Cyclic Nucleotides Suppress Tumor Necrosis Factor α-Mediated Apoptosis by Inhibiting Caspase Activation and Cytochrome cRelease in Primary Hepatocytes via a Mechanism Independent of Akt Activation
2000; Elsevier BV; Volume: 275; Issue: 17 Linguagem: Inglês
10.1074/jbc.275.17.13026
ISSN1083-351X
AutoresJianrong Li, Sufang Yang, Timothy R. Billiar,
Tópico(s)Acute Kidney Injury Research
ResumoCyclic nucleotides have been previously shown to modulate cell death processes in many cell types; however, the mechanisms by which cyclic nucleotides regulate apoptosis are unclear. In this study, we demonstrated that cAMP as well as cGMP analogs suppressed tumor necrosis factor α (TNFα) plus actinomycin D (ActD)-induced apoptosis in a dose-dependent manner in cultured primary hepatocytes. Furthermore, forskolin, which increases intracellular cAMP levels, also effectively suppressed TNFα+ActD-induced apoptosis. Activation of multiple caspases was suppressed in cells exposed to TNFα+ActD in the presence of cAMP or cGMP analogs. TNFα+ActD-induced cytochrome c release from mitochondria was also inhibited by cAMP or cGMP, reinforcing our conclusion that cyclic nucleotides interfere with the early signaling events of TNFα-mediated apoptosis. We evaluated the possibility that cAMP and cGMP inhibit apoptosis by activating the serine/threonine kinase Akt, which is known to promote cell survival. Both cAMP- and cGMP-elevating agents led to marked increases in Akt activation that was inhibited by the phosphatidylinositol 3′-kinase inhibitors, LY294002 and wortmannin. However, complete inhibition of cyclic nucleotide-induced Akt activation had little effect on cyclic nucleotide-mediated cell survival, indicating the existence of other survival pathways. Interestingly, the specific inhibitor of protein kinase A (PKA), KT5720, blocked cGMP-mediated protection but only partially prevented the anti-apoptotic effect of cAMP, indicating that both PKA-dependent and -independent mechanisms are involved in cAMP-mediated suppression of apoptosis signaling. Our data suggest that multiple survival signaling pathways coexist in cells and that cyclic nucleotides delay apoptosis by interfering with apoptosis signaling by both PKA-dependent and -independent mechanisms. Cyclic nucleotides have been previously shown to modulate cell death processes in many cell types; however, the mechanisms by which cyclic nucleotides regulate apoptosis are unclear. In this study, we demonstrated that cAMP as well as cGMP analogs suppressed tumor necrosis factor α (TNFα) plus actinomycin D (ActD)-induced apoptosis in a dose-dependent manner in cultured primary hepatocytes. Furthermore, forskolin, which increases intracellular cAMP levels, also effectively suppressed TNFα+ActD-induced apoptosis. Activation of multiple caspases was suppressed in cells exposed to TNFα+ActD in the presence of cAMP or cGMP analogs. TNFα+ActD-induced cytochrome c release from mitochondria was also inhibited by cAMP or cGMP, reinforcing our conclusion that cyclic nucleotides interfere with the early signaling events of TNFα-mediated apoptosis. We evaluated the possibility that cAMP and cGMP inhibit apoptosis by activating the serine/threonine kinase Akt, which is known to promote cell survival. Both cAMP- and cGMP-elevating agents led to marked increases in Akt activation that was inhibited by the phosphatidylinositol 3′-kinase inhibitors, LY294002 and wortmannin. However, complete inhibition of cyclic nucleotide-induced Akt activation had little effect on cyclic nucleotide-mediated cell survival, indicating the existence of other survival pathways. Interestingly, the specific inhibitor of protein kinase A (PKA), KT5720, blocked cGMP-mediated protection but only partially prevented the anti-apoptotic effect of cAMP, indicating that both PKA-dependent and -independent mechanisms are involved in cAMP-mediated suppression of apoptosis signaling. Our data suggest that multiple survival signaling pathways coexist in cells and that cyclic nucleotides delay apoptosis by interfering with apoptosis signaling by both PKA-dependent and -independent mechanisms. tumor necrosis factor-α acetyl- actinomycin D 3-isobutyl-1-methylxanthine nitric oxide p-nitroanilide 8-(4-chlorophenylthio)guanosine-3′,5′-cyclic monophosphate cAMP-dependent protein kinase cGMP-dependent protein kinase phenylmethanesulfonyl fluoride 8-bromoadenosine 3′,5′-cyclic monophosphorothioate, S p isomer benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone phosphatidylinositol 3′-kinase poly(ADP-ribose)polymerase enzyme-linked immunosorbent assay polyacrylamide gel electrophoresis dibutyryl cyclic AMP bromo phosphate-buffered saline protein kinase inhibitor mitogen-activated protein kinase glycogen synthase kinase-3 6-dichloro-1-β-d-ribofuranosylbenzimidazole-3′,5′-cyclic monophosphorothioate, Sp isomer Apoptosis plays fundamental roles in the development of multicellular organisms and the maintenance of homeostasis (reviewed in Refs. 1.Steller H. Science. 1995; 267: 1445-1462Crossref PubMed Scopus (2430) Google Scholar and 2.Jacobson M.D. Weil M. Raff M.C. Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (2409) Google Scholar). Our understanding of the underlying mechanism for apoptosis has advanced greatly. It is now well accepted that activation of a unique family of cysteine proteases named caspases plays a central role in the signaling and execution phase of apoptosis induced by a number of stimuli, including Fas, TNFα,1 TRAIL, radiation, and chemotherapeutic drugs (reviewed in Refs. 3.Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5152) Google Scholar, 4.Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 5.Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6157) Google Scholar). Activation of initiator caspases, such as caspase-8 and caspase-9, is known to transduce apoptotic signal and activate executioner caspases, such as caspase-3. Translocation of cytochrome c from mitochondria to cytoplasm has been viewed by many investigators as an irreversible step in committing cells to apoptotic cell death. We (6.Li J. Billiar T.R. Talanian R.V. Kim Y.M. Biochem. Biophys. Res. Commun. 1997; 240: 419-424Crossref PubMed Scopus (478) Google Scholar, 7.Kim Y.M. Talanian R.V. Billiar T.R. J. Biol. Chem. 1997; 272: 31138-31148Abstract Full Text Full Text PDF PubMed Scopus (798) Google Scholar, 8.Li J. Bombeck C.A. Yang S. Kim Y.M. Billiar T.R. J. Biol. Chem. 1999; 274: 17325-17333Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar) and others (9.Mannick J.B. Hausladen A. Liu L. Hess D.T. Zeng M. Miao Q.X. Kane L.S. Gow A.J. Stamler J.S. Science. 1999; 284: 651-654Crossref PubMed Scopus (704) Google Scholar, 10.Rossig L. Fichtlscherer B. Breitschopf K. Haendeler J. Zeiher A.M. Mulsch A. Dimmeler S. J. Biol. Chem. 1999; 274: 6823-6826Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 11.Tenneti L. D'Emilia D.M. Lipton S.A. Neurosci. Lett. 1997; 236: 139-142Crossref PubMed Scopus (149) Google Scholar, 12.Melino G. Bernassola F. Knight R.A. Corasaniti M.T. Nistico G. Finazzi-Agro A. Nature. 1997; 388: 432-433Crossref PubMed Scopus (375) Google Scholar, 13.Dimmeler S. Haendeler J. Nehls M. Zeiher A.M. J. Exp. Med. 1997; 185: 601-607Crossref PubMed Scopus (786) Google Scholar) previously demonstrated that nitric oxide (NO) prevents apoptosis by inhibiting caspase proteolytic activation as well as by directly suppressing caspase activity. NO is known to activate potently soluble guanylyl cyclase and results in production of intracellular cGMP (14.Murad F. J. Am. Med. Assoc. 1996; 276: 1189-1192Crossref PubMed Google Scholar). In some cell systems, the anti-apoptotic function of NO is mediated, at least partially, by NO-dependent generation of intracellular cGMP. For example, the protection of NO against apoptosis is found to be dependent on production of the secondary messenger cGMP in B lymphocytes (15.Genaro A.M. Hortelano S. Alvarez A. Martinez C. Bosca L. J. Clin. Invest. 1995; 95: 1884-1890Crossref PubMed Scopus (306) Google Scholar), eosinophils (16.Beauvais F. Michel L. Dubertret L. FEBS Lett. 1995; 361: 229-232Crossref PubMed Scopus (122) Google Scholar), embryonic motor neurons (17.Estevez A.G. Spear N. Thompson J.A. Cornwell T.L. Radi R. Barbeito L. Beckman J.S. J. Neurosci. 1998; 18: 3708-3714Crossref PubMed Google Scholar), PC12 cells (18.Farinelli S.E. Park D.S. Greene L.A. J. Neurosci. 1996; 16: 2325-2534Crossref PubMed Google Scholar, 19.Kim Y.M. Chung H.T. Kim S.S. Han J.A. Yoo Y.M. Kim K.M. Lee G.H. Yun H.Y. Green A. Li J. Simmons R.L. Billiar T.R. J. Neurosci. 1999; 19: 6740-6747Crossref PubMed Google Scholar), and ovarian follicles (20.Chun S.Y. Eisenhauer K.M. Kubo M. Hsueh A.J. Endocrinology. 1995; 136: 3120-3127Crossref PubMed Scopus (0) Google Scholar). However, it is unknown how cGMP prevents apoptosis signaling and supports survival. Recently, cAMP-elevating reagents have also been shown to suppress etoposide-, Fas-, or cycloheximide-induced apoptosis in neutrophils (21.Niwa M. Hara A. Kanamori Y. Matsuno H. Kozawa O. Yoshimi N. Mori H. Uematsu T. Eur. J. Pharmacol. 1999; 371: 59-67Crossref PubMed Scopus (23) Google Scholar, 22.Parvathenani L.K. Buescher E.S. Chacon-Cruz E. Beebe S.J. J. Biol. Chem. 1998; 273: 6736-6743Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), promonocytic leukemia cell lines (23.Garcia-Bermejo L. Perez C. Vilaboa N.E. de Blas E. Aller P. J. Cell Sci. 1998; 111: 637-644Crossref PubMed Google Scholar, 24.Jun C.D. Pae H.O. Kwak H.J. Yoo J.C. Choi B.M. Oh C.D. Chun J.S. Paik S.G. Park Y.H. Chung H.T. Cell Immunol. 1999; 194: 36-46Crossref PubMed Scopus (52) Google Scholar), hepatocytes (25.Fladmark K.E. Gjertsen B.T. Doskeland S.O. Vintermyr O.K. Biochem. Biophys. Res. Commun. 1997; 232: 20-25Crossref PubMed Scopus (58) Google Scholar, 26.Webster C.R. Anwer M.S. Hepatology. 1998; 27: 1324-1331Crossref PubMed Scopus (107) Google Scholar), nerve cells (18.Farinelli S.E. Park D.S. Greene L.A. J. Neurosci. 1996; 16: 2325-2534Crossref PubMed Google Scholar), endothelial cells (27.Polte T. Schroder H. Biochem. Biophys. Res. Commun. 1998; 251: 460-465Crossref PubMed Scopus (38) Google Scholar), and smooth muscle cells (28.Orlov S.N. Thorin-Trescases N. Dulin N.O. Dam T.V. Fortuno M.A. Tremblay J. Hamet P. Cell Death Differ. 1999; 6: 661-672Crossref PubMed Scopus (71) Google Scholar). However, the mechanism underlying this cyclic nucleotide-dependent suppression of apoptosis also remains elusive. Activation of phosphatidylinositol 3′-kinase (PI3′K) and its downstream effector Akt by growth factors, cytokines, and certain stress such as heat shock has been demonstrated to transduce signals regulating not only protein synthesis, proliferation, and glycogen metabolism but also cell survival (reviewed in Refs. 29.Hemmings B.A. Science. 1997; 275: 628-630Crossref PubMed Scopus (437) Google Scholar, 30.Alessi D.R. Cohen P. Curr. Opin. Genet. & Dev. 1998; 8: 55-62Crossref PubMed Scopus (675) Google Scholar, 31.Marte B.M. Downward J. Trends Biochem. Sci. 1997; 22: 355-358Abstract Full Text PDF PubMed Scopus (647) Google Scholar). Activation of the serine/threonine kinase Akt (also named protein kinase) has been shown to suppress apoptosis and promote cell survival (32.Crowder R.J. Freeman R.S. J. Neurosci. 1998; 18: 2933-2943Crossref PubMed Google Scholar, 33.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 (4946) Google Scholar, 34.del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1986) Google Scholar, 35.Songyang Z. Baltimore D. Cantley L.C. Kaplan D.R. Franke T.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11345-11350Crossref PubMed Scopus (322) Google Scholar). To date, fourin vivo substrates of Akt have been identified, including glycogen synthase kinase-3 (GSK3, Ref. 36.Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4375) Google Scholar), procaspase-9 (37.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 (2734) Google Scholar), the pro-apoptotic member of the Bcl-2 family, Bad (33.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 (4946) Google Scholar), and most recently the endothelial nitric oxide synthase (38.Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2231) Google Scholar, 39.Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3044) Google Scholar). It has been shown that phosphorylation of Bad by Akt results in dissociation of Bad from Bcl-2 permitting Bcl-2 to prevent apoptosis (32.Crowder R.J. Freeman R.S. J. Neurosci. 1998; 18: 2933-2943Crossref PubMed Google Scholar, 33.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 (4946) Google Scholar). Moreover, cAMP-elevating agents have recently been shown to activate Akt independently of PI3′K in transfected cells (40.Sable C.L. Filippa N. Hemmings B. Van Obberghen E. FEBS Lett. 1997; 409: 253-257Crossref PubMed Scopus (152) Google Scholar); however, the relationship between the anti-apoptotic action of cyclic nucleotides and the Akt pathway remains poorly understood. In this study, we investigated the anti-apoptotic mechanisms of cyclic nucleotides in preventing TNFα plus actinomycin D (TNFα+ActD)-induced apoptosis in cultured primary hepatocytes. Here we report that both cGMP- and cAMP-elevating agents suppressed TNFα+ActD-induced apoptosis via inhibiting activation and processing of caspases, including caspase-3, caspase-8, and caspase-9. This is consistent with the finding that cytochrome c release and DNA fragmentation, events downstream of caspase-8 and caspase-3, respectively, during TNFα+ActD-mediated apoptosis, were significantly inhibited when cells were cotreated with cyclic nucleotide-elevating agents and TNFα+ActD. Interestingly, we also found that both cyclic nucleotides rapidly activate endogenous Akt in cultured hepatocytes in a PI3′K-dependent manner. Inhibition of this Akt activation did not block the protective effect of cAMP or cGMP; however, inhibition of protein kinase A did. Our results indicate the existence of multiple survival pathways in cells and suggest that protein kinase A is involved in modulating the apoptotic signaling cascade by cyclic nucleotides. Williams Medium E, penicillin, streptomycin,l-glutamine, HEPES, and P81 phosphocellulose were purchased from Life Technologies, Inc. Insulin was from Lilly.N-Acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA), caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD-fmk), KT5823, and KT5720 were from Alexis Corp. (San Diego, CA). Stock solution of caspase inhibitor was prepared at 100 mm in dimethyl sulfoxide.N-Acetyl-Ile-Glu-Thr-Asp-p-nitroanilide (Ac-IETD-pNA), caspase-8 inhibitor Z-IETD-fmk, LY294002, and wortmannin were from Calbiochem. [γ-32P]ATP was from NEN Life Science Products. Mouse recombinant TNFα was obtained from R & D Systems (Minneapolis, MN). Antibodies used in this study were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, for caspase-3 H277), Oncogene Research Product (Cambridge, MA, for PARP), Sigma (for β-actin), StressGen (Victoria, British Columbia, Canada, for caspase-8), and New England Biolabs (Beverly, MA, for Akt, phospho-Akt, phospho-GSK, and caspase-9). ECL+PlusTM was from Amersham Pharmacia Biotech, and SupersignalTM chemiluminescence detection reagents were from Pierce. 8-(4-Chlorophenylthio)guanosine-3′,5′-cyclic monophosphate (8-pCPT-cGMP) and 8-bromoadenosine 3′,5′-cyclic monophosphorothioate,S p isomer ((S p)-cAMPS) were from Biolog Life Science Institute (La Jolla, CA). DNA fragmentation ELISA kit was from Roche Molecular Biochemicals. Unless indicated otherwise, all other chemicals were from Sigma. Primary rat hepatocytes were isolated and purified from male Harlan Sprague-Dawley rats and cultured as described previously (41.Stadler J. Bergonia H.A. Di Silvio M. Sweetland M.A. Billiar T.R. Simmons R.L. Lancaster Jr., J.R. Arch. Biochem. Biophys. 1993; 302: 4-11Crossref PubMed Scopus (115) Google Scholar). Highly purified hepatocytes (>98% purity and >95% viability by trypan blue exclusion) were suspended in Williams medium E supplemented with 10% calf serum, 2 mm l-glutamine, 15 mmHEPES, pH 7.4, 100 units/ml penicillin, and 100 μg/ml streptomycin. The cells were plated on collagen-coated tissue culture plates at a density of 2×105 cells/well in 12-well plates for cell viability analysis, 4 × 105 cells/well in 6-well plates for DNA fragmentation ELISA, or 3 × 106cells/60-mm dish for Western blot and enzyme assays. After attachment to the plates for 4 h, cells were rinsed once and cultured overnight with the same culture medium containing no serum. Apoptosis was induced by incubating the hepatocytes with the culture medium containing 2000 units/ml TNFα and 0.2 μg/ml actinomycin D for 6 h unless specified in the figure legends. Cells were then scraped off the plates and centrifuged, washed twice with cold phosphate-buffered saline (PBS), and resuspended in 5-fold volume of hypotonic buffer A (20 mm HEPES, pH 7.5, 10 mmKCl, 1.5 mm MgCl2, 1 mm EGTA, 1 mmEDTA, 0.5 mm PMSF, 5 μg/ml aprotinin, 5 μg/ml pepstatin, and 10 μg/ml leupeptin). After three cycles of freezing and thawing, cell debris was removed by centrifugation at 13,000 × g at 4 °C for 20 min. The supernatant was used as crude cytosol for caspase assays and Western blot for caspases, and the cell debris was used for Western blotting analysis of PARP. Protein concentration was determined with the BCA assay (Pierce) with bovine serum albumin as standard. Cell viability was determined by the crystal violet method as described previously (8.Li J. Bombeck C.A. Yang S. Kim Y.M. Billiar T.R. J. Biol. Chem. 1999; 274: 17325-17333Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Activation of Akt was evaluated by stimulating cells with indicated reagents for various times. Cells were first rinsed twice with ice-cold PBS, and 600 μl of whole cell lysis buffer (20 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Nonidet P-40, 2.5 mm sodium pyrophosphate, 1 mmβ-glycerophosphate, 1 mm Na3VO4, 1 μg/ml leupeptin, and 1 mm PMSF) was then added to each plate. After 5 min incubation on ice, cells were scraped off the plates and lysed at 4 °C for 30 min. The whole cell lysate was collected by centrifugation at 13,000 × g for 15 min to remove cell debris, frozen in liquid nitrogen, and stored in aliquots at −80 °C until use. Akt enzymatic activity was assayed with 1 μg per assay of recombinant glycogen synthase kinase (GSK3) fusion protein as substrate in a reaction mixture containing 25 mmTris, pH 7.5, 1 mm Na3VO4, 1 mm EDTA, and 0.5 mm PMSF, 10 mmMgCl2, 50 μm ATP, and 2 μCi of [γ-32P]ATP/per assay. The phosphorylation reaction was allowed to proceed for 30 min at 30 °C and stopped by adding 3× sample buffer. After boiling at 95 °C for 5 min, phosphorylated GSK3 was resolved by SDS-PAGE and quantitated with a ImageQuaNT system (Storm 860, Molecular Dynamics Inc., Sunnydale, CA). Caspase activity was evaluated by measuring proteolytic cleavage of the chromogenic substrate Ac-DEVD-pNA (for caspase-3-like protease activity) or Ac-IETD-pNA (for caspase-8 activity) as described previously (8.Li J. Bombeck C.A. Yang S. Kim Y.M. Billiar T.R. J. Biol. Chem. 1999; 274: 17325-17333Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Thirty to forty μg of protein was separated on 14% (caspase-3), 10% (Akt), or 8% (PARP) SDS-PAGE and transferred onto a nitrocellulose membrane. Nonspecific binding was blocked with TBS-T (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.1% Tween 20) containing 5% non-fat milk for 1 h at room temperature. Anti-caspase-3 polyclonal antibody H277 was diluted 1:500 in TBS-T containing 1% bovine serum albumin, anti-PARP antibody 1:500, anti-Akt, phospho (Ser473)-Akt antibody 1:1000, anti-caspases-9 antibody 1:500, anti-caspase-8 antibody 1:1000, and anti-actin antibody 1:30,000. After 1 h incubation at room temperature (caspase-3, caspase-8, PARP, Akt) or overnight at 4 °C (phospho-Akt together with actin, caspase-9) with agitation, membranes were washed three times with TBS-T. The secondary antibody was incubated at 1:5000 dilution for 1 h. Following 5 washes with TBS-T, the protein bands were visualized with SupersignalTM according to the manufacturer's instructions. The cell death detection enzyme-linked immunosorbent assay was used to quantitate changes in DNA fragmentation according to the manufacturer's instructions. cAMP levels were measured by using an iodinated assay system from NEN Life Science Products. Following exposure to indicated reagents for 30 min, the cells were lysed in lysis buffer supplemented with 1 mm3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor) to inhibit phosphodiesterases as described above. Intracellular cAMP contents were then determined with the cell lysate (10 μg of proteins) by radioimmunoassay according to the manufacturer's protocol. cAMP-dependent protein kinase activity was determined by in vitro phosphorylation of Kemptide. Cells were lysed as above, and aliquots of 10 μg of proteins were mixed with or without 1 μm protein kinase A inhibitor (PKI, 6–22) in 50 mm Tris, pH 7.5, in a volume of 30 μl. After incubation for 15 min at 25 °C to allow PKI to bind to PKA, 10 μl of 4× substrate solution containing 200 μm Kemptide, 40 mm MgCl2, 400 μm ATP and 1 μCi per assay of [γ-32P]ATP was added to each sample. Samples were incubated for 5 min at 30 °C, and the reactions were terminated by adding EDTA to a final concentration of 20 mm. 20 μl of the reaction mix was spotted onto P81 phosphocellulose paper disc. The P81 paper sheets were immersed in 1% (v/v) orthophosphoric acid, washed 5–6 times with the acid solution, and then 2 times with distilled water. The incorporated radioactivity was determined by scintillation counting. Background values obtained from a mixture lacking cell lysate were subtracted from all values. All measurements were performed in duplicate. TNFα rapidly triggers hepatocyte apoptotic cell death in the presence of the transcriptional inhibitor actinomycin D (8.Li J. Bombeck C.A. Yang S. Kim Y.M. Billiar T.R. J. Biol. Chem. 1999; 274: 17325-17333Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 42.Leist M. Gantner F. Bohlinger I. Germann P.G. Tiegs G. Wendel A. J. Immunol. 1994; 153: 1778-1788PubMed Google Scholar). Previously we have shown that the cell-permeable cGMP analog Br-cGMP partially suppresses TNFα+ActD-induced hepatocyte apoptosis (7.Kim Y.M. Talanian R.V. Billiar T.R. J. Biol. Chem. 1997; 272: 31138-31148Abstract Full Text Full Text PDF PubMed Scopus (798) Google Scholar). To investigate the underlying mechanism of the cyclic nucleotide-dependent inhibition of apoptosis, we first established the optimal concentration for the inhibition of apoptosis by Br-cGMP. When incubated with cells in the presence of TNFα+ActD, Br-cGMP suppressed apoptotic cell death in a dose-dependent manner (Fig. 1, A andB). The maximal inhibition was observed at 800 μm. Interestingly, cAMP analogs such as Br-cAMP also dose-dependently inhibited TNFα+ActD-induced cell death but at a much lower concentration when compared with the corresponding cGMP analog (Fig. 1, A and B). At the highest concentration used, Br-cAMP or Br-cGMP alone had no significant effect on cell viability and morphology in the absence of TNFα+ActD. Other cell permeable analogs of cGMP and cAMP, such as 8-pCPT-cGMP, dibutyryl-cGMP (Bt2cGMP), and dibutyryl-cAMP (Bt2cAMP) showed similar results for suppression of TNFα+ActD-induced hepatocyte cell death (data not shown). Treatment of hepatocytes with 10 μm forskolin, a reagent that activates adenylyl cyclase and thus increases intracellular cAMP level, also effectively suppressed TNFα+ActD-mediated apoptosis (Fig.1 B). TNFα+ActD-induced cell death and DNA fragmentation were completely inhibited by the pan caspase inhibitor, Z-VAD-fmk (Fig. 1 and Ref. 8.Li J. Bombeck C.A. Yang S. Kim Y.M. Billiar T.R. J. Biol. Chem. 1999; 274: 17325-17333Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Addition of cell-permeable Bt2cAMP or Br-cGMP at the same time with TNFα+ActD delayed the onset of apoptosis but did not completely inhibit cell death (Fig. 1 C). Since cell death was completely inhibited at the 6-h time point by cyclic nucleotides, this time point was thus chosen for subsequent studies to elucidate the mechanism by which cyclic nucleotides regulate apoptotic signaling events. Previously we have demonstrated that multiple caspases are activated in hepatocytes challenged with TNFα+ActD and that caspase activation is required for hepatocyte apoptosis in response to this stimulus (8.Li J. Bombeck C.A. Yang S. Kim Y.M. Billiar T.R. J. Biol. Chem. 1999; 274: 17325-17333Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). To examine whether cGMP and cAMP analogs suppress cell death by inhibiting activation of caspases, we first evaluated caspase-3-like activity of cells exposed to TNFα+ActD in the presence and absence of cyclic nucleotides. As shown in Fig. 2 A, cyclic nucleotides alone did not have any effect on caspase activity. However, concomitant exposure of cells to cyclic nucleotides with TNFα+ActD resulted in significant inhibition of caspase-3-like activity. The effect of cAMP and cGMP analogs on caspase-3-like protease activity closely correlated with their effect on hepatocyte DNA fragmentation (Fig. 2 B). Moreover, these cyclic nucleotide analogs suppressed proteolytic activation of procaspase-3 as determined by immunoblotting analysis (Fig. 2 C). Consistent with the finding that the caspase-3 activation was inhibited by the cyclic nucleotide analogs, cleavage of PARP, a well established intracellular substrate of caspase-3-like proteases, was also blocked (Fig. 2 C). These results demonstrate that cyclic nucleotides suppress TNFα+ActD-induced apoptosis at a site upstream of caspase-3 activation. The most proximal caspase activated by TNFα is believed to be caspase-8 (3.Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5152) Google Scholar). Activation of caspase-8 can lead to direct activation of other caspases or the release of cytochrome c from mitochondria. We next tested whether activation of procaspase-8 is suppressed by cyclic nucleotide-elevating agents. As shown in Fig. 3 A, caspase-8 activity in cytosolic extracts was markedly elevated in cells treated with TNFα+ActD. Addition of Br-cGMP or Bt2cAMP to cells significantly inhibited the caspase-8 activity. As expected, the pan caspase inhibitor Z-VAD-fmk completely prevented caspase-8 activation. Furthermore, forskolin also suppressed TNFα+ActD-induced caspase-8 activity. Immunoblotting analysis of procaspase-8 revealed that processing of this caspase was inhibited by cAMP and cGMP analog treatment (Fig. 3 B). These results indicate that elevation of cellular cyclic nucleotides interferes with the death signal leading to caspase-8 activation or amplification. We previously reported that cytochrome c translocates from mitochondria to cytoplasm in TNFα+ActD-treated hepatocytes (8.Li J. Bombeck C.A. Yang S. Kim Y.M. Billiar T.R. J. Biol. Chem. 1999; 274: 17325-17333Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Release of cytochromec is known to signal activation of procaspase-9 through interaction with Apaf-1 (43.Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6232) Google Scholar). As shown in Fig. 3 C, cytosol from untreated cells contained negligible cytochrome c. However, cytochrome c accumulated in cytosol of cells exposed to TNFα+ActD. This release of cytochrome c was significantly inhibited by cotreatment of cells with Bt2cAMP or Br-cGMP, demonstrating that cyclic nucleotides act at or above the level of cytochrome c release. Moreover, the activation of caspase-9 was suppressed by cyclic nucleotide-elevating agents (Fig. 3 C). These results are in agreement with our above findings that the cyclic nucleotide agents suppressed TNFα+ActD-induced caspase-8 activation. The PI3′K/Akt pathway plays an important role in inhibiting apoptosis. One mechanism by which Akt signaling suppresses apoptosis is by inhibiting cytochrome c release (44.Kennedy S.G. Kandel E.S. Cross T.K. Hay N. Mol. Cell. Biol. 1999; 19: 5800-5810Crossref PubMed Scopus (591) Google Scholar). To explore whether cyclic nucleotide may suppress apoptosis and apoptotic signaling via activating the Akt survival pathway, we then examined the effects of cyclic nucleotides on endogenous Akt activation. Since phosphorylation of Akt at Ser473 is required for its full activation (45.Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2517) Google Scholar, 46.Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar), we first examined the phosphorylation status of endogenous Akt using an antibody that specifically recognizes Akt phosphorylated at Ser473. We found rapid activation of endogenous Akt when cells were treated with Br-cGMP, and this activation was time- and concentration-dependent (Fig.4 A). Other cGMP analogs such as Bt2cGMP and the PKG activator 8-pCPT-cGMP also activated Akt in a dose- and time-dependent manner, revealing a link between cGMP signaling and the Akt signaling pathway. Similar results were observed for cAMP analogs. Treatment of cells with B
Referência(s)