Differential Involvement of Initiator Caspases in Apoptotic Volume Decrease and Potassium Efflux during Fas- and UV-induced Cell Death

Caspase activation and apoptotic volume decrease are fundamental features of programmed cell death; however, the relationship between these components is not well understood. Here we provide biochemical and genetic evidence for the differential involvement of initiator caspases in the apoptotic volume decrease during both intrinsic and extrinsic activation of apoptosis. Apoptosis induction in Jurkat T lymphocytes by Fas receptor engagement (intrinsic) or ultraviolet (UV)-C radiation (extrinsic) triggered the loss of cell volume, which was restricted to cells with diminished intracellular K+ ions. These characteristics kinetically coincided with the proteolytic processing and activation of both initiator and effector caspases. Although the polycaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone completely inhibited the Fas-mediated apoptotic volume decrease and K+ efflux, it was much less effective in preventing these processes during UV-induced cell death under conditions whereby caspase activities and DNA degradation were blocked. To define the roles of specific initiator caspases, we utilized Jurkat cells genetically deficient in caspase-8 or stably transfected with a dominant-negative mutant of caspase-9. The results show that the activation of caspase-8, but not caspase-9, is necessary for Fas-induced apoptosis. Conversely, caspase-9, but not caspase-8, is important for UV-mediated shrunken morphology and apoptosis progression. Together, these findings indicate that cell shrinkage and K+ efflux during apoptosis are tightly coupled, but are differentially regulated by either caspase-8 or caspase-9 depending on specific pathways of cell death. Caspase activation and apoptotic volume decrease are fundamental features of programmed cell death; however, the relationship between these components is not well understood. Here we provide biochemical and genetic evidence for the differential involvement of initiator caspases in the apoptotic volume decrease during both intrinsic and extrinsic activation of apoptosis. Apoptosis induction in Jurkat T lymphocytes by Fas receptor engagement (intrinsic) or ultraviolet (UV)-C radiation (extrinsic) triggered the loss of cell volume, which was restricted to cells with diminished intracellular K+ ions. These characteristics kinetically coincided with the proteolytic processing and activation of both initiator and effector caspases. Although the polycaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone completely inhibited the Fas-mediated apoptotic volume decrease and K+ efflux, it was much less effective in preventing these processes during UV-induced cell death under conditions whereby caspase activities and DNA degradation were blocked. To define the roles of specific initiator caspases, we utilized Jurkat cells genetically deficient in caspase-8 or stably transfected with a dominant-negative mutant of caspase-9. The results show that the activation of caspase-8, but not caspase-9, is necessary for Fas-induced apoptosis. Conversely, caspase-9, but not caspase-8, is important for UV-mediated shrunken morphology and apoptosis progression. Together, these findings indicate that cell shrinkage and K+ efflux during apoptosis are tightly coupled, but are differentially regulated by either caspase-8 or caspase-9 depending on specific pathways of cell death. ultraviolet-C benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone z-Ile-Glu-Thr-Asp-fmk N-acetyl-Asp-Glu-Val-Asp-p-nitroaniline Ac-Ile-Glu-Thr-Asp-p-nitroaniline Ac-Leu-glu-His-Asp-p-nitroaniline propidium iodide dominant-negative Apoptosis is a programmed form of cell death whose execution depends on the complex interplay of intracellular signaling molecules. This cell death is regulated by mechanisms that are both signal- and cell type-specific. During apoptosis, the cell undergoes a series of conserved biochemical and morphological changes, including the loss of cell volume, a defining morphological feature that distinguishes apoptosis from necrosis (1Cohen J.J. Duke R.C. Fadok V.A. Sellins K.S. Annu. Rev. Immunol. 1992; 10: 267-293Crossref PubMed Scopus (1101) Google Scholar, 2Benson R.S. Heer S. Dive C. Watson A.J. Am. J. Physiol. 1996; 270: C1190-C1203Crossref PubMed Google Scholar, 3Bortner C.D. Cidlowski J.A. Biochem. Pharmacol. 1998; 56: 1549-1559Crossref PubMed Scopus (214) Google Scholar). This process, also termed apoptotic volume decrease (4Maeno E. Ishizaki Y. Kanaseki T. Hazama A. Okada Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9487-9492Crossref PubMed Scopus (648) Google Scholar), is an early regulatory event that is essential for successful apoptosis progression (3Bortner C.D. Cidlowski J.A. Biochem. Pharmacol. 1998; 56: 1549-1559Crossref PubMed Scopus (214) Google Scholar, 4Maeno E. Ishizaki Y. Kanaseki T. Hazama A. Okada Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9487-9492Crossref PubMed Scopus (648) Google Scholar, 5Bortner C.D. Cidlowski J.A. Am. J. Physiol. 1996; 271: C950-C961Crossref PubMed Google Scholar). The signaling components modulating this event remains largely undefined, although previous studies have shown that cell shrinkage is associated with a dramatic loss of intracellular K+ and Na+ ions (2Benson R.S. Heer S. Dive C. Watson A.J. Am. J. Physiol. 1996; 270: C1190-C1203Crossref PubMed Google Scholar, 6Bortner C.D. Hughes Jr., F.M. Cidlowski J.A. J. Biol. Chem. 1997; 272: 32436-32442Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar,7Hughes Jr., F.M. Bortner C.D. Purdy G.D. Cidlowski J.A. J. Biol. Chem. 1997; 272: 30567-30576Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). Moreover, the efflux of K+ ions and the loss of cell volume correlate with several features of apoptosis, including mitochondrial membrane depolarization (8Dallaporta B. Hirsch T. Susin S.A. Zamzami N. Larochette N. Brenner C. Marzo I. Kroemer G. J. Immunol. 1998; 160: 5605-5615PubMed Google Scholar), activation of DEVDases and apoptotic nucleases (7Hughes Jr., F.M. Bortner C.D. Purdy G.D. Cidlowski J.A. J. Biol. Chem. 1997; 272: 30567-30576Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar), DNA degradation (5Bortner C.D. Cidlowski J.A. Am. J. Physiol. 1996; 271: C950-C961Crossref PubMed Google Scholar, 7Hughes Jr., F.M. Bortner C.D. Purdy G.D. Cidlowski J.A. J. Biol. Chem. 1997; 272: 30567-30576Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar), and ribosomal RNA degradation (9King K.L. Jewell C.M. Bortner C.D. Cidlowski J.A. Cell Death Differ. 2000; 7: 994-1001Crossref PubMed Scopus (31) Google Scholar). In addition to cell shrinkage, an incipient feature of many apoptotic paradigms is the activation of caspases. These proteases initiate and amplify a signaling proteolytic cascade that ultimately cleave a variety of cellular substrates and facilitate cellular disassembly (10Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6111) Google Scholar, 11Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2417) Google Scholar, 12Nicholson D.W. Cell Death Differ. 1999; 6: 1028-1042Crossref PubMed Scopus (1288) Google Scholar). Since caspase activation and the loss of cell volume are critical for execution of apoptosis, we wished to determine whether activation of initiator caspases is necessary for cell shrinkage during apoptosis. We explored the effects of Fas ligation and ultraviolet-C (UV-C)1 radiation, two distinct stimuli known to activate the extrinsic and intrinsic pathways of apoptosis, respectively. Activation of the Fas receptor by its cognate ligand or agonistic antibody ligation triggers clustering and/or trimerization of the receptor, interaction with adaptor molecules such as Fas-associated death domain adaptor protein, and the recruitment and activation of the initiator pro-caspase-8 (13Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2096) Google Scholar, 14Fernandes-Alnemri T. Armstrong R.C. Krebs J. Srinivasula S.M. Wang L. Bullrich F. Fritz L.C. Trapani J.A. Tomaselli K.J. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7464-7469Crossref PubMed Scopus (692) Google Scholar, 15Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Shevchenko A. Ni J. Scaffidi C. Bretz J.D. Zhang M. Gentz R. Mann M. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2714) Google Scholar). This death inducing signaling complex in turn activates a sequential caspase cascade, in which caspase-3 functions as a key effector caspase. In contrast, apoptosis induced by UV radiation entails a more complex system that in part involves signaling through DNA damage and cell surface receptors (16Sachsenmaier C. A. R. A. Cell. Full Text PDF PubMed Scopus Google Scholar, C. M. Science. 1996; PubMed Scopus Google Scholar, S. 1999; PubMed Scopus Google Scholar). in these pathways is the mitochondrial of that the of the which and D. I. Srinivasula S.M. M. Alnemri E.S. Wang Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, E. Science. 1997; PubMed Scopus Google Scholar, Y. L. G. J. 1999; PubMed Scopus Google Scholar, Y. Wang J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). processing of to its form a signaling cascade through effector caspases, including and Wang D.W. Alnemri E.S. J. Cell Biol. 1999; PubMed Scopus Google Scholar, M. J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Jr., Martins L.M. Srinivasula S.M. S. Alnemri E.S. W.C. Kaufmann S.H. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Here we the relationship between cell shrinkage and caspase activation in Jurkat T cells during and UV-mediated apoptosis. We show that the apoptotic volume decrease induced by both is tightly to K+ efflux, but the of the initiator caspase-8 and to the loss of cell volume and K+ ions depending on the specific apoptotic that is Jurkat T lymphocytes were from Jurkat cells genetically deficient in caspase-8 and the cells were from and J. Jurkat cells stably a dominant-negative mutant of caspase-9 were from the by a with either the or of Y. from cell were a and for cells were in with and were in a Cell in were with Fas or with a In the caspase benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone or z-Ile-Glu-Thr-Asp-fmk cells were with the for to apoptosis in cell volume were on a as C.D. Cidlowski J.A. 2000; PubMed Google Scholar). The of cell is by cell and were on to and shrunken The of shrunken cells was by of the of cell cells were with propidium iodide for to and cells were in intracellular of K+ were the as C.D. Hughes Jr., F.M. Cidlowski J.A. J. Biol. Chem. 1997; 272: 32436-32442Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar). was a and were on a to cells that a K+ to cells were from were in and with for of DNA as (5Bortner C.D. Cidlowski J.A. Am. J. Physiol. 1996; 271: C950-C961Crossref PubMed Google Scholar). with DNA were by on DNA were on either a or Caspase activities from were a to the In Jurkat cells were by for and were with and in on the was by and for the caspase of cell were in a the of of the was to of the from the was by a of was to the specific of caspases in were as of the were in for and by in a The were to membrane for as E. J. 1998; PubMed Scopus Google Scholar), that with were for caspase-3 or caspase-8, and were and specific were of the caspases were on a membrane with and for We have the of cell shrinkage during apoptosis induced by antibody or radiation by which cells of on decrease in the of with cell shrinkage C.D. Cidlowski J.A. 2000; PubMed Google Scholar). the volume of cells and induced to apoptosis with either antibody or UV the cells a and decrease in were on cells as shown in to cells with a and that were both the and shrunken cells were apoptotic induction for morphological that cells from the a well morphology that was distinct from the cells from the We the to determine the of shrunken cells in the apoptosis during which of cells were of Jurkat cells with either antibody or UV radiation induced both a loss of cell In both the apoptotic volume decrease was and as early as We the relationship between the apoptotic volume decrease and intracellular K+ in both and Jurkat the and intracellular in In contrast, with either or UV induced cell shrinkage and a decrease in that the shrunken of cells intracellular a relationship between cell and intracellular K+ is with both apoptotic To determine whether the loss of cell volume induced by antibody or UV with the activation of caspases, we the of caspase activation on the in of from the substrates or The is for caspase-8, and a for caspase-9, and and is by several caspases including and N.A. D.W. 2000; PubMed Google Scholar). The results in show that both Fas receptor ligation and UV radiation in and activities to the apoptotic volume and during Although the and UV-induced activation of and were with of a more activation of was was as a an of DEVDases in the caspase that activation of caspases is associated with the proteolytic processing of these (10Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6111) Google Scholar, 11Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2417) Google Scholar, 12Nicholson D.W. Cell Death Differ. 1999; 6: 1028-1042Crossref PubMed Scopus (1288) Google Scholar), we the of caspase-8, and from show that apoptosis induction by either antibody or UV radiation triggered a loss of pro-caspase-8 and the of two proteolytic processing was also with The of these caspases was and with the caspase and apoptotic volume In contrast, processing of both and UV these show that of Jurkat cells with these apoptotic stimuli induced a loss of cell volume, which was restricted to cells with K+ and with the of caspase To determine caspases are essential for the loss of intracellular K+ ions and cell volume, we the effects of an inhibitor of many but not caspases N.A. D.W. 2000; PubMed Google Scholar, Kaufmann S.H. W.C. Biochem. Sci. 1997; Full Text PDF PubMed Scopus Google Scholar, M. B. R. D.W. N.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Fritz L.C. 1999; PubMed Scopus Google Scholar, J. Cell Death Differ. 1999; 6: PubMed Scopus Google Scholar). The of was by its to caspase activities in and cell the completely inhibited the and UV-induced and activities as well as the processing of caspase-8, and not We also DNA degradation as a known event of apoptosis M. K. A. S. 1998; PubMed Scopus Google to the of in the by or both and UV-induced DNA both caspase activation and DNA degradation during either or UV-induced apoptosis. We the of to cell shrinkage and K+ loss during apoptosis. Although on cell volume, it completely cell shrinkage and K+ loss induced by Fas receptor ligation but was much less effective UV radiation a that to caspase activities or DNA degradation is important to that the to proteases such as S. R. 1999; PubMed Scopus Google and 1999; PubMed Google Scholar). these findings an essential of a for the apoptotic volume decrease and K+ efflux in the Fas but a involvement in the UV We that both the apoptotic volume decrease and K+ efflux induced by antibody a with the of caspase-8 in the Fas was the UV These that and proteases are critical for the Fas apoptotic volume decrease and K+ but a in the UV of on and UV-induced apoptotic volume decrease and Jurkat cells were with or for and to antibody or UV radiation for an Cell volume and intracellular were as in and results shown are the from Although caspase-8 an during the of Fas apoptosis (13Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2096) Google Scholar, 14Fernandes-Alnemri T. Armstrong R.C. Krebs J. Srinivasula S.M. Wang L. Bullrich F. Fritz L.C. Trapani J.A. Tomaselli K.J. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7464-7469Crossref PubMed Scopus (692) Google Scholar, 15Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Shevchenko A. Ni J. Scaffidi C. Bretz J.D. Zhang M. Gentz R. Mann M. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2714) Google Scholar), the of this caspase in the UV apoptotic is not well understood. have that UV radiation the Fas to apoptosis A. Chinnaiyan A.M. Dixit V.M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, R. J. Immunol. 1998; Google Scholar, Y. D. D. G. B. T. J. Cell Biol. 1998; PubMed Scopus Google Scholar, A. S. K. B. G. T. J. Immunol. 1998; 160: Google Scholar), and caspase-8 also in the UV To this we utilized Jurkat cells genetically deficient in caspase-8 J. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of the of caspase-8 in the cells but the of caspase-9 and to with cells processing of caspase-8, and in a the processing of caspase-9 or was in the the initiator of caspase-8 in the Fas-mediated Conversely, the cells were to UV radiation, caspase-9 and were to a as the Fas processing of caspase-9 and of caspase-8 UV We these findings by specific caspase activities in the and with the results in from cells with antibody a in and such were in the mutant cells In contrast, UV radiation induced activation of and DEVDases in both and caspase-8 mutant a in was in the cells UV but not This UV-induced from such as and caspase activities the of UV-induced caspase activation in the mutant cells to a of caspase-8 to the caspase To determine whether caspase-8 is for the apoptotic volume decrease and K+ efflux, and cells were to or UV radiation and the apoptotic volume decrease was by Although antibody induced a loss of cell volume and intracellular K+ in the cells were completely and and through These correlate with the caspase activation in the cells UV the cells shrunken in a with a loss of intracellular K+ ions and These were by to cells and were to the of caspase activities in caspase-8 to the UV apoptotic but is not necessary for cell shrinkage and K+ Although caspase-8 not an in the UV it is that an initiator caspase a is caspase-9, an initiator caspase of the of caspase activation D. 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A. 1998; PubMed Scopus Google Scholar). from the cells an of caspase-9 that as a distinct to the This to the mutant not Fas the processing of caspase-9, and was either or in cells the mutant with the These with the caspase of from cells In to however, the of caspase processing by caspase-9 was more and with the the activities of caspases and were largely with of caspase-9 to a was in from the mutant cells either Fas or UV The of this processing is the of the mutant was by the of the mutant cells to and UV-induced DNA degradation This was not to a in the caspase-9 mutant cells DNA degradation was the of the we utilized these cells to determine the of caspase-9 in the apoptotic volume decrease and K+ efflux during apoptosis. the mutant the loss of cell volume and intracellular K+ ions either or UV but this was the Fas-mediated the mutant cells to the apoptotic volume DNA and death to In contrast, the mutant cells these apoptotic features that caspase-9 a more in the UV We have shown that cell shrinkage by is to initiate apoptosis in cells (5Bortner C.D. Cidlowski J.A. Am. J. Physiol. 1996; 271: C950-C961Crossref PubMed Google Scholar). This apoptotic volume decrease is associated with the efflux of K+ and the shrunken cells DNA of mitochondrial membrane and activation of effector caspases (5Bortner C.D. Cidlowski J.A. Am. J. Physiol. 1996; 271: C950-C961Crossref PubMed Google Scholar, 6Bortner C.D. Hughes Jr., F.M. Cidlowski J.A. J. Biol. Chem. 1997; 272: 32436-32442Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar, 7Hughes Jr., F.M. Bortner C.D. Purdy G.D. Cidlowski J.A. J. Biol. Chem. 1997; 272: 30567-30576Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar), that the loss of cell volume is a necessary of the apoptotic with this and (4Maeno E. Ishizaki Y. Kanaseki T. Hazama A. Okada Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9487-9492Crossref PubMed Scopus (648) Google have in of cell the apoptotic volume decrease to DNA and activation of We have the of initiator caspases in the loss of cell volume and K+ efflux in both intrinsic and extrinsic signaling pathways of apoptosis. We show that the involvement of specific caspases in the apoptotic volume decrease is on the In the Fas the of the apoptotic volume decrease with the of and activation of caspase-8 and from show that the shrunken morphology during Fas-induced apoptosis is on the activation of and proteases and studies defining the Fas have caspase-8 as the which in the activation of effector caspases (13Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2096) Google Scholar, 14Fernandes-Alnemri T. Armstrong R.C. Krebs J. Srinivasula S.M. Wang L. Bullrich F. Fritz L.C. Trapani J.A. Tomaselli K.J. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. 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