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• W2012356167 abstract "We examined the mechanism of atrial natriuretic factor (ANF) transcription by isoproterenol (ISO), an agonist for the β-adrenergic receptor (βAR), in cardiac myocytes. ISO only modestly activated members of the mitogen-activated protein kinase family. ISO-induced ANF transcription was not affected by inhibition of mitogen-activated protein kinases, whereas it was significantly inhibited by KN93, an inhibitor of Ca2+/calmodulin-dependent kinase (CaM kinase II). Production of 3′-phosphorylated phosphatidylinositides (3 phosphoinositides) was also required for ISO-induced ANF transcription. ISO caused phosphorylation (Ser-473) and activation of Akt through CaM kinase II- and 3 phosphoinositides-dependent mechanisms. Constitutively active Akt increased myocyte surface area, total protein content, and ANF expression, whereas dominant negative Akt blocked ISO-stimulated ANF transcription. ISO caused Ser-9 phosphorylation and decreased activities of GSK3β. Overexpression of GSK3β inhibited ANF transcription, which was reversed by ISO. ISO failed to reverse the inhibitory effect of GSK3β(S9A), an Akt-insensitive mutant. Kinase-inactive GSK3β increased ANF transcription. Cyclosporin A partially inhibited ISO-stimulated ANF transcription, indicating that calcineurin only partially mediates ANF transcription. These results suggest that both CaM kinase II and 3 phosphoinositides mediate βAR-induced Akt activation and ANF transcription in cardiac myocytes. Furthermore, βAR-stimulated ANF transcription is predominantly mediated by activation of Akt and subsequent phosphorylation/inhibition of GSK3β. We examined the mechanism of atrial natriuretic factor (ANF) transcription by isoproterenol (ISO), an agonist for the β-adrenergic receptor (βAR), in cardiac myocytes. ISO only modestly activated members of the mitogen-activated protein kinase family. ISO-induced ANF transcription was not affected by inhibition of mitogen-activated protein kinases, whereas it was significantly inhibited by KN93, an inhibitor of Ca2+/calmodulin-dependent kinase (CaM kinase II). Production of 3′-phosphorylated phosphatidylinositides (3 phosphoinositides) was also required for ISO-induced ANF transcription. ISO caused phosphorylation (Ser-473) and activation of Akt through CaM kinase II- and 3 phosphoinositides-dependent mechanisms. Constitutively active Akt increased myocyte surface area, total protein content, and ANF expression, whereas dominant negative Akt blocked ISO-stimulated ANF transcription. ISO caused Ser-9 phosphorylation and decreased activities of GSK3β. Overexpression of GSK3β inhibited ANF transcription, which was reversed by ISO. ISO failed to reverse the inhibitory effect of GSK3β(S9A), an Akt-insensitive mutant. Kinase-inactive GSK3β increased ANF transcription. Cyclosporin A partially inhibited ISO-stimulated ANF transcription, indicating that calcineurin only partially mediates ANF transcription. These results suggest that both CaM kinase II and 3 phosphoinositides mediate βAR-induced Akt activation and ANF transcription in cardiac myocytes. Furthermore, βAR-stimulated ANF transcription is predominantly mediated by activation of Akt and subsequent phosphorylation/inhibition of GSK3β. adrenergic receptor protein kinase A atrial natriuretic factor luciferase hemagglutinin phenylephrine c-Jun N-terminal kinase isoproterenol mitogen-activated protein MAP kinase extracellular-regulated kinase 3′-phosphorylated phosphatidylinositides phosphatidylinositol 3-kinase glycogen synthase kinase 3 Ca2+/calmodulin-dependent protein kinase β-galactosidase 4-(2-aminoethyl)benzenesulfonyl fluoride The development of cardiac hypertrophy is a complex process mediated by mechanical forces, neurotransmitters, and hormonal factors (1.Chien K.R. Knowlton K.U. Zhu H. Chien S. FASEB J. 1991; 5: 3037-3046Crossref PubMed Scopus (692) Google Scholar, 2.Sadoshima J. Izumo S. Annu. Rev. Physiol. 1997; 59: 551-571Crossref PubMed Scopus (712) Google Scholar). In vivo and in vitro studies have shown that stimulation of myocardial β-adrenergic receptors (βARs)1 results in cardiac hypertrophy characterized by increases in the cell size, reexpression of the “fetal gene” program (atrial natriuretic factor (ANF) and skeletal α-actin), and organization of actin cytoskeleton (3.Boluyt M.O. Long X. Eschenhagen T. Mende U. Schmitz W. Crow M.T. Lakatta E.G. Am. J. Physiol. 1995; 269: H638-H647PubMed Google Scholar, 4.Iwaki K. Sukhatme V.P. Shubeita H.E. Chien K.R. J. Biol. Chem. 1990; 265: 13809-13817Abstract Full Text PDF PubMed Google Scholar, 5.Bogoyevitch M.A. Andersson M.B. Gillespie-Brown J. Clerk A. Glennon P.E. Fuller S.J. Sugden P.H. Biochem. J. 1996; 314: 115-121Crossref PubMed Scopus (158) Google Scholar, 6.Pinson A. Schler K. Zhou X. Schwartz P. Kessler-Icekson G. Piper H. J. Mol. Cell Cardiol. 1993; 25: 477-490Abstract Full Text PDF PubMed Scopus (74) Google Scholar, 7.Yamazaki T. Komuro I. Zou Y. Kudoh S. Shiojima I. Hiroi Y. Mizuno T. Aikawa R. Takano H. Yazaki Y. Circulation. 1997; 95: 1260-1268Crossref PubMed Scopus (130) Google Scholar, 8.Zou Y. Komuro I. Yamazaki T. Kudoh S. Uozumi H. Kadowaki T. Yazaki Y. J. Biol. Chem. 1999; 274: 9760-9770Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). 2C. Morisco, D. C. Zebrowski, D. E. Vatner, S. F. Vatner, and J. Sadoshima, submitted for publication. 2C. Morisco, D. C. Zebrowski, D. E. Vatner, S. F. Vatner, and J. Sadoshima, submitted for publication. The phenotype of cardiac hypertrophy stimulated by Gs-coupled βAR is similar, if not identical, to that by agonists for Gqα-coupled receptors, such as angiotensin II, phenylephrine (PE), and endothelin-1,2despite the fact that the intracellular signaling mechanism activated by Gsα and that by Gqα are substantially different. We have shown that increases in the protein content and transcription of ANF induced by βAR stimulation are predominantly mediated by the β1AR subtype and that both Gsα and Gβγ in concert mediate βAR-stimulated ANF transcription.2 The intracellular signaling mechanism remains unclear, however, as to how stimulation of the β1AR causes cardiac hypertrophy. Many protein kinases and protein phosphatases are activated by hypertrophic stimuli in the heart, and a series of protein phosphorylation and de-phosphorylation events are responsible for induction of specific phenotypes in cardiac hypertrophy. Among them, growing lines of evidence suggest that members of the MAP kinase family, including ERKs, JNKs, and p38-MAPK, play an important role in formation of cardiac hypertrophy caused by many stimuli (10.Sugden P.H. Clerk A. Circ. Res. 1998; 83: 345-352Crossref PubMed Scopus (439) Google Scholar, 11.Ramirez M.T. Sah V.P. Zhao X.-L. Hunter J.J. Chien K.R. Brown J.H. J. Biol. Chem. 1997; 272: 14057-14061Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 12.Force T. Pombo C.M. Avruch J.A. Bonventre J.V. Kyriakis J.M. Circ. Res. 1996; 78: 947-953Crossref PubMed Scopus (187) Google Scholar, 13.Zechner D. Thuerauf D.J. Hanford D.S. McDonough P.M. Glembotski C.C. J. Cell Biol. 1997; 139: 115-127Crossref PubMed Scopus (278) Google Scholar, 14.Wang Y. Huang S. Sah V.P. Ross Jr., J. Heller-Brown J. Harr J. Chien K.R. J. Biol. Chem. 1998; 273: 2161-2168Abstract Full Text Full Text PDF PubMed Scopus (742) Google Scholar, 15.Choukroun G. Hajjar R. Fry S. del Monte F. Haq S. Guerrero J.L. Picard M. Rosenzweig A. Force T. J. Clin. Invest. 1999; 104: 391-398Crossref PubMed Scopus (152) Google Scholar, 16.Nemoto S. Sheng Z. Lin A. Mol. Cell. Biol. 1998; 18: 3518-3526Crossref PubMed Scopus (209) Google Scholar, 17.Aoki H. Richmond M. Izumo S. Sadoshima J. Biochem. J. 2000; 347: 275-284Crossref PubMed Scopus (98) Google Scholar). Another recently emerged mechanism mediating cardiac hypertrophy involves calcineurin, a Ca2+-activated protein phosphatase (18.Olson E.N. Molkentin J.D. Circ. Res. 1999; 84: 623-632Crossref PubMed Scopus (103) Google Scholar, 19.Molkentin J.D. Lu J.-R. Antons C.L. Markham B. Richardson J. Robbins J. Grant S.R. Olson E.N. Cell. 1998; 93: 215-228Abstract Full Text Full Text PDF PubMed Scopus (2182) Google Scholar). It is unclear at present, however, whether these previously well studied signaling mechanisms play a major role in βAR-stimulated cardiac hypertrophy. By contrast with the wealth of knowledge as to the role of MAP kinases, the role of signaling molecules regulated by 3′-phosphorylated phosphatidylinositides (3 phosphoinositides) in cardiac hypertrophy has been less well understood. Akt (also called as protein kinase B) is a serine/threonine protein kinase activated by many growth factors, including platelet-derived growth factor and insulin (reviewed in Refs.20.Franke T.F. Kaplan D.R. Cantley L.C. Cell. 1997; 88: 435-437Abstract Full Text Full Text PDF PubMed Scopus (1510) Google Scholar and 21.Downward J. Curr. Opin. Cell Biol. 1998; 10: 262-267Crossref PubMed Scopus (1178) Google Scholar), and the activation of which is, in many cases, initiated by binding of 3 phosphoinositides to its pleckstrin homology domain, translocation from the cytoplasm to the plasma membrane, and subsequent phosphorylation by upstream kinases, including PDK1 (22.Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1290) Google Scholar). Akt phosphorylates various intracellular substrates, thereby affecting metabolism (23.Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4292) Google Scholar), protein synthesis (24.Burgering B.M. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1866) Google Scholar, 25.Gingras A.C. Kennedy S.G. O'Leary M.A. Sonenberg N. Hay N. Genes Dev. 1998; 12: 502-513Crossref PubMed Scopus (721) Google Scholar), and cell survival/apoptosis (26.Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4895) Google Scholar, 27.Cardone M.H. Roy N. Stennicke H.R. Salvesen G.S. Franke T.F. Stanbridge E. Frisch S. Reed J.C. Science. 1998; 282: 1318-1321Crossref PubMed Scopus (2704) Google Scholar) (reviewed in Ref. 28.Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3693) Google Scholar). Akt also regulates nuclear transcription factors (29.Brennan P. Babbage J.W. Burgering B.M. Groner B. Reif K. Cantrell D.A. Immunity. 1997; 7: 679-689Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 30.Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5333) Google Scholar, 31.Biggs 3rd, W.H. Meisenhelder J. Hunter T. Cavenee W.K. Arden K.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7421-7426Crossref PubMed Scopus (936) Google Scholar, 32.Du K. Montminy M. J. Biol. Chem. 1998; 273: 32377-32379Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar, 33.Kane L.P. Shapiro V.S. Stokoe D. Weiss A. Curr. Biol. 1999; 9: 601-604Abstract Full Text Full Text PDF PubMed Scopus (726) Google Scholar), thereby controlling gene expression (reviewed in Ref. 21.Downward J. Curr. Opin. Cell Biol. 1998; 10: 262-267Crossref PubMed Scopus (1178) Google Scholar). Among cellular substrates of Akt, glycogen synthase kinase 3 (GSK3) is a ubiquitously expressed serine/threonine kinase the activity of which is inhibited by Akt (23.Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4292) Google Scholar). GSK3 phosphorylates a range of substrates, including glycogen synthase, β-catenin (34.Brown J.D. Moon R.T. Curr. Opin. Cell. Biol. 1998; 10: 182-187Crossref PubMed Scopus (98) Google Scholar), several transcriptional factors (reviewed in Ref. 35.Beals C.R. Sheridan C.M. Turck C.W. Gardner P. Crabtree G.R. Science. 1997; 275: 1930-1934Crossref PubMed Scopus (633) Google Scholar), a translation initiation factor (36.Welsh G.I. Miyamoto S. Price N.T. Safer B. Proud C.G. J. Biol. Chem. 1996; 271: 11410-11413Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), and a cell cycle regulator (37.Diehl J.A. Cheng M. Roussel M.F. Sherr C.J. Genes Dev. 1998; 12: 3499-3511Crossref PubMed Scopus (1837) Google Scholar). Interestingly, GSK3β phosphorylates NF-AT3, thereby causing its nuclear exit in COS cells. By contrast, calcineurin dephosphorylates NF-AT3 and causes its nuclear accumulation, which in turn mediates cardiac hypertrophy (18.Olson E.N. Molkentin J.D. Circ. Res. 1999; 84: 623-632Crossref PubMed Scopus (103) Google Scholar, 19.Molkentin J.D. Lu J.-R. Antons C.L. Markham B. Richardson J. Robbins J. Grant S.R. Olson E.N. Cell. 1998; 93: 215-228Abstract Full Text Full Text PDF PubMed Scopus (2182) Google Scholar). Thus, it is possible that GSK3β may counteract the effect of calcineurin in the heart, and more importantly, Akt-induced inhibition of GSK3β may serve as an alternative mechanism to stimulate transcriptional events promoting cardiac hypertrophy. Little information is available, however, with regard to the role of the Akt-GSK3β pathway in cardiac hypertrophy. The present investigation was conducted to elucidate signaling mechanisms of cardiac hypertrophy induced by βAR stimulation. We studied the mechanism of ANF transcription, because ANF transcription in ventricular myocytes is frequently activated in many types of cardiac hypertrophy, and the result of this investigation may be compared with those of previous studies regarding the mechanism of ANF transcription by stimulation of the Gq-coupled receptors. In particular, we examined the role of MAP kinases, the Akt-GSK3β pathway, and other signaling mechanisms in βAR-mediated ANF transcription. Our results suggest that members of the MAP kinase family, including ERKs, JNKs, and p38-MAPK, do not play a central role in βAR-mediated ANF transcription. By contrast, a signaling molecule activated by both Ca2+/calmodulin-dependent protein kinase (CaM kinase II) and 3 phosphoinositides, namely Akt, plays a critical role in βAR-stimulated ANF transcription. Furthermore, we provide evidence that phosphorylation and subsequent inhibition of GSK3β by Akt is essential for βAR-stimulated ANF transcription. KN93, wortmannin, and LY 290042 were purchased from Biomol. Rapamycin was provided by Wyeth-Ayerst Research. SB203580 was purchased from Calbiochem. Cyclosporin A was from Sigma. Primary cultures of ventricular cardiac myocytes were prepared from 1-day-old Crl:(WI)BR-Wistar rats (Charles River Laboratories) as described previously (38.Aoki H. Izumo S. Sadoshima J. Circ. Res. 1998; 81: 666-676Crossref Scopus (201) Google Scholar). In brief, cardiac myocytes were dispersed from the ventricles by digestion with collagenase type IV(Sigma), 0.1% trypsin (Life Technologies, Inc.), and 15 μg/ml DNase I (Sigma). Cell suspensions were applied on a discontinuous Percoll gradient (1.060/1.086 g/ml) made up in Ads buffer (116 mm NaCl, 20 mm HEPES, 1 mmNaH2PO4, 5.5 mm glucose, 5.4 mm KCl, 0.8 mm MgSO4, pH 7.35) and subjected to centrifugation at 3000 rpm for 30 min (4.Iwaki K. Sukhatme V.P. Shubeita H.E. Chien K.R. J. Biol. Chem. 1990; 265: 13809-13817Abstract Full Text PDF PubMed Google Scholar). Cells were cultured in the cardiac myocyte culture medium containing Dulbecco's modified Eagle's medium/Ham's F-12 medium supplemented with 5% horse serum, 4 μg/ml transferrin, 0.7 ng/ml sodium selenite (Life Technologies, Inc.), 2 g/liter bovine serum albumin (fraction V), 3 mmol/liter pyruvic acid, 15 mmol/liter HEPES, 100 μmol/liter ascorbic acid, 100 μg/ml ampicillin, 5 μg/ml linoleic acid, and 100 μmol/liter 5-bromo-2′-deoxyuridine (Sigma). We obtained myocyte cultures in which more than 95% were myocytes, as assessed by immunofluorescence staining with a monoclonal antibody against sarcomeric myosin (MF20). Culture media were changed to serum-free at 24 h. Myocytes were cultured in serum-free conditions for 48 h before experiments. Plasmids encoding constitutively active MEK1 (MEK1(ΔN3-S218E-S222D)) and dominant negative MEK1 (MEK1(K97M)) were provided by Dr. K.-L. Guan (University of Michigan). Dominant negative MEKK1 (MEKK1(K1257M)), constitutively active MKK3, constitutively active Ras (L61Ras), and dominant negative Ras (N17Ras) were provided by Dr. L. B. Holzman (University of Michigan). Dominant negative p38-MAPK (human CSBP1 (39.Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3113) Google Scholar), T180A, and Y182F) was provided by Dr. Y. Takuwa (Kanazawa University). Plasmid encoding the catalytic subunit of protein kinase A (PKA) was purchased from Stratagene. Plasmids encoding an epitope-tagged Akt (HA-Akt), constitutively active Akt (HA-myr Akt and HA-Akt(E40K)), and dominant negative Akt (HA-Akt(K179M) and HA-CAAX-Akt) have been described (40.Bellacosa A. Chan T.O. Ahmed N.N. Datta K. Malstrom S. Stokoe D. McCormick F. Feng J. Tsichlis P. Oncogene. 1998; 17: 313-325Crossref PubMed Scopus (450) Google Scholar). For some experiments, recombinant adenovirus harboring constitutively active Akt (Ad5·CMV 6-myr-Akt) was used. Adenovirus harboring lacZ (Ad5·CMV-β-galactosidase) was used as control. Plasmids expressing constitutively active PI3K (p110*) and its kinase-deficient mutant (p110*Δkin) (41.Klippel A. Reinhard C. Kavanaugh W.M. Apell G. Escobedo M.A. Williams L.T. Mol. Cell. Biol. 1996; 16: 4117-4127Crossref PubMed Scopus (415) Google Scholar) were provided by Dr. L.T. Williams (Chiron Corporation). GSK3β was amplified by reverse transcription-polymerase chain reaction from the rat skeletal muscle mRNA, subcloned into pCR3.1 (Invitrogen) and the entire open reading frame was sequenced to confirm the correct sequence. Kinase-inactive GSK3β (GSK3βKI, K85M, and K86I) (42.He X. Saint-Jeannet J.P. Woodgett J.R. Varmus H.E. Dawid I.B. Nature. 1995; 374: 617-622Crossref PubMed Scopus (446) Google Scholar) and GSK3β(S9A) were prepared by site-directed mutagenesis using a Quick Change mutagenesis kit from Stratagene. PTEN and PDK1 were amplified by polymerase chain reaction from a human heart cDNA library (CLONTECH) subcloned into pCR3.1, and the entire open reading frame was sequenced. For transient transfection, myocytes were plated at a density of 1 × 106 per well in six-well plates. Twenty-four h after plating, the medium was changed to Dulbecco's modified Eagle's medium/Ham's F-12 medium without supplement. Transfections were carried out using 10 μl/ml of LipofectAMINE (Life Technologies, Inc.) in 1 ml/well Dulbecco's modified Eagle's medium medium. To determine whether the MAP kinases are activated by a given stimulus, the PathDetectTM in vivo signal transduction pathway trans-reporting system (Stratagene) was used, in which a plasmid (1 μg/ml) containing five tandem repeats of the yeast GAL4 binding sites linked to firefly luciferase (pFR-Luc) and another plasmid (50 ng/ml), consisting of the activation domain of a transcription factor, Elk1, c-Jun, ATF2, or CHOP fused with the DNA binding domain of the yeast GAL4, were cotransfected. To determine the activity of the ANF promoter, a plasmid (1 μg/ml) containing a 638- or 3003-base pair fragment of the rat ANF promoter linked to firefly luciferase (ANF-Luc (638), courtesy of Dr. K. R. Chien, University of California, San Diego, CA (4.Iwaki K. Sukhatme V.P. Shubeita H.E. Chien K.R. J. Biol. Chem. 1990; 265: 13809-13817Abstract Full Text PDF PubMed Google Scholar) or ANF-Luc (3003)2) was used. In some experiments, myocytes were co-transfected with various doses of expression plasmid encoding signaling molecules. Total amounts of DNA were adjusted to 2 μg/ml by adding an empty vector. Twenty-four h after the transfection, the culture medium was changed to the cardiac myocyte culture medium without serum. Myocytes were further cultured in the presence or absence of isoproterenol (ISO) (10 μm), PE (10 μm), or fetal bovine serum (20%) for an additional 24 h. Myocytes were then lysed with the Reporter lysis buffer (Promega) and luciferase activities measured. An SV40 promoter-driven β-galactosidase construct (SV40-β-gal, 0.5 μg/ml) was co-transfected, and the β-gal activity was determined by using Lumi-Gal 530 (Lumigen). The luciferase values were divided by the β-gal values to correct for differences in the transfection efficiency. Cardiac myocytes were grown in six-well plates (1 × 106 cells/well). Myocytes were lysed with 100 μl of lysis buffer. For Akt, lysis buffer contained 50 mm HEPES (pH 7.6), 1 mm EDTA, 5 mmEGTA, 10 mm MgCl2, 50 mmβ-glycerophosphate, 1 mm Na3VO4, 10 mm NaF, 30 mm sodium pyrophosphate, 2 mm dithiothreitol, 1 mm AEBSF. For GSK3β, lysis buffer contained 20 mm Tris (pH 7.5), 25 mm β-glycerophosphate, 100 mm NaCl, 1 mm Na3VO4, 2 mm EGTA, 2 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mm AEBSF. Samples were subjected to SDS-PAGE, and transferred onto polyvinylidene difluoride membranes. The membranes were probed with anti-phospho-Akt (Ser-473) (New England BioLabs) or anti-phospho-GSK3β (Ser-9) (Oncogene Research Product) antibodies. Horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies were used as secondary antibodies. The bound secondary antibody was detected by the enhanced chemiluminescence (Amersham Pharmacia Biotech). The activity of Akt and GSK3β was measured by the immune complex kinase assay. For Akt, the cells were lysed with 1 ml of a lysis buffer (1% Nonidet P-40, 10% glycerol, 137 mm NaCl, 20 mmTris (pH 7.4), 1 μg/ml aprotinin, 2 μg/ml leupeptin, 1 mm AEBSF, 20 mm NaF, 1 mmNa3VO4, 30 mm sodium pyrophosphate). Akt was immunoprecipitated with rabbit anti-Akt antibody coupled to protein A-Sepharose (New England BioLabs). After washing the immune complex, kinase activities were assayed with GSK3 fusion protein (GSK3α peptide 7–25 fused to paramyosin) (New England BioLabs) as a substrate, in a reaction buffer, containing 25 mm Tris (pH 7.5), 5 mm β-glycerophosphate, 2 mm dithiothreitol, 0.1 mmNa3VO4, 10 mm MgCl2, 200 μm ATP. The phosphorylation reactions were allowed to proceed for 30 min at 30 °C and then were stopped by adding 20 μl of 3× SDS sample buffer. The samples were loaded on SDS-PAGE gel (15%). The extent of GSK3 phosphorylation caused by immunoprecipitated Akt was determined by Western blot analyses using anti-phospho-GSK3α/β (Ser-21/Ser-9) antibody (New England BioLabs). For GSK3β kinase activity assays, the cells were lysed with 1 ml of lysis buffer (20 mm Tris (pH 7.5), 25 mmβ-glycerophosphate, 100 mm NaCl, 1 mmNa3VO4, 2 mm EGTA, 2 μg/ml leupeptin, 1 μg/ml aprotinin,1 mm AEBSF) (43.Ueki K. Yamamoto-Honda R. Kaburagi Y. Yamauchi T. Tobe K. Burgering B.M. Coffer P.J. Komuro I. Akanuma Y. Yazaki Y. Kadowaki T. J. Biol. Chem. 1998; 273: 5315-5322Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). GSK3β was immunoprecipitated with anti-mouse GSK3β antibody (Calbiochem) coupled to protein G-Sepharose. After washing the immune complex, the kinase activity was assayed by using 1 μg of GS peptide-2 (Upstate Biotechnology) as a substrate in a reaction buffer containing 25 mm β-glycerophosphate, 40 mm HEPES (pH 7.2), 10 mm MgCl2, 1 mm protein kinase inhibitory peptide (rabbit sequence), 50 μm ATP, 5 μCi of [γ-32P]ATP. After 20 min of incubation at 30 °C, 15 μl of radiolabeled peptide was spotted onto Whatman P81 filter papers. Samples were washed five times for 5 min each in a large volume of 180 mm of phosphoric acid and then once for 3 min in 99% ethanol. After drying, the samples were counted using a beta counter. Myocytes were grown on gelatin-coated glass coverslips. Twenty-four h after plating, myocytes were transfected with either HA-Akt (E40K) or HA-myrAkt and cultured for 48 h in serum-free conditions. Myocytes were then fixed with paraformaldehyde (3.6%) for 10 min. Double staining was performed by using affinity purified anti-HA monoclonal antibody (3F10, Roche Molecular Biochemicals) at a 1:200 dilution and anti-ANF polyclonal antibody (Peninsula) at a 1:100 dilution. Rhodamine-conjugated anti-rat IgG and FITC conjugated anti-rabbit IgG were used as secondary antibodies. The myocyte surface area and ANF expression in HA staining-positive myocytes were compared with those in HA-negative myocytes. To determine the myocyte surface area, microscope images (× 400) were captured by a digital camera, and analyses were performed by using Adobe Photoshop. Cardiac myocytes were plated on six-well plates and cultured in a serum-free condition for 48 h before experiments. Myocytes were infected with adenovirus harboring myr Akt or β-gal and further cultured for 48 h. After stimulation, each well was rinsed three times with phosphate-buffered saline. The cell layer was scraped with 1 ml of 1x standard sodium citrate containing 0.25% (w/v) SDS and vortexed extensively. The total cell protein and the DNA content were determined by the Lowry method and the Hoechst dye method, as described previously (44.Sadoshima J. Jahn L. Takahashi T. Kulik T.J. Izumo S. J. Biol. Chem. 1992; 267: 10551-10560Abstract Full Text PDF PubMed Google Scholar). The protein content was normalized by the DNA content to correct for differences in the cell number. Data are given as mean ± S.D. Statistical analyses were performed using the analysis of variance. The posttest comparison was performed by the method of Tukey. Significance was accepted at p < 0.05 level. Because the Ras-MAP kinase (ERKs, JNKs, and p38-MAPK) pathways mediate cardiac hypertrophy initiated by many growth factors (10.Sugden P.H. Clerk A. Circ. Res. 1998; 83: 345-352Crossref PubMed Scopus (439) Google Scholar, 45.Sugden P.H. Clerk A. Adv. Enzyme Regul. 1998; 38: 87-98Crossref PubMed Scopus (26) Google Scholar), we examined whether these mechanisms are critical for ANF transcription by βAR stimulation. We first asked whether stimulation of the βAR activates MAP kinases sufficiently enough to affect nuclear transcription factors. We used sensitive reporter gene assays, which detect transcriptional activities of GAL4-Elk1, c-Jun, ATF2, and CHOP fusion proteins. Activation domains of Elk1, c-Jun, ATF2, and CHOP are phosphorylated by distinct members of the MAP kinase family (Elk1: ERKs, JNKs, and p38-MAPK; c-Jun: JNKs; ATF2: JNKs, p38-MAPK; CHOP: p38-MAPK), which in turn increases their transcriptional activities (46.Karin M. Hunter T. Curr. Biol. 1995; 5: 747-757Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). As expected, these transcription factors were strongly activated by upstream regulators of MAP kinases, including constitutively active Ras, MEKK1, and MKK3, in cardiac myocytes (Fig.1 A). By contrast, stimulation of the βAR by ISO only modestly increased transcriptional activities of GAL4-Elk1 and GAL4-ATF2 and decreased those of GAL4-cJun and GAL4-CHOP (Fig. 1 A). The effect of PE, an agonist for the α1 adrenergic receptor and a hypertrophic stimulus, was also examined. PE potently activated GAL4-Elk1 and GAL4-cJun. PE only modestly stimulated ATF2-GAL4, whereas it slightly decreased GAL4-CHOP. These results suggest that ligand binding to the βAR stimulates some members of the MAP kinase family only modestly to affect activities of the nuclear transcription factors. We have previously shown that stimulation of the βAR increases ANF expression through increases in transcription.2 Selective inhibition of MAP kinases by co-transfection of dominant negative MEK, dominant negative MEKK1, or dominant negative p38-MAPK failed to significantly inhibit βAR-stimulated increases in activities of the ANF promoter, which is determined by using the ANF-Luc reporter gene (Fig. 1 B). SB203580, a specific inhibitor for p38-MAPK/p38β1-MAPK and some isoforms of JNK (47.Clerk A. Sugden P.H. FEBS Lett. 1998; 426: 93-96Crossref PubMed Scopus (205) Google Scholar) also failed to significantly inhibit ISO-induced increases in ANF transcription (10 μm ISO alone, 4.57 ± 1.63; 10 μmSB203580 + 10 μm ISO 4.25 ± 0.81, n= 6). Furthermore, inhibition of Ras, an upstream regulator of MAP kinases by dominant negative Ras (N17Ras) failed to inhibit βAR-stimulated increases in ANF transcription (Fig. 1 B). We have recently shown that dominant negative MEK and N17Ras significantly suppresses ANF transcription stimulated by angiotensin II, whereas dominant negative MEKK1, SB203580, and N17Ras significantly suppress ANF transcription stimulated by phenylephrine in cardiac myocytes (17.Aoki H. Richmond M. Izumo S. Sadoshima J. Biochem. J. 2000; 347: 275-284Crossref PubMed Scopus (98) Google Scholar). These results suggest that the Ras-MAP kinase pathway is not required for increases in ANF transcription by βAR stimulation. It has been shown that CaM kinase II is able to stimulate ANF transcription (48.Ramirez M.T. Zhao X.L. Schulman H. Brown J.H. J. Biol. Chem. 1997; 272: 31203-31208Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Because stimulation of the βAR enhances Ca2+ influx throughl-type Ca2+ channels, we examined whether Ca2+-dependent mechanisms are involved in ANF transcription by βAR stimulation. Both nicardipine, a Ca2+ channel antagonist, and KN93, a specific inhibitor for CaM kinase II (48.Ramirez M.T. Zhao X.L. Schulman H. Brown J.H. J. Biol. Chem. 1997; 272: 31203-31208Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), inhibited increases in ANF transcription by βAR stimulation (Fig. 2 A). Thus, Ca2+-dependent mechanisms play an important role in βAR-stimulated ANF transcription. Because stimulation of the βAR increases cAMP and activates PKA in cardiac myocytes, we tested whether or not stimulation of PKA activates ANF transcription in" @default.
• W2012356167 created "2016-06-24" @default.
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• W2012356167 date "2000-05-01" @default.
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• W2012356167 title "The Akt-Glycogen Synthase Kinase 3β Pathway Regulates Transcription of Atrial Natriuretic Factor Induced by β-Adrenergic Receptor Stimulation in Cardiac Myocytes" @default.
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• W2012356167 doi "https://doi.org/10.1074/jbc.275.19.14466" @default.
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