Dephosphorylation-induced decrease of anti-apoptotic function of Bcl-2 in neuronally differentiated P19 cells following ischemic insults

Brain Research 857 Ž2000. 78–86 www.elsevier.comrlocaterbres

Research report

Dephosphorylation-induced decrease of anti-apoptotic function of Bcl-2 in neuronally differentiated P19 cells following ischemic insults Hideyuki Yokote ) , Tomoaki Terada, Hiroyuki Matsumoto, Kohji Kakishita, Yasunobu Kinoshita, Naoyuki Nakao, Kunio Nakai, Tohru Itakura Department of neurosurgery, Wakayama Medical College, Kimiidera 811-1, Wakayama 641-0012, Japan Accepted 23 November 1999

Abstract It is known that Bcl-2 has a protective effect against neuronal ischemia. Some reports speculate anti-apoptotic function of Bcl-2 depends not on the expression level but on the phosphorylation state. We found induction of apoptosis and CPP32 activation by energy impairment Ž3-nitropropionic acid Ž3-NP.-treatment or glucose-deprivation. in the neuronally differentiated P19 cells. Time course study of cell viability following ischemic insults showed that the number of viable cells decreased along with the increase in the amount of dephosphorylated Bcl-2 without obvious quantitative alteration of the protein. Then, we generated differentiated P19 cells overexpressing wild-type Bcl-2 ŽP19rwt.Bcl-2. or phosphorylation-negative Bcl-2 mutant ŽP19rmut.Bcl-2., in which alanine was substituted for serine 70. When the cell viability was examined within 24 h, P19rmut.Bcl-2 was more vulnerable to energy impairment as compared with P19rwt.Bcl-2. In addition, overexpression of wild-type Bcl-2 inhibited DNA laddering and CPP32 activation induced by the insults, while that of mutant Bcl-2 did not. These findings suggest that the phosphorylation state, as well as the expression level, of Bcl-2 plays an important role to modulate its protective effect against ischemic insults. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Ischemia; Neuronal cell death; Apoptosis; Bcl-2; Phosphorylation; CPP32

1. Introduction Oxygen and glucose are essential factors for neurons to stay alive. If blood supply to a part of the brain is stopped, neurons in the most severely affected area immediately die of oxygen starvation, as exemplified by cerebral infarction. Recent experiments suggest that apoptosis contributes to neuronal cell death after cerebral ischemia induced by cutting off blood flow to the animals’ brains w4,12,14, 30,38,41x. Behrens et al. w4x reported that 3-nitropropionic acid Ž3-NP., an inhibitor of the mitochondrial enzyme succinate dehydrogenase, induced apoptosis in cultured neurons, suggesting that apoptosis was associated with neuronal cell death induced by chemical inhibition of energy metabolism. Apoptosis is a form of cell death resulting from activation of a genetically determined cell-suicide program w22x. Many genes responsible for apoptosis were identified in the nematode Caenorhabditis elegans Ž C. elegans cell )

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death, CED genes. w16x. At least, 13 members of the interleukin 1b-converting enzyme ŽICE. protease family have been reported to mediate apoptosis in many cell types w2x. CPP32 Žnow designated caspase-3., a member of ICE family w50x, is expressed as a 32-kDa of proenzyme that is proteolytically cleaved to 20 and 10 kDa molecules, and reassembled to an active heterodimer when cells are signaled to die w40,43,46x. A recent study demonstrated that CPP32 was activated during apoptosis induced by experimental cerebral ischemia w9,15,17,39x. Bcl-2, a 26-kDa membrane-anchored proto-oncoprotein, was the first gene product discovered as an apoptosis suppressor acting in various cells w27,42x, although the mechanism is not clearly analyzed. Overexpression of Bcl-2 prevents cells from progressing to apoptosis in several cell systems w45,47x. Martinou et al. w34x have reported that Bcl-2 protects cortical neurons of adult mice from ischemic insults induced by permanent middle cerebral artery occlusion. Yang et al. w48x also report that Bcl-2 overexpression protects neurons from damage by 1methyl-4-phenyl-1,2,3,6-tetrahydropyride ŽMPTP ., a chemical inactivator of the complex I in the mitochondrial

0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 4 1 4 - 2

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electron transport system. However, a recent report demonstrates that the quantity of Bcl-2 is not always correlated with the cell survival against death-promoting stimuli w27x. The Bcl-2-phosphorylation was first reported by Alnemri et al. w3x in SF9 cells, although its functional implication was not unclear. Thereafter, May et al. w36x demonstrated that treatment of murine myeloid factor-dependent FDC-P1rER cells with interleukin-3 and bryostatin-1, a protein kinase C activator, resulted in the induction of Bcl-2-hyperphosphorylation and prevention of apoptosis. P19 embryonal carcinoma cells are pluripotent stem cell line which can be induced to differentiate into the cell types similar to CNS cells with retinoic acid w33x. The irreversibly post-mitotic neurons obtained under this culture condition show a typical neuronal morphology, and express a number of neuron-specific markers. Therefore, P19 cells have been used as an in vitro model of the CNS cells after differentiation by retinoic acid w1,35,49x. Further advantage in using these cells is easy transfectability of DNA in their undifferentiated state, which could be attributed to their rapid growth. In this study, we examined the significance of Bcl-2 dephosphorylation in the death-process induced by energy starvation in neuron-differentiated P19 cells.

2. Materials and methods 2.1. Cell culture and neuronal differentiation P19 cells derived from mouse embryonal carcinoma were grown in a MEM ŽGibco BRL, Rockville, MD. plus 10% fetal calf serum. Neuronal differentiation was performed as described previously w33x. Briefly, P19 cells were cultured in the medium containing 0.5 mM retinoic acid ŽSigma. for 3 days. The cells were, then, passaged and seeded at 500 cellsrmm2 into collagen I-coated dishes ŽIwaki, Tokyo, Japan.. Twenty-four hours later, the serum-supplemented media was replaced with serum-reduced opti-MEM medium ŽGibco BRL.. The cultures were maintained in opti-MEM ŽGibco BRL. for 7 days. To confirm neuronal differentiation, cells were fixed with 4% paraformaldehyde, and examined for the presence of MAP2, a neuron-specific antigen, by immunocytochemistry using anti-MAP2 antibody followed by DAB staining. Then, neuronally differentiated P19 cells were exposed to ischemic insults. 2.2. Establishment of transfectants oÕerexpressing Bcl-2 and mutant Bcl-2 (S70A) Full length cDNA of mouse Bcl-2 was obtained from mouse brain by RT-PCR method using the following forward and reverse primers which contain EcoRI and XhoI sites, respectively ŽNisshinbo Bio-Service, Tokyo,

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Japan.. Forward primer, 5X-GAATTCATGGCGCAAGCCGGGAGA-3X ; reverse primer, 5X-CTCGAGTCACTTGTGGCCCAGGTA-3X . The PCR product was subcloned into a pCR2 plasmid vector ŽInvitrogen, San Diego, CA.. The insert was then digested with EcoRI and XhoI, and cloned into an eukaryotic expression plasmid vector, pCR3 ŽInvitrogen., to construct pCR3rBcl-2. The cDNA sequence was confirmed by the dideoxy sequencing analysis ŽNisshinbo Bio-Service.. A plasmid construct to express a mutant Bcl-2 in which 209 T of Bcl-2 cDNA sequence was converted to G ŽpCR3rmutant Bcl-2. was generated using a site-directed mutagenesis kit ŽStratagene, La Jolla, CA.. For this point mutation, the following complementary oligonucleotides were used. Sense oligonucleotide, 5XGATGGCTGCCAGGACGGCTCCTCTCAG-3X ; antisense oligonucleotide, 5X -CTGAGAGGAGCCGTCCTG GCAGCCATC-3X Žunderlined G and C indicate the inserted mutation.. The cDNA of Bcl-2 with the point mutation encodes a mutant protein ŽS70A. in which alanine was substituted for serine 70 in the amino-sequence of Bcl-2. The point mutation was confirmed by the sequencing analysis. Two plasmid constructs, pCR3rBcl-2 and pCR3rmutant Bcl-2, were transfected into undifferentiated P19 cells by the lipofection method w18x. The transfected P19 cells were selected by 0.7 mgrml G418 ŽSigma.. The overexpression of Bcl-2 and mutant Bcl-2 was confirmed by RT-PCR method using the RNA LAPCR kit ŽTakara, Tokyo, Japan.. Total RNA from each transfectant Ž0.5 mg. was used for reverse transcription at 428C for 40 min. The cDNA was amplified by 15 cycles of PCR, a cycle of which consists of 30 s at 968C, 30 s at 608C and 90 s at 728C, using the primers for Bcl-2 described above. 2.3. Ischemic insults and cell Õiability assay We used two types of ischemic insults, chemical injury by 3-NP which exerts its cytotoxic effect as a succinyl dehydrogenase inhibitor, and glucose-deprivation injury. The neuronally differentiated cells were treated with 3NP-containing or glucose-deprived medium. For 3-NP injury, the chemical was added to opti-MEM medium, while the cells were cultured in glucose-deprived DMEM ŽGibco BRL. for glucose-deprivation injury. Cell viability was examined by 3-Ž4,5-dimethyl-thiazole-2-yl.-2,5-diphenyl tetrozolium bromide ŽMTT. assay. Briefly, the retinoic acid-treated P19 cells were seeded at 500 cellsrmm2 into collagen I-coated 96 well plates ŽIwaki.. After neuron-differentiation and ischemic insults, MTT solution Ž5 mgrml in PBS. was added into each well at a final concentration of 0.5 mgrml, and the cultures were maintained further for 4 h. The culture medium was removed, and 100 ml of DMSO was added into each well to resolve the reagent. An OD590 in each well was counted by a microplate reader, ELNX 96 ŽToray-Fuji Bio., Tokyo, Japan., and the value was corrected by subtracting the blank. Cell viability

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was expressed as a percentage for control Žnon-treated cells.. Quadruplicate or quintuplicate samples were used for each condition in each experiment. Results were expressed as the mean " S.D. Statistical significance was evaluated by Student’s t-test. 2.4. Characterization of cell death For observation of nuclear morphology, 5 = 10 5 cells were scraped off the culture plates, and collected by centrifugation at 400 = g for 5 min. The cells were resuspended and fixed with 1% glutaraldehyde at room temperature for 30 min. After washing with PBS, the cells were stained with 30 ml of PBS containing 0.25 mM Hoechst 33258. The cell suspension, 5 ml, was examined with a fluorescent microscope. For detection of DNA fragmentation by apoptosis, DNA was extracted as follows. A total of 5 = 10 5 cells was lysed with 200 ml solution containing 1% SDS, 0.5 mgrml proteinase K and 0.5 mgrml RNase A, and incubated at 378C for 30 min. The lysate was mixed with 300 ml of NaI solution containing 6 M NaI, 13 mM EDTA, 0.5% sodium-N-lauroylsarcosinate, 10 mgrml glycogen and 26 mM Tris–HCl ŽpH 8.0., and incubated at 608C for 15 min. Then, isopropanol, 500 ml, was added, and the mixture was incubated at room temperature for 15 min. After centrifugation at 20,000 = g for 15 min, DNA pellet was rinsed twice with 50% isopropanol and once with 100% isopropanol. The pellet was washed with diethyl ether, air-dried, and dissolved in 20 ml solution containing 10 mM Tris–HCl and 1 mM EDTA ŽpH 7.5.. The sample was fractionated by 2% agarose gel in TAE buffer. DNA was visualized by ethidium bromide staining. 2.5. Western blot analysis Cells treated with 3-NP or glucose-deprivation were washed with phosphate-buffered saline once, and lysed in lysis buffer containing 10 mM Tris–HCl, pH 7.4, 1% Žvrv. Noidet P40, 0.1% Žwrv. sodium deoxycholate, 0.1% Žwrv. SDS, 0.15 M NaCl, and protease inhibitor cocktail ŽBoehringer Mannheim.. The whole cell lysates were clarified by centrifugation at 12,000 = g for 30 min. Lysates containing 50 mg of protein were fractionated by SDS-2.5% polyacrylamide gel and transferred onto PVDF membrane ŽATTO, Tokyo, Japan.. The blots were incubated with antibody specific for Bcl-2 ŽSanta Cruz Biotech., Delaware Avenue, CA. or CPP32 ŽSanta Cruz Biotech.., and with the second antibody for detection of the protein Bcl-2 using the ECL detection system ŽAmersham, Arlington Heights, IL.. 2.6. Metabolic labeling and immunoprecipitation Differentiated cells were washed once with phosphatefree DMEM. After culturing in phosphate-free DMEM containing 100 mCirml 32 P-orthophosphate for 2 h, the

cells were washed with phosphate-free DMEM followed by ischemic insults. The cells were cultured either in phosphate-free DMEM containing 10 mM 3-NP or in phosphate- and glucose-free DMEM. After incubation, the cells were washed with phosphate- and glucose-free medium, and lysed in 500 ml of lysis buffer described above. Extracts were clarified by centrifugation at 12,000 = g for 30 min. Each sample was subjected to immunoprecipitation using anti-Bcl-2 antibody ŽSanta Cruz Biotech... The immunoprecipitates were fractionated by an SDS-12.5% polyacrylamide gel, and immunoblotted using anti-Bcl-2 antibody as described above or analyzed directly using an imaging analyzer BAS 2000 ŽFuji Photo, Tokyo, Japan..

3. Results 3.1. Neuronal differentiation of P19 cells and induction of apoptosis by energy impairment Treatment with retinoic acid induced differentiation of P19 cells to neuronal cells. The differentiation was confirmed by positive staining of a neuron-specific marker, MAP2 as reported w33x Ždata not shown.. Either treatment with 10 mM 3-NP or glucose-deprivation induced cell death as determined by MTT assay, and nuclear fragmentation typical to apoptosis as shown by staining with Hoechst 33258 in a time-dependent manner. To confirm that apoptosis was induced by ischemic insults, we examined DNA fragmentation using agarose gel electrophoresis. DNA extracted from the differentiated P19 cells treated with 3-NP or glucose-deprivation for 12 or 24 h was fractionated by electrophoresis. The amount of fragmented DNA increased with duration of energy-impairment treatment with either 3-NP or glucose-deprivation as shown in Fig. 1. These

Fig. 1. Analysis of DNA fragmentation by agarose gel electrophoresis. Each DNA sample extracted from 5=10 5 cells was analyzed by 2% agarose gel electrophoresis followed by ethidium bromide staining as described in Section 2. Lanes 1 and 2 show molecular weight markers and control DNA extracted from non-treated cells, respectively. DNA samples were extracted from the cells treated with either 10 mM 3-NP Žlanes 3 and 4. or glucose-deprivation Žlanes 5 and 6. for 12 h Žlanes 3 and 5. or 24 h Žlanes 4 and 6..

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Fig. 2. Western blot analysis of CPP32 activation by 3-NP treatment or glucose-deprivation. Whole cell lysates were analyzed by SDS-12.5% polyacrylamide gel electrophoresis ŽPAGE. followed by Western blotting using anti-CPP32 antibody which recognizes the amino terminal p20 subunit. Lanes 3–6 show samples from differentiated P19 cells treated with 10 mM 3-NP for 3 h Žlanes 3., 6 h Žlanes 4., 9 h Žlanes 5. or 12 h Žlanes 6.. Lanes 7–10 show samples from the cells treated with glucose-deprivation for 3 h Žlanes 7., 6 h Žlanes 8., 9 h Žlanes 9. or 12 h Žlanes 10.. Lanes 1 and 2 show molecular weight markers and control cell lysate from non-treated cells, respectively.

findings indicate that apoptosis participated in the ischemic neuronal cell death. 3.2. Western blot analysis of CPP32 We examined whether CPP32 was activated in the process to apoptosis of neuron-differentiated P19 cells induced by 3-NP or glucose-deprivation. CPP32 was known to be proteolytically cleaved to generate amino and carboxy terminal subunits of approximately 20 and 10 kDa molecular weight, respectively. In this experiment, we

used anti-CPP32 antibody which recognizes the amino terminal p20 subunit. The p20 subunit was not detected in the cells treated with 10 mM 3-NP for 3 h ŽFig. 2, lanes 3. or glucose-deprivation for 3 h ŽFig. 2, lanes 7.. However, either treatment for more than 6 h activated CPP32 to generate the p20 subunit. The amount of p20 subunit increased time-dependently, and became prominent at 12 h of treatment ŽFig. 2, lanes 6 and 10., indicating that apoptotic signal via CPP32 was activated either by 3-NPtreatment or by glucose-deprivation.

Fig. 3. Quantitative analysis of the total and phosphorylated Bcl-2 by Western blotting. ŽA. Western blot analysis of the Bcl-2 protein. Whole cell lysates were analyzed by SDS-12.5% Žwrv. PAGE followed by Western blotting using anti-mouse Bcl-2 antibody. Differentiated P19 cells were treated with 10 mM 3-NP for 3 h Žlanes 3., 6 h Žlanes 4. or 9 h Žlanes 5.. The cells were treated with glucose-deprivation for 3 h Žlanes 6., 6 h Žlanes 7. or 9 h Žlanes 8.. Lanes 1 and 2 represent molecular weight markers and cell lysate from non-treated P19 cells Žcontrol., respectively. ŽB. State of Bcl-2 phosphorylation. 32 P-labeled samples immunoprecipitated by anti-Bcl-2 antibody were fractionated by SDS-PAGE, and analyzed by Western blotting Župper panel. or by a BAS 2000 imaging analyzer for radioactivity. The cells were treated with either 10 mM 3-NP Žlanes 4–6. or glucose-deprivation Žlanes 7–9. for 3 h Žlanes 4 and 7., 6 h Žlanes 5 and 8. or 9 h Žlanes 6 and 9.. Lane 1 shows molecular weight markers. Lanes 2 and 3 represent control samples prepared from the cells immediately before treatment and from those incubated without any treatment for 9 h, respectively.

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3.3. Dephosphorylation of Bcl-2 induced by energy impairment It was suggested that phosphorylated condition was important for Bcl-2 to play an anti-apoptotic action w19x. Therefore, we examined whether phosphorylation of Bcl-2 was modified by the ischemic insults using metabolic labeling. There was no quantitative alteration of Bcl-2 protein induced by both insults ŽFig. 3A.. While 32 P-labeled Bcl-2 did not decrease during the 9-h culture without ischemic insults ŽFig. 3B, lower panel, lanes 2 and 3., 32 P-labeled Bcl-2 decreased remarkably after 3-NP-treatment or glucose-deprivation in a time-dependent fashion ŽFig. 3B, lower panel, lanes 4–9.. On the contrary, total immunoprecipitated Bcl-2 did not detectably decrease by the insults ŽFig. 3B, upper panel.. These findings indicate that inhibition of energy conservation dephosphorylated Bcl-2 protein without major quantitative alteration of the protein. 3.4. Difference in Õiability between P19 cells oÕerexpressing wild-type and mutant Bcl-2 It has been reported that IL-mediated suppression of apoptosis is associated with hyperphosphorylation of Bcl-2

which is induced exclusively on serine residues, and that phosphorylation at serine 70 of Bcl-2 is required for functional suppression of apoptosis by Bcl-2 in murine growth factor-dependent cell lines w23x. To examine the relationship between cell viability and dephosphorylation of Bcl-2, we produced two types of stable transfectants, P19rwt.Bcl-2 and P19rmut.Bcl-2, P19 cells overexpressing wild-type Bcl-2 and mutant Bcl-2 ŽS70A., respectively. Overexpression of wild-type Bcl-2 and mutant Bcl-2 was confirmed by RT-PCR ŽFig. 4A.. Although P19 cells express intrinsic Bcl-2, expression of the intrinsic Bcl-2 mRNA was not detectable under the conditions used here for RT-PCR ŽFig. 4A, lane 2.. P19rwt.Bcl-2 and P19rmut.Bcl-2 overexpressed mRNA of the wild-type Bcl-2 and the mutant Bcl-2, respectively ŽFig. 4A, lanes 3 and 4.. Then, we examined differences in cell viability after ischemic insults among neuron-differentiated P19, P19rpCR Žvector alone transfectant., P19rwt.Bcl-2, and P19rmut.Bcl-2 ŽFig. 4B.. No significant difference was found between P19 and P19rpCR cells in either treatment with 3-NP ŽFig. 4B, left panel. or glucose-deprivation ŽFig. 4B, right panel.. P19rwt.Bcl-2 was most tolerant for both treatment among these groups up to 24 h. P19rmut.Bcl-2 had intermediate viability for both treatments between P19rpCR and P19rwt.Bcl-2. However, the viability of

Fig. 4. Comparison of survival rate in wild-type and mutant Bcl-2 ŽS70A. P19 cells after energy impairment. ŽA. RT-PCR analysis of Bcl-2 mRNA in the transfectants, P19rpCR, P19rwt.Bcl-2 and P19rmut.Bcl-2. Amplification by PCR was carried out as described in Section 2. Lanes 2, 3 and 4 represent P19 cells transfected with the pCR3 vector ŽP19rpCR. alone, wild-type Bcl-2 ŽP19rwt.Bcl-2., and mutant Bcl-2 ŽP19rmut.Bcl-2., respectively. Lane 1 shows molecular weight markers. ŽB. Time-course of cell viability of P19 Žopen bar., P19rpCR Ždotted bar., P19rmut.Bcl-2 Žshaded bar. and P19rwt.Bcl-2 Žblack bar. during the treatment with 10 mM 3-NP Žleft panel. or glucose-deprivation Žright panel.. Cell viability was calculated by MTT assay and expressed in a percentage as described in Section 2. Single and double asterisks show statistical significance in viability Ž p-values- 0.001 and 0.05, respectively..

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Fig. 5. Induction of apoptosis in the nerve-differentiated P19 cells overexpressing wild-type or mutant Bcl-2 ŽS70A.. ŽA. Inhibition of DNA laddering by wild-type Bcl-2 but not by mutant Bcl-2 ŽS70A.. DNA samples were analyzed by 2% agarose gel electrophoresis followed by ethidium bromide staining. Lane 1 shows molecular weight markers. DNA samples were extracted from either P19rpCR Žlanes 2 and 3., P19rmut.Bcl-2 Žlanes 4 and 5. or P19rwt.Bcl-2 Žlanes 6 and 7.. Each transfectant was treated with either 10 mM 3-NP for 24 h Žlanes 2, 4 and 6. or glucose-deprivation for 24 h Žlanes 3, 5 and 7.. ŽB. Inhibition of CPP32 activation by wild-type Bcl-2 but not by mutant Bcl-2. Whole cell lysates were analyzed by SDS-12.5% Žwrv. PAGE followed by Western blotting. Lanes 1 shows molecular weight markers, and lanes 2 and 6 represent cell lysates from non-treated controls. Upper and lower panels show the samples from P19rmut.Bcl-2 ŽS70A. and P19rwt.Bcl-2, respectively. Differentiated transfectants were treated with either 10 mM 3-NP Žlanes 3–5. or glucose-deprivation Žlanes 7–9. for 6 h Žlanes 3 and 7., 9 h Žlanes 4 and 8. or 12 h Žlanes 5 and 9..

P19rwt.Bcl-2 was similar to that of P19rmut.Bcl-2 when the cells were treated with ischemic insults for an extended period of more than 36 h. These results suggest that serine 70 of Bcl-2 was important to exert an protective effects on the differentiated P19 cells undergoing death due to energy impairment.

the other hand, in P19rmut.Bcl-2, p20 subunit was visualized remarkably after 12 h by either treatment. This results indicate that the mutant Bcl-2 ŽS70A., which could not be phosphorylated at substituted alanine 70 residue, had reduced inhibitory effect on CPP32 activation.

3.5. Decreased inhibitory effect of mutant Bcl-2 (S70A) on caspase actiÕation

4. Discussion

It is highly likely that Bcl-2 exerts its anti-apoptotic function through inhibiting the upstream of CPP32-activating pathway w11,37x. Therefore, we examined DNA laddering and activation of CPP32 in P19rpCR, P19rwt.Bcl-2 and P19rmut.Bcl-2 following energy impairment. As shown in Fig. 5A, DNA laddering was induced by treatment with 10 mM 3-NP or glucose-deprivation for 24 h. Although DNA laddering by either treatment for 24 h was not clear in P19rwt.Bcl-2 ŽFig. 5A, lanes 6 and 7., it was clearly shown in P19rpCR ŽFig. 5A, lanes 2 and 3. and P19rmut.Bcl-2 ŽFig. 5A, lanes 4 and 5., suggesting that phosphorylation at serine 70 was important to block apoptosis induced by energy impairment. Fig. 5B shows the results of CPP32 activation in P19rwt.Bcl-2 and P19rmut.Bcl-2. In P19rwt.Bcl-2 treated with 3-NP or glucose-deprivation, cleaved p20 subunit was not detectable, at least, within 12 h ŽFig. 5B, lower panel.. On

It has been presumed that ischemia causes the death of neurons through necrotic mechanisms. In fact, majority of neurons in the most severely affected area, known as ischemic core, die immediately from necrosis. However, recent experiments w24,32,41x suggest that apoptosis contributes to neuronal cell death after cerebral ischemia, especially in the moderate or mild ischemic area, known as ischemic penumbra. Some others also report that the degree of apoptotic contribution depends on the severity of ischemia in the lesion w5,14x or that ischemic neuronal cell death shows both apoptotic and necrotic characteristics w6x. Indeed, we have shown in the present study that neuronally differentiated P19 cells can be induced to apoptosis by energy impairment. We have also shown that CPP32 was cleaved to produce p20 subunit, implying that CPP32mediated apoptotic pathway was activated by energy impairment. These findings are compatible with recent reports w10,20,39x showing that caspase inhibitors reduce

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infarction, and suggest that blockade of the apoptotic signaling pathway prevents neurons from ischemic cell death. We have demonstrated here that overexpression of Bcl-2 protects cells from apoptosis induced by 3-NP or glucosedeprivation. Bcl-2 is well known to block apoptosis by various stimuli, although the mechanism is still unknown. After cerebral ischemia, Bcl-2 can be induced in surviving neurons w6–8,13x, suggesting its protective effect on ischemic brain injury. Overexpression of Bcl-2 by gene transfer or in transgenic mice reduces the volume of infarction after cerebral ischemia w28,31,34x. Two mechanisms can be taken into account to explain the pro-survival effect of Bcl-2 against ischemic insults. The first is the anti-apoptotic effect of Bcl-2, which may be the major action. Indeed, our data demonstrate that overexpression of Bcl-2 prevented cells from CPP32 activation and DNA degradation. This hypothesis is supported by a report showing that the protection by overexpressed Bcl-2 correlates with the decrease of the number of apoptotic cells w6x. Therefore, we speculate that the anti-apoptotic effect of Bcl-2 plays an important role for protection against cell death induced by energy impairment. The second mechanism might be through another function of Bcl-2 as an antioxidant as reported w21,25,38x. Oxygen free radicals are known to be produced in ischemic tissue w26x. In our current study, P19 cells overexpressing mutant Bcl-2 ŽS70A. were more resistant to energy impairment than control cells, although they were unable to inhibit DNA degradation and CPP32 activation. These findings indicate that the phosphorylation of serine 70 regulates some but all of the protective effects of Bcl-2 and suggest that the protective mechanism might partly be attributed to the function of Bcl-2 that is not related with the apoptotic pathway itself. The postulated anti-oxidant function of Bcl-2 fits well with this observation, and serine 70 might not be required for this function of Bcl-2. Our results show that ischemic insults dephosphorylated Bcl-2 in a time-dependent fashion without affecting the total amount of protein, and suggest that phosphorylation of serine 70 is one of the critical factors in augmenting the function of Bcl-2. The neuronally differentiated P19 cells overexpressing mutant Bcl-2 ŽS70A. exhibit reduced activity against ischemic insults in pro-survival functions and in preventing the activation of apoptotic pathways, as compared to the cells overexpressing wild-type Bcl-2. These findings are compatible with several reports showing that the cellular Bcl-2 amount is not always correlated with increased ability of cells to resist death-promoting stimuli w19,29,47x, and also well supported by another report showing that interleukin-3 and bryostatin-1, a potent activator of PKC, suppress apoptosis through the mechanism which appears to be associated with serine-phosphorylation of Bcl-2 w36x. The serine 70 residue is conserved throughout the species, including human, rat, chicken, and mouse w44x, suggesting that this residue has important

functions. The importance of the serine residue was recently demonstrated by Ito et al. w23x, who showed that serine 70, among other serine residues composing Bcl-2, is the critical Bcl-2 phosphorylation site to activate the protein for anti-apoptotic function in IL-3 dependent NSFrN1.H7 cells. From our results, the same serine 70 is also likely to be the site for Bcl-2 phosphorylation in the neuronally differentiated P19 cells, and the phosphorylation appears to be critical for activation of Bcl-2 which protects neuronal cells from apoptosis induced by energy impairment. In summary, our in vitro study utilized succinyl dehydrogenase inhibitor and glucose-deprivation to simulate ischemia in vitro. The neuronally differentiated P19 cells exposed to these ischemic insults reduced the amount of phosphorylated Bcl-2 in a time-dependent fashion, and underwent apoptosis. Overexpression of wild-type Bcl-2 rescued this apoptosis, while the mutant Bcl-2 ŽS70A. had limited function in rescuing apoptosis. In conclusion, we have demonstrated an important role of the total amount of Bcl-2 and its serine-70 phosphorylated form for preventing neuronal cells from apoptotic death due to ischemic insults.

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