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• W2120342771 abstract "Little is known about the role of Rho proteins in apoptosis produced by stimuli evolved specifically to produce apoptosis, such as granzymes from cytotoxic T lymphocytes (CTLs) and Fas. Here we demonstrate that all three Rho family members are involved in CTL- and Fas-induced killing. Dominant-negative mutants of each Rho family member and Clostridium difficile toxin B, an inhibitor of all family members, strongly inhibited the susceptibility of cells to CTL- and Fas-induced apoptosis. Fas-induced caspase-3 activation was inhibited by C. difficile toxin. Activated mutants of each GTPase increased susceptibility to apoptosis, and activation of Cdc42 increased within 5 min of Fas stimulation. In contrast, during the time required for CTL and Fas killing, no apoptosis was produced by dominant-negative or activated mutants or byC. difficile toxin alone. Inhibition of actin polymerization using latrunculin A reduced the ability of constitutively active GTPase mutants to stimulate apoptosis and blocked Fas-induced activation of caspase-3. Furthermore, the ability of Rac to enhance apoptosis was decreased by point mutations reported to block Rac induction of actin polymerization. Rho family proteins may regulate apoptosis through their effects on the actin cytoskeleton. Little is known about the role of Rho proteins in apoptosis produced by stimuli evolved specifically to produce apoptosis, such as granzymes from cytotoxic T lymphocytes (CTLs) and Fas. Here we demonstrate that all three Rho family members are involved in CTL- and Fas-induced killing. Dominant-negative mutants of each Rho family member and Clostridium difficile toxin B, an inhibitor of all family members, strongly inhibited the susceptibility of cells to CTL- and Fas-induced apoptosis. Fas-induced caspase-3 activation was inhibited by C. difficile toxin. Activated mutants of each GTPase increased susceptibility to apoptosis, and activation of Cdc42 increased within 5 min of Fas stimulation. In contrast, during the time required for CTL and Fas killing, no apoptosis was produced by dominant-negative or activated mutants or byC. difficile toxin alone. Inhibition of actin polymerization using latrunculin A reduced the ability of constitutively active GTPase mutants to stimulate apoptosis and blocked Fas-induced activation of caspase-3. Furthermore, the ability of Rac to enhance apoptosis was decreased by point mutations reported to block Rac induction of actin polymerization. Rho family proteins may regulate apoptosis through their effects on the actin cytoskeleton. c-Jun N-terminal kinase cytotoxic T lymphocyte Chinese hamster ovary green fluorescent protein major histocompatibility complex guanosine 5′-3-O-(thio)triphosphate Apoptosis is a form of “cell suicide” in which cells fragment their cytoplasm, nucleus, and DNA for orderly disposal (1.Martin S.J. Green D.R. Cotter T.G. Trends Biochem. Sci. 1994; 19: 26-30Abstract Full Text PDF PubMed Scopus (399) Google Scholar, 2.McConkey D.J. Orrenius S. Trends Cell. Biol. 1994; 4: 370-374Abstract Full Text PDF PubMed Scopus (159) Google Scholar). It is ubiquitous and essential to normal homeostasis in higher animals and is important in modulating immune cell populations for defense against cancer and virus-infected cells (3.Glinsky G.V. Glinsky V.V. Ivanova A.B. Hueser C.J. Cancer Lett. 1997; 115: 185-193Crossref PubMed Scopus (134) Google Scholar, 4.Amiesem J.C. Estaquier J. Idziorek T. Immunol. Rev. 1994; 142: 9-34Crossref PubMed Scopus (85) Google Scholar). Apoptosis is induced through activation of caspases, which activate one another in a proteolytic cascade (5.Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6133) Google Scholar), leading to cleavage of cytoskeletal and signaling molecules that result in irreversible changes in cell morphology and the plasma membrane (6.Rudel T. Bokoch G.M. Science. 1997; 276: 1571-1574Crossref PubMed Scopus (602) Google Scholar, 7.Janicke R.U. Ng P. Sprengart M.L. Porter A.G. J. Biol. Chem. 1998; 273: 15540-15545Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 8.Kothakota S. Azuma T. Reinhard C. Klippel A. Tang J. Chu K. McGarry T.J. Kirschner M.W. Koths K. Kwiatkowski D.J. Williams L.T. Science. 1997; 278: 294-298Crossref PubMed Scopus (1031) Google Scholar, 9.Caulin C. Salvesen G.S. Osima R.G. J. Cell Biol. 1997; 138: 1379-1394Crossref PubMed Scopus (542) Google Scholar, 10.Wen L.P. Fahrni J.A. Troie S. Guan J.L. Orth K. Rosen G.D. J. Biol. Chem. 1997; 272: 26056-26061Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar).The Rho family of small GTPases plays a role in the terminal morphological changes of apoptosis (11.Mills J.C. Stone N.L. Erhardt J. Pittman R.N. J. Cell Biol. 1998; 140: 627-636Crossref PubMed Scopus (407) Google Scholar), but previous reports present conflicting data about its role in apoptosis induction. Apoptotic signaling pathways can be grouped into two distinct categories (12.Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5112) Google Scholar): mechanisms that induce rapid apoptosis through specific molecules that have evolved to produce cell death (i.e. Fas ligand or cytotoxic T cell granzymes) (12.Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5112) Google Scholar) and pathways that lead to apoptosis when normal homeostatic signaling is disrupted (13.Rawson C.L. Loo D.T. Duimstra J.R. Hedstrom O.R. Schmidt E.E Barnes D.W. J. Cell Biol. 1991; 113: 671-680Crossref PubMed Scopus (63) Google Scholar, 14.Frisch S.M. Francis H. J. Cell Biol. 1994; 124: 619-626Crossref PubMed Scopus (2749) Google Scholar, 15.Evan G. Littlewood T. Science. 1998; 281: 1317-1322Crossref PubMed Scopus (1360) Google Scholar). Examples of the latter type of perturbation include serum withdrawal (13.Rawson C.L. Loo D.T. Duimstra J.R. Hedstrom O.R. Schmidt E.E Barnes D.W. J. Cell Biol. 1991; 113: 671-680Crossref PubMed Scopus (63) Google Scholar), detachment from extracellular matrix (14.Frisch S.M. Francis H. J. Cell Biol. 1994; 124: 619-626Crossref PubMed Scopus (2749) Google Scholar), and an imbalance of survival homeostatic signaling pathways (15.Evan G. Littlewood T. Science. 1998; 281: 1317-1322Crossref PubMed Scopus (1360) Google Scholar). Numerous studies of Rho family signaling in such pathways have revealed that the GTPases play a complex role, participating in both stimulatory and inhibitory paradigms (16.Bazenet C.E. Mota M.A. Rubin L.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3984-3989Crossref PubMed Scopus (114) Google Scholar, 17.Nishida K. Kaziro Y. Takaya S. Oncogene. 1999; 18: 407-415Crossref PubMed Scopus (80) Google Scholar, 18.Bobak D. Moorman J. Guanzon A. Gilmer L. Hahn C. Oncogene. 1997; 15: 2179-2189Crossref PubMed Scopus (70) Google Scholar). Downstream mediators for positive (i.e.c-Jun N-terminal kinase (JNK)1) and negative (i.e. Akt) effects have been identified. In contrast, much less is known about Rho family signaling in apoptosis produced by CTLs, Fas, and other molecules directly inducing apoptosis. Here we show that Rho family GTPases play a stimulatory role in CTL- and Fas-induced apoptosis.The Rho family of small GTPases consists of three proteins (Cdc42, Rac, and Rho) that interact with downstream effectors upon binding to GTP. Their nucleotide state and localization are controlled by >40 currently identified regulatory factors, including guanine nucleotide exchange factors, GDP dissociation inhibitors, and GTPase-activating proteins (19.Bokoch G.M. Bohl B.P. Chuang T.-H. J. Biol. Chem. 1994; 269: 31674-31679Abstract Full Text PDF PubMed Google Scholar, 20.Lamarche N. Hall A. Trends Genet. 1994; 10: 346-440Abstract Full Text PDF Scopus (210) Google Scholar, 21.Cerione R.A. Zheng Y. Curr. Opin. Cell Biol. 1996; 8: 216-222Crossref PubMed Scopus (464) Google Scholar, 22.Zhou K. Wang Y. Gorski J.L. Nomura N. Collard J. Bokoch G.M. J. Biol. Chem. 1998; 273: 16782-16786Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 23.Van Aelst L. D'Sousa-Scorey C. Gen. Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2083) Google Scholar). Rho family proteins have been implicated in controlling both transcriptional activation and a wide range of cell morphological changes, including mitosis, nerve outgrowth, and immune cell motility (24.Minden A. Lin A. Claret F.-X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1444) Google Scholar, 25.Coso O.A. Cjoaroe M., Yu, J.-C. Teramot H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1559) Google Scholar, 26.Vojtek A.B. Cooper J.A. Cell. 1995; 82: 527-529Abstract Full Text PDF PubMed Scopus (253) Google Scholar, 27.Dreschel D.N. Hyman A.A. Hall A. Glotzer M. Curr. Biol. 1997; 7: 12-23Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 28.Dutartre H. Davoust J. Gorvel J.P. Chavrier P. J. Cell Sci. 1996; 109: 367-377Crossref PubMed Google Scholar, 29.Luo L. Hensch T.K. Ackerman L. Barbel S. Jan L.Y. Jan Y.N. Nature. 1996; 379: 837-840Crossref PubMed Scopus (392) Google Scholar, 30.Allen W.E. Zicha D. Ridley A.J. Jones G.E. J. Cell Biol. 1998; 141: 1147-1157Crossref PubMed Scopus (445) Google Scholar). Their effects on cell shape are mediated by regulation of the actin-myosin cytoskeleton, and each of the family members has been shown to have different specific effects on actin in studies of fibroblast morphology (31.Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5185) Google Scholar, 32.Nobes C.D. Hall A. J. Cell Biol. 1999; 144: 1235-1244Crossref PubMed Scopus (1204) Google Scholar, 33.Nobes C. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3700) Google Scholar). Rho stimulates formation of stress fibers and impacts on focal adhesion formation; Rac promotes cell ruffles and lamellipodia; and Cdc42 controls the extension of filopodia (34.Chant J. Stowers L. Cell. 1995; 81: 1-4Abstract Full Text PDF PubMed Scopus (260) Google Scholar). The Rho GTPases also influence each other's activity: Cdc42 activates Rac and can antagonize Rho activation, and in more limited cases, Rac can activate Rho (33.Nobes C. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3700) Google Scholar, 34.Chant J. Stowers L. Cell. 1995; 81: 1-4Abstract Full Text PDF PubMed Scopus (260) Google Scholar).Rho family proteins can coordinate multiple signaling pathways through their ability to regulate both the cytoskeleton and transcription. They induce rearrangements of the actin scaffolding to control the organization and interactions of signaling molecules (35.Kong Y.Y. Fischer K.D. Mariathasan S. Bachmann M. Kozieradski I. Nghiem M.P. Bouchard D. Bernstein A. Ohashi P.S. Penninger J.M. J. Exp. Med. 1998; 188: 2099-2111Crossref PubMed Scopus (84) Google Scholar, 36.Penninger J. Crabtree G.R. Cell. 1999; 96: 9-12Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), whereas they directly stimulate transcriptional cascades (24.Minden A. Lin A. Claret F.-X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1444) Google Scholar, 25.Coso O.A. Cjoaroe M., Yu, J.-C. Teramot H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1559) Google Scholar, 26.Vojtek A.B. Cooper J.A. Cell. 1995; 82: 527-529Abstract Full Text PDF PubMed Scopus (253) Google Scholar). They also control the formation of “signaling organelles” such as focal adhesion complexes that affect both extracellular matrix signaling and anchor actin fibers (37.Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-519Crossref PubMed Scopus (1647) Google Scholar). Proteins that modulate Rho family nucleotide state contain both catalytic and localization domains (19.Bokoch G.M. Bohl B.P. Chuang T.-H. J. Biol. Chem. 1994; 269: 31674-31679Abstract Full Text PDF PubMed Google Scholar, 20.Lamarche N. Hall A. Trends Genet. 1994; 10: 346-440Abstract Full Text PDF Scopus (210) Google Scholar, 21.Cerione R.A. Zheng Y. Curr. Opin. Cell Biol. 1996; 8: 216-222Crossref PubMed Scopus (464) Google Scholar, 22.Zhou K. Wang Y. Gorski J.L. Nomura N. Collard J. Bokoch G.M. J. Biol. Chem. 1998; 273: 16782-16786Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 23.Van Aelst L. D'Sousa-Scorey C. Gen. Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2083) Google Scholar), indicating that control of cell morphology through localization of Rho family activity is coordinated with activation of downstream pathways. This information suggested to us that the Rho proteins might also regulate the signaling events controlling rapid apoptosis through control of the actin cytoskeleton. We show here that, although Rho family GTPases do not themselves induce rapid cell death, their activity is critical for Fas and CTL death signaling. We also show that Rho proteins modulate CTL and Fas signaling through their effects on the actin cytoskeleton.DISCUSSIONOur results demonstrate that the Rho family proteins play a role in the induction of apoptosis by CTLs and Fas. C. difficiletoxin B, a specific inhibitor of all Rho proteins (48.Just J. Selzer J. Wilson M. von Eichel-Streiber C. Mann M. Aktories K. Nature. 1995; 375: 500-503Crossref PubMed Scopus (872) Google Scholar), blocks both Fas- and CTL-induced apoptosis. Furthermore, transfection with mutants of each family member affects the ability of Fas and CTLs to induce apoptosis; activated mutants stimulate killing, and dominant-negative mutants inhibit it. We also demonstrated that Fas stimulation increases the level of Cdc42 activation within 5 min. Rho proteins are known to control some aspects of apoptotic morphological changes (11.Mills J.C. Stone N.L. Erhardt J. Pittman R.N. J. Cell Biol. 1998; 140: 627-636Crossref PubMed Scopus (407) Google Scholar), so we were careful to establish that the GTPases were not affecting only late stage apoptotic morphological events, but were in fact regulating the induction of apoptosis. This was most conclusively demonstrated by inhibiting Fas activation of caspase-3 using Clostridiumtoxin B. Caspase-3 cleavage and activation are considered to be critical steps in the commitment to apoptosis in Fas-induced cell death (50.Zheng T.S. Schlosser S.F. Dao T. Hingorani R. Crispe I.N. Boyer J.L. Flavell R., A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13618-13623Crossref PubMed Scopus (226) Google Scholar). More important, we observed that both activated and inhibitory GTPase mutants had no effect on cell death in the absence of apoptotic stimuli, despite their strong influence on CTL- and Fas-induced killing. In summary, these results demonstrate that the Rho family GTPases regulate the induction of apoptosis produced by CTLs and Fas, but that activation alone is not sufficient to produce cell death during the time required for killing by CTLs and Fas.There have been previous reports indicating that Rac plays a role in Fas-induced apoptosis. Rac antisense oligonucleotides and dominant-negative Rac mutants have been shown to inhibit Fas-induced killing of T cells after 3 h (52.Gulbins E. Coggeshall K.M. Brenner B. Sclottmann K. Linderkamp O. Lang F. J. Biol. Chem. 1996; 271: 26389-26394Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Rac activation by Fas ligand has been demonstrated (52.Gulbins E. Coggeshall K.M. Brenner B. Sclottmann K. Linderkamp O. Lang F. J. Biol. Chem. 1996; 271: 26389-26394Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Dominant-negative Rac inhibited apoptosis produced by tumor necrosis factor, whereas dominant-negative Rho had no effect (53.Esteve P. Embade N. Perona R. Jimenez B. del Peso L. Leon J. Arends M. Miki T. Lacal J.C. Oncogene. 1998; 17: 1855-1869Crossref PubMed Scopus (88) Google Scholar). In contrast to these and the present data, other studies have shown that transfection with activated Cdc42 and Rac alone is sufficient to induce apoptosis in some cells (16.Bazenet C.E. Mota M.A. Rubin L.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3984-3989Crossref PubMed Scopus (114) Google Scholar, 51.Chuang T.-H. Hahn K.M. Lee J.-D. Danley D.E. Bokoch G.M. Mol. Biol. Cell. 1997; 8: 1687-1698Crossref PubMed Scopus (82) Google Scholar). Ashkenazi and Dixit (12.Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5112) Google Scholar) and Evan and Littlewood (12.Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5112) Google Scholar, 15.Evan G. Littlewood T. Science. 1998; 281: 1317-1322Crossref PubMed Scopus (1360) Google Scholar) have pointed out that apoptotic stimuli can be divided into two groups: perturbations of normal homeostasis or stimuli that interact with cellular molecules specifically evolved to induce apoptosis. Perturbations of homeostasis such as growth factor withdrawal (13.Rawson C.L. Loo D.T. Duimstra J.R. Hedstrom O.R. Schmidt E.E Barnes D.W. J. Cell Biol. 1991; 113: 671-680Crossref PubMed Scopus (63) Google Scholar), loss of cell-matrix attachment (14.Frisch S.M. Francis H. J. Cell Biol. 1994; 124: 619-626Crossref PubMed Scopus (2749) Google Scholar), and DNA damage (15.Evan G. Littlewood T. Science. 1998; 281: 1317-1322Crossref PubMed Scopus (1360) Google Scholar) produce apoptosis much more slowly than direct interaction with apoptosis-inducing molecules such as CTL granzymes, Fas ligand, or tumor necrosis factor. This direct stimulation of apoptotic pathways occurs in minutes to hours. Our measurements of apoptosis due to activation of Rho proteins alone were made at the short times required for CTL and Fas killing. Previous reports examined effects of activated GTPase mutants after 24 h or longer. Thus, our data do not contradict earlier work, but instead indicate that Rho family proteins affect direct induction of apoptosis by mechanisms different from those in apoptosis produced by perturbation of homeostasis.In studies where apoptosis was produced through perturbation of normal homeostasis, both inhibitory and stimulatory activities of Rho GTPases have been reported. Activation of Rac and Rho inhibits apoptosis produced by growth factor deprivation (17.Nishida K. Kaziro Y. Takaya S. Oncogene. 1999; 18: 407-415Crossref PubMed Scopus (80) Google Scholar, 54.Gomez J. Martinez C. Giry M. Garcia A. Rebollo A. Eur. J. Immunol. 1997; 27: 2793-2799Crossref PubMed Scopus (62) Google Scholar), and Rho activation inhibits apoptosis induced by DNA damage (55.Fiorentini C. Matarrese P. Straface E. Falzano L. Fabbri A. Donelli G. Cossarizza A. Boquet P. Malorni W. Exp. Cell Res. 1998; 242: 341-350Crossref PubMed Scopus (81) Google Scholar) and loss of cell adhesion (18.Bobak D. Moorman J. Guanzon A. Gilmer L. Hahn C. Oncogene. 1997; 15: 2179-2189Crossref PubMed Scopus (70) Google Scholar). Previously published experiments using activated or inhibitory mutants of Rac, Rho, or Cdc42 indicate that these proteins potentiate apoptosis produced by withdrawal of serum or nerve growth factor (16.Bazenet C.E. Mota M.A. Rubin L.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3984-3989Crossref PubMed Scopus (114) Google Scholar,53.Esteve P. Embade N. Perona R. Jimenez B. del Peso L. Leon J. Arends M. Miki T. Lacal J.C. Oncogene. 1998; 17: 1855-1869Crossref PubMed Scopus (88) Google Scholar). Mechanistic studies have revealed that Cdc42 and Rac induce apoptosis through activation of JNK and/or p38 mitogen-activated protein kinase signaling cascades (16.Bazenet C.E. Mota M.A. Rubin L.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3984-3989Crossref PubMed Scopus (114) Google Scholar, 51.Chuang T.-H. Hahn K.M. Lee J.-D. Danley D.E. Bokoch G.M. Mol. Biol. Cell. 1997; 8: 1687-1698Crossref PubMed Scopus (82) Google Scholar) and that inhibition by Rac proceeds through activation of phosphatidylinositide 3′-hydroxykinase and Akt (17.Nishida K. Kaziro Y. Takaya S. Oncogene. 1999; 18: 407-415Crossref PubMed Scopus (80) Google Scholar).The Rho family proteins control the actin cytoskeleton via multiple downstream effectors, resulting in complex localized changes in actin structures and focal adhesions (23.Van Aelst L. D'Sousa-Scorey C. Gen. Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2083) Google Scholar, 31.Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5185) Google Scholar, 37.Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-519Crossref PubMed Scopus (1647) Google Scholar). Our data show that the Rho family proteins alter apoptotic signaling, but cannot themselves induce apoptosis. This is consistent with a mechanism in which Rho proteins alter the cytoskeletal scaffolding required for apoptosis induction and/or affect pathways by which cytoskeletal changes induce apoptosis (35.Kong Y.Y. Fischer K.D. Mariathasan S. Bachmann M. Kozieradski I. Nghiem M.P. Bouchard D. Bernstein A. Ohashi P.S. Penninger J.M. J. Exp. Med. 1998; 188: 2099-2111Crossref PubMed Scopus (84) Google Scholar, 36.Penninger J. Crabtree G.R. Cell. 1999; 96: 9-12Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). This is in agreement with recent studies showing that caspases assemble into filamentous cytoplasmic structures during apoptosis (56.Siegel R.M. Martin D.A. Zheng L. Ng S.Y. Bertin J. Cohen J. Lenardo M.J. J. Cell Biol. 1998; 141: 1243-1253Crossref PubMed Scopus (214) Google Scholar). Furthermore, the extracellular matrix controls apoptotic susceptibility by interacting with focal adhesion components. Rho influences the number and nature of focal adhesions, and both Cdc42 and Rho can induce focal complexes and alter the filamentous actin network anchored to focal adhesions.We tested the involvement of actin in Rho family modulation of apoptotic signaling. Latrunculin, an inhibitor of actin polymerization, strongly affected caspase activation by Fas. Recently published studies have shown that jasplakinolide, a molecule that stabilizes polymerized actin structures, increases apoptosis (57.Posey S.C. Bierer B.E. J. Biol. Chem. 1999; 274: 4259-4265Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Latrunculin blocked the ability of constitutively active Rho family members to enhance CTL- and Fas-induced killing. These experiments indicate that polymerized actin structures are required for apoptosis induction and that Rho family proteins do not operate on a pathway independent of their ability to regulate the actin cytoskeleton. This evidence is consistent with a mechanism in which Rho modulation of actin affects Fas and CTL signaling, possibly through rearrangement of actin scaffolds, alterations in focal adhesion signaling, or cytoskeletal changes accompanying Fas receptor capping. These models are supported by our experiments in which point mutations affecting the ability of Rac to alter cortical actin interfered with its ability to increase apoptosis. The facts that Cdc42 is stimulated rapidly, within 5 min of Fas addition, and that simulation recedes well before caspase activation suggest that Rho proteins participate in early events of receptor-mediated signaling. Previous reports also show rapid activation of Rac by Fas (52.Gulbins E. Coggeshall K.M. Brenner B. Sclottmann K. Linderkamp O. Lang F. J. Biol. Chem. 1996; 271: 26389-26394Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar).Our data show a strong correlation between morphological features produced by Rho proteins and susceptibility to apoptosis. This further supports a mechanism in which the Rho GTPases affect apoptosis by modulating the cytoskeleton. Our observations are consistent with previous studies demonstrating a connection between cell shape and susceptibility to apoptosis. Ingber and co-workers (58.Chen C.S. Mrksich M. Huang S. Whitesides G.M. Ingber D.E. Science. 1997; 276: 1425-1428Crossref PubMed Scopus (4144) Google Scholar, 59.Chicurel M.E. Chen C.S. Ingber D.E. Curr. Opin. Cell Biol. 1998; 10: 232-239Crossref PubMed Scopus (447) Google Scholar) have elucidated mechanisms by which mechanical stresses control cell death. It was shown that cell shape strongly influences the extent of apoptosis regardless of the total area of contact with cell-surface molecules. Using a system distinct from that utilized in our studies, these workers observed that spread cells were less susceptible to apoptosis. This apparent contradiction suggests that apoptosis requires specific cytoskeletal structures whose formation does not correlate with gross morphological features like the extent of cell spreading.The work described here further dissects the complex multifunctional role of the Rho proteins in apoptosis induction. The mechanisms by which cytoskeletal dynamics impact upon cell signaling remain poorly understood. In future studies, we hope to identify specific actin structures and dynamics involved in apoptosis induction and elucidate their control by Rho family signaling. Apoptosis is a form of “cell suicide” in which cells fragment their cytoplasm, nucleus, and DNA for orderly disposal (1.Martin S.J. Green D.R. Cotter T.G. Trends Biochem. Sci. 1994; 19: 26-30Abstract Full Text PDF PubMed Scopus (399) Google Scholar, 2.McConkey D.J. Orrenius S. Trends Cell. Biol. 1994; 4: 370-374Abstract Full Text PDF PubMed Scopus (159) Google Scholar). It is ubiquitous and essential to normal homeostasis in higher animals and is important in modulating immune cell populations for defense against cancer and virus-infected cells (3.Glinsky G.V. Glinsky V.V. Ivanova A.B. Hueser C.J. Cancer Lett. 1997; 115: 185-193Crossref PubMed Scopus (134) Google Scholar, 4.Amiesem J.C. Estaquier J. Idziorek T. Immunol. Rev. 1994; 142: 9-34Crossref PubMed Scopus (85) Google Scholar). Apoptosis is induced through activation of caspases, which activate one another in a proteolytic cascade (5.Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6133) Google Scholar), leading to cleavage of cytoskeletal and signaling molecules that result in irreversible changes in cell morphology and the plasma membrane (6.Rudel T. Bokoch G.M. Science. 1997; 276: 1571-1574Crossref PubMed Scopus (602) Google Scholar, 7.Janicke R.U. Ng P. Sprengart M.L. Porter A.G. J. Biol. Chem. 1998; 273: 15540-15545Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 8.Kothakota S. Azuma T. Reinhard C. Klippel A. Tang J. Chu K. McGarry T.J. Kirschner M.W. Koths K. Kwiatkowski D.J. Williams L.T. Science. 1997; 278: 294-298Crossref PubMed Scopus (1031) Google Scholar, 9.Caulin C. Salvesen G.S. Osima R.G. J. Cell Biol. 1997; 138: 1379-1394Crossref PubMed Scopus (542) Google Scholar, 10.Wen L.P. Fahrni J.A. Troie S. Guan J.L. Orth K. Rosen G.D. J. Biol. Chem. 1997; 272: 26056-26061Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). The Rho family of small GTPases plays a role in the terminal morphological changes of apoptosis (11.Mills J.C. Stone N.L. Erhardt J. Pittman R.N. J. Cell Biol. 1998; 140: 627-636Crossref PubMed Scopus (407) Google Scholar), but previous reports present conflicting data about its role in apoptosis induction. Apoptotic signaling pathways can be grouped into two distinct categories (12.Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5112) Google Scholar): mechanisms that induce rapid apoptosis through specific molecules that have evolved to produce cell death (i.e. Fas ligand or cytotoxic T cell granzymes) (12.Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5112) Google Scholar) and pathways that lead to apoptosis when normal homeostatic signaling is disrupted (13.Rawson C.L. Loo D.T. Duimstra J.R. Hedstrom O.R. Schmidt E.E Barnes D.W. J. Cell Biol. 1991; 113: 671-680Crossref PubMed Scopus (63) Google Scholar, 14.Frisch S.M. Francis H. J. Cell Biol. 1994; 124: 619-626Crossref PubMed Scopus (2749) Google Scholar, 15.Evan G. Littlewood T. Science. 1998; 281: 1317-1322Crossref PubMed Scopus (1360) Google Scholar). Examples of the latter type of perturbation include serum withdrawal (13.Rawson C.L. Loo D.T. Duimstra J.R. Hedstrom O.R. Schmidt E.E Barnes D.W. J. Cell Biol. 1991; 113: 671-680Crossref PubMed Scopus (63) Google Scholar), detachment from extracellular matrix (14.Frisch S.M. Francis H. J. Cell Biol. 1994; 124: 619-626Crossref PubMed Scopus (2749) Google Scholar), and an imbalance of survival homeostatic signaling pathways (15.Evan G. Littlewood T. Science. 1998; 281: 1317-1322Crossref PubMed Scopus (1360) Google Scholar). Numerous studies of Rho family signaling in such pathways have revealed that the GTPases play a complex role, participating in both stimulatory and inhibitory paradigms (16.Bazenet C.E. Mota M.A. Rubin L.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3984-3989Crossref PubMed Scopus (114) Google Scholar, 17.Nishida K. Kaziro Y. Takaya S. Oncogene. 1999; 18: 407-415Crossref PubMed Scopus (80) Google Scholar, 18.Bobak D. Moorman J. Guanzon A. Gilmer L. Hahn C. Oncogene. 1997; 15: 2179-2189Crossref PubMed Scopus (70) Google Scholar). Downstream mediators for positive (i.e.c-Jun N-terminal kinase (JNK)1) and negative (i.e. Akt) effects have been identified. In contrast, much less is known about Rho family signaling in apoptosis produced by CTLs, Fas, and other molecules directly inducing apoptosis. Here we show that Rho family GTPases play a stimulatory role in CTL- and Fas-induced apoptosis. The Rho family of small GTPases consists of three proteins (Cdc42, Rac, and Rho) that interact with downstream effectors upon binding to GTP. Their nucleotide state and localization are controlled by >40 currently identified regulatory factors, including guanine nucleotide exchange factors, GDP dissociation inhibitors, and GTPase-activating proteins (19.Bokoch G.M. Bohl B.P. Chuang T.-H. J. Biol. Chem. 1994; 269: 31674-31679Abst" @default.
• W2120342771 created "2016-06-24" @default.
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• W2120342771 title "Rho Family Proteins Modulate Rapid Apoptosis Induced by Cytotoxic T Lymphocytes and Fas" @default.
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