- Email: [email protected]
NO INDUCED APOPTOSIS ACCOMPANYING THE CHANGE OF ONCOPROTEIN EXPRESSION AND THE ACTIVATION OF CPP32 PROTEASE
Life Sciences, Vol. 62, No. 3, pp. 239–245, 1998 Copyright 01997 EIsevier Science Inc. Printed in the USA. All rights reserved 0024-3205&8 $19.00 + .OU
E
PII S0024-3205(97)01092-8
NO INDUCED APOPTOSIS ACCOMPANYING THE CHANGE OF ONCOPROTEIN EXPRESSION AND THE ACTIVATION OF CPP32 PROTEASE Eisuke Nishio* and Yasuhiro Watanabe Departmentof Pharmacology, NationalDefense MedicalCollege 3-2, Namiki, Tokorozawa, Saitama, Japan 359 (Received in final form October 31, 1997)
Previously we have shown that nitric oxide (NO) donors induced apoptosis in vascular smooth muscle cells (VSMCS).However, the mechanisms by which NO induced apoptosis in VSMCSare entirely unknown. In the present study, we intended to identify the mechanism by which NO donors induce apoptosis in VSMCS. Firs~ we evaluatedthe expression of c-Myc, P53, and BcI-2 proteins in VSMCStreated by NO donors. c-Myc and P53 protein expression increased after VSMCSwere incubatedwith NO donors for 6 hr and reached a maximumlevel at 24 hr, while Bc1-2protein decreased after 12 hr incubation. Next we investigated to see whether the CPP32 protease activation was involved in NO donorsinduced apoptosis. In VSMCS treated by NO donors, the increase of CPP32 protease activity was observed and specific inhibition of CPP32 activity significantly prevented apoptosis induced by NO donors in a dose-dependent manner. These results suggest that NO donors induced apoptosis through protooncoproteinexpression and CPP32-like protease activation.
Key Wor&: nitric oxide, c-Myc,P53, Bc1-2,CPP32
It is now well established that apoptosis is a physical cell death process, important for normal developmentand involved in many pathologicalconditions (1). Recently, in the advanced atheroma lesion, apoptosis of VSMCShas been reported to accompanyproliferationof VSMCS, macrophages and T lymphocytes (2). Previously, we demonstrated that NO donors induce apoptosis in VSMCSthrough a cGMPindependent mechanism (3). But the precise mechanism is unknown. The present study was performed to investigate the mechanism by which NO donors induced apoptosis in VSMCS, particularly through the expression of the apoptosis-related gene product or the activation of protease. NO is known to act as an intracellularmediator in several cells (4). Further, it is known that VSMC, microphage, and endothelial cells, which are components of atherosclerotic lesion, release NO through nitric oxide synthase (5). Therefore it is important to investigate the mechanism by . . whi..&NO induces a.poptos]sm VSMCSin alherog nesis. * To whom correspondenceshould be addressed Fea: (0429)-%-5191, Tel: (0429)-95-1484
240
NO DonorsandApoptosisin VSMCS
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62, No. 3, 199S
Two gene families are known to be important in apoptosis control (6). These are, first the genes encoding the interleukin-l~-converting enzyme family of cystein protease and, second, those related to proto-oncogene Bc1-2. Both of these families are homologous to cell death genes in C. elegans (7). In mammaliancells the number of members of both families is growing rapidly and an effort is being directedtowards establishing the roles played by each member in apoptosis. Other genes with established roles in the regulation of proliferation and differentiation are also important in controlling apoptosis. Several of these are known proto-oncogenes, e.g. c-Myc, or tumor suppressers, e.g. P53. P53 induced apoptosis through the positive regulation of Bax (8) and negative regulation of Bc1-2(9). Despite the evidence that c-Myc protein in tissue culture leads to proliferation, overexpression of c-Myc can accelerate cell death via apoptosis (10). This is not surprising since the death signaling domain of c-Myc is identical to that for proliferation (11). In certain conditions, the dysregulation of the P53, c-Myc, and Bc1-2 proto-oncogene can cause apoptosis in human malignancies (12). In this contex~ we investigated whether these proteins change in NOdonors induced apoptosis or not, and whether protease activationis involved in NO induced apoptosis. MATERIALS AND ME@HQQS
S-nitroso-N-acetyl penicillamine (SNAP) was obtained from BIOMOL Research Laboratories inc. [3H] thymidine and ECL Western blotting detection kit were from Amersham. AUcell culture materials were from Life Technologies. Ac-Asp-Glu-Val-Asp-CHO (inhibitor of caspase-3 (CPP32)), Ac-Tyr-Val-Ala-Asp-CHO(inhibitorof caspase-1 (ICE)) and Ac-Val-Glu-Ile-Asp-CHO (inhibitor of caspase-6 (Mch2)) were from Peptide Institute, INC. (Osaka). Anti BcL2, c-Myc and P53 antibodies were from OncogeneScience. Anti-CPP32 was from Santa Cruz Biochemical. Cell Cb Aortic VSMCS were obtained from thoracic aorta of the Japanese white rabbit by the method described previously (13). The ce11s(lx105) were seeded into 35-mm diameter dishes and maintainedin 2 ml of Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum at 37~ in a humidifiedatmosphere of 5?Z0 COZ/ 95?Z0air. The cells were used between the third and fifth passage. Cells were grown to confluence, at which time they were rendered quiescent by serum deprivationand maintainedin serum-free mediumfor 24h before experimentation.
Subconfluent plates of SMCS were labeledwith [’H]thymidine (lmCi/ ml) for 24hr. Then after the cells were incubated with the indicated media, DNA fragmentation was quantitated as follows: 0.5 ml of Iysis buffer was added to each culture well and mixed by pipetting, and the suspension was transferred to an Eppendorf tube, incubated on melting ice for 10 rnin, and centrifuged at 8,000g for 5 min at 4“C. Subsequently, fragmented radiolabeled DNA was counted in the supernatant by liquid scintillation counting. Radioactivity of cells treated with lysis buffer and ultrasound homogenator was used as total activity. Results are expressed as fragmented DNA as a percentageof total DNA ( 14). LDH was measured as previously described (15). CPP37.like protease acti“vity The fluorescent substrate MCA-DEVDAPK (drip) was incubated at 30”C for 30min with cytosolic extracts prepared from NCMreatedVSMCS, and the fluorescence of the cleaved substrates was determined using a spectrofluorometer set at an excitation wavelength of 325nm and an
Vol. 62
No.
NO Donors and Apoptosis in VSMCS
3, 1998
emission wavelength of 392nm ( 16).
Cell lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred electrophoreticallyto PVDF membranes. The membranes were probed with anti-Bcl-2 , P53, and cMyc mouse antibodies. After treating the membrane with peroxidase-conjugated goat anti-mouse secondary antibodies, peroxidase activity was detected using ECL reagents (17). CPP32/p17 was detected with the primary antibody against human CPP32/p17 and peroxidase-conjugatedanti-goat antibody, followed by enhanced ECL reagents.
All the results are expressed as the mean t SD and statistical analysis was performed with ANOVAfollowed by a modified Bonferroni LSD test (SPSS Software). Differences with a p value of less than 0.05 were considered significant. ULTSND
DISCUSSION
ated VSMCS Previously, we demonstratedthat two commonly used NO donors, SNAP and SIN-1, induced apoptosis (3) by demonstrating DNA fragmentation through agarose gel electrophoresis and TUNEL method, but the underlying mechanism is unknown. Fig. 1 shows that SNAP induced apoptosis in a time-dependentmanner. A significant apoptosis was induced by SNAP (100pM) at 8 hr incubation. LDH release did not increase = IOOpMSNAP (105*8% compared with control cells) excludingthe inductionof necrosis. Several gene products are known to be important in controlling the apoptotic process. To determine if NO donors have any effects on the expression level of these products, the levels of the proteins were studied by Western blotting with antibodies against P53, c-Myc, and BcI-2 proteins. Results are shown in Figure 2. The P53 protein began to increase after 6hr incubation, reached a maximum level after 24hr incubationand thereafterdecreased. The c-Myc protein began to increase after 6hr incubation, reached a maximum level after 24hr and subsequently decreased. In contrast, the expression of BcI-2 protein decreased after 12hr incubation. It decreased to a minimum level after 24hr incubation. Two gene products, c-Myc and P53 (12), were shown to be implicated in the etiology of most human tumors. Bc1-2was shown to block cell death initiated by many different stimuli. The precise biochemicalmechanisms by which Bc1-2family proteins exert their influence on cell death remain far from clear, though several theories have been advanced, including cysteine protease activation (18) and targeting the protein kinase Raf-1 to mitochondria(19). Increased expression of both P53 and c-Myc protein appeared before the occurrence of significantapoptosis (Fig. 1). These data may suggest that up-regulationof P53 and c-Myc proteins, as well as signals triggering cleavage of ribosomal RNA (20), may be a pre-requisite for DNA fragmentation and apoptosis. One possible mechanism to be considered is a direct modulatory action of NO on P53 activity or the protein stability by targeting cysteine residues normally implicatedin zinc binding (21). However, further studies are required to find a mechanism whereby NO regulatesthe apoptotic-regulatorygene product, Also, it is necessary to elucidatethe cause-andeffect relationship between NO and the apoptosis-regulatorygene product. . . . . NO-do~ by CPP32 Md2mr.r Evidence is growing that CPP32 may play an importantrole in the apoptotic pathway. CPP32 is expressed as a 32-KDa precursor which is processed into active p17 and p12 subunits upon activation(22). Therefore, we determined whether CPP32 is activatedduring apoptosis induced by NO donors. VSMCS expressed the 32-KDa preform of CPP32, and treatment with NO-donors generated p17 subunits (Fig.3). NO also significantly increased CPP32-like protease activity
242
NO Donors and Apoptosis in VSMCS
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(128*1 1% of controls). Furthermore, we investigated whether NOinduced apoptosis is antagonizedby the inhibitionof CPP32-like activityby the specifictetrapeptideinhibitor Ac-DEVD CHO. Figure 4 shows that the Ac-DEVD-CHOinhibits apoptosis induced by NOdonors in a dosedepcndent manner. In contrast, both AC-YVA~CHO, the inhibitor of ICE, and Ac-VEID-CHO, the inhibitor of Mch2, does not significantlyinhibit apoptosis induced by NO donors. These results suggest that CPP32 activation is involved in apoptosis in NOdonor treated VSMCS. However, further studies are required to elucidatethe mechanismwhereby NO activatesCPP32 protease. One possible mechanismis that the downregulationof Bc1-2may result in the CPP32 activation( 18). In summary, these results show that NOinduced apoptosis is accompaniedby the accumulation of P53 and c-Myc, the downregulation of BcI-2 and the activation of CPP32 protease. However, further study is needed to answer the question concerning the significance of gene product regulationand protease activationin regulatingof apoptosis.
50
-—o—
**
40
30
20
10
o~ 0
10
5
(hr) Fig. 1. Time-dependentinduction of apoptosis in VSMCSby NO donor, SNAP (1OOPM). Serum deprived VSMCS were treated with SNAP (100@1) for the indicated time. DNA fragmentation is expressed as a percentage of total DNA by [3H] thymidine assay as described in Methods and represents the average and range of three independent experiments in duplicate. Significantlydifferentfor the control at the indicatedtime (*P-4.05, **P
—
NODonorsandApoptosisin VSMCS
Vol.62 No.3, 1998
243
P 53-
c – Myc +
Bcl – 2 + Fig.2. Differentialmodulation of P53, c-Myc, and Bc1-2during apoptosis induced by NO donor, SNAP (1OOPM). Serum deprived VSMCStreated with SNAP (100pM) for indicated times were subjected to Iysis and Western blot analysis and probed with antibodies specific proteins. Each experimentwas repeatedthree times and a representativeblot is shown above.
KD
The induction of CPP32 protease activatii;g;~”VSMCs by NO donor, SNAP (1OOPM). Serum deprived VSMCS treated with SNAP (100pM) for 24hr were subjected to Iysis and Western blot analysis and probed with antibodies specific CPP32 protease proteins. Each experiments was repeatedthree and a representativeblot is shown above.
244
::: ,\< ,., ::: 1
NO Donors and Apoptosis in VSMCS
1
;:; ::: ::: ::: ::: ::: ::: :.:
8 10 0
Vol. 62, No. 3, 1998
❑ no addition or SNAP(100pM) ❑ +Ac-DEVD (10- 1OOOYM) El +Ac-YVAD (10- 1OOOPM) E +Ac-VEID (10-1OOOPM)
z
$$ Zz ram Effect of CPP32 inhibitor on the induction of apoptosis in VSMC by SNAP (1OOJJM).Serum deprived VSMCS treated with SNAP (100p@ ad various tetrap~ptideinhibitors for 24hr. DNA fragmentation is expressed as a percentage of total DNA by [3H] thymidine assay as described in Methods and represents the average and range of three independent experiments in duplicate. Significantly different for SNAP (100@1) at the indicated time (*P
3. 4. 5. 6.
D.A. CARSON and J. M. RIBEIRO, Lancet3..4-L1251-1254(1993). K.M.H. DAVID,C.H. CHRISTIAN, K.H. MUN, T.T. BRAD, B.L. MARTIN and L. GENE, Am. J. Pathol. W7 267-277 (1995). E. NISHIO, K. FUKUSHIMA, M. SHIOZAKI and Y. WATANABE, Biochem. Biophys. Res. Comm. W 163-168(1996). V.M. DANIEL, Proc. Natl. Acad. Sci. USA ,$l!l4329-4331 (1993). M. HECKER, M. BOESE., V.B. SCHINI-KERTH, A. MULSCH and R. BUSSE, Proc. Nat]. Acad. Sci. USA %2 4671-4675 (1995). A.J. HALE, C.A. SMITH, L.C. SUTHERLAND, V.E. STONEMAN, V.L. LONGTHORNE., A.C. CULHANE and G. T. WILLIAMS, Eur. J. Biochem. 236
Vol. 62 No. 3, 1998
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245
1-26 (1996). J. YUAN, S. SHAHAM, S. LEDOUX, H.M. ELLIS and H. R. HORVITZ, Cell H 641-652 (1993). T. MIYASHITAand J. C. REED, Cell ~ 293-299 (1995). T. MIYASHITA, S. KRAJEWSKI, M. KRAJEWSKA, H.G. WANG, H.K. LIN, :: D.A. LIEBERMANN, B. HOFFMAN and J. C. REED, Oncogene9 1799-1805(1994). 10. G.I. EVAN, A.H. WYLLIE, C.S. GILBERT, T.D. LITTLEWOOD,H. LAND, M. BROOKS., C.M. WATERS, L.Z. PENN and D. C. HANCOCK, Cell U 119-128 (1992). B. AMATI,T.D. LITTLEWOOD,G.I. EVAN and H. LAND, EMBOJ. 1.2508311. ~ø• 5087 (1993). 12. E. YONISH-ROUACH., D. RESNITZKY, J. L~EM, L. SACHS., A. KIMCHI. AND M. OREN, Nature W 345-347 (1991). 13. E. NISHIO, H. NAKATA., S. ARIMURA,andY. WATANABE,Biochem. Biophys. Res. Comm. U277-282 (1996). 14. E. NISHIO., S. ARIMURAand Y. WATANABE,Biochem. Biophys. Res. Comm. 2L23 413-418 (1996). 15. N.J. BUMP, M. HACKETT, M. HUGUNIN., S. SESHAGIRI, K. BRADY., P. CHEN, C. FERENZ, S. FRANKLIN., T. GHAYUR, P. LI, P. LICARI, J. MANKOVICH, L. SHI, A.H. GREENBERG, L.K. MILLERand W.W. WONG, Science2.69 1885-1888(1995). 16. M. ENARI, V.T. ROBERT, W.W. WINNIE and S. NAGATA, Nature w 723-726
7.
17. l!!??~~HIO. and Y. WATANABE,Biochem. Biophys. Res. Comm.226 928-934 (1996). 18. C.A. BOULAKIA, G. CHEN, F.W. NG, J.G. TEODORO,P.E. BRATON, D.W. NICHOLSON, G.G. POIRIER and G. C. SHORE, Oncogenel-2 529-535 (1996). 19. W. HONG-GANG, U.R. RAPP and J. C. REED, Cell&7 629-638 (1996). 20. G. HOUGE, S.0. DOSKELAND,R. BOE and M. LANOITE, FEBS lettersw 16-20 (1993). 21. R. RAINWATER, S.D. PARK, M.E. ANDERSON, P. TEGTMEYERand K. MANN, Mol. Cell. Biol. u 3892-3903 (1995). 22. A.J. DARMON, D.W. NICHOLSON and R. C. BLEACKLEY, Nature Z7 446-448 (1995).
E
PII S0024-3205(97)01092-8
NO INDUCED APOPTOSIS ACCOMPANYING THE CHANGE OF ONCOPROTEIN EXPRESSION AND THE ACTIVATION OF CPP32 PROTEASE Eisuke Nishio* and Yasuhiro Watanabe Departmentof Pharmacology, NationalDefense MedicalCollege 3-2, Namiki, Tokorozawa, Saitama, Japan 359 (Received in final form October 31, 1997)
Previously we have shown that nitric oxide (NO) donors induced apoptosis in vascular smooth muscle cells (VSMCS).However, the mechanisms by which NO induced apoptosis in VSMCSare entirely unknown. In the present study, we intended to identify the mechanism by which NO donors induce apoptosis in VSMCS. Firs~ we evaluatedthe expression of c-Myc, P53, and BcI-2 proteins in VSMCStreated by NO donors. c-Myc and P53 protein expression increased after VSMCSwere incubatedwith NO donors for 6 hr and reached a maximumlevel at 24 hr, while Bc1-2protein decreased after 12 hr incubation. Next we investigated to see whether the CPP32 protease activation was involved in NO donorsinduced apoptosis. In VSMCS treated by NO donors, the increase of CPP32 protease activity was observed and specific inhibition of CPP32 activity significantly prevented apoptosis induced by NO donors in a dose-dependent manner. These results suggest that NO donors induced apoptosis through protooncoproteinexpression and CPP32-like protease activation.
Key Wor&: nitric oxide, c-Myc,P53, Bc1-2,CPP32
It is now well established that apoptosis is a physical cell death process, important for normal developmentand involved in many pathologicalconditions (1). Recently, in the advanced atheroma lesion, apoptosis of VSMCShas been reported to accompanyproliferationof VSMCS, macrophages and T lymphocytes (2). Previously, we demonstrated that NO donors induce apoptosis in VSMCSthrough a cGMPindependent mechanism (3). But the precise mechanism is unknown. The present study was performed to investigate the mechanism by which NO donors induced apoptosis in VSMCS, particularly through the expression of the apoptosis-related gene product or the activation of protease. NO is known to act as an intracellularmediator in several cells (4). Further, it is known that VSMC, microphage, and endothelial cells, which are components of atherosclerotic lesion, release NO through nitric oxide synthase (5). Therefore it is important to investigate the mechanism by . . whi..&NO induces a.poptos]sm VSMCSin alherog nesis. * To whom correspondenceshould be addressed Fea: (0429)-%-5191, Tel: (0429)-95-1484
240
NO DonorsandApoptosisin VSMCS
vol.
62, No. 3, 199S
Two gene families are known to be important in apoptosis control (6). These are, first the genes encoding the interleukin-l~-converting enzyme family of cystein protease and, second, those related to proto-oncogene Bc1-2. Both of these families are homologous to cell death genes in C. elegans (7). In mammaliancells the number of members of both families is growing rapidly and an effort is being directedtowards establishing the roles played by each member in apoptosis. Other genes with established roles in the regulation of proliferation and differentiation are also important in controlling apoptosis. Several of these are known proto-oncogenes, e.g. c-Myc, or tumor suppressers, e.g. P53. P53 induced apoptosis through the positive regulation of Bax (8) and negative regulation of Bc1-2(9). Despite the evidence that c-Myc protein in tissue culture leads to proliferation, overexpression of c-Myc can accelerate cell death via apoptosis (10). This is not surprising since the death signaling domain of c-Myc is identical to that for proliferation (11). In certain conditions, the dysregulation of the P53, c-Myc, and Bc1-2 proto-oncogene can cause apoptosis in human malignancies (12). In this contex~ we investigated whether these proteins change in NOdonors induced apoptosis or not, and whether protease activationis involved in NO induced apoptosis. MATERIALS AND ME@HQQS
S-nitroso-N-acetyl penicillamine (SNAP) was obtained from BIOMOL Research Laboratories inc. [3H] thymidine and ECL Western blotting detection kit were from Amersham. AUcell culture materials were from Life Technologies. Ac-Asp-Glu-Val-Asp-CHO (inhibitor of caspase-3 (CPP32)), Ac-Tyr-Val-Ala-Asp-CHO(inhibitorof caspase-1 (ICE)) and Ac-Val-Glu-Ile-Asp-CHO (inhibitor of caspase-6 (Mch2)) were from Peptide Institute, INC. (Osaka). Anti BcL2, c-Myc and P53 antibodies were from OncogeneScience. Anti-CPP32 was from Santa Cruz Biochemical. Cell Cb Aortic VSMCS were obtained from thoracic aorta of the Japanese white rabbit by the method described previously (13). The ce11s(lx105) were seeded into 35-mm diameter dishes and maintainedin 2 ml of Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum at 37~ in a humidifiedatmosphere of 5?Z0 COZ/ 95?Z0air. The cells were used between the third and fifth passage. Cells were grown to confluence, at which time they were rendered quiescent by serum deprivationand maintainedin serum-free mediumfor 24h before experimentation.
Subconfluent plates of SMCS were labeledwith [’H]thymidine (lmCi/ ml) for 24hr. Then after the cells were incubated with the indicated media, DNA fragmentation was quantitated as follows: 0.5 ml of Iysis buffer was added to each culture well and mixed by pipetting, and the suspension was transferred to an Eppendorf tube, incubated on melting ice for 10 rnin, and centrifuged at 8,000g for 5 min at 4“C. Subsequently, fragmented radiolabeled DNA was counted in the supernatant by liquid scintillation counting. Radioactivity of cells treated with lysis buffer and ultrasound homogenator was used as total activity. Results are expressed as fragmented DNA as a percentageof total DNA ( 14). LDH was measured as previously described (15). CPP37.like protease acti“vity The fluorescent substrate MCA-DEVDAPK (drip) was incubated at 30”C for 30min with cytosolic extracts prepared from NCMreatedVSMCS, and the fluorescence of the cleaved substrates was determined using a spectrofluorometer set at an excitation wavelength of 325nm and an
Vol. 62
No.
NO Donors and Apoptosis in VSMCS
3, 1998
emission wavelength of 392nm ( 16).
Cell lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred electrophoreticallyto PVDF membranes. The membranes were probed with anti-Bcl-2 , P53, and cMyc mouse antibodies. After treating the membrane with peroxidase-conjugated goat anti-mouse secondary antibodies, peroxidase activity was detected using ECL reagents (17). CPP32/p17 was detected with the primary antibody against human CPP32/p17 and peroxidase-conjugatedanti-goat antibody, followed by enhanced ECL reagents.
All the results are expressed as the mean t SD and statistical analysis was performed with ANOVAfollowed by a modified Bonferroni LSD test (SPSS Software). Differences with a p value of less than 0.05 were considered significant. ULTSND
DISCUSSION
ated VSMCS Previously, we demonstratedthat two commonly used NO donors, SNAP and SIN-1, induced apoptosis (3) by demonstrating DNA fragmentation through agarose gel electrophoresis and TUNEL method, but the underlying mechanism is unknown. Fig. 1 shows that SNAP induced apoptosis in a time-dependentmanner. A significant apoptosis was induced by SNAP (100pM) at 8 hr incubation. LDH release did not increase = IOOpMSNAP (105*8% compared with control cells) excludingthe inductionof necrosis. Several gene products are known to be important in controlling the apoptotic process. To determine if NO donors have any effects on the expression level of these products, the levels of the proteins were studied by Western blotting with antibodies against P53, c-Myc, and BcI-2 proteins. Results are shown in Figure 2. The P53 protein began to increase after 6hr incubation, reached a maximum level after 24hr incubationand thereafterdecreased. The c-Myc protein began to increase after 6hr incubation, reached a maximum level after 24hr and subsequently decreased. In contrast, the expression of BcI-2 protein decreased after 12hr incubation. It decreased to a minimum level after 24hr incubation. Two gene products, c-Myc and P53 (12), were shown to be implicated in the etiology of most human tumors. Bc1-2was shown to block cell death initiated by many different stimuli. The precise biochemicalmechanisms by which Bc1-2family proteins exert their influence on cell death remain far from clear, though several theories have been advanced, including cysteine protease activation (18) and targeting the protein kinase Raf-1 to mitochondria(19). Increased expression of both P53 and c-Myc protein appeared before the occurrence of significantapoptosis (Fig. 1). These data may suggest that up-regulationof P53 and c-Myc proteins, as well as signals triggering cleavage of ribosomal RNA (20), may be a pre-requisite for DNA fragmentation and apoptosis. One possible mechanism to be considered is a direct modulatory action of NO on P53 activity or the protein stability by targeting cysteine residues normally implicatedin zinc binding (21). However, further studies are required to find a mechanism whereby NO regulatesthe apoptotic-regulatorygene product, Also, it is necessary to elucidatethe cause-andeffect relationship between NO and the apoptosis-regulatorygene product. . . . . NO-do~ by CPP32 Md2mr.r Evidence is growing that CPP32 may play an importantrole in the apoptotic pathway. CPP32 is expressed as a 32-KDa precursor which is processed into active p17 and p12 subunits upon activation(22). Therefore, we determined whether CPP32 is activatedduring apoptosis induced by NO donors. VSMCS expressed the 32-KDa preform of CPP32, and treatment with NO-donors generated p17 subunits (Fig.3). NO also significantly increased CPP32-like protease activity
242
NO Donors and Apoptosis in VSMCS
Vol. 6& No. 3,1998
(128*1 1% of controls). Furthermore, we investigated whether NOinduced apoptosis is antagonizedby the inhibitionof CPP32-like activityby the specifictetrapeptideinhibitor Ac-DEVD CHO. Figure 4 shows that the Ac-DEVD-CHOinhibits apoptosis induced by NOdonors in a dosedepcndent manner. In contrast, both AC-YVA~CHO, the inhibitor of ICE, and Ac-VEID-CHO, the inhibitor of Mch2, does not significantlyinhibit apoptosis induced by NO donors. These results suggest that CPP32 activation is involved in apoptosis in NOdonor treated VSMCS. However, further studies are required to elucidatethe mechanismwhereby NO activatesCPP32 protease. One possible mechanismis that the downregulationof Bc1-2may result in the CPP32 activation( 18). In summary, these results show that NOinduced apoptosis is accompaniedby the accumulation of P53 and c-Myc, the downregulation of BcI-2 and the activation of CPP32 protease. However, further study is needed to answer the question concerning the significance of gene product regulationand protease activationin regulatingof apoptosis.
50
-—o—
**
40
30
20
10
o~ 0
10
5
(hr) Fig. 1. Time-dependentinduction of apoptosis in VSMCSby NO donor, SNAP (1OOPM). Serum deprived VSMCS were treated with SNAP (100@1) for the indicated time. DNA fragmentation is expressed as a percentage of total DNA by [3H] thymidine assay as described in Methods and represents the average and range of three independent experiments in duplicate. Significantlydifferentfor the control at the indicatedtime (*P-4.05, **P
—
NODonorsandApoptosisin VSMCS
Vol.62 No.3, 1998
243
P 53-
c – Myc +
Bcl – 2 + Fig.2. Differentialmodulation of P53, c-Myc, and Bc1-2during apoptosis induced by NO donor, SNAP (1OOPM). Serum deprived VSMCStreated with SNAP (100pM) for indicated times were subjected to Iysis and Western blot analysis and probed with antibodies specific proteins. Each experimentwas repeatedthree times and a representativeblot is shown above.
KD
The induction of CPP32 protease activatii;g;~”VSMCs by NO donor, SNAP (1OOPM). Serum deprived VSMCS treated with SNAP (100pM) for 24hr were subjected to Iysis and Western blot analysis and probed with antibodies specific CPP32 protease proteins. Each experiments was repeatedthree and a representativeblot is shown above.
244
::: ,\< ,., ::: 1
NO Donors and Apoptosis in VSMCS
1
;:; ::: ::: ::: ::: ::: ::: :.:
8 10 0
Vol. 62, No. 3, 1998
❑ no addition or SNAP(100pM) ❑ +Ac-DEVD (10- 1OOOYM) El +Ac-YVAD (10- 1OOOPM) E +Ac-VEID (10-1OOOPM)
z
$$ Zz ram Effect of CPP32 inhibitor on the induction of apoptosis in VSMC by SNAP (1OOJJM).Serum deprived VSMCS treated with SNAP (100p@ ad various tetrap~ptideinhibitors for 24hr. DNA fragmentation is expressed as a percentage of total DNA by [3H] thymidine assay as described in Methods and represents the average and range of three independent experiments in duplicate. Significantly different for SNAP (100@1) at the indicated time (*P
3. 4. 5. 6.
D.A. CARSON and J. M. RIBEIRO, Lancet3..4-L1251-1254(1993). K.M.H. DAVID,C.H. CHRISTIAN, K.H. MUN, T.T. BRAD, B.L. MARTIN and L. GENE, Am. J. Pathol. W7 267-277 (1995). E. NISHIO, K. FUKUSHIMA, M. SHIOZAKI and Y. WATANABE, Biochem. Biophys. Res. Comm. W 163-168(1996). V.M. DANIEL, Proc. Natl. Acad. Sci. USA ,$l!l4329-4331 (1993). M. HECKER, M. BOESE., V.B. SCHINI-KERTH, A. MULSCH and R. BUSSE, Proc. Nat]. Acad. Sci. USA %2 4671-4675 (1995). A.J. HALE, C.A. SMITH, L.C. SUTHERLAND, V.E. STONEMAN, V.L. LONGTHORNE., A.C. CULHANE and G. T. WILLIAMS, Eur. J. Biochem. 236
Vol. 62 No. 3, 1998
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