Hormonal Regulation of the Phosphoenolpyruvate Carboxykinase Gene

The CCAAT/enhancer-binding protein α (C/EBP) is a transcription factor that trans-activates a number of metabolically important genes. Previous work has demonstrated that C/EBPα and C/EBPβ have the potential to mediate the cAMP responsiveness of phosphoenolpyruvate carboxykinase (PEPCK) in liver cells. However, these studies used GAL4 fusion proteins and artificial promoter-reporter gene vectors in transfection experiments; as a result, these studies only indicated that both isoforms had the potential to mediate the hormonal response and not which isoform actually participated in vivo. To address this issue, we produced hepatoma cell lines that stably expressed either a dominant negative inhibitor or antisense RNA for these two main liver C/EBP isoforms. Inhibition of all C/EBP isoforms via expression of the dominant negative protein eliminated cAMP responsiveness, and reduced glucocorticoid responsiveness, of the endogenous PEPCK gene in hepatoma cells. Antisense directed against C/EBPα mRNA, which reduced C/EBPα protein levels by nearly 80%, also significantly reduced the cAMP responsiveness of the endogenous PEPCK promoter, whereas antisense directed against C/EBPβ was without effect. There was no major alteration in cAMP signaling in the C/EBPα antisense cells, as cAMP induction of the C/EBPβ gene was similar to that in wild-type H4IIE cells. These data suggest that the α-isoform of C/EBP is specifically utilized for mediating the cAMP responsiveness of the PEPCK gene. The CCAAT/enhancer-binding protein α (C/EBP) is a transcription factor that trans-activates a number of metabolically important genes. Previous work has demonstrated that C/EBPα and C/EBPβ have the potential to mediate the cAMP responsiveness of phosphoenolpyruvate carboxykinase (PEPCK) in liver cells. However, these studies used GAL4 fusion proteins and artificial promoter-reporter gene vectors in transfection experiments; as a result, these studies only indicated that both isoforms had the potential to mediate the hormonal response and not which isoform actually participated in vivo. To address this issue, we produced hepatoma cell lines that stably expressed either a dominant negative inhibitor or antisense RNA for these two main liver C/EBP isoforms. Inhibition of all C/EBP isoforms via expression of the dominant negative protein eliminated cAMP responsiveness, and reduced glucocorticoid responsiveness, of the endogenous PEPCK gene in hepatoma cells. Antisense directed against C/EBPα mRNA, which reduced C/EBPα protein levels by nearly 80%, also significantly reduced the cAMP responsiveness of the endogenous PEPCK promoter, whereas antisense directed against C/EBPβ was without effect. There was no major alteration in cAMP signaling in the C/EBPα antisense cells, as cAMP induction of the C/EBPβ gene was similar to that in wild-type H4IIE cells. These data suggest that the α-isoform of C/EBP is specifically utilized for mediating the cAMP responsiveness of the PEPCK gene. phosphoenolpyruvate carboxykinase cytosolic form of PEPCK cAMP response element cAMP response element-binding protein 8-chlorophenylthio-cAMP The cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC4.1.1.32) (PEPCK-C)1 is generally considered to be the rate-limiting enzyme of gluconeogenesis (1.Rognstad R. J. Biol. Chem. 1979; 254: 1875-1878Abstract Full Text PDF PubMed Google Scholar), and accordingly it is expressed at highest levels in liver and kidney (1.Rognstad R. J. Biol. Chem. 1979; 254: 1875-1878Abstract Full Text PDF PubMed Google Scholar, 2.Alleyne G.A.O. Scullard G.H. J. Clin. Invest. 1969; 48: 364-370Crossref PubMed Scopus (96) Google Scholar, 3.Hanson R.W. Reshef L. Annu. Rev. Biochem. 1997; 66: 581-611Crossref PubMed Scopus (621) Google Scholar). The promoter of PEPCK-C is hormonally responsive and contains a number of cis-elements that bindtrans-acting factors capable of mediating multiple hormonal signals (for review, see Ref. 4.Hanson R.W. Patel Y.M. Adv. Enzymol. Relat. Areas Mol. Biol. 1994; 69: 203-281PubMed Google Scholar). In liver, the PEPCK-C promoter is negatively regulated by insulin but induced by glucocorticoids, thyroid hormones, and glucagon, via cAMP (2.Alleyne G.A.O. Scullard G.H. J. Clin. Invest. 1969; 48: 364-370Crossref PubMed Scopus (96) Google Scholar, 5.Tilghman S.M. Hanson R.W. Reshef L. Hopgood M.F. Ballard F.J. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 1304-1308Crossref PubMed Scopus (83) Google Scholar, 6.Loose D.S. Cameron D.K. Short H.P. Hanson R.W. Biochemistry. 1985; 24: 4509-4512Crossref PubMed Scopus (61) Google Scholar). The responsiveness of the PEPCK-C promoter to cAMP is relatively liver-specific and is mediated by a group of cis-elements collectively known as a cAMP response unit. The cAMP response unit includes a cAMP response element (CRE), which is capable of binding molecules of the cAMP response element-binding protein (CREB); three binding sites for CCAAT/enhancer-binding protein (C/EBP) isoforms; and one binding site for AP-1 (7.Roesler W.J. Simard J. Graham J.G. McFie P.J. J. Biol. Chem. 1994; 269: 14276-14283Abstract Full Text PDF PubMed Google Scholar, 8.Yeagley D. Agati J.M. Quinn P.G. J. Biol. Chem. 1998; 273: 18743-18750Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). This complex organization of DNA sequences and DNA-binding proteins likely allows for intricate control of tissue-specific expression and the integration of various signaling pathways. For example, the response of the PEPCK-C promoter to thyroid hormones is mediated not only by the binding of an activated thyroid hormone receptor to a typical thyroid hormone response element (9.Giralt M. Park E.A. Liu J.S. Gurney A.L. Hakimi P. Hanson R.W. J. Biol. Chem. 1991; 266: 21991-21996Abstract Full Text PDF PubMed Google Scholar, 10.Schmidt E.D.L van Beeren M. Glass C.K. Wiersinga W.M. Lamers W.H. Biochim. Biophys. Acta. 1993; 1172: 82-88Crossref PubMed Scopus (6) Google Scholar), but also requires one of the C/EBP binding sites utilized in the CRU (11.Park E.A. Song S. Vinson C. Roesler W.J. J. Biol. Chem. 1999; 274: 211-217Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Thus, there is a potential integration of the thyroid hormone response and the cAMP response of the PEPCK-C promoter, brought about by this C/EBP molecule, which may account for the synergistic activation of the promoter by these two signaling pathways. CCAAT/enhancer-binding proteins are members of the basic region-leucine zipper (bZIP) family of transcription factors (12.Landshultz W.H. Johnson P.F. Adashi E.Y. Graves B.J. McKnight S.L. Genes Dev. 1988; 2: 786-800Crossref PubMed Scopus (626) Google Scholar, 13.Landschultz W.H. Johnson P.F. McKnight S.L. Science. 1988; 240: 1759-1763Crossref PubMed Scopus (2512) Google Scholar). The two main C/EBP isoforms expressed in liver are C/EBPα and C/EBPβ (14.Alam T. An M.R. Papaconstantinou J. J. Biol. Chem. 1992; 267: 5021-5024Abstract Full Text PDF PubMed Google Scholar). The α-isoform of C/EBP is considered to be a central regulator of energy homeostasis (15.McKnight S.L. Lane M.D. Gluecksohn-Waelsch S. Genes Dev. 1989; 3: 2021-2024Crossref PubMed Scopus (195) Google Scholar), being involved in the trans-activation of a number of metabolically important genes (16.Park E.A. Gurney A.L. Nizielski S.E. Hakimi P. Cao Z. Moorman A. Hanson R.W. J. Biol. Chem. 1993; 268: 613-619Abstract Full Text PDF PubMed Google Scholar, 17.Park E.A. Roesler W.J. Liu J.S. Klemm D.J. Gurney A.L. Thatcher J.D. Shuman J. Friedman A. Hanson R.W. Mol. Cell. Biol. 1990; 10: 6264-6272Crossref PubMed Scopus (172) Google Scholar). The importance of C/EBPα in the maintenance of metabolic homeostasis has been particularly well demonstrated by whole animal and tissue-specific knockout studies (18.Wang N. Finegold M.J. Bradley A. Ou C.N. Abdelsayed S.V. Wilde N.M. Taylor L.R. Wilson D.R. Darlington G.J. Science. 1995; 269: 1108-1112Crossref PubMed Scopus (826) Google Scholar, 19.Lee Y. Sauer B. Johnson P.F. Gonzalez F.J. Mol. Cell. Biol. 1997; 17: 6014-6022Crossref PubMed Google Scholar). The β-isoform of C/EBP also appears to have some role in metabolism (20.Liu S. Croniger C. Arizmendi C. Harada-Shiba M. Ren J. Poli V. Hanson R.W. Friedman J.E. J. Clin. Invest. 1999; 103: 207-213Crossref PubMed Scopus (79) Google Scholar), as well as regulating the immune response (21.Tanaka T. Akira S. Yoshida K. Umemoto M. Yoneda Y. Shirafuji N. Fujiwara H. Suematsu S. Yoshida N. Kishimoto T. Cell. 1995; 80: 353-361Abstract Full Text PDF PubMed Scopus (462) Google Scholar). Prior work by our laboratory using GAL4-C/EBP fusion proteins suggested that either C/EBP isoform has the potential to mediate the cAMP response of PEPCK-C in liver (11.Park E.A. Song S. Vinson C. Roesler W.J. J. Biol. Chem. 1999; 274: 211-217Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). However, to date there has been no evidence in regards to which specific isoform mediates the cAMP responsiveness of the endogenous PEPCK-C gene in liver cells. The goal of this present study was to inhibit C/EBP activity in hepatoma cells and assess what effect it had on the cAMP responsiveness of the endogenous PEPCK-C gene. DNA-modifying enzymes were purchased from New England BioLabs, Promega Corp., and U. S. Biochemical Corp. Gentacin™ and tissue culture supplies were obtained from Life Technologies, Inc. 8-Chlorophenylthio-cAMP (8-CPT-cAMP) was obtained from Sigma. [α-32P]Uridine triphosphate (3000 Ci/mmol) was purchased from Mandel. Antisera specific for C/EBPα, C/EBPβ, and TFIIEα were obtained from Upstate Biotechnology, Inc. TRIzol™ reagent was obtained from Life Technologies, Inc. The expression vector for GBF-F (MSV-GBF-F), utilized for the production of the MSV-GBF-F stable H4IIE cell line, was constructed by ligating anEcoRI/BamHI fragment of pRGX-GBF-F (22.Olive M. Williams S.C. Dezan C. Johnson P.F. Vinson C. J. Biol. Chem. 1996; 271: 2040-2047Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), corresponding to full-length GBF-F, into theEcoRI/BamHI sites of MSV-C/EBPβ (23.Cao Z. Umek R.M. McKnight S.L. Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1329) Google Scholar), thereby displacing the full-length C/EBPβ insert. The GBF-FEcoRI/BamHI fragment was obtained from pRGX-GBF-F by polymerase chain reaction amplification utilizing a forward polymerase chain reaction primer designed to introduce anEcoRI site into the 5′-untranslated region of the GBF-F cDNA sequence. The forward and reverse polymerase chain reaction primers utilized to amplify the approximately 400-base pair GBF-FEcoRI/BamHI fragment from pRGX-GBF-F were as follows: GBF-F forward, 5′-TATCGAATTCATGCCAGTGAAGGAT-3′ and GBF-F reverse, 5′-TATCGGATCCAAGCTTGCCGTC-3′. The plasmid designed to express C/EBPα antisense RNA (MSV-C/EBPα anti) was constructed by subcloning thePstI/BamHI fragment of pMSV-C/EBPα (24.Friedman A.D. Landshultz W.H. McKnight S.L. Genes Dev. 1989; 3: 1314-1322Crossref PubMed Scopus (363) Google Scholar), corresponding to nucleotides +782 to +131 of the rat C/EBPα cDNA sequence, into the PstI/BamHI sites of the multiple cloning site of pBluescript® SK+ (Stratagene) to produce SK+-C/EBPα anti. The EcoRI/BamHI fragment of SK+-C/EBPα anti was then ligated into theEcoRI/BamHI sites of MSV-C/EBPβ to produce MSV-C/EBPα anti. The plasmid designed to express C/EBPβ antisense RNA (MSV-C/EBPβ anti) was constructed by subcloning the PstI fragment of pSCt-LAP (25.Descombes P. Chojkier M. Lichtsteiner S. Falvey E. Schibler U. Genes Dev. 1990; 4: 1541-1551Crossref PubMed Scopus (419) Google Scholar), corresponding to nucleotides +736 to +246 of the rat C/EBPβ cDNA sequence, into the PstI site of pBluescript® SK+ to produce SK+-C/EBPβ anti. The EcoRI/BamHI fragment of pBluescript® SK+-C/EBPβ anti was then ligated into theEcoRI/BamHI sites of MSV-C/EBPβ to produce MSV-C/EBPβ anti. The plasmid designed to express C/EBPα sense RNA (MSV-GαN175) was constructed by ligating the EcoRI/BamHI fragment of pGαN175 (26.Roesler W.J. Park E.A. McFie P.J. J. Biol. Chem. 1998; 273: 14950-14957Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), corresponding to nucleotides +117 to +658 of the rat C/EBPα cDNA sequence (coding for amino acids 6–175 of C/EBPα) into the EcoRI/BamHI sites of MSV-C/EBPβ to produce MSV-C/EBPα sense. The plasmids designed for the production of ribonuclease protection assay probes for detection of C/EBPα, C/EBPβ, and PEPCK mRNA have been described previously (27.Crosson S.M. Davies G.F. Roesler W.J. Diabetologia. 1997; 40: 1117-1124Crossref PubMed Scopus (24) Google Scholar). The plasmid designed for the production of ribonuclease protection assay probes for detection of C/EBPα antisense RNA was produced by ligating theEcoRI/BamHI fragment of pGαN175, corresponding to nucleotides +117 to +658 of the rat C/EBPα cDNA sequence, into the EcoRI/BamHI sites of pTZ18R (U. S. Biochemical Corp., Inc.). This vector was linearized for probe production by digestion with HindIII, which cuts at position +310 of the pTZ18R multiple cloning site, producing a 573-nucleotide probe that protects 541 nucleotides of the C/EBPα antisense RNA. The vector utilized as a template for ribonuclease protection analysis probes for the detection of C/EBPβ antisense RNA (19R-LAP) was constructed by ligating the PstI/PstI fragment of pSCt-LAP, corresponding to nucleotides +246 to +736 of the rat C/EBPβ cDNA sequence, into the PstI site of pTZ19R. This vector was linearized for probe production by digestion withEcoRI, which cuts at nucleotide +315 of the multiple cloning site of pTZ19R, producing a 550-nucleotide probe, which protects 490 nucleotides of the C/EBPβ antisense RNA. Cells were grown in Dulbecco's modified Eagle's medium/F-12 medium containing 5% fetal bovine serum and 5% calf serum. Where indicated, cells were treated with 200 μm 8-CPT-cAMP or 1 μm dexamethasone for the times indicated and then harvested for RNA isolation. Rat hepatoma H4IIE cells were transfected by the calcium phosphate precipitate method (28.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 16.32-16.38Google Scholar). Cells were transfected with the appropriate expression vector and pSV2neo (InforMax, Inc.) at a ratio of 10:1, respectively. The total amount of DNA introduced per transfection was 15 μg/100-mm plate. After 3 days, cells were placed in medium containing 400 μg/ml gentamicin. Individual clones, once isolated and expanded, were maintained in medium containing 100 μg/ml gentamicin. Total cellular RNA was extracted from cultured cells using TRIzol™ reagent (Life Technologies, Inc.). RNase protection assays and Northern analysis were conducted as described previously (27.Crosson S.M. Davies G.F. Roesler W.J. Diabetologia. 1997; 40: 1117-1124Crossref PubMed Scopus (24) Google Scholar, 29.Davies G.F. Khandelwal R.L. Roesler W.J. Biochim. Biophys. Acta. 1999; 1451: 122-131Crossref PubMed Scopus Google Scholar). of signals was by of using the Inc.). that an amount of RNA was from was by RNA analysis using a probe Biochim. Biophys. Acta. Scholar). The for of cellular and analysis have been described previously G.F. S.M. Khandelwal R.L. Roesler W.J. Biochem. Biophys. 1995; PubMed Scopus Google Scholar). were by as described To address which C/EBP isoform in mediating the cAMP responsiveness of the PEPCK promoter, H4IIE cell lines stably either a dominant negative molecule, or antisense to C/EBPα or C/EBPβ were A number of of cell were for the levels of expression not The highest was for The or in expression levels in RNA or are the S.E. of data from at three To the of C/EBP isoforms for PEPCK-C and cAMP we a H4IIE cell that expressed a dominant negative inhibitor of GBF-F (22.Olive M. Williams S.C. Dezan C. Johnson P.F. Vinson C. J. Biol. Chem. 1996; 271: 2040-2047Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). GBF-F is an of the DNA-binding of the protein to the a designed to form with C/EBP isoforms. levels of GBF-F expression the cell inhibit the activity of all C/EBP isoforms by of The of GBF-F mRNA in the GBF-F and in wild-type H4IIE cells, is in A. 1 a that the of GBF-F expression on C/EBPα and C/EBPβ mRNA levels in the GBF-F cell to wild-type H4IIE cells. The of GBF-F in a in C/EBPα mRNA levels and a in C/EBPβ mRNA the effect of GBF-F expression on C/EBPβ mRNA no in C/EBPβ protein GBF-F cells and wild-type H4IIE cells was 1 However, the levels of C/EBPα protein were reduced by in GBF-F cells in with wild-type H4IIE cells. be that RNase protection analysis of C/EBP mRNA, the of this either one or two This is a of the of the in utilized to produce the antisense in which two probe of were what appears to be only one have not been to as an to two GBF-F the activity of all C/EBP we cell lines antisense RNA for either the or A the levels of expression of the C/EBPα antisense RNA in the H4IIE cell in with wild-type H4IIE cells. The expression of C/EBPα antisense RNA reduced the mRNA of C/EBPα by 5% in with H4IIE cells the levels of C/EBPα protein were also in the cell by in to wild-type cells An was the of C/EBPα antisense RNA expression on C/EBPα and C/EBPβ The expression of C/EBPα antisense RNA an of C/EBPβ in that it was to both C/EBPβ mRNA and protein levels and to wild-type H4IIE cells. it that antisense expression in the of C/EBPα, there is a corresponding in the expression of the β-isoform of In the H4IIE cell line, which expressed C/EBPβ antisense RNA 3 the of C/EBPβ mRNA was reduced by 3 and protein levels by 3 to wild-type H4IIE cells. The levels of C/EBPα protein in the cell were by to wild-type H4IIE cells 3 the levels of C/EBPα no corresponding in C/EBPα mRNA was in these cells 3 a control for these a H4IIE stable cell a sense C/EBPα corresponding to the region for amino acids was in C/EBPα or C/EBPβ protein levels to wild-type H4IIE cells was as a of this sense C/EBPα RNA expression not the of cAMP responsiveness in the cell lines described of wild-type H4IIE cells with 200 μm 8-CPT-cAMP produced a in PEPCK-C The responsiveness of PEPCK-C to an with 8-CPT-cAMP was in the GBF-F cell GBF-F the and of C/EBP isoforms for the induction of PEPCK-C expression by the expression of the PEPCK-C gene was reduced by in the GBF-F cell to wild-type H4IIE cells. The specific of C/EBPα activity by antisense in the cell also PEPCK-C cAMP responsiveness The levels of PEPCK-C mRNA were also reduced in the cell by with wild-type cells. alteration in PEPCK-C responsiveness to 8-CPT-cAMP was in C/EBPβ cells In the induction of PEPCK-C gene expression levels by cAMP in cells was that in wild-type H4IIE cells. in PEPCK-C expression levels was in cells to wild-type cells. of cAMP induction of PEPCK mRNA levels was in the C/EBPα sense RNA cell This that the of stable integration and not with the of cAMP induction of endogenous was that the of C/EBPα antisense were an by the levels activity of a protein for cAMP responsiveness, as Thus, it was to the C/EBPα antisense cells had a in cAMP the of GBF-F and C/EBPα antisense RNA expression on the cAMP responsiveness of an endogenous H4IIE gene The expression of C/EBPβ is induced by cAMP (27.Crosson S.M. Davies G.F. Roesler W.J. Diabetologia. 1997; 40: 1117-1124Crossref PubMed Scopus (24) Google Scholar), by a that is M. C. Mol. Cell. Biol. 1997; 17: PubMed Google Scholar). in the levels of C/EBPβ protein were induced by in cells and by in GBF-F cells by which is with the induction in H4IIE cells. the levels of protein were not significantly to wild-type cells in either GBF-F or cells not Thus, it that cAMP signaling in the C/EBPα antisense RNA and GBF-F lines was not the that the in cAMP responsiveness of the PEPCK-C gene was to a specific of C/EBPα activity in these cells. also utilized these stable cell lines to assess the of C/EBP for the glucocorticoid responsiveness of the PEPCK-C gene. in the induction of PEPCK-C mRNA obtained by dexamethasone of wild-type H4IIE cells was not by antisense RNA for either C/EBPα or C/EBPβ However, in cells GBF-F, which the activity of all C/EBP the of induction of PEPCK-C mRNA in response to dexamethasone was reduced to from The data in this C/EBPα as the specific C/EBP isoform that in the cAMP responsiveness of the PEPCK-C gene. This is by the of cAMP induction of PEPCK-C in both the GBF-F and cell and the cAMP response that in the cell for this is from the that the levels of C/EBPα protein are reduced in the GBF-F cell in which PEPCK-C cAMP responsiveness is the levels of C/EBPβ protein are in the cell the cAMP responsiveness of the PEPCK-C gene was the levels of C/EBPα protein are in the cell which an response of the PEPCK-C gene to for our from the that C/EBPβ knockout no alteration in the expression or cAMP of the PEPCK-C gene (20.Liu S. Croniger C. Arizmendi C. Harada-Shiba M. Ren J. Poli V. Hanson R.W. Friedman J.E. J. Clin. Invest. 1999; 103: 207-213Crossref PubMed Scopus (79) Google Scholar). these data that C/EBPα in this hormonal whereas C/EBPβ be that C/EBPα knockout have been the cAMP responsiveness of the PEPCK-C gene has the (18.Wang N. Finegold M.J. Bradley A. Ou C.N. Abdelsayed S.V. Wilde N.M. Taylor L.R. Wilson D.R. Darlington G.J. Science. 1995; 269: 1108-1112Crossref PubMed Scopus (826) Google Scholar). is to the an in for which PEPCK is the rate-limiting The that GBF-F expression of C/EBPα expression is with the of the C/EBPα gene C. P. P. 1993; PubMed Scopus Google Scholar, N. Wilson D.R. Taylor L.R. Abdelsayed S. Wilde M. M. Darlington G.J. Mol. Cell. Biol. 1995; PubMed Google Scholar). The C/EBPα promoter is known to C/EBPα and in transfection of C/EBPα the C/EBPα promoter C. P. P. 1993; PubMed Scopus Google Scholar). The work of N. Wilson D.R. Taylor L.R. Abdelsayed S. Wilde M. M. Darlington G.J. Mol. Cell. Biol. 1995; PubMed Google also suggested of the C/EBPα gene. of either C/EBPα or C/EBPβ in hepatoma cells by antisense the expression of both main C/EBP isoforms. In hepatoma cells C/EBPα not only was the expression of C/EBPα as there was also a corresponding in the expression of C/EBPβ cells C/EBPβ antisense the in C/EBPβ with an of C/EBPα was not by in C/EBPα mRNA These suggest that there be some in which to a of C/EBP protein in the liver there is evidence that these two isoforms have some and in specific gene it is also that these two isoforms and to the sequences in Thus, the of one isoform and a in the of the genes that are on for of activity be to expression at or The in suggest a for C/EBP isoforms in the of the glucocorticoid responsiveness of the endogenous PEPCK-C gene. The that of one isoform by antisense had no on glucocorticoid responsiveness, whereas GBF-F expression reduced the responsiveness, is with the that either isoform in this hormonal This is from that by K. D.K. J. D.K. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), specifically C/EBPβ as the factor in the glucocorticoid However, it be that this was on using promoter-reporter gene and GAL4-C/EBP of the C/EBPβ fusion protein was not to glucocorticoid responsiveness, which had been reduced to the of the C/EBP binding site for a GAL4 These in the our study and that of K. D.K. J. D.K. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). However, are in that both to a C/EBP protein as an important factor in the glucocorticoid The data in this study evidence that C/EBP particularly the in mediating the of hormones on gene In the of the PEPCK promoter, in mediating the of cAMP this this and (9.Giralt M. Park E.A. Liu J.S. Gurney A.L. Hakimi P. Hanson R.W. J. Biol. Chem. 1991; 266: 21991-21996Abstract Full Text PDF PubMed Google Scholar, E.A. Song S. Vinson C. Roesler W.J. J. Biol. Chem. 1999; 274: 211-217Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The production of stable rat hepatoma H4IIE cell molecules designed to inhibit the activity of C/EBP have to assess the of these transcription factors in various cellular This work has also demonstrated the to promoter-reporter studies with that the role that C/EBP isoforms in the of endogenous genes that a and Vinson for also for for the H4IIE stable transfection

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