and sodium arsenite), and the cytokines TNF-α, IL-6, and IL-1 (18Sasaki K. Cripe T.P. Koch S.R. Andreone T.L. Petersen D.D. Beale E.G. Granner D.K. J. Biol. Chem. 1984; 259: 15242-15251Abstract Full Text PDF PubMed Google Scholar, 19Chu D.T. Stumpo D.J. Blackshear P.J. Granner D.K. Mol. Endocrinol. 1987; 1: 53-59Crossref PubMed Scopus (32) Google Scholar, 20Sutherland C. Tebbey P.W. Granner D.K. Diabetes. 1997; 46: 17-22Crossref PubMed Scopus (37) Google Scholar, 21Hill M.R. McCallum R.E. Infect. Immun. 1992; 60: 4040-4050Crossref PubMed Google Scholar, 22Christ B. Nath A. Heinrich P.C. Jungermann K. Hepatology. 1994; 20: 1577-1583Crossref PubMed Scopus (31) Google Scholar, 23Christ B. Nath A. Biochem. J. 1996; 320: 161-166Crossref PubMed Scopus (27) Google Scholar) repress PEPCK gene transcription. Although the hormone response units that confer stimulation of PEPCK gene transcription are well characterized, the mechanisms that lead to repression of PEPCK transcription are not fully understood. An insulin response sequence (IRS) involved in both the insulin and phorbol ester responses is positioned between −413 and −407 relative to the transcription start site within the PEPCK gene promoter (24O'Brien R.M. Lucas P.C. Forest C.D. Magnuson M.A. Granner D.K. Science. 1990; 249: 533-537Crossref PubMed Scopus (289) Google Scholar, 25O'Brien R.M. Bonovich M.T. Forest C.D. Granner D.K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6580-6584Crossref PubMed Scopus (71) Google Scholar) (Fig. 1). However, insulin still represses PEPCK gene expression when this IRS is deleted or mutated (24O'Brien R.M. Lucas P.C. Forest C.D. Magnuson M.A. Granner D.K. Science. 1990; 249: 533-537Crossref PubMed Scopus (289) Google Scholar). This observation led to the suggestion that another element, acting more proximal to this IRS, is also involved in this response (designated by X in Fig.1). Alternatively, insulin and the other negative regulators of PEPCK gene transcription could also work by disrupting protein·protein interactions necessary for communication between transcription factors and coactivators with the basal transcriptional machinery. Insulin stimulates signaling pathways that lead to the activation of phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), and p38 MAPK in hepatoma cells (20Sutherland C. Tebbey P.W. Granner D.K. Diabetes. 1997; 46: 17-22Crossref PubMed Scopus (37) Google Scholar, 26Gabbay R.A. Sutherland C. Gnudi L. Kahn B.B. O'Brien R.M. Granner D.K. Flier J.S. J. Biol. Chem. 1996; 271: 1890-1897Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Two well-characterized PI3K inhibitors block the action of insulin on PEPCK gene expression, so this enzyme is assumed to be involved in insulin-mediated repression of the PEPCK gene (27Sutherland C. O'Brien R.M. Granner D.K. J. Biol. Chem. 1995; 270: 15501-15506Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). A variety of experimental approaches were used to show that neither MAPK nor p38 MAPK is involved in the insulin response of PEPCK gene transcription (20Sutherland C. Tebbey P.W. Granner D.K. Diabetes. 1997; 46: 17-22Crossref PubMed Scopus (37) Google Scholar, 26Gabbay R.A. Sutherland C. Gnudi L. Kahn B.B. O'Brien R.M. Granner D.K. Flier J.S. J. Biol. Chem. 1996; 271: 1890-1897Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Insulin also stimulates signaling pathways that result in the activation of the transcription factor NF-κB in the liver (28Zhou G. Kuo M.T. J. Biol. Chem. 1997; 272: 15174-15183Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 29Bertrand F. Philippe C. Antoine P.J. Baud L. Groyer A. Capeau J. Cherqui G. J. Biol. Chem. 1995; 270: 24435-24441Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 30Bertrand F. Atfi A. Cadoret A. L'Allemain G. Robin H. Lascols O. Capeau J. Cherqui G. J. Biol. Chem. 1998; 273: 2931-2938Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Interestingly, many of the other repressors of PEPCK gene transcription (such as phorbol esters, oxidative stress, and various cytokines) stimulate the nuclear localization of NF-κB wherein it serves as a transcription factor (31Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4602) Google Scholar, 32Baeuerle P.A. Curr. Biol. 1998; 8: R19-R22Abstract Full Text Full Text PDF PubMed Google Scholar, 33Sen C.K. Packer L. FASEB J. 1996; 10: 709-720Crossref PubMed Scopus (1781) Google Scholar). Conversely, activators of PEPCK gene transcription, such as glucocorticoids and cAMP, inhibit nuclear localization and activation of NF-κB (34Neumann M. Grieshammer T. Chuvpilo S. Kneitz B. Lohoff M. Schimpl A. Franza Jr., B.R. Serfling E. EMBO J. 1995; 14: 1991-2004Crossref PubMed Scopus (181) Google Scholar). NF-κB is a ubiquitous transcription factor whose properties have been characterized primarily in cells of the immune system. In addition to its role in the immune response and inflammation, NF-κB is involved in cell cycle progression (35Perkins N.D. Felzien L.K. Betts J.C. Leung K. Beach D.H. Nabel G.J. Science. 1997; 275: 523-527Crossref PubMed Scopus (666) Google Scholar) and in liver development and regeneration (36Cressman D.E. Greenbaum L.E. Haber B.A. Taub R. J. Biol. Chem. 1994; 269: 30429-30435Abstract Full Text PDF PubMed Google Scholar). The critical role NF-κB plays in hepatic development is underscored by the observation that p65 knockout mice die before birth from massive degeneration of the liver due to apoptosis (37Beg A.A. Sha W.C. Bronson R.T. Ghosh S. Baltimore D. Nature. 1995; 376: 167-170Crossref PubMed Scopus (1640) Google Scholar). The NF-κB/Rel family of transcription factors includes the subunits p50, p52, p65, c-Rel, and RelB. These proteins share an N-terminal region of homology, known as the rel homology domain, that is important for DNA binding and dimerization. Two transactivation domains are found in the C-terminal region of p65, c-Rel, and RelB, but these are lacking in p50 and p52 (31Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4602) Google Scholar, 38Grimm S. Baeuerle P.A. Biochem. J. 1993; 290: 297-308Crossref PubMed Scopus (404) Google Scholar). In the cytoplasm, NF-κB binds to the inhibitory protein, IκB, to form an inactive NF-κB·IκB complex (39Beg A.A. Ruben S.M. Scheinman R.I. Haskill S. Rosen C.A. Baldwin Jr., A.S. Genes Dev. 1992; 6: 1899-1913Crossref PubMed Scopus (614) Google Scholar). Most agents that activate NF-κB do so by stimulating the phosphorylation and subsequent degradation of IκB, thus allowing NF-κB to translocate to the nucleus and regulate transcription (40Beg A.A. Baldwin Jr., A.S. Genes Dev. 1993; 7: 2064-2070Crossref PubMed Scopus (740) Google Scholar, 41Henkel T. Machleidt T. Alkalay I. Kronke M. Ben-Neriah Y. Baeuerle P.A. Nature. 1993; 365: 182-185Crossref PubMed Scopus (1039) Google Scholar). NF-κB regulates transcription by several different mechanisms. In most cases, NF-κB activates transcription by binding to an NF-κB element and interacting with the basal transcription machinery (38Grimm S. Baeuerle P.A. Biochem. J. 1993; 290: 297-308Crossref PubMed Scopus (404) Google Scholar,42Kerr L.D. Ransone L.J. Wamsley P. Schmitt M.J. Boyer T.G. Zhou Q. Berk A.J. Verma I.M. Nature. 1993; 365: 412-419Crossref PubMed Scopus (131) Google Scholar). However, NF-κB represses transcription in the presence of a corepressor, such as the Drosophila proteins dorsal switch or Groucho (43Lehming N. Thanos D. Brickman J.M. Ma J. Maniatis T. Ptashne M. Nature. 1994; 371: 175-179Crossref PubMed Scopus (204) Google Scholar, 44Kirov N. Zhelnin L. Shah J. Rushlow C. EMBO J. 1993; 12: 3193-3199Crossref PubMed Scopus (87) Google Scholar, 45Dubnicoff T. Valentine S.A. Chen G. Shi T. Lengyel J.A. Paroush Z. Courey A.J. Genes Dev. 1997; 11: 2952-2957Crossref PubMed Scopus (130) Google Scholar). NF-κB also represses transcription by interacting directly with transcription factors, such as GR, or by competing with other factors for binding to coactivators, such as p300, CBP, or SRC-1 (46Sheppard K.A. Phelps K.M. Williams A.J. Thanos D. Glass C.K. Rosenfeld M.G. Gerritsen M.E. Collins T. J. Biol. Chem. 1998; 273: 29291-29294Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 47Hottiger M.O. Felzien L.K. Nabel G.J. EMBO J. 1998; 17: 3124-3134Crossref PubMed Scopus (127) Google Scholar, 48Zhong H. Voll R.E. Ghosh S. Mol Cell. 1998; 1: 661-671Abstract Full Text Full Text PDF PubMed Scopus (1024) Google Scholar, 49Parry G.C. Mackman N. J. Immunol. 1997; 159: 5450-5456PubMed Google Scholar, 50Ray A. Prefontaine K.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 752-756Crossref PubMed Scopus (919) Google Scholar, 51McKay L.I. Cidlowski J.A. Mol. Endocrinol. 1998; 12: 45-56Crossref PubMed Scopus (304) Google Scholar). We now have evidence that the p65 subunit of NF-κB represses PEPCK gene transcription, in a DNA-independent fashion, by disrupting the coactivation function of CBP. These data suggest that the NF-κB signaling pathway contributes to the repression of hormone-activated PEPCK gene expression. H4IIE or HepG2 hepatoma cells were grown to confluence in Dulbecco's modified Eagle's medium (DMEM) containing 2.5% (v/v) newborn calf serum and 2.5% (v/v) fetal calf serum (3Petersen D.D. Magnuson M.A. Granner D.K. Mol. Cell. Biol. 1988; 8: 96-104Crossref PubMed Scopus (86) Google Scholar, 5Hall R.K. Scott D.K. Noisin E.L. Lucas P.C. Granner D.K. Mol. Cell. Biol. 1992; 12: 5527-5535Crossref PubMed Scopus (77) Google Scholar). Cells were pelleted and incubated for 30 min at room temperature with 2 ml of a calcium phosphate:DNA co-precipitate containing plasmid DNA. Cells were then plated in 10-cm2 culture dishes and incubated at 37 °C. After 4 h, cells were treated with 20% (v/v) dimethyl sulfoxide in serum-containing medium for 5 min, washed with phosphate-buffered saline, and incubated in serum-free media for 18 h. In certain experiments, cells were treated for 18 h with 500 nmdexamethasone, 0.1 mm 8-(4-chlorophenylthio)-cAMP (8CPT-cAMP), 10 nm insulin, 1 μm PMA, 1 ng/ml TNF-α, or 160 ng/ml IL-6. Cells were harvested by trypsin digestion and sonicated in 200 μl of 250 mm Tris-HCl at pH 7.8. Following sonication, the extracts were heated for 10 min at 65 °C, and cellular debris was removed by centrifugation. CAT assays were performed using the supernatant as described previously (7Imai E. Stromstedt P.E. Quinn P.G. Carlstedt-Duke J. Gustafsson J.A. Granner D.K. Mol. Cell. Biol. 1990; 10: 4712-4719Crossref PubMed Scopus (244) Google Scholar). CAT activity was normalized for the protein concentration in the cell lysate by using the Pierce BCA assay. EMSA was performed as described previously (28Zhou G. Kuo M.T. J. Biol. Chem. 1997; 272: 15174-15183Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). H4IIE cells were incubated in serum-free media with 500 nmdexamethasone and 0.1 mm 8CPT-cAMP, in the presence or absence of 10 nm insulin, 1 mmH2O2, or 1 μm PMA for 4 h. Subsequently, cells were washed twice in 5 ml of ice-cold, phosphate-buffered saline and were harvested by centrifugation. The cell pellet was resuspended in buffer A (20 mm HEPES, pH 7.9, 1 mm EDTA, 1 mm EGTA, 1 mmdithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 20 mm NaF, 1 mm Na3VO4, 1 mm Na4P2O7, 0.4 mm Na2MoO4, 0.125 μmokadaic acid, 1 μg/ml aprotinin, 1 μg/ml pepstatin, and 1 μg/ml leupeptin). After homogenization in a tight-fitting Dounce homogenizer, cell lysates were centrifuged at 2000 × g for 10 min, and nuclear pellets were resuspended in buffer B (buffer A containing 420 mm NaCl and 20% glycerol). The resuspended nuclei were exposed to consistent agitation for 30 min and centrifuged at 13,000 × g for 20 min. The supernatant was used for DNA-binding reactions. Protein amounts were determined using the Bio-Rad reagent. Oligonucleotides corresponding to the NF-κB binding site were annealed and end-labeled using polynucleotide kinase in the presence of [γ-32P]dATP: 5′-TCAGAGGGGACTTTCCGAGAGG-3′ and 5′-CCTCTCGGAAAGTCCCCTCTGA-3′. DNA-binding reactions were performed in a buffer containing 10 mm Tris-HCl, pH 7.6, 50 mm NaCl, 0.5 mm dithiothreitol, 10% glycerol, 0.2% Nonidet P-40, and 3 μg of poly(dI-dC) using 5 μg of nuclear protein and the labeled NF-κB probe (approximately 60,000–75,000 counts/min per reaction). For supershift assays, reactions were performed in the presence or absence of 1 μg of antibody specific for p65, c-Rel, RelB, p50, or p52. Samples were incubated at room temperature for 25 min and analyzed by electrophoresis on a 6% nondenaturing polyacrylamide gel in TAE buffer (7 mm Tris, pH 7.6, 3 mm sodium acetate, 1 mm EDTA). The plasmid pPL32 contains the PEPCK promoter sequence from −467 to +69, relative to the transcription start site, ligated to the CAT reporter gene, as described previously (3Petersen D.D. Magnuson M.A. Granner D.K. Mol. Cell. Biol. 1988; 8: 96-104Crossref PubMed Scopus (86) Google Scholar). The mutation of pPL32 to make the construct pPL32 (mκB) was performed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and the oligonucleotides 5′-CCCTTGGCCAACAGCTCAAATCCGGCGAGAC-3′ and 5′-TCTCGCCGGATTTGAGCTGTTGGCCAAGGG-3′. The p65 mutants were also constructed using the QuikChange kit. The following oligonucleotides were used to construct these mutants: ΔRHD, 5′-GCCTCTGGCCCCTATGTGGCCATTGTGTTCCGG-3′ and 5′-CCGGAACACAATGGCCACATAGGGGCCAGAGGC-3′; ΔTA, 5′-CGTAAAAGGACATAAGGGGGTGAC-3′ and 5′-GTCACCCCCTTATGTCCTTTTACG-3′; S276A, 5′-CTGCGGCGGCCTGCCGACCGGGAGCTC-3′ and 5′-GAGCTCCCGGTCGGCAGGCCGCCGCAG-3′. The DNA sequence of each mutant was verified. Radioisotopes ([γ-32P]dATP and [3H]sodium acetate) were obtained from Amersham Pharmacia Biotech and ICN, respectively. Insulin was purchased from Collaborative Bioproducts. 8CPT-cAMP was purchased from Roche Molecular Biochemicals. Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma. Antibodies specific to p50 or p65 were purchased from Santa Cruz Biotechnology. Dexamethasone and H2O2 were purchased from Sigma, IL-6 was purchased from Promega, and TNF-α from RD Research. The ability of insulin and phorbol esters (PMA) to repress PEPCK mRNA expression has been extensively documented (18Sasaki K. Cripe T.P. Koch S.R. Andreone T.L. Petersen D.D. Beale E.G. Granner D.K. J. Biol. Chem. 1984; 259: 15242-15251Abstract Full Text PDF PubMed Google Scholar, 19Chu D.T. Stumpo D.J. Blackshear P.J. Granner D.K. Mol. Endocrinol. 1987; 1: 53-59Crossref PubMed Scopus (32) Google Scholar, 20Sutherland C. Tebbey P.W. Granner D.K. Diabetes. 1997; 46: 17-22Crossref PubMed Scopus (37) Google Scholar, 25O'Brien R.M. Bonovich M.T. Forest C.D. Granner D.K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6580-6584Crossref PubMed Scopus (71) Google Scholar, 52O'Brien R.M. Noisin E.L. Granner D.K. Biochem. J. 1994; 303: 737-742Crossref PubMed Scopus (21) Google Scholar). Given the central role of PEPCK in maintaining gluconeogenesis, several investigators postulated that the hypoglycemia commonly associated with endotoxemia could result from decreased expression of this hepatic enzyme. In rats, endotoxin treatment is believed to result in the release of the proinflammatory cytokines, IL-6, IL-1, and TNF-α, all of which reduce PEPCK mRNA in rat hepatocytes and H4IIE hepatoma cells (21Hill M.R. McCallum R.E. Infect. Immun. 1992; 60: 4040-4050Crossref PubMed Google Scholar, 22Christ B. Nath A. Heinrich P.C. Jungermann K. Hepatology. 1994; 20: 1577-1583Crossref PubMed Scopus (31) Google Scholar, 23Christ B. Nath A. Biochem. J. 1996; 320: 161-166Crossref PubMed Scopus (27) Google Scholar). Interestingly, each of the agents that repress PEPCK gene expression promotes the nuclear localization and activation of NF-κB (28Zhou G. Kuo M.T. J. Biol. Chem. 1997; 272: 15174-15183Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 30Bertrand F. Atfi A. Cadoret A. L'Allemain G. Robin H. Lascols O. Capeau J. Cherqui G. J. Biol. Chem. 1998; 273: 2931-2938Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 31Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4602) Google Scholar, 32Baeuerle P.A. Curr. Biol. 1998; 8: R19-R22Abstract Full Text Full Text PDF PubMed Google Scholar, 33Sen C.K. Packer L. FASEB J. 1996; 10: 709-720Crossref PubMed Scopus (1781) Google Scholar). In view of these results, we decided to determine whether the different NF-κB subunits have an effect on PEPCK gene transcription. Transient transfection experiments were conducted to determine if the p65 or p50 subunits of NF-κB repress PEPCK gene expression. We chose to focus on these subunits, because they are expressed and activated in the cell lines used in our experiments (see below). Experiments designed to examine PEPCK gene expression were performed by co-transfecting pPL32 (a plasmid with the wild type −467 to +69 sequence of the PEPCK promoter located upstream of a CAT reporter gene) with either the catalytic subunit of PKA or the glucocorticoid receptor (GR), to enhance the expression of the gene. HepG2 cells were transfected with the PKA expression vector, which results in a 10- to 12-fold induction of PEPCK gene expression in response to this effector. The expression of p65 effectively repressed the PKA response in a concentration-dependent manner, whereas p50 expression had no effect (Fig. 2 A). The same experiments were performed to examine the effects of p65 or p50 on glucocorticoid-mediated induction of pPL32. H4IIE hepatoma cells were used for these experiments, because they give a more robust response to glucocorticoids than do HepG2 cells. Increasing amounts of p65 also repressed the glucocorticoid response, whereas p50 had a small effect (Fig. 2 B). We were not able to compare the level of protein expression of p65 and p50 due to the low transfection efficiency of H4IIE and HepG2 cells. We were able to show by Western blot in COS-1 cells, however, that the p50 expression vector does express protein (data not shown). It is possible that p50 is expressed at a lower level than p65 and that higher p50 protein levels would further repress PEPCK gene transcription. We chose not to transfect higher amounts of the p50 expression vector, however, because higher amounts of the empty expression vector had a repressive effect on PEPCK gene expression. For subsequent studies, we chose to examine the role of p65 on PEPCK gene expression. To confirm that the effects of p65 on PEPCK gene expression were not due to a direct repressive effect on transcription, or to a direct inhibition of CAT activity, p65 was co-transfected with a reporter plasmid that has six HIV long terminal repeat κB elements positioned upstream of a minimal thymidine kinase promoter and the CAT reporter gene (referred to as 6xκB/CAT). A 2- to 3-fold stimulation of transcription was observed, whereas no stimulation (or inhibition) of the parent vector TK-CAT was seen (Fig. 2 C). We were unable to use p50 for these experiments, because this protein lacks a transactivation domain and so does not activate 6xκB/CAT. As mentioned above, NF-κB can mediate transcription in a DNA binding-dependent or -independent manner. Therefore, the PEPCK promoter was examined for potential κB elements, which consist of the consensus sequence GGGRNNYYCC (38Grimm S. Baeuerle P.A. Biochem. J. 1993; 290: 297-308Crossref PubMed Scopus (404) Google Scholar). A site resembling this consensus κB element (GGGGAAATCC) was identified at position +40 to +49 relative to the transcription start site in the PEPCK promoter. Electrophoretic mobility shift assays (EMSAs) were performed using oligonucleotides that correspond to this site, or to a consensus κB site, and nuclear extracts isolated from H4IIE cells (Fig.3 A). DNA binding activity by NF-κB was detected in cells incubated in serum-free DMEM. This is consistent with other reports that describe the presence of NF-κB in the nucleus of untreated hepatoma cells (53Chang K.S. Hsu M.L. Josephs S.F. Cancer Lett. 1993; 74: 75-83Crossref PubMed Scopus (5) Google Scholar). As expected, nuclear extracts obtained after the treatment of H4IIE cells with a combination of dexamethasone and cAMP (Dex/cAMP) provided a reduced formation of the NF-κB·DNA complex, as compared with untreated cells. However, DNA binding by the NF-κB in nuclear extracts was increased in response to treatment of cells with insulin, hydrogen peroxide, and PMA (all of which are inhibitors of PEPCK gene transcription), even in cells that had been treated with Dex/cAMP, showing that these compounds overcome the repressive effect of Dex/cAMP on DNA binding. The consensus sequence oligonucleotide bound one protein complex, whereas the oligonucleotide corresponding to the PEPCK sequence bound two protein complexes (Fig. 3 A). Supershift analyses using antibodies specific for the p50 or p65 subunits of NF-κB revealed that the consensus oligonucleotide bound to a p50/p65 heterodimer. The c-Rel, RelB, and p52 subunits w
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