Pancreatic β-Cell-specific Repression of Insulin Gene Transcription by CCAAT/Enhancer-binding Protein β
1997; Elsevier BV; Volume: 272; Issue: 45 Linguagem: Inglês
10.1074/jbc.272.45.28349
ISSN1083-351X
AutoresMing Lu, Jochen Seufert, Joel F. Habener,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoChronic exposure of β-cells to supraphysiologic glucose concentrations results in decreased insulin gene transcription. Here we identify the basic leucine zipper transcription factor, CCAAT/enhancer-binding protein β (C/EBPβ), as a repressor of insulin gene transcription in conditions of supraphysiological glucose levels. C/EBPβ is expressed in primary rat islets. Moreover, after exposure to high glucose concentrations the β-cell lines HIT-T15 and INS-1 express increased levels of C/EBPβ. The rat insulin I gene promoter contains a consensus binding motif for C/EBPβ (CEB box) that binds C/EBPβ. In non-β-cells C/EBPβ stimulates the activity of the rat insulin I gene promoter through the CEB box. Paradoxically, in β-cells C/EBPβ inhibits transcription, directed by the promoter of the rat insulin I gene by direct protein-protein interaction with a heptad leucine repeat sequence within activation domain 2 of the basic helix-loop-helix transcription factor E47. This interaction leads to the inhibition of both dimerization and DNA binding of E47 to the E-elements of the insulin promoter, thereby reducing functionally the transactivation potential of E47 on insulin gene transcription. We suggest that the induction of C/EBPβ in pancreatic β-cells by chronically elevated glucose levels may contribute to the impaired insulin secretion in severe type II diabetes mellitus. Chronic exposure of β-cells to supraphysiologic glucose concentrations results in decreased insulin gene transcription. Here we identify the basic leucine zipper transcription factor, CCAAT/enhancer-binding protein β (C/EBPβ), as a repressor of insulin gene transcription in conditions of supraphysiological glucose levels. C/EBPβ is expressed in primary rat islets. Moreover, after exposure to high glucose concentrations the β-cell lines HIT-T15 and INS-1 express increased levels of C/EBPβ. The rat insulin I gene promoter contains a consensus binding motif for C/EBPβ (CEB box) that binds C/EBPβ. In non-β-cells C/EBPβ stimulates the activity of the rat insulin I gene promoter through the CEB box. Paradoxically, in β-cells C/EBPβ inhibits transcription, directed by the promoter of the rat insulin I gene by direct protein-protein interaction with a heptad leucine repeat sequence within activation domain 2 of the basic helix-loop-helix transcription factor E47. This interaction leads to the inhibition of both dimerization and DNA binding of E47 to the E-elements of the insulin promoter, thereby reducing functionally the transactivation potential of E47 on insulin gene transcription. We suggest that the induction of C/EBPβ in pancreatic β-cells by chronically elevated glucose levels may contribute to the impaired insulin secretion in severe type II diabetes mellitus. Insulin is a hormone essential for the control of mammalian glucose homeostasis and is produced predominantly in pancreatic β-cells of adult animals (1Giddings S.J. Chirgwin J. Permutt M.A. Diabetologia. 1985; 28: 343-347Crossref PubMed Scopus (58) Google Scholar). The expression of the insulin gene occurs to a large extent at the level of transcription. Control elements residing in the 5′ 350-base pair sequence flanking exon 1 of the rat insulin I gene are sufficient to direct β-cell-specific expression (2Philippe J. Endocr. Rev. 1994; 2: 21-27Google Scholar) (Fig. 1 A). Arrays of A and E elements 1Throughout this report, the revised nomenclature of the cis-acting elements of the insulin promoters (65German M. Ashcroft S. Docherty K. Edlund H. Edlund T. Goodison S. Imura H. Kennedy G. Madsen O. Melloul D. Moss L. Olson L.K. Permutt M.A. Philippe J. Robertson R.P. Rutter W.J. Serup P. Stein R. Steiner D. Tsai M.J. Walker M.D. Diabetes. 1995; 44: 1002-1004Crossref PubMed Scopus (148) Google Scholar) is used, and the former indications are given in parentheses. 1Throughout this report, the revised nomenclature of the cis-acting elements of the insulin promoters (65German M. Ashcroft S. Docherty K. Edlund H. Edlund T. Goodison S. Imura H. Kennedy G. Madsen O. Melloul D. Moss L. Olson L.K. Permutt M.A. Philippe J. Robertson R.P. Rutter W.J. Serup P. Stein R. Steiner D. Tsai M.J. Walker M.D. Diabetes. 1995; 44: 1002-1004Crossref PubMed Scopus (148) Google Scholar) is used, and the former indications are given in parentheses. (Far-FLAT, Nir-P1) constitute symmetrical enhansons that cooperatively account for >90% of the transcriptional activity of the insulin gene promoter (3Ohlsson H. Karlsson O. Edlund T. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4228-4231Crossref PubMed Scopus (73) Google Scholar). The E elements are recognition motifs for transcription factors in the basic helix-loop-helix (bHLH) 2The abbreviations used are: bHLH, basic helix-loop-helix; IDX-1, islet-duodenum-homeobox factor; RIPE, rat insulin II promoter element; BETA-2, β-cell E-box transactivator 2; C/EBPα and -β, CCAAT/enhancer-binding protein α and β, respectively; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; bp, base pair(s); CRE, cAMP response element; CREB, CRE-binding protein; AD1 and AD2, activation domains 1 and 2, respectively. 2The abbreviations used are: bHLH, basic helix-loop-helix; IDX-1, islet-duodenum-homeobox factor; RIPE, rat insulin II promoter element; BETA-2, β-cell E-box transactivator 2; C/EBPα and -β, CCAAT/enhancer-binding protein α and β, respectively; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; bp, base pair(s); CRE, cAMP response element; CREB, CRE-binding protein; AD1 and AD2, activation domains 1 and 2, respectively.family, such as E12 and E47, which activate the insulin promoter in close synergism with A element binding homeobox transcription factors, such as IDX-1. Chronic hyperglycemia may contribute to the pancreatic β-cell dysfunction observed in patients with type II diabetes, a phenomenon attributed to the concept of glucose toxicity (4Robertson R.P. Olson L.K. Zhang H.J. Diabetes. 1994; 43: 1085-1089Crossref PubMed Google Scholar). Studies usingin vivo animal models and in vitro β-cell lines have demonstrated that a reduction of insulin gene transcription by glucose toxicity is associated with the loss of transactivator proteins such as IDX-1/IPF-1/STF-1 and RIPE3b1-binding protein (5Robertson R.P. Zhang H.J. Pyzdrowski K.L. Walseth T.F. J. Clin. Invest. 1992; 90: 320-325Crossref PubMed Scopus (190) Google Scholar, 6Olson L.K. Redmon J.B. Towle H.C. Robertson R.P. J. Clin. Invest. 1993; 92: 514-519Crossref PubMed Scopus (173) Google Scholar, 7Olson L.K. Sharma A. Peshavaria M. Wright C.V. Towle H.C. Robertson R.P. Stein R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9127-9131Crossref PubMed Scopus (130) Google Scholar, 8Poitout V. Olson L.K. Robertson R.P. J. Clin. Invest. 1996; 97: 1041-1046Crossref PubMed Scopus (127) Google Scholar, 9Sharma A. Olson L.K. Robertson R.P. Stein R. Mol. Endocrinol. 1995; 9: 1127-1134Crossref PubMed Google Scholar, 10Zangen D.H. Bonner-Weir S. Lee C.H. Latimer J.B. Miller C.P. Habener J.F. Weir G.C. Diabetes. 1997; 46: 258-264Crossref PubMed Scopus (126) Google Scholar). Because insulin gene transcription is both positively and negatively regulated, we sought to identify repressors that might also mediate the effects of glucose toxicity on insulin gene transcription. In this report we describe CCAAT/enhancer binding protein-β (C/EBPβ) as a glucose-induced repressor of insulin gene transcription. C/EBPs are a family of transcription factors that regulate genes of the acute phase response, cell growth, differentiation, and the expression of cell type-specific genes (11Vasseur-Cognet M. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7312-7316Crossref PubMed Scopus (35) Google Scholar, 12Poli V. Mancini F.P. Cortese R. Cell. 1990; 63: 643-653Abstract Full Text PDF PubMed Scopus (454) Google Scholar, 13Mandrup S. Lane M.D. J. Biol. Chem. 1997; 272: 5367-5370Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, 14Descombes P. Chojkier M. Lichtsteiner S. Falvey E. Schibler U. Genes Dev. 1990; 4: 1541-1551Crossref PubMed Scopus (419) Google Scholar, 15Pope R.M. Leutz A. Ness S.A. J. Clin. Invest. 1994; 94: 1449-1455Crossref PubMed Scopus (164) Google Scholar, 16Trautwein C. Rakemann T. Pietrangelo A. Plumpe J. Montosi G. Manns M.P. J. Biol. Chem. 1996; 271: 22262-22270Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The C/EBPs consist of the activators C/EBP α, β, γ, δ, and ε and the repressors CHOP, LIP, and C/EBP-30; the latter two repressors arise by alternative downstream translation of the mRNAs (17Ossipow V. Descombes P. Schibler U. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8219-8223Crossref PubMed Scopus (320) Google Scholar). The C/EBPs bind to DNA exclusively as dimers and contain a conserved C-terminal basic region-leucine zipper domain that is characterized by a DNA-contacting basic region linked to a leucine zipper dimerization motif (18Landschulz W.H. Johnson P.F. McKnight S.L. Science. 1989; 243: 1681-1688Crossref PubMed Scopus (418) Google Scholar). They bind preferentially to a consensus DNA sequence T(T/G)NNGNAA(T/G) (19Akira S. Isshiki H. Nakajima T. Kinoshita S. Nishio Y. Hashimoto S. Natsuka S. Kishimoto T. Chem. Immunol. 1992; 51: 299-322Crossref PubMed Scopus (31) Google Scholar, 20Osada S. Yamamoto H. Nishihara T. Imagawa M. J. Biol. Chem. 1996; 271: 3891-3896Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). The founding member of the family of C/EBP transcription factors, C/EBPα, is expressed during terminal differentiation of cells such as adipocytes (13Mandrup S. Lane M.D. J. Biol. Chem. 1997; 272: 5367-5370Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar) and keratinocytes. C/EBPβ is abundant in liver, is expressed in response to stress-activated signaling pathways, and activates the expression of genes involved in the acute phase response such as cytokine genes. It has been shown that the expression of C/EBPβ transactivates the transcription of genes encoding the insulin receptor and glucose transporter-2 (21Webster N.J. Kong Y. Cameron K.E. Resnik J.L. Diabetes. 1994; 43: 305-312Crossref PubMed Google Scholar, 22Kaestner K.H. Christy R.J. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 251-255Crossref PubMed Scopus (225) Google Scholar), suggesting that C/EBPβ may play an important role in glucose homeostasis and the metabolic stress associated with diabetes mellitus. The promoters of both of the rat insulin I and II gene, as well as the human insulin gene, contain sequence elements that closely resemble the consensus C/EBP-binding site (see Fig. 1). The sequence similarities among the elements imply that the C/EBP family of DNA-binding proteins may regulate the expression of the insulin gene. In addition, the activity of the insulin gene promoter is regulated by glucose and hormones, which elevate β-cell [Ca2+] and cAMP levels and possibly protein kinase C activity (2Philippe J. Endocr. Rev. 1994; 2: 21-27Google Scholar, 23Goodison S. Kenna S. Ashcroft S.J. Biochem. J. 1992; 285: 563-568Crossref PubMed Scopus (70) Google Scholar). C/EBPβ may mediate the effects of multiple second messengers on insulin gene expression, since its activity can be influenced by Ca2+, cAMP, and protein kinase C signaling pathways (24Metz R. Ziff E. Genes Dev. 1991; 5: 1754-1766Crossref PubMed Scopus (301) Google Scholar, 25Trautwein C. van der Geer P. Karin M. Hunter T. Chojkier M. J. Clin. Invest. 1994; 93: 2554-2561Crossref PubMed Scopus (128) Google Scholar, 26Wegner M. Cao Z. Rosenfeld M.G. Science. 1992; 256: 370-373Crossref PubMed Scopus (304) Google Scholar). In the present study, we find that C/EBPβ is expressed in pancreatic β-cells and is up-regulated by supraphysiologic glucose concentrations in the culture media of pancreatic β-cell lines. C/EBPβ inhibits insulin promoter activity in β-cell lines, but not in the non-β-cell HeLa and BHK cell lines. In pancreatic β-cells C/EBPβ specifically interacts with a heptad leucine repeat sequence within activation domain 2 (AD2) of the basic helix-loop-helix transcription factor E47, thereby inhibiting the DNA binding activity and the transactivation potential of E47. DNA-modifying enzymes were purchased from New England Biolabs (Beverly, MA) or Boehringer Mannheim; radioactive compounds were from NEN Life Science Products;d-luciferin-potassium was from the Analytical Luminescence Laboratory (San Diego, CA); RPMI 1640 and DMEM medium and fetal bovine serum (FBS) were purchased from Life Technologies, Inc. Nucleotides were obtained from Pharmacia Biotech Inc. All other reagents were purchased from Sigma. The pancreatic β-cell line HIT-T15 (27Santerre R.F. Cook R.A. Crisel R.M. Sharp J.D. Schmidt R.J. Williams D.C. Wilson C.P. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4339-4343Crossref PubMed Scopus (305) Google Scholar) at passage 64 and COS-7 cells were purchased from the ATCC. Ins-1 (28Asfari M. Janjic D. Meda P. Li G. Halban P.A. Wollheim C.B. Endocrinology. 1992; 130: 167-178Crossref PubMed Scopus (748) Google Scholar) cells at passage 99 were a gift from Dr. Claes B. Wollheim (University of Geneva, Switzerland). HIT-T15 cells were maintained in RPMI 1640 medium (Life Technologies, Inc.) with 10% FBS at 37 °C in a 5% CO2, 95% air atmosphere as described (7Olson L.K. Sharma A. Peshavaria M. Wright C.V. Towle H.C. Robertson R.P. Stein R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9127-9131Crossref PubMed Scopus (130) Google Scholar). Ins-1 cells were grown in RPMI 1640 medium with 10% FBS, 50 μmβ-mercaptoethanol, 1 mm sodium pyruvate, and 10 mm HEPES as reported (28Asfari M. Janjic D. Meda P. Li G. Halban P.A. Wollheim C.B. Endocrinology. 1992; 130: 167-178Crossref PubMed Scopus (748) Google Scholar). Both cell lines were passaged weekly. For the model of long term exposure, HIT cells were cultured from passage 64 to passage 82 in 11.1 mmd-glucose or 0.8 mmd-glucose with adjustment of osmolality by the addition of mannitol to the low glucose medium. Glucose concentrations for HIT-T15 cells were chosen according to the left-shifted insulin response curve as previously reported (29Zhang H.J. Walseth T.F. Robertson R.P. Diabetes. 1989; 38: 44-48Crossref PubMed Scopus (78) Google Scholar). Ins-1 cells were grown in 25 mm or 5.6 mmd-glucose, respectively, with mannitol adjustment as reported previously (30Olson L.K. Poitout V. Robertson R.P. Diabetes. 1996; 45 (abstr.): 243Google Scholar). The βTC-6 cell line (31Efrat S. Linde S. Kofod H. Spector D. Delannoy M. Grant S. Hanahan D. Baekkeskov S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9037-9041Crossref PubMed Scopus (473) Google Scholar) was a gift from Dr. Shimon Efrat (Albert Einstein University College of Medicine, New York). The βTC-6 cells were cultured in Dulbecco's modified Eagle's medium (25 mm glucose) supplemented with 10% FBS. Passages from 23 to 33 were used for transfection experiments. Male Sprague-Dawley rats (150–200 g) were anesthetized with 100 mg/kg intraperitoneal pentobarbital sodium. Islets were isolated from the pancreata using an adaptation for rat islets of the method of Gotoh et al. (32Gotoh M. Maki T. Kiyoizumi T. Satomi S. Monaco A.P. Transplantation. 1985; 40: 437-438Crossref PubMed Scopus (534) Google Scholar). Briefly, after cannulation of the common bile duct and instillation of 10 ml of a prewarmed (37 °C) solution containing 1 mg/ml Collagenase P and 0.5 mg/ml DNase I, the pancreas was removed and digested for 30 min at 37 °C in a shaking water bath followed by dilution and washing of the digest and hand picking of the released islets under a dissecting microscope. Liver nuclei were prepared by the method of Gorski et al. (33Gorski K. Carneiro M. Schibler U. Cell. 1986; 47: 767-776Abstract Full Text PDF PubMed Scopus (969) Google Scholar). Polyclonal rabbit antisera for C/EBPβ and E47 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The IDX-1 antiserum was described previously (34Miller C.P. McGehee Jr., R.E. Habener J.F. EMBO J. 1994; 13: 1145-1156Crossref PubMed Scopus (374) Google Scholar). Western immunoblot analysis was performed on nuclear extracts prepared from the cell lines according to standard techniques (35Schreiber E. Matthias P. Müller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3903) Google Scholar). Extracts of pancreatic islet whole cells and liver nuclei were prepared by lysing isolated rat islets and liver nuclei in SDS-PAGE sample buffer (36Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). In each lane, a sample containing 100 μg of protein was loaded. A C/EBPβ protein fragment containing the basic region-leucine zipper binding domain that starts at an internal methionine site (12Poli V. Mancini F.P. Cortese R. Cell. 1990; 63: 643-653Abstract Full Text PDF PubMed Scopus (454) Google Scholar) was made by insertion into the BamHI-XhoI site of the pRSET-A vector (Invitrogen Inc., Carlsbad, CA). The protein was transcribed and translated and was purified with a nickel-chelating resin column and eluted by a pH gradient. Electrophoretic mobility shift assays (EMSAs) were performed as described (34Miller C.P. McGehee Jr., R.E. Habener J.F. EMBO J. 1994; 13: 1145-1156Crossref PubMed Scopus (374) Google Scholar), using bacterially expressed C/EBPβ or nuclear extracts prepared from HIT-T15, βTC-6, and Ins-1 cells (37Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9132) Google Scholar). The oligonucleotides used for EMSA are as follows: CEB oligonucleotide, 5′-GATCTGAGGGGCTGAAGCTGTAATTTCCAAACACTTGCCTAGTGCTAAGTG-3′ (antisense strand); CEB mutated oligonucleotide, 5′-GATCTGAGGGGCTGAAGCCTCGGCTTCCAAACACTGCCTAGTGCTAAGTG-3′; P1 element oligonucleotide, 5′-GATCCTACCTACCCCTCCTAGAGCCCTTAATGGGCCAAACGGCAAAGTCCAGGGGGCAGA-3′; CRE oligonucleotide, 5′-GATCTAGAGTTGTTGACGTCCAATGAGCG-3′; FLAT oligonucleotide, 5′-GATCCTTCATCAGGCCATCTGGCCCCTTGTTAATAATCTAATTACCCTAGGTCTA-3′. Double-stranded oligonucleotide probes comprising the E1 elements (Nir, RIPE3) of the rat insulin I and II promoters were as follows: Nir, 5′-GATCCAGCTTCAGCCCCTCTCGCCATCTGCCTACCTACCCCTCCTAGAGCCCTT-3′; RIPE3, 5′-GATCTGGAAACTGCAGCTTCAGCCCCTCTGGCCATCTGCTGATCCG-3′. DNA-binding probes were incubated in a 20-μl volume with the indicated combinations of in vitro translated proteins in a buffer containing 20 mm Tris (pH 7.5), 100 mmNaCl, 2 mm EDTA, 2 mm dithiothreitol, 10% glycerol, 200 ng of poly(dI-dC). To adjust the salt concentration, each reaction was equilibrated to a mock reticulocyte lysate reaction. Binding complexes were resolved by electrophoresis on 7% polyacrylamide gels in 0.5 × TBE buffer. C/EBPβ isolated from bacteria was incubated with a fragment of the 5′-flanking region of the rat insulin I gene (nucleotides −280 to +1). The fragment was end-labeled on the coding strand by the polymerase chain reaction. The DNase I footprinting analysis was carried out as described previously (38Vallejo M. Miller C.P. Habener J.F. J. Biol. Chem. 1992; 267: 12868-12875Abstract Full Text PDF PubMed Google Scholar). The plasmid −410INS-LUC contains a fragment of the rat insulin I gene promoter from base pairs −410 to +49 cloned into the pXP2 vector containing the coding sequence of firefly luciferase cDNA. The luciferase-reporter plasmid DNA containing the CEB mutation within the rat insulin I gene promoter was created by mutation of the CEB box from−128TGTAAT−133 to−128CTCGGC−133 using oligonucleotide-directed mutagenesis (39Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4540) Google Scholar). The luciferase plasmid containing 190 bp of the promoter of IDX-1 (−190IDX-LUC) was a gift from Dr. Marc Montminy (The Salk Institute, La Jolla, CA) and has been described previously (40Sharma S. Leonard J. Lee S. Chapman H.D. Leiter E.H. Montminy M.R. J. Biol. Chem. 1996; 271: 2294-2299Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The luciferase reporter construct containing the proximal 180 bp of the promoter sequence of the human islet sulfonylurea receptor (−180SUR-LUC) 3J. Ferrer, unpublished results. was a gift from Dr. Jorge Ferrer (Harvard Medical School, Boston, MA). All insulin promoter deletions were generated by polymerase chain reaction mutagenesis and subsequently sequenced. For expression of C/EBPβ in COS-7 cells and in vitro transcription/translation, the plasmid C/EBPβ-pcDNA I was used (41Ron D. Habener J.F. Genes Dev. 1992; 6: 439-453Crossref PubMed Scopus (969) Google Scholar). For bHLH factors, the plasmid shPanI.pBAT14 (42German M.S. Blanar M.A. Nelson C. Moss L.G. Rutter W.J. Mol. Endocrinol. 1991; 5: 292-299Crossref PubMed Scopus (111) Google Scholar) (hamster homolog of E47; a gift from Dr. M. German, University of California, San Francisco, CA) was used for expression in COS cells. Pan I (rat homolog of E47) was produced byin vitro transcription/translation from the plasmid pARP5/P2 (43Nelson C. Shen L.P. Meister A. Fodor E. Rutter W.J. Genes Dev. 1990; 4: 1035-1043Crossref PubMed Scopus (122) Google Scholar) (gift from C. Nelson, University of California, San Francisco). To produce the leucine zipper minus mutant form of Pan I, two point-mutations were introduced into the plasmid pARP5/P2 by polymerase chain reaction-based site-directed mutagenesis (QuikChange® site-directed mutagenesis kit, Stratagene, La Jolla, CA) to replace the second and the third leucines within the heptad leucine repeat of the AD2 of Pan I/E47 by phenylalanines. The mutated sequences were confirmed by sequencing using the dideoxy chain termination method (44Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 12: 5463-5467Crossref Scopus (52251) Google Scholar) with Sequenase version 2.0 (U.S. Biochemical Corp.). The plasmid for bacterial expression of the GST-C/EBPβ-fusion protein (pGEX-KG) has been described (41Ron D. Habener J.F. Genes Dev. 1992; 6: 439-453Crossref PubMed Scopus (969) Google Scholar). At 50% confluence (10-cm culture dishes), HIT-T15 or βTC-6 cells were transfected with 3 μg of the rat insulin I promoter-luciferase plasmid DNA using the DEAE-dextran method in cell suspension (45Vallejo M. Penchuk L. Habener J.F. J. Biol. Chem. 1992; 267: 12876-12884Abstract Full Text PDF PubMed Google Scholar). HeLa cells were transfected using CaPO4 (46$$Google Scholar) with 3 μg of the −410INS-LUC (or deletion constructs) and 0.5 μg of C/EBPβ-pcDNA I expression plasmid. After a 48-h incubation, the cells were harvested, and the luciferase activity was determined as described previously (47Brasier A.R. Tate J.E. Habener J.F. Biotechniques. 1989; 7: 1116-1122PubMed Google Scholar). The plasmids containing fusions between the GAL4 DNA-binding domain and either AD1 or AD2 of E47 (48Robinson G.L. Henderson E. Massari M.E. Murre C. Stein R. Mol. Cell Biol. 1995; 15: 1398-1404Crossref PubMed Google Scholar) were a gift from Dr. Roland Stein (Vanderbilt University, Nashville, TN). At 50% confluence (10-cm culture dishes), HIT-T15 cells were cotransfected with 10 μg of a luciferase reporter-construct containing a multimerized Gal4-binding site ((GBS)3-p59RLG) and one of each of the GAL4 constructs with a C/EBPβ expression plasmid (C/EBPβ-pcDNA I) or empty vector (pcDNA I) using the DEAE-dextran method in cell suspension (45Vallejo M. Penchuk L. Habener J.F. J. Biol. Chem. 1992; 267: 12876-12884Abstract Full Text PDF PubMed Google Scholar). Rous sarcoma virus-CAT was used as an internal control for monitoring of transfection efficiency. After a 48-h incubation, the cells were harvested, and the luciferase activity was determined as described previously (47Brasier A.R. Tate J.E. Habener J.F. Biotechniques. 1989; 7: 1116-1122PubMed Google Scholar). Values were expressed as means ± S.E. of three independent experiments. Transfection of COS-7 cells for protein expression was performed by liposomal transfer (Lipofectin®, Life Technologies, Inc.) according to the manufacturer's manual. Recombinant proteins were produced by in vitro transcription/translation with the TNT-coupled reticulocyte lysate system (Promega, Madison, WI) according to the manufacturer's instructions using T7 polymerase for all plasmids transcribed. Each translation reaction was performed in duplicate with and without inclusion of [35S]methionine, and protein identity was confirmed by autoradiography of products separated by SDS-polyacrylamide electrophoresis or Western immunoblot analysis. Proteins from transfected COS-7 cells were labeledin vivo by incubation inl-methionine/l-cysteine-free Dulbecco's modified Eagle's medium (Life Technologies) containing 200 μCi/ml [35S]methionine/[35S]cysteine (NEN Life Science Products) and 10% dialyzed FBS for 4 h. Nuclear extracts from COS-7 cells and from the β-cell lines HIT-T15 and Ins-1 were prepared as described previously (35Schreiber E. Matthias P. Müller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3903) Google Scholar). For immunoprecipitation, nuclear extracts and in vitro translated protein solutions were adjusted to 200 mm NaCl, 0.1% Nonidet P-40, 50 mm HEPES, 1 mm phenylmethylsulfonyl fluoride, 5 mm EDTA, 0.5 mm dithiothreitol and precleared with protein A-Sepharose (Pharmacia Biotech, Uppsala, Sweden). After the addition of the respective antisera for C/EBPβ and E47 (Santa Cruz Biotechnology) and incubation at 4 °C for 15 h, the immune complexes were precipitated with protein A-Sepharose, washed, and subjected to SDS-PAGE followed by autoradiography or Western immunoblot analysis. For GST pull down analysis, the GST fusion proteins of C/EBPβ were prepared as described by Ron and Habener (41Ron D. Habener J.F. Genes Dev. 1992; 6: 439-453Crossref PubMed Scopus (969) Google Scholar), except the proteins were not eluted from the glutathione-Sepharose. Interacting proteins were precipitated with the glutathione-Sepharose-coupled fusion proteins in the same buffer used for immunoprecipitation and analyzed by SDS-PAGE autoradiography and Western immunoblot as described. All values were expressed as means ± S.E. Statistical analysis was performed via Student's t test for paired and unpaired values (49Baillar III, J.C. Mosteller F. Medical Uses of Statistics. The New England Journal of Medicine, Waltham, MA1992Google Scholar). Potential C/EBPβ-binding Sites in the Promoters of the Rat and Human Insulin Genes—Inspection of the promoters of the rat insulin I, rat insulin II, and human insulin genes reveals nucleotide sequence elements that resemble the consensus motif that binds the C/EBP family of transcription factors (Fig.1, A and B). Isolated rat pancreatic islets and several β- and non-β-cell lines were assayed for C/EBPβ expression by Western immunoblot using liver nuclei extracts as a positive control, since C/EBPβ was originally defined as the liver-enriched activator protein, LAP (14Descombes P. Chojkier M. Lichtsteiner S. Falvey E. Schibler U. Genes Dev. 1990; 4: 1541-1551Crossref PubMed Scopus (419) Google Scholar). The 32-kDa C/EBPβ protein was detected in isolated rat islet whole cell extracts (Fig.2 A), although the antiserum also recognized a more abundant protein with an apparent molecular mass of 42 kDa. C/EBPβ was also detected in the nuclear extracts from Ins-1, βTC-6, and HIT-T15 cells, which are islet β-cell lines derived from rat, mouse, and hamster, respectively. To determine whether the expression of C/EBPβ is altered in β-cells during chronic or short term exposure to supraphysiologic concentrations of glucose, we used standard in vitro glucose desensitization models (6Olson L.K. Redmon J.B. Towle H.C. Robertson R.P. J. Clin. Invest. 1993; 92: 514-519Crossref PubMed Scopus (173) Google Scholar, 30Olson L.K. Poitout V. Robertson R.P. Diabetes. 1996; 45 (abstr.): 243Google Scholar). HIT-T15 cells were serially passaged in RPMI 1640 medium containing 11.1 mm glucose or 0.8 mm glucose for 16 weeks. Since the EC50 for glucose-stimulated insulin secretion is left-shifted to 1 mm in HIT-T15 cells rather than about 8 mm in normal islets (50Regazzi R. Li G.D. Deshusses J. Wollheim C.B. J. Biol. Chem. 1990; 265: 15003-15009Abstract Full Text PDF PubMed Google Scholar), 11.1 mm glucose was chosen as a supraphysiological concentration, and 0.8 mm was considered a physiological concentration of glucose for the HIT-T15 cells. As shown in Fig. 2 B, the expression of IDX-1 decreased from week 4 to 16, confirming the published observations (9Sharma A. Olson L.K. Robertson R.P. Stein R. Mol. Endocrinol. 1995; 9: 1127-1134Crossref PubMed Google Scholar). In contrast, the expression of C/EBPβ was markedly enhanced from week 8 to 16. These observations indicate that the level of C/EBPβ in HIT-T15 cells is up-regulated by prolonged exposure to high glucose concentrations and that C/EBPβ might serve as a repressor of insulin gene transcription. The increased expression of C/EBPβ in HIT-T15 cells after chronic exposure to high glucose was prevented by culturing HIT-T15 cells in the RPMI 1640 medium containing 0.8 mmglucose (Fig. 2 B). To validate the in vitro long term model and to show an effect of high glucose concentrations on the regulation of insulin secretion, we measured the insulin concentration in the culture medium in response to increasing glucose concentrations in HIT-T15 cells at different passages in 2-h static incubation intervals. Whereas HIT-T15 cells cultured in high glucose displayed a passage-dependent decrease in glucose-responsive insulin secretion (50 ± 7.7% after 16 weeks in 11.1 mmglucose), no such decrease was seen in cells cultured in low glucose (data not shown). The findings of long term high glucose exposure on C/EBPβ expression were
Referência(s)