Abstract
When PC12 cells are deprived of trophic support they undergo apoptosis. We have previously shown that survival of trophic factor-deprived PC12M1 cells can be promoted by activation of the G protein-coupled muscarinic receptors. The mechanism whereby muscarinic receptors inhibit apoptosis is poorly understood. In the present study we investigated this mechanism by examining the effect of muscarinic receptor activation on the serum deprivation-induced activity of key players in apoptosis, the caspases, in PC12M1 cells. The results showed that m1 muscarinic activation inhibits caspase activity induced by serum deprivation. This effect appeared to be caused by the prevention of activation of caspases such as caspase-2 and caspase-3, and not by the inhibition of existing activity. Muscarinic receptor activation also stimulated the mitogen-activated protein kinase/extracellular signaling-regulated kinase (MAPK/ERK) and phosphoinositide (PI) 3-kinase signaling pathways. The PI 3-kinase pathway inhibitors wortmannin and LY294002, as well as the MAPK/ERK pathway PD98059 inhibitor, did not however suppress the inhibitory effect of the muscarinic receptors on caspase activity. The results therefore suggested that the muscarinic survival effect is mediated by a pathway that leads to caspase inhibition by MAPK/ERK- and PI 3-kinase-independent signaling cascades. Cell Death and Differentiation (2000) 7, 825–833
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Abbreviations
- Ac-DEVD-7AMC:
-
Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin
- ERK:
-
extracellular signal-regulated kinase
- G-protein:
-
GTP-binding protein
- MAPK:
-
mitogen-activated protein kinase
- mAChRs:
-
muscarinic acetylcholine receptors
- MTT:
-
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
- NGF:
-
nerve growth factor
- PBS:
-
phosphate-buffered saline
- PI:
-
phosphoinositide
- PKB:
-
protein kinase B
References
Barinaga M . (1998) Is apoptosis key in Alzheimer's disease? Science 281: 1303–1304
Barinaga M . (1998) Stroke-damaged neurons may commit cellular suicide. Science 281: 1302–1303
Bergeron L and Yuan J . (1998) Sealing one's fate: control of cell death in neurons. Curr. Opin. Neurobiol. 8: 55–63
Thornberry NA and Lazebnik Y . (1998) Caspases: enemies within. Science 281: 1312–1316
Raff MC, Barres BA, Burne JF, Coles HS, Ishizaki Y and Jacobson MD . (1994) Programmed cell death and the control of cell survival. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 345: 265–268
Rukenstein A, Rydel RE and Green LA . (1991) Multiple agents rescue PC12 cells from serum-free cell death by translation- and transcription-independent mechanisms. J. Neurosci. 11: 2552–2563
Smeyne RJ, Klein R, Schnapp A, Long LK, Bryant S, Lewin A, Lira SA and Barbacid M . (1994) Severe sensory and sympathetic neuropathies in mice carrying a disrupted Trk/NGF receptor gene. Nature 368: 246–249
Parrizas M, Saltiel AR and LeRoith D . (1997) Insulin-like growth factor 1 inhibits apoptosis using the phosphatidylinositol 3′-kinase and mitogen-activated protein kinase pathways. J. Biol. Chem. 272: 154–161
Armant M, Delespesse G and Sarfati M . (1995) IL-2 and IL-7 but not IL-12 protect natural killer cells from death by apoptosis and up-regulate bcl-2 expression. Immunology 85: 331–337
Williams GT, Smith CA, Spooncer E, Dexter TM and Taylor DR . (1990) Haemopoietic colony stimulating factors promote cell survival by suppressing apoptosis. Nature 343: 76–79
Vella A, Teague TK, Ihle J, Kappler J and Marrack P . (1997) Interleukin 4 (IL-4) or IL-7 prevents the death of resting T cells: stat6 is probably not required for the effect of IL-4. J. Exp. Med. 186: 325–330
Demoulin JB, Uyttenhove C, Van Roost E, DeLestre B, Donckers D, Van Snick J and Renauld JC . (1996) A single tyrosine of the interleukin-9 (IL-9) receptor is required for STAT activation, antiapoptotic activity, and growth regulation by IL-9. Mol. Cell. Biol. 16: 4710–1716
Xia Z, Dickens M, Raingeaud J, Davis RJ and Greenberg ME . (1995) Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270: 1326–1331
Anderson CNG and Tolkovsky AM . (1999) A role for MAPK/ERK in sympathetic neuron survival: protection against a p53-dependent, JNK-independent induction of apoptosis by cytosine arabinoside. J. Neurosci. 19: 664–673
Yao R and Cooper GM . (1995) Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science 267: 2003–2006
Dudek H, Datta SR, Franke TF, Birnbaum MJ, Yao R, Cooper GM, Segal RA, Kaplan DR and Greenberg ME . (1997) Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science 275: 661–665
Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, Frisch S and Reed JC . (1998) Regulation of cell death protease caspase-9 by phosphorylation. Science 282: 1318–1321
Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y and Greenberg ME . (1997) Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91: 231–241
del Peso L, Gonzalez-Garcia M, Page C, Herrara R and Nunez G . (1997) Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science 278: 687–689
Filippa N, Sable CL, Filloux C, Hemmings B and Van Obberghen E . (1999) Mechanism of protein kinase B activation by cyclic AMP-dependent protein kinase. Mol. Cell. Biol. 19: 4989–5000
Yano S, Tokumitsu H and Soderling TR . (1998) Calcium promotes cell survival through CaM-K kinase activation of the protein-kinase-B pathway. Nature 396: 584–587
Scheid MP, Schubert KM and Duronio V . (1999) Regulation of bad phosphorylation and association with Bcl-x(L) by the MAPK/Erk kinase. J. Biol. Chem. 274: 31108–31113
Bonni A, Brunet A, West AE, Datta SR, Takasu MA and Greenberg ME . (1999) Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms [see comments]. Science 286: 1358–1362
Harada H, Becknell B, Wilm M, Mann M, Huang LJ, Taylor SS, Scott JD and Korsmeyer SJ . (1999) Phosphorylation and inactivation of BAD by mitochondria-anchored protein kinase A. Mol. Cell 3: 413–422
Yan GM, Lin SZ, Irwin RP and Paul SM . (1995) Activation of muscarinic cholinergic receptors blocks apoptosis of cultured cerebellar granule neurons. Mol. Pharmacol. 47: 248–257
Lindenboim L, Pinkas-Kramarski R, Sokolovsky M and Stein R . (1995) Activation of muscarinic receptors inhibits apoptosis in PC12M1 cells. J. Neurochem. 64: 2491–2499
Wess J . (1993) Molecular basis of muscarinic acetylcholine receptor function. Trends Pharmacol. Sci. 14: 308–313
Wess J . (1996) Molecular biology of muscarinic acetylcholine receptors. Crit. Rev. Neurobiol. 10: 69–99
Fukuda K, Higashida H, Kubo T, Maeda A, Akiba I, Bujo H, Mishina M and Numa S . (1988) Selective coupling with K+ currents of muscarinic acetylcholine receptor subtypes in NG108-15 cells. Nature 335: 355–358
Fukuda K, Kubo T, Akiba I, Maeda A, Mishina M and Numa S . (1987) Molecular distinction between muscarinic acetylcholine receptor subtypes. Nature 327: 623–625
Felder CC, Poulter MO and Wess J . (1992) Muscarinic receptor-operated Ca2+ influx in transfected fibroblast cells is independent of inositol phosphates and release of intracellular Ca2+. Proc. Natl. Acad. Sci. U.S.A. 89: 509–513
Sandmann J, Peralta EG and Wurtman RJ . (1991) Coupling of transfected muscarinic acetylcholine receptor subtypes to phospholipase D. J. Biol. Chem. 266: 6031–6034
Marais R, Light Y, Mason C, Paterson H, Olson MF and Marshall CJ . (1998) Requirement of Ras-GTP-Raf complexes for activation of Raf-1 by protein kinase C. Science 280: 109–112
Lopez-Ilasaca M, Crespo P, Pellici PG, Gutkind JS and Wetzker R . (1997) Linkage of G protein-coupled receptors to the MAPK signaling pathway through PI 3-kinase gamma. Science 275: 394–397
van Biesen T, Hawes BE, Raymond JR, Luttrell LM, Koch WJ and Lefkowitz RJ . (1996) G(o)-protein alpha-subunits activate mitogen-activated protein kinase via a novel protein kinase C-dependent mechanism. J. Biol. Chem. 271: 1266–1269
Schmidt M, Voss M, Thiel M, Bauer B, Grannass A, Tapp E, Cool RH, de Gunzburg J, von Eichel-Streiber C and Jakobs KH . (1998) Specific inhibition of phorbol ester-stimulated phospholipase D by Clostridium sordellii lethal toxin and Clostridium difficile toxin B-1470 in HEK-293 cells. Restoration by Ral GTPases. J. Biol. Chem. 273: 7413–7422
Lindenboim L, Diamond R, Rothenberg E and Stein R . (1995) Apoptosis induced by serum deprivation of PC12 cells is not preceded by growth arrest and can occur at each phase of the cell cycle. Cancer Res. 55: 1242–1247
Park DS, Stefanis L, Yan CYL, Farinelli SE and Greene LA . (1996) Ordering the cell death pathway. Differential effects of BCL2, an interleukin-1-converting enzyme family protease inhibitor, and other survival agents on JNK activation in serum/nerve growth factor-deprived PC12 cells. J. Biol. Chem. 271: 21898–21905
Ochu EE, Rothwell NJ and Waters CM . (1998) Caspases mediate 6-hydroxydopamine-induced apoptosis but not necrosis in PC12 cells. J. Neurochem. 70: 2637–2640
Suzuki A . (1997) Amyloid beta-protein induces necrotic cell death mediated by ICE cascade in PC12 cells. Exp. Cell Res. 234: 507–511
Yen C, Mar M and Zeisel SH . (1999) Choline deficiency-induced apoptosis in PC12 cells is associated with diminished membrane phosphatidylcholine and sphingomyelin, accumulation of ceramide and diacylglycerol, and activation of a caspase. FASEB J. 13: 135–142
Kruman I, Guo Q and Mattson MP . (1998) Calcium and reactive oxygen species mediate staurosporine-induced mitochondrial dysfunction and apoptosis in PC12 cells. J. Neurosci. Res. 51: 293–308
Troy CM, Stefanis L, Prochiantz A, Greene LA and Shelanski ML . (1996) The contrasting roles of ICE family proteases and interleukin-1beta in apoptosis induced by trophic factor withdrawal and by copper/zinc superoxide dismutase down-regulation. Proc. Natl. Acad. Sci. U.S.A. 93: 5635–5640
Troy CM, Stefanis L, Greene LA and Shelanski ML . (1997) Nedd2 is required for apoptosis after trophic factor withdrawal, but not superoxide dismutase (SOD1) downregulation, in sympathetic neurons and PC12 cells. J. Neurosci. 17: 1911–1918
Stefanis L, Park DS, Yan CY, Farinelli SE, Troy CM, Shelanski ML and Greene LA . (1996) Induction of CPP32-like activity in PC12 cells by withdrawal of trophic support. Dissociation from apoptosis. J. Biol. Chem. 271: 30663–30671
Haviv R, Lindenboim L, Li H, Yuan J and Stein R . (1997) Need for caspases in apoptosis of trophic factor-deprived PC12 cells. J. Neurosci. Res. 50: 69–80
Haviv R, Lindenboim L, Yuan J and Stein R . (1998) Need for caspase-2 in apoptosis of growth-factor-deprived PC12 cells. J. Neurosci. Res. 52: 491–497
Schulz JB, Bremen D, Reed JC, Lommatzsch J, Takayama S, Wullner U, Loschmann PA, Klockgether T and Weller M . (1997) Cooperative interception of neuronal apoptosis by BCL-2 and BAG-1 expression: prevention of caspase activation and reduced production of reactive oxygen species. J. Neurochem. 69: 2075–2086
Gurwitz D, Haring R, Pinkas Kramarski R, Stein R, Heldman E, Karton Y and Fisher A . (1995) NGF-dependent neurotrophic-like effects of AF102B, an M1 muscarinic agonist, in PC12M1 cells. Neuroreport 6: 485–488
Kimura K, Hattori S, Kabuyama Y, Shizawa Y, Takayanagi J, Nakamura S, Toki S, Matsuda Y, Onodera K and Fukui Y . (1994) Neurite outgrowth of PC12 cells is suppressed by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase. J. Biol. Chem. 269: 18961–18967
Murga C, Laguinge L, Wetzker R, Cuadrado A and Gutkind JS . (1998) Activation of Akt/protein kinase B by G protein-coupled receptors. A role for alpha and beta gamma subunits of heterotrimeric G proteins acting through phosphatidylinositol-3-OH kinasegamma. J. Biol. Chem. 273: 19080–19085
Haring R, Fisher A, Marciano D, Pittel Z, Kloog Y, Zuckerman A, Eshhar N and Heldman E . (1998) Mitogen-activated protein kinase-dependent and protein kinase C-dependent pathways link the m1 muscarinic receptor to beta-amyloid precursor protein secretion. J. Neurochem. 71: 2094–2103
Desdouits-Magnen J, Desdouits F, Takeda S, Syu LJ, Saltiel AR, Buxbaum JD, Czernik AJ, Nairn AC and Greengard P . (1998) Regulation of secretion of Alzheimer amyloid precursor protein by the mitogen-activated protein kinase cascade. J. Neurochem. 70: 524–530
Andjelkovic M, Suidan HS, Meier R, Frech M, Alessi DR and Hemmings BA . (1998) Nerve growth factor promotes activation of the alpha, beta and gamma isoforms of protein kinase B in PC12 pheochromocytoma cells. Eur. J. Biochem. 251: 195–200
Talanian RV, Quinlan C, Trautz S, Hackett MC, Mankovich JA, Banach D, Ghayur T, Brady KD and Wong WW . (1997) Substrate specificities of caspase family proteases. J. Biol. Chem. 272: 9677–9682
Thornberry NA, Rano TA, Peterson EP, Rasper DM, Timkey T, Garcia-Calvo M, Houtzager VM, Nordstrom PA, Roy S, Vaillancourt JP, Chapman KT and Nicholson DW . (1997) A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J. Biol. Chem. 272: 17907–17911
Janicke RU, Ng P, Sprengart ML and Porter AG . (1998) Caspase-3 is required for alpha-fodrin cleavage but dispensable for cleavage of other death substrates in apoptosis. J. Biol. Chem. 273: 15540–15545
Stefanis L, Troy CM, Qi H, Shelanski ML and Greene LA . (1998) Caspase-2 (Nedd-2) processing and death of trophic factor-deprived PC12 cells and sympathetic neurons occur independently of caspase-3 (CPP32)-like activity. J. Neurosci. 18: 9204–9215
Lan L and Wong NS . (1999) Phosphatidylinositol 3-kinase and protein kinase C are required for the inhibition of caspase activity by epidermal growth factor. FEBS Lett. 444: 90–96
Kennedy SG, Wagner AJ, Conzen SD, Jordan J, Bellacosa A, Tsichlis PN and Hay N . (1997) The PI 3-kinase/Akt signaling pathway delivers an anti-apoptotic signal. Genes Dev. 11: 701–713
Spear N, Estevez AG, Barbeito L, Beckman JS and Johnson GV . (1997) Nerve growth factor protects PC12 cells against peroxynitrite-induced apoptosis via a mechanism dependent on phosphatidylinositol 3-kinase. J. Neurochem. 69: 53–59
Kummer JL, Rao PK and Heidenreich KA . (1997) Apoptosis induced by withdrawal of trophic factors is mediated by p38 mitogen-activated protein kinase. J. Biol. Chem. 272: 20490–20494
Yan CYI and Greene LA . (1998) Prevention of PC12 cell death by N-acetylcysteine requires activation of the Ras pathway. J. Neurosci. 18: 4042–4049
Hartfield PJ, Bilney AJ and Murray AW . (1998) Neurotrophic factors prevent ceramide-induced apoptosis downstream of c-Jun N-terminal kinase activation in PC12 cells. J. Neurochem. 71: 161–169
Philpott KL, McCarthy MJ, Klippel A and Rubin LL . (1997) Activated phosphatidylinositol 3-kinase and Akt kinase promote survival of superior cervical neurons. J. Cell Biol. 139: 809–815
Ulrich E, Duwel A, Kauffmann-Zeh A, Gilbert C, Lyon D, Rudkin B, Evan G and Martin-Zanca D . (1998) Specific TrkA survival signals interfere with different apoptotic pathways. Oncogene 16: 825–832
Scheid MP and Duronio V . (1998) Dissociation of cytokine-induced phosphorylation of Bad and activation of PKB/akt: involvement of MEK upstream of Bad phosphorylation. Proc. Natl. Acad. Sci. U.S.A. 95: 7439–7444
Edsall LC, Pirianov GG and Spiegel S . (1997) Involvement of sphingosine 1-phosphate in nerve growth factor-mediated neuronal survival and differentiation. J. Neurosci. 17: 6952–6960
Meyer zu Heringdorf D, Lass H, Alemany R, Laser KT, Neumann E, Zhang C, Schmidt M, Rauen U, Jakobs KH and van Koppen CJ . (1998) Sphingosine kinase-mediated Ca2+ signalling by G-protein-coupled receptors. EMBO J. 17: 2830–2837
Xiang J, Chao DT and Korsmeyer SJ . (1996) BAX-induced cell death may not require interleukin 1 beta-converting enzyme-like proteases. Proc. Natl. Acad. Sci. U.S.A. 93: 14559–14563
Le-Niculescu H, Bonfoco E, Kasuya Y, Claret FX, Green DR and Karin M . (1999) Withdrawal of survival factors results in activation of the JNK pathway in neuronal cells leading to fas ligand induction and cell death. Mol. Cell. Biol. 19: 751–763
Ashkenazi A and Dixit VM . (1998) Death receptors: signaling and modulation. Science 281: 1305–1308
Varadhachary AS, Peter ME, Perdow SN, Krammer PH and Salgame P . (1999) Selective up-regulation of phosphatidylinositol 3′-kinase activity in Th2 cells inhibits caspase-8 cleavage at the death-inducing complex: a mechanism for Th2 resistance from Fas-mediated apoptosis. J. Immunol. 163: 4772–4779
Pinkas Kramarski R, Stein R, Lindenboim L and Sokolovsky M . (1992) Growth factor-like effects mediated by muscarinic receptors in PC12M1 cells. J. Neurochem. 59: 2158–2166
Stefanis L, Troy CM, Qi H and Greene LA . (1997) Inhibitors of trypsin-like serine proteases inhibit processing of the caspase Nedd-2 and protect PC12 cells and sympathetic neurons from death evoked by withdrawal of trophic support. J. Neurochem. 69: 1425–1437
Mosmann T . (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65: 55–63
Acknowledgements
This work was supported by the German-Israeli Foundation for Scientific Research and Development and the Joint German-Israeli Research Program. We thank Dr. Lloyd Greene for providing the anti-N-Nedd antibody and Ms. Shirley Smith for excellent editorial assistance.
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Leloup, C., Michaelson, D., Fisher, A. et al. M1 muscarinic receptors block caspase activation by phosphoinositide 3-kinase- and MAPK/ERK-independent pathways. Cell Death Differ 7, 825–833 (2000). https://doi.org/10.1038/sj.cdd.4400713
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DOI: https://doi.org/10.1038/sj.cdd.4400713
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