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• W2000669888 abstract "To maintain blood glucose levels within narrow limits, the synthesis and secretion of pancreatic islet hormones is controlled by a variety of extracellular signals. Depolarization-induced calcium influx into islet cells has been shown to stimulate glucagon gene transcription through the transcription factor cAMP response element-binding protein that binds to the glucagon cAMP response element. By transient transfection of glucagon-reporter fusion genes into islet cell lines, this study identified a second calcium response element in the glucagon gene (G2 element, from −165 to −200). Membrane depolarization was found to induce the binding of a nuclear complex with NFATp-like immunoreactivity to the G2 element. Consistent with nuclear translocation, a comigrating complex was found in cytosolic extracts of unstimulated cells, and the induction of nuclear protein binding was blocked by inhibition of calcineurin phosphatase activity by FK506. A mutational analysis of G2 function and nuclear protein binding as well as the effect of FK506 indicate that calcium responsiveness is conferred to the G2 element by NFATp functionally interacting with HNF-3β binding to a closely associated site. Transcription factors of the NFAT family are known to cooperate with AP-1 proteins in T cells for calcium-dependent activation of cytokine genes. This study shows a novel pairing of NFATp with the cell lineage-specific transcription factor HNF-3β in islet cells to form a novel calcium response element in the glucagon gene. To maintain blood glucose levels within narrow limits, the synthesis and secretion of pancreatic islet hormones is controlled by a variety of extracellular signals. Depolarization-induced calcium influx into islet cells has been shown to stimulate glucagon gene transcription through the transcription factor cAMP response element-binding protein that binds to the glucagon cAMP response element. By transient transfection of glucagon-reporter fusion genes into islet cell lines, this study identified a second calcium response element in the glucagon gene (G2 element, from −165 to −200). Membrane depolarization was found to induce the binding of a nuclear complex with NFATp-like immunoreactivity to the G2 element. Consistent with nuclear translocation, a comigrating complex was found in cytosolic extracts of unstimulated cells, and the induction of nuclear protein binding was blocked by inhibition of calcineurin phosphatase activity by FK506. A mutational analysis of G2 function and nuclear protein binding as well as the effect of FK506 indicate that calcium responsiveness is conferred to the G2 element by NFATp functionally interacting with HNF-3β binding to a closely associated site. Transcription factors of the NFAT family are known to cooperate with AP-1 proteins in T cells for calcium-dependent activation of cytokine genes. This study shows a novel pairing of NFATp with the cell lineage-specific transcription factor HNF-3β in islet cells to form a novel calcium response element in the glucagon gene. CRE-binding protein cAMP response element interleukin 2 nuclear factor of activated T cells reverse transcription and PCR amplification 12-O-tetradecanoylphorbol-13-acetate Activation of gene transcription allows cells to adapt to changes in environmental conditions through a new pattern of expressed proteins. In cells that are electrically excitable, calcium is an important intracellular second messenger that directs the genomic response of the cell. Transcription factors that have been shown to mediate calcium-induced gene transcription include CREB1 (1Sheng M. Thompson M. Greenberg M.E. Science. 1991; 252: 1427-1430Crossref PubMed Scopus (1289) Google Scholar, 2Ghosh A. Greenberg M.E. Science. 1995; 268: 239-247Crossref PubMed Scopus (1247) Google Scholar, 3Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar, 4Sun P. Lou L. Maurer R.A. J. Biol. Chem. 1996; 271: 3066-3073Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), serum response factor (2Ghosh A. Greenberg M.E. Science. 1995; 268: 239-247Crossref PubMed Scopus (1247) Google Scholar, 5Misra R.P. Bonni A. Miranti C.K. Rivera V.M. Sheng M. Greenberg M.E. J. Biol. Chem. 1994; 269: 25483-25493Abstract Full Text PDF PubMed Google Scholar), and C/EBPβ (6Wegner M. Cao Z. Rosenfeld M.G. Science. 1992; 256: 370-373Crossref PubMed Scopus (308) Google Scholar). Like neurons, endocrine cells of the pancreatic islets are electrically excitable and express L-type voltage-dependent calcium channels (7Yaney G.C. Wheeler M.B. Wei X. Perez-Reyes E. Birnbaumer L. Boyd III, A.E. Moss L.G. Mol. Endocrinol. 1992; 6: 2143-2152PubMed Google Scholar, 8Holz G.G. Habener J.F. Trends Biochem. Sci. 1992; 17: 388-393Abstract Full Text PDF PubMed Scopus (112) Google Scholar). By virtue of stimulation of glycogenolysis and gluconeogenesis in the liver, the islet hormone glucagon is an important regulator of blood glucose levels (9Unger R.H. Orci L. N. Engl. J. Med. 1981; 304: 1518-1524Crossref PubMed Scopus (211) Google Scholar). Glucagon-producing islet cells show spontaneous electrical activity (10Rorsman P. Hellman B. J. Gen Physiol. 1988; 91: 223-242Crossref PubMed Scopus (79) Google Scholar). Membrane electrical activity and calcium influx into glucagon-producing pancreatic islet cells is tightly controlled by extracellular messengers. Whereas l-arginine increases spike frequency (10Rorsman P. Hellman B. J. Gen Physiol. 1988; 91: 223-242Crossref PubMed Scopus (79) Google Scholar), β-adrenergic cell-surface receptor stimulation by catecholamines through cAMP enhances the L-type calcium current, increasing the influx of calcium associated with each action potential (11Rorsman P. J. Gen. Physiol. 1988; 91: 243-254Crossref PubMed Scopus (29) Google Scholar). It is well known that membrane depolarization and calcium influx increase the cytosolic free calcium concentration, which stimulates hormone secretion by exocytosis (8Holz G.G. Habener J.F. Trends Biochem. Sci. 1992; 17: 388-393Abstract Full Text PDF PubMed Scopus (112) Google Scholar, 12Johansson H. Gylfe E. Hellman B. Cell Calcium. 1989; 10: 205-211Crossref PubMed Scopus (52) Google Scholar). The question is whether, and if so, how, this calcium signal reaches also into the nucleus and regulates gene transcription. Previous experiments have shown that membrane depolarization and calcium influx stimulate glucagon gene transcription (3Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar). A mechanism involved has been characterized. Through a calcium-/calmodulin-dependent protein kinase calcium stimulates the phosphorylation of the transcription factor CREB on the same serine residue that is also phosphorylated by cAMP-dependent protein kinase A. CREB binds to a CRE in the 5′-flanking region of the glucagon gene and stimulates transcription (3Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar, 13Schwaninger M. Blume R. Oetjen E. Lux G. Knepel W. J. Biol. Chem. 1993; 268: 23111-23115Abstract Full Text PDF PubMed Google Scholar, 14Schwaninger M. Blume R. Oetjen E. Knepel W. Naunyn-Schmiedeberg's Arch. Pharmacol. 1993; 348: 541-545Crossref PubMed Scopus (29) Google Scholar, 15Schwaninger M. Blume R. Krüger M. Lux G. Oetjen E. Knepel W. J. Biol. Chem. 1995; 270: 8860-8866Crossref PubMed Scopus (80) Google Scholar). The present study addressed the question whether there are additional calcium-responsive elements in the glucagon gene. By the results obtained, the G2 element is identified as a second calcium response element. The further characterization suggests that calcium responsiveness is conferred by the calcium/calcineurin-regulated transcription factor NFATp functionally synergizing with the cell-specifically expressed transcription factor HNF-3β. NFAT family proteins are known to cooperate with newly synthetized AP-1 proteins in T cells (16Jain J. McCaffrey P.G. Valge-Archer V.E. Rao A. Nature. 1992; 356: 801-804Crossref PubMed Scopus (429) Google Scholar, 17Northrop J.P. Ullman K.S. Crabtree G.R. J. Biol. Chem. 1993; 268: 2917-2923Abstract Full Text PDF PubMed Google Scholar, 18Nolan G.P. Cell. 1994; 77: 795-798Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 19Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2227) Google Scholar). This study shows a novel pairing of NFATp with the constitutively expressed cell-specific transcription factor HNF-3β forming a novel calcium response element in the glucagon gene. The plasmids −350GluLuc (3Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar), −350(−297/−292)GluLuc (3Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar), −350ΔGluLuc (20Fürstenau U. Schwaninger M. Blume R. Kennerknecht I. Knepel W. Mol. Cell. Biol. 1997; 17: 1805-1816Crossref PubMed Scopus (22) Google Scholar), −292GluLuc (20Fürstenau U. Schwaninger M. Blume R. Kennerknecht I. Knepel W. Mol. Cell. Biol. 1997; 17: 1805-1816Crossref PubMed Scopus (22) Google Scholar), pT81Luc (21Nordeen S.K. BioTechniques. 1988; 6: 454-457PubMed Google Scholar), 4xG3T81Luc (22Diedrich T. Fürstenau U. Knepel W. Biol. Chem. 1997; 378: 89-98Crossref PubMed Scopus (10) Google Scholar), 4xCST81Luc (23Hochhuth C. Neubauer A. Knepel W. Endocrine. 1994; 2: 833-839Google Scholar), 4xGluCRET81Luc (24Oetjen E. Diedrich T. Eggers A. Eckert B. Knepel W. J. Biol. Chem. 1994; 269: 27036-27044Abstract Full Text PDF PubMed Google Scholar), 4xG2T81Luc, 4xG2 m1T81Luc, 4xG2 m3T81Luc, and 4xG2 m5T81Luc (20Fürstenau U. Schwaninger M. Blume R. Kennerknecht I. Knepel W. Mol. Cell. Biol. 1997; 17: 1805-1816Crossref PubMed Scopus (22) Google Scholar) have been described previously. The plasmid −350(2Δ)GluLuc was prepared by subcloning the SstI fragment of −350ΔGluLuc into theSstI site of pT81ΔN (pT81Luc (21Nordeen S.K. BioTechniques. 1988; 6: 454-457PubMed Google Scholar) with theAatII site deleted); four bases in the CRE octamer (from −296 to −293) were then deleted with the restriction enzymeAatII and T4 DNA polymerase; after religation theSstI fragment was subcloned into the SstI site of pXP2 (21Nordeen S.K. BioTechniques. 1988; 6: 454-457PubMed Google Scholar). An expression vector encoding the DNA-binding domain of NFATp (amino acids 398–584) (19Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2227) Google Scholar, 25Luo C. Burgeon E. Rao A. J. Exp. Med. 1996; 184: 141-147Crossref PubMed Scopus (87) Google Scholar) (pBK-CMV-NFATpDBD) was prepared by PCR using pLGPmNFAT1-B (26Luo C. Burgeon E. Carew J.A. McCaffrey P.G. Badalian T.M. Lane W.S. Hogan P.G. Rao A. Mol. Cell. Biol. 1996; 16: 3955-3966Crossref PubMed Scopus (173) Google Scholar) as template and the oligonucleotides 5′-GATTGAGCTCGCAGCTCCACGGCTACAT-3′ and 5′-GCGCGAATTCTCCACCGTAGCTTCCATC-3′ as upstream and downstream primers, respectively; the PCR product was digested with SacI andEcoRI and subcloned into theSacI-EcoRI sites of pBK-CMV (Stratagene, Heidelberg, Germany). All constructs were confirmed by sequencing. The pancreatic islet cell line HIT-T15 (27Santerre R.F. Cook R.A. Crisel R.M.D. 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 (307) Google Scholar) was grown in RPMI 1640 medium supplemented with 10% fetal calf serum, 5% horse serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. αTC2 cells (28Powers A.C. Efrat S. Mojsov S. Spector D. Habener J.F. Hanahan D. Diabetes. 1990; 39: 406-414Crossref PubMed Scopus (116) Google Scholar, 29Hamaguchi K. Leiter E.H. Diabetes. 1990; 39: 415-425Crossref PubMed Google Scholar) were grown in Dulbecco's modified Eagle's medium (4.5 g of glucose/liter) supplemented with 2.5% fetal calf serum, 15% horse serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were trypsinized and transfected in suspension by the DEAE-dextran method (3Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar) with 2 μg of indicator plasmid per 6-cm dish. Rous sarcoma virus-chloramphenicol acetyltransferase plasmid (0.4 μg/6-cm dish) was added as a second reporter to check for transfection efficiency. When indicated, 0.7 μg of pBK-CMV-NFATpDBD was co-transfected per 6-cm dish; these co-transfections were done with a constant DNA concentration, which was maintained by adding Bluescript (Stratagene, Heidelberg, Germany). Cells were stimulated with high KCl (45 mm final concentration) or TPA (300 nm) for 6 h before harvest. FK506 (167 nm, unless noted otherwise) was added 1 h before stimulation. Cell extracts (3Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar) were prepared 48 h after transfection. A chromatographic chloramphenicol acetyltransferase assay was performed as described previously (30Knepel W. Jepeal L. Habener J.F. J. Biol. Chem. 1990; 265: 8725-8735Abstract Full Text PDF PubMed Google Scholar). Thin layer chromatography plates were analyzed with a Fuji PhosphorImager. The luciferase assay was performed as described previously (3Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar). Nuclear extracts were prepared from αTC2 cells by the method of Schreiber et al. (31Schreiber E. Matthias P. Müller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3918) Google Scholar), except for the experiments shown in Fig. 3 G. In the experiments shown in Fig. 3 G, separate nuclear and cytosolic extracts were prepared as described previously (32McCaffrey P.G. Luo C. Kerppola T.K. Jain J. Badalian T.M. Ho A.M. Burgeon E. Lane W.S. Lambert J.N. Curran T. Verdine G.L. Rao A. Hogan P.G. Science. 1993; 262: 750-754Crossref PubMed Scopus (379) Google Scholar, 33McCaffrey P.G. Perrino B.A. Soderling T.R. Rao A. J. Biol. Chem. 1993; 268: 3747-3752Abstract Full Text PDF PubMed Google Scholar, 34McCaffrey P.G. Jain J. Jamieson C. Sen R. Rao A. J. Biol. Chem. 1992; 267: 1864-1871Abstract Full Text PDF PubMed Google Scholar). Synthetic complementary oligonucleotides with 5′-GATC overhangs were annealed and labeled by a fill-in reaction with [α-32P]dCTP and Klenow enzyme. By using 15 μg of protein from cell extracts, the electrophoretic mobility shift assay was performed as described by Klemsz et al. (35Klemsz M.J. McKercher S.R. Celada A. Van Beveren C. Maki R.A. Cell. 1990; 61: 113-124Abstract Full Text PDF PubMed Scopus (759) Google Scholar). In some binding reactions, a specific anti-NFATp antiserum (kindly provided by A. Rao, Harvard Medical School, Boston) was used. This antiserum (anti-67.1) is directed against the 67.1 peptide of murine NFATp; it does not cross-react with NFATc, NFAT3, or NFATx (36Ho A.M. Jain J. Rao A. Hogan P.G. J. Biol. Chem. 1994; 269: 28181-28186Abstract Full Text PDF PubMed Google Scholar). Cell extracts were incubated with 1 μl of a 1:10 dilution of preimmune serum or anti-NFATp antiserum in the reaction buffer with probe for 15 min at room temperature. Following a 15-min incubation on ice, the samples were loaded onto the gels and electrophoresed as described above. The sequences of the G2 oligonucleotides (wild type and mutants 1, 3, and 5) were as described previously (20Fürstenau U. Schwaninger M. Blume R. Kennerknecht I. Knepel W. Mol. Cell. Biol. 1997; 17: 1805-1816Crossref PubMed Scopus (22) Google Scholar) and read as follows (only one strand with the 5′-GATC overhang is shown, and mutated bases are underlined): G2, 5′-GATCCAGGCACAAGAGTAAATAAAAAGTTTCCGGGCCTCTGA-3′; G2 m1, 5′-GATCCAGGCACAGTTCGAAATAAAAAGTTTCCGGGCCTCTGA-3′; G2 m3, 5′-GATCCAGGCACAAGAGTAAATAAGGGATTTCCGGGCCTCTGA-3′; G2 m5, 5′-GATCCAGGCACAAGAGTAAATAAAAAGTTTGGGGGCCTCTGA-3′. The sequence of the oligonucleotide (NFATcons) containing a well characterized NFAT-binding site was as described (36Ho A.M. Jain J. Rao A. Hogan P.G. J. Biol. Chem. 1994; 269: 28181-28186Abstract Full Text PDF PubMed Google Scholar) except thatBamHI (upstream) and BglII ends (downstream) with 5′-GATC overhangs were added. Poly (A)+ RNA was extracted from αTC2 cells using a commercial kit (Fast Track 2.0TM, Invitrogen). RT-PCR was performed using a commercial kit (Gene AmpTM Thermostable rTth Reverse Transcriptase RNA PCR Kit, Roche Molecular Systems) with primers and PCR reaction conditions as follows: upstream primer 5′-AGTCCCCAAGACGAGCT-3′, downstream primer 5′-CGGGCTCAGAAAGTTCTGG-3′; 15 s at 95 °C, 30 s at 57 °C, 45 s at 72 °C, for 34 cycles; the expected product is 234 base pairs long. After agarose gel electrophoresis, the product obtained was verified by extraction, subcloning (pCR© 2.1, Invitrogen), and cycle sequencing (Thermo Sequenase fluorescent labeled primer cycle sequencing kit, Amersham, Braunschweig, Germany; M13 fluorescent primer). Nuclear extracts were resolved on a 5.5% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, incubated for 2 h in 10% nonfat dry milk dissolved in TBST (10 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.2% Tween 20), incubated for 1 h in 2% gelatin dissolved in TBST, and then incubated with anti-NFATp antiserum (anti-67.1) (36Ho A.M. Jain J. Rao A. Hogan P.G. J. Biol. Chem. 1994; 269: 28181-28186Abstract Full Text PDF PubMed Google Scholar), diluted 1:3,000 in TBST, overnight at 4 °C. Antibody-antigen complexes were detected with ECL reagents (Amersham). Using the same samples, immunoblots were performed with an anti-CREB antiserum as has been described previously (15Schwaninger M. Blume R. Krüger M. Lux G. Oetjen E. Knepel W. J. Biol. Chem. 1995; 270: 8860-8866Crossref PubMed Scopus (80) Google Scholar). A stock solution of TPA (1 mm) was prepared in dimethyl sulfoxide and further diluted in cell culture medium. FK506 (provided by Fujisawa) was dissolved in ethanol. Controls received the solvent only. To study the calcium responsiveness of the glucagon gene in the absence of a functional CRE, the CRE in the 5′-flanking region of the rat glucagon gene was removed by either 5′-deletion to −292 or by internal deletion of four bases within the CRE octamer motif, yielding the constructs −292GluLuc and −350(−297/−292)GluLuc, respectively (Fig.1). The corresponding glucagon-reporter fusion genes were transiently transfected into two pancreatic islet cell lines, HIT and αTC2. These cell lines have been used previously to demonstrate that membrane depolarization and calcium influx induce glucagon gene transcription through the CRE of the glucagon gene (3Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar,13Schwaninger M. Blume R. Oetjen E. Lux G. Knepel W. J. Biol. Chem. 1993; 268: 23111-23115Abstract Full Text PDF PubMed Google Scholar, 14Schwaninger M. Blume R. Oetjen E. Knepel W. Naunyn-Schmiedeberg's Arch. Pharmacol. 1993; 348: 541-545Crossref PubMed Scopus (29) Google Scholar, 15Schwaninger M. Blume R. Krüger M. Lux G. Oetjen E. Knepel W. J. Biol. Chem. 1995; 270: 8860-8866Crossref PubMed Scopus (80) Google Scholar). HIT cells (27Santerre R.F. Cook R.A. Crisel R.M.D. 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 (307) Google Scholar) express glucagon, although only at a low level (37Knepel W. Chafitz J. Habener J.F. Mol. Cell. Biol. 1990; 10: 6799-6804Crossref PubMed Scopus (68) Google Scholar, 38Diem P. Walseth T.F. Zhang H.-J. Robertson R.P. Endocrinology. 1990; 127: 1609-1612Crossref PubMed Scopus (10) Google Scholar), and have the advantage to respond to second messenger stimulation and to be well characterized with respect to electrical activity, voltage-dependent calcium channels, cytosolic calcium concentration, and secretion (see Ref. 3Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar). Increases in extracellular potassium concentration to 40 mm have been shown to depolarize HIT cells with action potentials continuing at the peak of the depolarizing phase of the spontaneous activity; at the same time, high potassium initiates the influx of calcium through dihydropyridine-sensitive, L-type Ca2+ channels in the cell membrane and elevates the cytosolic calcium concentration (39Keahey H.H. Rajan A.S. Boyd III, A.E. Kunze D.L. Diabetes. 1989; 38: 188-193Crossref PubMed Google Scholar). Membrane depolarization was induced by elevating the potassium chloride concentration in the incubation medium from 5 to 45 mm. As shown in Fig. 1 B, the high potassium-induced increase in glucagon gene transcription was decreased in HIT cells by 77% by the 4-base deletion in the CRE octamer motif, as has been reported previously (3Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar). It was decreased by 46% when the CRE was removed by 5′-deletion to −292 (Fig. 1 B). Basal reporter activity was not changed by these deletions (data not shown). Thus, although the depolarization-induced increase in glucagon gene transcription was reduced using both constructs, glucagon gene transcription did still respond to membrane depolarization, resulting in a 2.0- and 3.3-fold stimulation of transcription in the absence of the CRE, respectively (Fig. 1 B). After transfection of −292GluLuc, the stimulation of transcription by high potassium was abolished when extracellular calcium was bound by 1.5 mmEGTA added to the medium (data not shown), suggesting that gene induction by membrane depolarization depends on calcium influx elevating intracellular calcium levels. The pancreatic α-like cell line αTC2 has been established from a glucagonoma arising in transgenic mice expressing the SV40 large T-antigen oncogene (28Powers A.C. Efrat S. Mojsov S. Spector D. Habener J.F. Hanahan D. Diabetes. 1990; 39: 406-414Crossref PubMed Scopus (116) Google Scholar, 29Hamaguchi K. Leiter E.H. Diabetes. 1990; 39: 415-425Crossref PubMed Google Scholar). These cells express predominantly glucagon in a rather uniform pattern (28Powers A.C. Efrat S. Mojsov S. Spector D. Habener J.F. Hanahan D. Diabetes. 1990; 39: 406-414Crossref PubMed Scopus (116) Google Scholar). In αTC2 cells, the depolarization-induced stimulation of glucagon gene transcription was only slightly reduced by 5′-deletion to −292 or by the internal deletion of four bases within the CRE octamer (Fig. 1 C). When compared with −350GluLuc, basal reporter activity of −350(−297/−292)GluLuc was not changed, and basal reporter activity of −292GluLuc was reduced to 38 ± 7% (n = 12). The results obtained in both pancreatic islet cell lines thus indicate that besides the CRE the rat glucagon gene 5′-flanking region contains at least one more calcium-responsive element within 292 bases in front of the transcription start site. Known elements within the enhancer region of the glucagon gene include the G2 element (20Fürstenau U. Schwaninger M. Blume R. Kennerknecht I. Knepel W. Mol. Cell. Biol. 1997; 17: 1805-1816Crossref PubMed Scopus (22) Google Scholar, 40Philippe J. Drucker D.J. Knepel W. Jepeal L. Misulovin Z. Habener J.F. Mol. Cell. Biol. 1988; 8: 4877-4888Crossref PubMed Scopus (116) Google Scholar, 41Philippe J. Morel C. Prezioso V.R. Mol. Cell. Biol. 1994; 14: 3514-3523Crossref PubMed Scopus (58) Google Scholar), the G3 element (30Knepel W. Jepeal L. Habener J.F. J. Biol. Chem. 1990; 265: 8725-8735Abstract Full Text PDF PubMed Google Scholar, 40Philippe J. Drucker D.J. Knepel W. Jepeal L. Misulovin Z. Habener J.F. Mol. Cell. Biol. 1988; 8: 4877-4888Crossref PubMed Scopus (116) Google Scholar, 42Knepel W. Vallejo M. Chafitz J.A. Habener J.F. Mol. Endocrinol. 1991; 5: 1457-1466Crossref PubMed Scopus (42) Google Scholar, 43Wrege A. Diedrich T. Hochhuth C. Knepel W. Gene Expr. 1995; 4: 205-216PubMed Google Scholar, 44Sander M. Neubüser A. Kalamaras J. Ee H.C. Martin G.R. German M.S. Genes Dev. 1997; 11: 1662-1673Crossref PubMed Scopus (468) Google Scholar), as well as a binding site for C/EBP proteins (CS) (23Hochhuth C. Neubauer A. Knepel W. Endocrine. 1994; 2: 833-839Google Scholar) (Fig.2). To study their role, four copies of oligonucleotides containing the G2 element (from −200 to −165), the G3 element (from −274 to −234), or the CS element (from −241 to −212) were placed in front of the truncated viral thymidine kinase promoter of herpes simplex virus (from −81 to +52) fused to the luciferase reporter gene (plasmid pT81Luc) (21Nordeen S.K. BioTechniques. 1988; 6: 454-457PubMed Google Scholar). As shown in Fig. 2, the promoter alone did not respond to membrane depolarization in αTC2 cells. The G3 element and the binding site for C/EBP proteins were also inactive (Fig. 2). However, the G2 element did confer depolarization responsiveness to the promoter (Fig. 2). The blocker of L-type, voltage-dependent calcium channels, diltiazem, inhibited the high potassium-induced transcriptional activation of the G2 element (data not shown). Depolarization responsiveness conferred by the G2 element to the nonresponsive thymidine kinase promoter was only somewhat less than that conferred by the glucagon CRE. When in separate experiments the plasmids 4xG2T81Luc and 4xGluCRET81Luc were transfected in parallel into αTC2 cells, membrane depolarization stimulated transcription to 182 ± 14% of controls (n = 6) through the G2 element and to 225 ± 18% of controls (n = 6) through the glucagon CRE. These data indicate that the G2 element is a calcium-responsive element of the glucagon gene. The G2 element has been shown before to confer cell-specific basal activity (20Fürstenau U. Schwaninger M. Blume R. Kennerknecht I. Knepel W. Mol. Cell. Biol. 1997; 17: 1805-1816Crossref PubMed Scopus (22) Google Scholar, 40Philippe J. Drucker D.J. Knepel W. Jepeal L. Misulovin Z. Habener J.F. Mol. Cell. Biol. 1988; 8: 4877-4888Crossref PubMed Scopus (116) Google Scholar, 41Philippe J. Morel C. Prezioso V.R. Mol. Cell. Biol. 1994; 14: 3514-3523Crossref PubMed Scopus (58) Google Scholar) and Ras/protein kinase C responsiveness (20Fürstenau U. Schwaninger M. Blume R. Kennerknecht I. Knepel W. Mol. Cell. Biol. 1997; 17: 1805-1816Crossref PubMed Scopus (22) Google Scholar) to the glucagon gene. The binding of two transcription factors is required for both of these activities in αTC2 cells, HNF-3β and an Ets-like protein (20Fürstenau U. Schwaninger M. Blume R. Kennerknecht I. Knepel W. Mol. Cell. Biol. 1997; 17: 1805-1816Crossref PubMed Scopus (22) Google Scholar). However, the precise role of HNF-3β in the islet-specific, basal transcriptional activity of G2 remains to be defined (20Fürstenau U. Schwaninger M. Blume R. Kennerknecht I. Knepel W. Mol. Cell. Biol. 1997; 17: 1805-1816Crossref PubMed Scopus (22) Google Scholar, 41Philippe J. Morel C. Prezioso V.R. Mol. Cell. Biol. 1994; 14: 3514-3523Crossref PubMed Scopus (58) Google Scholar). To study the effect of membrane depolarization and calcium influx on nuclear protein binding to the G2 element, electrophoretic mobility shift assays were performed. Nuclear extracts were prepared from pancreatic islet αTC2 cells with or without stimulation by membrane depolarization. It has been shown previously that under basal nonstimulated conditions band h (Fig.3 A) represents the binding of HNF-3β, whereas several weak bands collectively labeled e(Fig. 3 A) represent proteins with a binding specificity related to the Ets family of transcription factors (20Fürstenau U. Schwaninger M. Blume R. Kennerknecht I. Knepel W. Mol. Cell. Biol. 1997; 17: 1805-1816Crossref PubMed Scopus (22) Google Scholar). Membrane depolarization for 15 or 120 min induced the binding of a protein complex that migrated among the bands e (Fig. 3 A, marked by an arrow). Whereas the intensities of some bands e varied somewhat between the experiments, the depolarization-induced nuclear protein binding was consistently observed (Fig. 3,A–F). The binding specificity of the depolarization-induced protein complex was studied using G2 mutants. The depolarization-induced nuclear protein complex did bind to labeled mutant 1 (Fig. 3 B, lanes 7 and 8) but not to labeled mutant 5 (Fig.3 B, lanes 11 and 12). In mutant 1 and mutant 5, 5 and 2 bases, respectively, have been mutated that are required for the binding of HNF-3β and the Ets-like proteins, respectively (see Ref. 20Fürstenau U. Schwaninger M. Blume R. Kennerknecht I. Knepel W. Mol. Cell. Biol. 1997; 17: 1805-1816Crossref PubMed Scopus (22) Google Scholar; see also top of Fig.3 A). Consequently, band h was not formed when G2 mutant 1 was used as probe, and bands e were not detectable when G2 mutant 5 was used as probe (Fig. 3 B). This indicates that the depolarization-induced nuclear complex binds to a site within G2 that is distinct from the HNF-3β-binding site; in contrast, the two guanine bases (noncoding strand) exchanged in mutant 5 are essential for binding (see top of Fig. 3 A). The binding specificity was further characterized in competition experiments using G2 wild type, mutant 1, mutant 5, and also mutant 3. In mutant 3, 4 bases have been mutated (see top of Fig.3 A). As shown in Fig. 3 C, the binding of HNF-3β (band h) was competed away by mutants 5 and 3 but not by mutant 1, which carries mutations within the HNF-3β-binding sequence. In contrast, mutant 1 competed for the binding of the depo" @default.
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• W2000669888 title "Characterization of a Novel Calcium Response Element in the Glucagon Gene" @default.
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