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• W2016602122 abstract "Gene transcription can be induced by cAMP and Ca2+through distinct protein kinases phosphorylating the transcription factor CREB, which binds to cAMP response elements (CREs) in various genes. Induction of gene transcription by Ca2+has been shown recently to depend on the Ca2+/calmodulin-dependent protein phosphatase calcineurin in pancreatic islet cells. This study investigates the role of calcineurin in CRE-directed gene transcription after stimulation by cAMP. Reporter fusion genes under the transcriptional control of CREs were transiently transfected into the cell line HIT. Pharmacological evidence suggests that cAMP stimulates CRE-mediated transcription through a Ca2+-dependent mechanism. The immunosuppressive drugs cyclosporin A and FK506 inhibited CRE-mediated transcription stimulated by cAMP. At the same concentrations they also inhibited calcineurin phosphatase activity. Reversal of calcineurin inhibition by rapamycin or overexpression of calcineurin led to disinhibition of CRE-mediated gene transcription. Immunoblots with a phosphoCREB-specific antibody showed that cyclosporin A and FK506 do not interfere with CREB phosphorylation at serine 119 stimulated with cAMP or membrane depolarization. These results indicate that in HIT cells stimulation of CRE-mediated transcription depends not only on the activity of protein kinases phosphorylating CREB but also on the Ca2+/calmodulin-dependent protein phosphatase calcineurin that is necessary for the transcriptional competence of phosphorylated CREB. Gene transcription can be induced by cAMP and Ca2+through distinct protein kinases phosphorylating the transcription factor CREB, which binds to cAMP response elements (CREs) in various genes. Induction of gene transcription by Ca2+has been shown recently to depend on the Ca2+/calmodulin-dependent protein phosphatase calcineurin in pancreatic islet cells. This study investigates the role of calcineurin in CRE-directed gene transcription after stimulation by cAMP. Reporter fusion genes under the transcriptional control of CREs were transiently transfected into the cell line HIT. Pharmacological evidence suggests that cAMP stimulates CRE-mediated transcription through a Ca2+-dependent mechanism. The immunosuppressive drugs cyclosporin A and FK506 inhibited CRE-mediated transcription stimulated by cAMP. At the same concentrations they also inhibited calcineurin phosphatase activity. Reversal of calcineurin inhibition by rapamycin or overexpression of calcineurin led to disinhibition of CRE-mediated gene transcription. Immunoblots with a phosphoCREB-specific antibody showed that cyclosporin A and FK506 do not interfere with CREB phosphorylation at serine 119 stimulated with cAMP or membrane depolarization. These results indicate that in HIT cells stimulation of CRE-mediated transcription depends not only on the activity of protein kinases phosphorylating CREB but also on the Ca2+/calmodulin-dependent protein phosphatase calcineurin that is necessary for the transcriptional competence of phosphorylated CREB. Much information has accumulated about the pathway by which cAMP stimulates transcription of a number of genes that contain a cAMP-responsive element (CRE) 1The abbreviations used are:CREcAMP-responsive elementCaM kinaseCa2+/calmodulin-dependent protein kinase8-Br-cAMP8-bromo cyclic AMPCREBCRE-binding protein. 1The abbreviations used are:CREcAMP-responsive elementCaM kinaseCa2+/calmodulin-dependent protein kinase8-Br-cAMP8-bromo cyclic AMPCREBCRE-binding protein. in their promoter with the core octamer sequence TGACGTCA. cAMP binds to the regulatory subunits of protein kinase A and thereby induces the dissociation of two catalytic subunits that are translocated to the cell nucleus (1Nigg E.A. Hilz H. Eppenberger H.M. Dutly F. EMBO J. 1985; 4: 2801-2806Crossref PubMed Scopus (198) Google Scholar, 2Hagiwara M. Brindle P. Harootunian A. Armstrong R. Rivier J. Vale W. Tsien R. Montminy M.R. Mol. Cell. Biol. 1993; 13: 4852-4859Crossref PubMed Scopus (379) Google Scholar). The catalytic subunit then phosphorylates the CRE-binding transcription factor CREB (3Hoeffler J.P. Meyer T.E. Yun Y. Jameson J.L. Habener J.F. Science. 1988; 242: 1430-1433Crossref PubMed Scopus (525) Google Scholar, 4Gonzalez G.A. Yamamoto K.K. Fischer W.H. Karr D. Menzel P. Biggs III, W. Vale W.W. Montminy M.R. Nature. 1989; 337: 749-752Crossref PubMed Scopus (648) Google Scholar) at serine 119 (in CREB-327, corresponding to serine 133 in CREB-341), which leads to an increase in its transcriptional activity (5Meyer T.E. Habener J.F. Endocr. Rev. 1993; 14: 269-290PubMed Google Scholar). The effect of cAMP is limited as CREB is dephosphorylated at serine 119 by phosphatase-1 or −2a (6Hagiwara M. Alberts A. Brindle P. Meinkoth J. Feramisco J. Deng T. Karin M. Shenolikar S. Montminy M. Cell. 1992; 70: 105-113Abstract Full Text PDF PubMed Scopus (406) Google Scholar, 7Wadzinski B.E. Wheat W.H. Jaspers S. Peruski Jr., L.F. Lickteig R.L. Johnson G.L. Klemm D.J. Mol. Cell. Biol. 1993; 13: 2822-2834Crossref PubMed Scopus (284) Google Scholar). cAMP-responsive element Ca2+/calmodulin-dependent protein kinase 8-bromo cyclic AMP CRE-binding protein. cAMP-responsive element Ca2+/calmodulin-dependent protein kinase 8-bromo cyclic AMP CRE-binding protein. Recently the induction of gene transcription by membrane depolarization and Ca2+influx in neuronal PC12 pheochromocytoma and endocrine pancreatic islet cell lines has also been mapped to the CRE of the c- fos (8Sheng M. Dougan S.T. McFadden G. Greenberg M.E. Mol. Cell. Biol. 1988; 8: 2787-2796Crossref PubMed Scopus (297) Google Scholar, 9Sheng M. McFadden G. Greenberg M.E. Neuron. 1990; 4: 571-582Abstract Full Text PDF PubMed Scopus (878) Google Scholar, 10Sheng M. Thompson M.A. Greenberg M.E. Science. 1991; 252: 1427-1430Crossref PubMed Scopus (1282) Google Scholar), tyrosine hydroxylase (11Kilbourne E.J. Nankova B.B. Lewis E.J. McMahon A. Osaka H. Sabban D.B. Sabban E.L. J. Biol. Chem. 1992; 267: 7563-7569Abstract Full Text PDF PubMed Google Scholar), and glucagon genes (12Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar). Elevation of the intracellular free Ca2+concentration activates CREB (10Sheng M. Thompson M.A. Greenberg M.E. Science. 1991; 252: 1427-1430Crossref PubMed Scopus (1282) Google Scholar, 12Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar) and leads to an increased phosphorylation of CREB at serine 119, the same site that is also phosphorylated by protein kinase A (10Sheng M. Thompson M.A. Greenberg M.E. Science. 1991; 252: 1427-1430Crossref PubMed Scopus (1282) Google Scholar). A CaM kinase probably mediates the effect of Ca2+as CaM kinases are activated in the course of membrane depolarization (13Jefferson A.B. Travis S.M. Schulman H. J. Biol. Chem. 1991; 266: 1484-1490Abstract Full Text PDF PubMed Google Scholar), CaM kinases I, II, and IV can phosphorylate CREB in vitro (10Sheng M. Thompson M.A. Greenberg M.E. Science. 1991; 252: 1427-1430Crossref PubMed Scopus (1282) Google Scholar, 14Dash P.K. Karl K.A. Colicos M.A. Prywes R. Kandel E.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5061-5065Crossref PubMed Scopus (474) Google Scholar, 15Enslen H. Sun P. Brickey D. Soderling S.H. Klamo E. Soderling T.R. J. Biol. Chem. 1994; 269: 15520-15527Abstract Full Text PDF PubMed Google Scholar), and a pharmacological blocker of CaM kinase activity decreases the effect of membrane depolarization on gene transcription (12Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar, 16Bading H. Ginty D.D. Greenberg M.E. Science. 1993; 260: 181-186Crossref PubMed Scopus (958) Google Scholar). However, a model according to which CaM kinase mediates the stimulatory effect of membrane depolarization on CREB/CRE-directed transcription may be incomplete. In pancreatic islet cells the immunosuppressive drugs cyclosporin A and FK506 inhibited CRE-mediated transcription stimulated by membrane depolarization and Ca2+influx (17Schwaninger M. Blume R. Oetjen E. Lux G. Knepel W. J. Biol. Chem. 1993; 268: 23111-23115Abstract Full Text PDF PubMed Google Scholar). Evidence suggests that cyclosporin A and FK506 act through inhibition of the Ca2+/calmodulin-dependent protein phosphatase calcineurin (17Schwaninger M. Blume R. Oetjen E. Lux G. Knepel W. J. Biol. Chem. 1993; 268: 23111-23115Abstract Full Text PDF PubMed Google Scholar). Hence, in addition to CaM kinase, the phosphatase calcineurin seems to be necessary for stimulation of CRE-mediated transcription by membrane depolarization in pancreatic islet cells (17Schwaninger M. Blume R. Oetjen E. Lux G. Knepel W. J. Biol. Chem. 1993; 268: 23111-23115Abstract Full Text PDF PubMed Google Scholar). It is unknown whether also cAMP induction of transcription relies on calcineurin phosphatase activity. The aim of the present study was to investigate in pancreatic islet cells whether calcineurin is involved in cAMP induction of CRE-mediated transcription. The results obtained suggest the existence of a calcineurin-sensitive phosphorylation event that interferes with the transcriptional competence of CREB phosphorylated on serine 119, offering a new mechanism through which CREB/CRE-dependent transcription could be regulated. The plasmids −350GluLuc, 5xGluCRET81Luc, 4xCGαCRET81Luc, Gal4-Luc (12Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar, 17Schwaninger M. Blume R. Oetjen E. Lux G. Knepel W. J. Biol. Chem. 1993; 268: 23111-23115Abstract Full Text PDF PubMed Google Scholar), 4xSomCRET81Luc (18Oetjen E. Diedrich T. Eggers A. Eckert B. Knepel W. J. Biol. Chem. 1994; 269: 27036-27044Abstract Full Text PDF PubMed Google Scholar), and pT81 (19Nordeen S.K. BioTechniques. 1988; 6: 454-457PubMed Google Scholar) have been described previously. The pancreatic islet cell line HIT-T15 (20Santerre 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 and transiently transfected as described (12Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar, 21Knepel W. Chafitz J. Habener J.F. Mol. Cell. Biol. 1990; 10: 6799-6804Crossref PubMed Scopus (67) Google Scholar, 22Knepel W. Jepeal L. Habener J.F. J. Biol. Chem. 1990; 265: 8725-8735Abstract Full Text PDF PubMed Google Scholar, 23Knepel W. Vallejo M. Chafitz J.A. Habener J.F. Mol. Endocrinol. 1991; 5: 1457-1466Crossref PubMed Scopus (41) Google Scholar). GH3cells were transfected in suspension by the DEAE-dextran method (5 μg of indicator plasmid/6-cm dish). JEG-3 cells were transfected by the calcium phosphate precipitation method (10 μg of indicator plasmid/6-cm dish). Transfection efficiency was checked by cotransfection of CMV-βGal. When indicated, 2 μg of pBJ5-CNA plus 2 μg of pBJ5-CNB (24Kincaid R.L. Giri P.R. Higuchi S. Tamura J. Dixon S.C. Marietta C.A. Amorese D.A. Martin B.M. J. Biol. Chem. 1990; 265: 11312-11319Abstract Full Text PDF PubMed Google Scholar, 25Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1473) Google Scholar), 5 μg of RSV-KCREB (26Walton K.M. Rehfuss R.P. Chrivia J.C. Lochner J.E. Goodman R.H. Mol. Endocrinol. 1992; 6: 647-655PubMed Google Scholar), or 5 μg of pZ1 (12Schwaninger 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 cotransfected as described (12Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar). Cell extracts were prepared 48 h after transfection and 6 h after stimulation. Test drugs were added 1 h before stimulation. The luciferase assay was performed as described previously (12Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar, 21Knepel W. Chafitz J. Habener J.F. Mol. Cell. Biol. 1990; 10: 6799-6804Crossref PubMed Scopus (67) Google Scholar). The β-galactosidase activity was measured using a commercial kit for chemoluminescent detection (Serva, Germany). The calcineurin phosphatase activity in the cultured cells was measured as described (27Fruman D.A. Klee C.B. Bierer B.E. Burakoff S.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3686-3690Crossref PubMed Scopus (755) Google Scholar, 28Schwaninger M. Blume R. Oetjen E. Knepel W. Naunyn-Schmiedeberg's Arch. Pharmacol. 1993; 348: 541-545Crossref PubMed Scopus (29) Google Scholar). HIT cell lysates (in 50 m M Tris-HCl, pH 7.0, 1% SDS, 2% 2-mercaptoethanol) were resolved on a 10% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, rinsed in TBST (10 m M Tris-HCl, pH 8.0, 150 m M NaCl, 0.05% Tween 20), incubated in 10% nonfat dry milk dissolved in TBST for 1 h, and then incubated with either anti-CREB antiserum (29Waeber G. Meyer T.E. LeSieur M. Hermann H.L. Gérard N. Habener J.F. Mol. Endocrinol. 1991; 5: 1418-1430Crossref PubMed Scopus (142) Google Scholar), diluted 1:50,000 in TBST, or with anti-phosphoCREB antibodies (0.2 μg/ml in TBST) (30Ginty D.D. Kornhauser J.M. Thompson M.A. Bading H. Mayo K.E. Takahashi J.S. Greenberg M.E. Science. 1993; 260: 238-241Crossref PubMed Scopus (743) Google Scholar) for 2 h. Antibody-antigen complexes were detected with ECL reagents (Amersham Corp.). Insulin was measured by radioimmunoassay (31Joost H.G. Horm. Metab. Res. 1979; 11: 104-106Crossref PubMed Scopus (11) Google Scholar). FK506 and rapamycin were dissolved in ethanol. KN-62 was dissolved in dimethyl sulfoxide. A stock solution of cyclosporin A (10 mg/ml) was prepared in ethanol with 20% Tween 80 and further diluted in medium. Controls received the solvent only. The CRE of the rat glucagon gene has the typical consensus sequence TGACGTCA and confers responsiveness to cAMP (21Knepel W. Chafitz J. Habener J.F. Mol. Cell. Biol. 1990; 10: 6799-6804Crossref PubMed Scopus (67) Google Scholar) and Ca2+influx following membrane depolarization induced by high KCl concentration in the pancreatic islet cell line HIT (12Schwaninger 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 reporter gene under control of five copies of the rat glucagon CRE in front of the truncated viral thymidine kinase promoter (from −81 to +52) was transiently transfected into HIT cells. The glucagon CRE did not increase basal activity (data not shown). However, transcription was stimulated 7.3-fold by 1.5 m M 8-Br-cAMP (Fig. 1). Lowering the extracellular Ca2+concentration by addition of EGTA had no effect on basal gene transcription, but inhibited 8-Br-cAMP-induced transcription by about 80% and abolished the stimulation by high KCl (Fig. 1). The effect of EGTA seems to be specific since a dye exclusion test showed no change in cell viability after EGTA treatment compared to controls. Furthermore, the effect of EGTA was reversible, since EGTA treatment followed by washing of the cells and re-addition of Ca2+had no significant effect on the subsequent stimulation by forskolin (10 μM) of insulin secretion (120.5 ± 17.0 ng ml−1h−1versus 159.5 ± 26.0 ng ml−1h−1in controls) or CRE-mediated transcription (6.1 ± 0.7-fold versus 5.8 ± 0.6-fold in controls). Therefore, these data suggest that cAMP-induced gene transcription depends on extracellular Ca2+. Verapamil (10 μM), a blocker of L-type voltage-dependent Ca2+channels, inhibited CRE-mediated transcription by 27% after stimulation by 8-Br-cAMP (p < 0.05, t test), whereas depolarization-induced transcription was completely blocked (data not shown), indicating that the stimulation of CRE-mediated transcription by cAMP depends in part on Ca2+influx through voltage-dependent Ca2+channels. KN-62 is a selective inhibitor of CaM kinases that spares other protein kinases, e.g. protein kinase A, and calmodulin-dependent enzymes (32Tokumitsu H. Chijiwa T. Hagiwara M. Mizutani A. Terasawa M. Hidaka H. J. Biol. Chem. 1990; 265: 4315-4320Abstract Full Text PDF PubMed Google Scholar). KN-62 (1 μM) had no effect on 8-Br-cAMP stimulation of CRE-mediated transcription, whereas it completely abrogated the stimulatory effect of membrane depolarization (not shown). Therefore, all these data suggest that a Ca2+-dependent enzyme other than CaM kinase is involved in cAMP-induced transcription through the CRE. Cyclosporin A and FK506 are inhibitors of the Ca2+/calmodulin-dependent protein phosphatase calcineurin (33Liu J. Trends Pharmacol. Sci. 1993; 14: 182-188Abstract Full Text PDF PubMed Scopus (108) Google Scholar). Fig. 2 shows the effects of cyclosporin A and FK506 on gene transcription stimulated by cAMP through the CRE. Both drugs inhibited cAMP-induced transcription (Fig. 2); in parallel experiments they also inhibited Ca2+-induced transcription (Fig. 2) as reported previously (17Schwaninger M. Blume R. Oetjen E. Lux G. Knepel W. J. Biol. Chem. 1993; 268: 23111-23115Abstract Full Text PDF PubMed Google Scholar). The effect was concentration-dependent. The IC50 for cyclosporin A was about 60 n M or 15 n M after 8-Br-cAMP or KCl stimulation, respectively, for FK506 about 1 n M after both stimuli. The maximal inhibition by cyclosporin A or FK506 was about 60-70% after stimulation with 8-Br-cAMP and about 80% after stimulation with KCl. The effective concentrations are consistent with the reported affinities of both drugs to their immunophilin receptors and are similar to those concentrations effective in T cells (25Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1473) Google Scholar, 34O'Keefe S.J. Tamura J. Kincaid R.L. Tocci M.J. O'Neill E.A. Nature. 1992; 357: 692-694Crossref PubMed Scopus (788) Google Scholar, 35Dumont F.J. Melino M.R. Staruch M.J. Koprak S.L. Fischer P.A. Sigal N.H. J. Immunol. 1990; 144: 1418-1424PubMed Google Scholar). Cyclosporin A also reduced transcription stimulated by forskolin through the well characterized CREs of the rat somatostatin or the human choriogonadotropin α genes (Fig. 3 A). The somatostatin, choriogonadotropin α, and glucagon gene CREs contain the CRE octamer consensus sequence but differ in the bases flanking the CRE octamer. These data thus indicate that susceptibility to cyclosporin A and FK506 is conferred by the CRE octamer and does not depend on the sequence context. Although a number of transcription factors can bind to the CRE consensus sequence, only CREB and the highly homologous factors ATF-1 and CREMτ are known to mediate cAMP responsiveness (5Meyer T.E. Habener J.F. Endocr. Rev. 1993; 14: 269-290PubMed Google Scholar). The notion that cyclosporin A and FK506 interfere with the action of CREB or a homologous factor is supported by the observation that overexpression of a dominant repressor of CREB, KCREB (26Walton K.M. Rehfuss R.P. Chrivia J.C. Lochner J.E. Goodman R.H. Mol. Endocrinol. 1992; 6: 647-655PubMed Google Scholar), inhibited the cyclosporin A-sensitive transcription stimulated by forskolin through the somatostatin CRE (Fig. 3 B). The stimulation by cAMP of transcription mediated by a GAL4-CREB fusion protein, which contains the CREB transactivation domain (12Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar), was inhibited by cyclosporin A (Fig. 3 B), indicating that CREB is responsive to cyclosporin A. Ca2+and cAMP stimulate CRE-mediated transcription synergistically (12Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar). While CRE-mediated transcription was stimulated by 8-Br-cAMP 8.3-fold and by KCl 3.7-fold, the combination of both induced a 38.8-fold increase in gene transcription (Fig. 4). This synergism was almost completely inhibited by cyclosporin A (Fig. 4). Thus, the synergism, like the effect of the individual stimuli, is sensitive to inhibition by cyclosporin A. To test whether cyclosporin A and FK506 are effective inhibitors of calcineurin phosphatase activity in HIT cells, calcineurin activity was measured. Untreated cells had a calcineurin activity of 127.3 ± 8.4 pmol of phosphate released min−1mg−1(n = 7). Cyclosporin A and FK506 inhibited calcineurin phosphatase activity in HIT cells in a concentration-dependent manner (Fig. 5). The IC50 values for calcineurin inhibition were about 3 and 40 n M for FK506 and cyclosporin A, respectively. Thus, at concentrations that inhibited CRE-directed transcription, cyclosporin A and FK506 also inhibited calcineurin phosphatase activity in HIT cells. To test the relationship between the inhibition of calcineurin activity and CRE-directed transcription stimulated by cAMP, rapamycin and calcineurin expression vectors were used. Rapamycin is an analogue of FK506. Rapamycin binds to the cytosolic FK506 receptor, the FK506-binding protein FKBP-12, but in contrast to the FK506/FKBP-12 complex, the rapamycin/FKBP-12 complex does not inhibit calcineurin, resulting in a competitive antagonism by rapamycin of FK506 action with respect to calcineurin inhibition (33Liu J. Trends Pharmacol. Sci. 1993; 14: 182-188Abstract Full Text PDF PubMed Scopus (108) Google Scholar). Rapamycin does not, however, bind to cyclophilins, the intracellular receptors of cyclosporin A (33Liu J. Trends Pharmacol. Sci. 1993; 14: 182-188Abstract Full Text PDF PubMed Scopus (108) Google Scholar). Rapamycin alone had no effect on calcineurin activity in HIT cells (data not shown). Inhibition by cyclosporin A (60 n M) of calcineurin activity was not reduced by rapamycin (1.1 μM); it was even somewhat enhanced (Fig. 6 A). In contrast, inhibition of calcineurin activity by FK506 (5 n M) was completely reversed by rapamycin (Fig. 6 A). This indicates that in HIT cells as well, inhibition by FK506 of calcineurin depends on a FKBP that binds FK506 and rapamycin. Rapamycin had no effect on gene transcription stimulated by cAMP through the CRE (data not shown) and did not prevent its inhibition by cyclosporin A (Fig. 6 B); it was even somewhat enhanced (Fig. 6 B). In contrast, the inhibition by FK506 was disinhibited by rapamycin (Fig. 6 B). Thus, disinhibition of calcineurin activity was accompanied by disinhibition of CRE-mediated transcription. If inhibition of calcineurin activity is indeed the mechanism by which cyclosporin A and FK506 inhibit CRE-directed transcription after stimulation by cAMP, then overexpression of calcineurin should render HIT cells more resistant to the action of these compounds. Fig. 7 shows the effect of overexpression of the subunits A and B of murine calcineurin (24Kincaid R.L. Giri P.R. Higuchi S. Tamura J. Dixon S.C. Marietta C.A. Amorese D.A. Martin B.M. J. Biol. Chem. 1990; 265: 11312-11319Abstract Full Text PDF PubMed Google Scholar, 25Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1473) Google Scholar) on gene transcription stimulated by cAMP through the CRE. Cotransfection of the expression vectors for calcineurin had no effect on basal transcription (not shown) and increased 8-Br-cAMP-induced gene transcription only slightly (Fig. 7). However, after overexpression of calcineurin FK506 (1.6 n M) or cyclosporin A (60 n M) no longer inhibited the 8-Br-cAMP-induced increase in gene transcription (Fig. 7). These results suggest that cyclosporin A and FK506 inhibit cAMP-induced transcription through inhibition of calcineurin, implying that gene transcription stimulated by cAMP through the CRE depends on calcineurin phosphatase activity in HIT cells. The cAMP and Ca2+signaling pathways converge on serine 119 of CREB-327 (corresponding to serine 133 in CREB-341), which is phosphorylated in response to both signals initiating an increased transcriptional activity. Since cyclosporin A and FK506 inhibit CRE-directed transcription stimulated by Ca2+(17Schwaninger M. Blume R. Oetjen E. Lux G. Knepel W. J. Biol. Chem. 1993; 268: 23111-23115Abstract Full Text PDF PubMed Google Scholar) and cAMP (this study), it was tested whether cyclosporin A and FK506 change the phosphorylation of CREB at serine 119. The phosphorylation of CREB at serine 119 was investigated by immunoblots of HIT cell lysates using an antibody that recognizes CREB phosphorylated at serine 119 but fails to recognize CREB that is not phosphorylated at serine 119 (30Ginty D.D. Kornhauser J.M. Thompson M.A. Bading H. Mayo K.E. Takahashi J.S. Greenberg M.E. Science. 1993; 260: 238-241Crossref PubMed Scopus (743) Google Scholar). After treatment of HIT cells with forskolin or KCl, a band of 43 kDa comigrating with CREB as stained by another anti-CREB antiserum and a band of 38 kDa were increased in intensity (Fig. 8). The 38-kDa protein could correspond to ATF-1 (36Liu F. Thompson M.A. Wagner S. Greenberg M.E. Green M.R. J. Biol. Chem. 1993; 268: 6714-6720Abstract Full Text PDF PubMed Google Scholar), which is highly homologous to CREB and seems to cross-react with the anti-phosphoCREB antiserum (30Ginty D.D. Kornhauser J.M. Thompson M.A. Bading H. Mayo K.E. Takahashi J.S. Greenberg M.E. Science. 1993; 260: 238-241Crossref PubMed Scopus (743) Google Scholar). Pretreatment of HIT cells with cyclosporin A and FK506 did not change the intensities of the bands corresponding to phosphoCREB or phospho-p38 after stimulation by cAMP as well as Ca2+(Fig. 8). Therefore, inhibition of calcineurin by cyclosporin A and FK506 interferes with CRE-mediated transcription without reducing cAMP- or Ca2+-induced CREB phosphorylation at serine 119. To determine if the requirement for calcineurin occurs in cell types other than HIT cells, JEG-3 and GH3cells were tested; these have been used for analysis of cAMP-dependent activation of CREB (3Hoeffler J.P. Meyer T.E. Yun Y. Jameson J.L. Habener J.F. Science. 1988; 242: 1430-1433Crossref PubMed Scopus (525) Google Scholar, 5Meyer T.E. Habener J.F. Endocr. Rev. 1993; 14: 269-290PubMed Google Scholar, 37Drust D.S. Troccoli N.M. Jameson J.L. Mol. Endocrinol. 1991; 5: 1541-1551Crossref PubMed Scopus (40) Google Scholar). In JEG-3 cells, FK506 severely inhibited calcineurin phosphatase activity whereas CRE-directed transcription induced by cAMP was not changed by FK506 (Fig. 9). EGTA (3 m M), added 1 h before cAMP stimulation, did also not change cAMP-induced transcription (not shown). In GH3cells, FK506 inhibited calcineurin phosphatase activity and severely blunted CRE-mediated transcription induced by cAMP (Fig. 9). EGTA treatment (3 m M) reduced transcription by about 80% (not shown). Consistent with other experiments in which additional cell lines were tested, 2M. Krüger, M. Schwaninger, and W. Knepel, unpublished results. these data indicate that, under the experimental conditions used, transcription stimulated by cAMP through CREs depends on calcineurin phosphatase activity in some but not all cell types. This study shows that cyclosporin A and FK506 inhibit cAMP-induced transcription mediated by the CRE in a pancreatic islet cell line. At the same concentrations, these structurally unrelated agents also inhibit calcineurin phosphatase activity. Furthermore, when inhibition of calcineurin by FK506 or cyclosporin A was reduced by rapamycin or overexpression of calcineurin, CRE-dependent transcription was disinhibited. These data strongly suggest that cyclosporin A and FK506 inhibit CRE-directed transcription after stimulation by cAMP through inhibition of calcineurin phosphatase activity. This implies that after stimulation by cAMP, CRE-directed transcription depends not only on protein kinase A but also on calcineurin phosphatase activity. Together with previous evidence that also after membrane depolarization and Ca2+influx transcription mediated through the CRE depends on calcineurin (17Schwaninger M. Blume R. Oetjen E. Lux G. Knepel W. J. Biol. Chem. 1993; 268: 23111-23115Abstract Full Text PDF PubMed Google Scholar), this suggests a central role for calcineurin phosphatase activity in CRE-mediated transcription in pancreatic islet cells. The cAMP- and Ca2+-induced signaling pathways converge on the transcription factor CREB, phosphorylation of CREB at serine 119 by cAMP- or Ca2+/calmodulin-dependent kinases being the initial event of CREB activation (9Sheng M. McFadden G. Greenberg M.E. Neuron. 1990; 4: 571-582Abstract Full Text PDF PubMed Scopus (878) Google Scholar, 10Sheng M. Thompson M.A. Greenberg M.E. Science. 1991; 252: 1427-1430Crossref PubMed Scopus (1282) Google Scholar). Activation of CREB is thought to involve an increase in transcriptional activity (5Meyer T.E. Habener J.F. Endocr. Rev. 1993; 14: 269-290PubMed Google Scholar) and possibly DNA binding (2Hagiwara M. Brindle P. Harootunian A. Armstrong R. Rivier J. Vale W. Tsien R. Montminy M.R. Mol. Cell. Biol. 1993; 13: 4852-4859Crossref PubMed Scopus (379) Google Scholar, 38Nichols M. Weih F. Schmid W. DeVack C. Kowenz-Leutz E. Luckow B. Boshart M. Schütz G. EMBO J. 1992; 11: 3337-3346Crossref PubMed Scopus (276) Google Scholar). Although the mechanism by which phosphorylation at serine 119 stimulates CREB transcriptional activity remains elusive, this modification may stimulate interaction with one or more of the general transcription factors or, alternatively, allow recruitment of a co-activator (39Chrivia J.C. Kwok R.P.S. Lamb N. Hagiwara M. Montminy M.R. Goodman R.H. Nature. 1993; 365: 855-859Crossref PubMed Scopus (1766) Google Scholar, 40Ferreri K. Gill G. Montminy M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1210-1213Crossref PubMed Scopus (164) Google Scholar, 41Quinn P.G. J. Biol. Chem. 1993; 268: 16999-17009Abstract Full Text PDF PubMed Google Scholar). To date, it has not been possible to determine whether the single phosphorylation of CREB at serine 119 is sufficient for CREB activation. Based on the finding that recombinant CREB phosphorylated in vitro on serine 119 was transcriptionally active upon its microinjection into fibroblast nuclei, it was suggested that phosphorylation of CREB at serine 119 is sufficient to stimulate CRE-dependent transcription in intact cells (42Alberts A.S. Arias J. Hagiwara M. Montminy M.R. Feramisco J.R. J. Biol. Chem. 1994; 269: 7623-7630Abstract Full Text PDF PubMed Google Scholar). In the present experiments, cAMP- and Ca2+-induced CREB phosphorylation at serine 119 was unimpaired in the presence of cyclosporin A and FK506, while CRE-dependent transcription was largely reduced. This provides direct evidence for the notion that under these conditions the phosphorylation of CREB at serine 119 is not sufficient for activity. Furthermore, since cyclosporin A and FK506 seem to act through inhibition of a phosphatase (calcineurin), these results suggest the existence of an additional phosphorylation event that directly or indirectly interferes with the transcriptional competence of serine 119-phosphorylated CREB; this inhibitory phosphorylation can be removed by calcineurin phosphatase activity. The nature of the kinase involved is unclear. Its activity may be constitutive or regulated by stimuli including cAMP and Ca2+. Its substrate could be any protein that directly or indirectly can inhibit transactivation by CREB phosphorylated on serine 119. Serine 119 in CREB is flanked by multiple consensus sites for potential phosphorylation by protein kinase C, casein kinase II, and glycogen synthase kinase-3 (5Meyer T.E. Habener J.F. Endocr. Rev. 1993; 14: 269-290PubMed Google Scholar) within a region of CREB that is required for interaction with CBP (39Chrivia J.C. Kwok R.P.S. Lamb N. Hagiwara M. Montminy M.R. Goodman R.H. Nature. 1993; 365: 855-859Crossref PubMed Scopus (1766) Google Scholar), a potential CREB co-activator that together with a dTAFII 110-like protein (40Ferreri K. Gill G. Montminy M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1210-1213Crossref PubMed Scopus (164) Google Scholar) may be required for the interaction of serine 119-phosphorylated CREB with the TFIID complex. Although the functional significance of these phosphorylation sites has not yet been demonstrated, mutation of serine 128 of CREB to alanine (CREB-327 corresponding to serine 142 in CREB-341) in overexpressed GAL4-CREB fusion proteins enhanced transcriptional activation in response to CaM kinase II1–290 (43Sun P. Enslen H. Myung P.S. Maurer R.A. Genes & Dev. 1994; 8: 2527-2539Crossref PubMed Scopus (646) Google Scholar), suggesting that phosphorylation of CREB at serine 128 may inhibit its transcriptional activity. Furthermore, activation by protein kinase C through phorbol esters in hepatoma cells reduced the occupancy of the CRE in the tyrosine aminotransferase gene in vivo (44Reik A. Stewart A.F. Schütz G. Mol. Endocrinol. 1994; 8: 490-497PubMed Google Scholar), and overexpression of p34 kinase, which also can phosphorylate CREB in vitro, repressed transactivation by CREB in JEG choriocarcinoma cells (45de Groot R.P. Derua R. Goris J. Sassone-Corsi P. Mol. Endocrinol. 1993; 7: 1495-1501PubMed Google Scholar). Characterization of the calcineurin-sensitive phosphorylation event that, as suggested by the present study, interferes with the transcriptional competence of serine 119-phosphorylated CREB may add to the understanding of the transactivating mechanism of CREB. The implied inhibitory phosphorylation offers another mechanism in addition to serine 119 phosphorylation through which CREB/CRE-dependent transcription could be regulated according to functional, developmental, and cell-specific cues. Consistent with this view, induction of CRE-directed transcription by cAMP was found by this study and additional experiments2to depend on calcineurin phosphatase activity in some but not all cell types, under the experimental conditions used. Calcineurin is a Ca2+/calmodulin-dependent enzyme (33Liu J. Trends Pharmacol. Sci. 1993; 14: 182-188Abstract Full Text PDF PubMed Scopus (108) Google Scholar). Consistent with a central role for calcineurin, the Ca2+-chelating agent EGTA reduced the stimulation by cAMP of CRE-dependent transcription in HIT cells. EGTA treatment prevents influx of extracellular Ca2+and also lowers basal cytosolic free Ca2+concentration in these cells (46Rajan A.S. Hill R.S. Boyd III, A.E. Diabetes. 1989; 38: 874-880Crossref PubMed Scopus (47) Google Scholar). The effect of verapamil suggests that part of the Ca2+needed for cAMP-induced transcription enters the cell through L-type voltage-dependent Ca2+channels. This may be accounted for by the stimulation by cAMP of Ca2+influx through L-type voltage-dependent Ca2+channels (46Rajan A.S. Hill R.S. Boyd III, A.E. Diabetes. 1989; 38: 874-880Crossref PubMed Scopus (47) Google Scholar, 47Li G. Hidaka H. Wollheim C.B. Mol. Pharmacol. 1992; 42: 489-498PubMed Google Scholar). As the inhibition by EGTA or cyclosporin A and FK506 exceeds that by verapamil, Ca2+from other sources may participate (48Satin L.S. Cook D.L. Pflügers Arch. 1988; 411: 401-409Crossref PubMed Scopus (54) Google Scholar, 49Sussman K.E. Leitner J.W. Draznin B. Diabetes. 1987; 36: 571-577Crossref PubMed Google Scholar, 50Baldari C.T. Macchia G. Heguy A. Melli M. Telford J.L. J. Biol. Chem. 1991; 266: 19103-19108Abstract Full Text PDF PubMed Google Scholar). Cyclosporin A and FK506 are of great clinical importance as powerful immunosuppressive drugs. Through inhibition of calcineurin, these drugs interfere with the nuclear translocation of a subunit of the T-cell-specific transcription factor NF-AT, which enhances the transcription of genes essential for T-cell-activation, such as the interleukin-2 gene (25Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1473) Google Scholar, 34O'Keefe S.J. Tamura J. Kincaid R.L. Tocci M.J. O'Neill E.A. Nature. 1992; 357: 692-694Crossref PubMed Scopus (788) Google Scholar). This may explain part of cyclosporin A/FK506-mediated immunosuppression (33Liu J. Trends Pharmacol. Sci. 1993; 14: 182-188Abstract Full Text PDF PubMed Scopus (108) Google Scholar). The functional disruption of CREB/CRE-directed transcription at concentrations that block T-cell activation represents a novel mechanism of cyclosporin A/FK506 action, which may underlie some of their pharmacological effects, both desired and undesired. We appreciate the following generous gifts: calcineurin expression vectors from Dr. Gerald R. Crabtree, Stanford; expression vector for KCREB from Dr. Richard H. Goodman, Oregon; anti-phosphoCREB antibody from Dr. Michael Greenberg, Boston; KN-62 from Dr. Hiroyoshi Hidaka, Nagoya. Insulin was measured by Dr. Arnold Hasselblatt, Göttingen. Cyclosporin A was provided by Sandoz, FK506 by Fujisawa, rapamycin by Wyeth-Ayerst, and verapamil by Knoll." @default.
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