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Vascular System Research Center and Department of Molecular and Cellular Biochemistry, Kangwon National University, School of Medicine, Chunchon, Kangwon-do, Korea;
* Department of Surgery, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania, USA; and
Department of Microbiology and Immunology, Wonkwang University, School of Medicine, Iksan, Chonbuk, Korea
1Correspondence: Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chunchon, Kangwon-do, Korea. E-mail: ymkim{at}kangwon.ac.kr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: caspase • mitochondria • cytochrome c • Bad phosphorylation • protein kinase G
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Ligation of tumor necrosis factor family receptors induces autoactivation of caspase-8 through the formation of death-inducing signaling complexes (3)
. Active caspase-8 can induce mitochondrial apoptosis signaling, including the release of cytochrome c, apoptosis-inducing factor, and endonuclease G, as well as direct activation of downstream caspases (4
, 5)
. Nonreceptor-mediated apoptotic stimuli induce the translocation of mitochondrial cytochrome c into the cytosol. Cytosolic cytochrome c binds with procaspase-9 and Apaf-1 in the presence of dATP (6)
, resulting in caspase-9 activation, which leads to activation of caspase-3, -6, and -7. Caspase-3 cleaves terminal death substrates leading to systematic destruction of cells and activation of caspase-activated DNase, resulting in DNA fragmentation and apoptotic cell death.
Neuronal apoptosis occurs in pathological processes (7)
such as stroke (8)
, trauma (9)
, Parkinson’s disease (10)
, and Alzheimer’s disease (11)
. 6-Hydroxydopamine (6-OHDA) is a neurotoxin used to induce experimental Parkinson’s disease in animals (12)
by a model that destroys catecholaminergic neurons, probably by the production of reactive oxygen species (13)
. Recently, much attention has been paid to the role of caspase activation in neuronal cell apoptosis induced by the treatment with 6-OHDA as an in vitro experimental model for the study of Parkinson’s disease (14
15
16)
. These observations suggest that regulation of caspase activation/activity may assist in the treatment of the diseases related to defects in the regulation of neuronal apoptosis.
Nitric oxide (NO) is produced by a family of NO synthases composed of three isoforms, including the constitutive endothelial (eNOS) and neuronal (nNOS) isoforms and the inducible isoform (iNOS). NO induces apoptosis in some cells and prevents apoptosis in others (17)
. NO synthesized by neuronal NOS (nNOS) was identified originally as a neurotransmitter (18)
and later was found to be an anti-apoptotic modulator in the nervous system (19
, 20)
. However, NO can lead to neuronal cell death when produced in excess (21
, 22)
. The physiological level of NO produced from NOS or NO donors can function as an endogenous regulator of apoptosis in primary cultured neurons (19
, 23)
, PC12 cells (20)
, as well as a hepatotoxic animal model (24)
. The protective effect probably occurs at least by inhibiting the proteolytic activation and activity of caspases. NO is known to activate soluble guanylyl cyclase, resulting in the production of intracellular cGMP (25)
. In many neuronal cells, the anti-apoptotic function of NO is mediated at least partially by NO-dependent generation of intracellular cGMP. For example, the protective effect of NO against apoptosis is dependent on production of the second messenger cGMP in embryonic motor neurons (19)
and PC12 cells (20
, 23)
. However, the mechanism underlying cGMP-dependent suppression of apoptosis remains elusive.
Activation of the serine/threonine kinase Akt by phosphatidylinositol 3-kinase (PI3K) serves as a multifunctional regulator of apoptotic cell death, cell growth, and glucose metabolism. With respect to neuronal cell function, Akt has been shown to be required for the prevention of apoptosis and the promotion of cell survival through the phosphorylation of proapoptotic Bad (26)
, procaspase-9 (27)
, and eNOS (28)
and through activation of NF-
B (29)
. However, the relationship between the anti-apoptotic action of the NO/cGMP and Akt pathway remains poorly understood.
In this study, we investigated the anti-apoptotic mechanisms of NO/cGMP in the protection of 6-OHDA-induced apoptosis in pheochromocytoma PC12 cells. We report that both NO and cGMP suppressed 6-OHDA-induced apoptosis by inhibiting cytochrome c release and caspase activation. The anti-apoptotic activity of NO was also suppressed by the inhibition of guanylyl cyclase, protein kinase G (PKG), and PI3K. Our results indicate that NO prevents 6-OHDA-induced PC12 cell apoptosis by inhibiting cytochrome c release and caspase activation through a cGMP-dependent activation of the PI3K/Akt signaling pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-32P]ATP was obtained from NEN Life Science Products (Boston, MA, USA). N-Acetyl-Asp-Glu-Val-Asp-P-nitroanilide (Ac-DEVD-pNA), N-acetyl-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-cho), and N-acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-cho) were obtained from Alexis Corporation (San Diego, CA, USA) and 1H-(1,2,4)-oxadiazole[4,3-a]quinoxalon-1-one (ODQ) was purchased from Promega (Madison, WI, USA). S-Nitroso-N-acetyl-D,L-penicillamine (SNAP) was synthesized as described previously (30)
. All other reagents were purchased from Sigma (St. Louis, MO, USA), unless otherwise indicated.
Cell culture and transfection with nNOS
Culture dishes were coated by spreading collagen solution [bovine skin collagen, 100 g/250 mL in distilled water) over the dish and allowing it to dry at room temperature in a sterile laminar flow hood. PC12 cells were cultured on the plates and maintained in RPMI 1640 medium containing 10% horse serum and 5% fetal bovine serum (FBS) supplemented with 2 mM glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin at 37°C with 95% air/5% CO2. PC12 cells were differentiated by treating with nerve growth factor (NGF, 50 ng/mL) in RPMI 1640 containing 1% horse serum for 7days. PC12 cells cultured in 6-well plates until 
70% confluence were transfected with pCMV-nNOS or pCMV-LacZ by the lipofectamine method. Stable transfectants were selected by culturing with G418 (800 µg/mL) followed by serial dilution of the cells in 96-well plates.
Cell viability assay
PC12 cells were washed extensively with serum-free RPMI 1640 medium and replated onto new collagen-coated 24-well plates at a density of 2 x 105 cells per well in a volume of 1 mL. Cells were preincubated with SNAP or 8-Br-cGMP in RPMI 1640 medium containing 1% FBS for 30 min, followed by pretreatment of 6-OHDA for 6 h. For determination of cell viability, cells were washed with phosphate-buffered saline (PBS) and incubated for 3 h in medium containing neutral red (0.005%) as described previously (31)
. Damaged or dead cells lose the ability to retain neutral red. Cells were washed with PBS and the dye was extracted with a solution of acetic acid (1%)/methanol (50%) (300 µL/well). The plate was agitated for 10 min and the absorbance of the extracted dye was determined in an ELISA reader at 540 nm. Counts were performed on triplicate wells.
Enzyme assays
The cell pellets were washed with ice-cold PBS and resuspended in 100 mM HEPES buffer, pH 7.4, containing protease inhibitors (5 mg/mL aprotinin and pepstatin, 10 mg/mL leupeptin, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). The cell suspension was lysed by three freeze-thaw cycles, and the cytosolic fraction was obtained by centrifugation at 13,000 x g for 20 min at 4°C. DEVDase activity was assayed by colorimetric assay using the substrate-specific tetrapeptide Ac-DEVD-pNA (32)
. To assay Akt activity, cells were first rinsed twice with ice-cold PBS; 600 µL of whole cell lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4, 1 µg/mL leupeptin, and 1 mM PMSF) was then added to each plate. After 5 min incubation on ice, cells were collected from the plates and lysed at 4°C for 30 min. The whole cell lysate was collected by centrifugation at 13,000 x g for 20 min to remove cell debris. Akt enzymatic activity was assayed with 1 µg per assay of recombinant glycogen synthase kinase (GSK3) fusion protein as substrate in a reaction mixture containing 25 mM Tris, pH 7.5, 1 mM Na3VO4, 1 mM EDTA, and 0.5 mM PMSF, 10 mM MgCl2, 50 µM ATP, and 2 µCi of [-32P]ATP per assay. The phosphorylation reaction was allowed to proceed for 30 min at 30°C and stopped by adding 3x sample buffer. The resulting solution was boiled at 95°C for 5 min, and phosphorylated GSK3 was resolved by SDS-PAGE and quantitated with PhosphorImager.
Intracellular cGMP measurement
cGMP levels were measured by using an iodinated assay system from Perkin Elmer Life Sciences (Boston, MA, USA). After exposure to SNAP for 1 h, cells were lysed in the lysis buffer supplemented with 1 mM 3-isobutyl-1-methylxanthin to inhibit phosphodiesterases. Intracellular cGMP contents were then determined with the whole cell lysate (20 µg of protein) by radioimmunoassay according to the manufacturer’s protocol.
DNA fragmentation
Cytosolic DNA was prepared according to the method of Kim et al. (33)
. Cell pellets were resuspended in 750 µL of lysis buffer (20 mM Tris-HCl, 10 mM EDTA, and 0.5% Triton X-100, pH 8.0) and shaken occasionally while on ice for 45 min. DNA was extracted with phenol and precipitated with alcohol. The pellet was dried and resuspended in 100 µL of 20 mM Tris-HCl, pH 8.0. After digesting RNA with RNase (0.1 mg/mL) at 37°C for 1 h, samples (15 µL) were electrophoresed through a 1.2% agarose gel in 450 mM Tris borate-EDTA buffer, pH 8.0. DNA was photographed under visualization with UV light.
Western blot analyses
Cell pellets were suspended in ice-cold sterilized water and kept on ice for 1 min. The suspension was mixed with an equal volume of 500 mM sucrose solution and homogenized in a Dounce tissue grinder with a loose pestle (Wheaton, Millville, NJ, USA). Nuclei and cell debris were removed by centrifugation at 500 x g for 10 min. The supernatant was further centrifuged at 130,000 x g for 1 h to separate cytosol from mitochondrial fraction. These cellular fractions were used for assays of mitochondrial cytochrome c release and subcellular Bad localization. Some cells were lysed by three freeze/thaw cycles and cell lysates were obtained by centrifugation at 13,000 x g for 20 min for assays of caspase activation and PARP cleavage. This was used for Western blot analyses of caspase activation and PARP cleavage. Whole cell lysates prepared for Akt activity assay as described above were used for Akt phosphorylation. Forty microgram samples of protein were separated on 8% (PARP), 12% (caspase-9), or 14% (caspase-3 and cytochrome c) SDS-PAGE, then transferred to nitrocellulose. The membranes were hybridized with antibodies for caspase-3, cytochrome c, PARP, phospho-Akt, and Akt, and protein bands were visualized by exposing to X-ray film, as described previously (20)
.
Other analyses
Protein concentrations were determined with the BCA assay (Pierce, Rockford IL, USA). Quantitative data represent the mean ± SD of at least three separate experiments. The results of blots were representative of two or three independent experiments. Comparisons between two values were analyzed using Student’s t test. Differences were considered significant when P 
0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 34)
. Consistent with these earlier observations, we found that treatment of dopaminergic PC12 cells for 6 h with up to 100 µM 6-OHDA induced cell death in a dose-dependent manner (Fig. 1
A). Since activation of the caspase cascade is required for neuronal cell apoptosis (16
, 35)
, we examined whether inhibiting caspase family proteases affects 6-OHDA-induced PC12 cell apoptosis. The inhibitor of caspase-3-like protease, Ac-DEVD-cho, protected PC12 cells from 6-OHDA-induced cell death whereas the inhibitor of caspase-1-like protease, Ac-YVAD-cho, did not (Fig. 1B
). The activity of the caspase-3-like enzyme (DEVDase) was measured by a colorimetric assay using the substrate-specific tetrapeptide Ac-DEVD-pNA (Fig. 1C
). The cytosols of 6-OHDA-treated PC12 cells increased DEVDase activity. This activity was decreased by Ac-DEVD-cho but not by the addition of Ac-YVAD-cho.
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NO prevents 6-OHDA-induced PC12 cell apoptosis by inhibiting DEVDase activity
It has been shown that NO, produced either by NOS or NO donors, protected PC12 cells (20)
, rat embryonic motor neurons (19)
, and sympathetic neurons (23)
from apoptotic cell death induced by trophic factor deprivation. We examined whether NO protects PC12 cells from 6-OHDA-induced apoptosis by treating PC12 cells with the NO donor SNAP. SNAP prevented 6-OHDA-induced apoptosis in a dose-dependent manner up to 100 µM (Fig. 2
A), but decreased cell viability at doses higher than 250 µM (data not shown). This protection was observed when cells were pretreated with SNAP at least 30 min before treatment of 6-OHDA and gradually decreased by post-treatment of SNAP (Fig. 2B
). Since caspase activation is essential for the execution of the signal cascade that results in apoptotic cell death, we examined whether NO regulates DEVDase activity in PC12 cells treated with 6-OHDA. DEVDase activity was increased in the cytosol from 6-OHDA-treated cells, and this activity was decreased in a dose-dependent manner by SNAP treatment (Fig. 2C
). These results indicate that the cytoprotective effect of NO on 6-OHDA-induced apoptosis in PC12 cells may be caused by an inhibition of DEVDase activation and/or activity.
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The anti-apoptotic effect of NO is associated with cGMP production
NO interacts with heme-containing, soluble guanylyl cyclase-producing cGMP (25)
. The anti-apoptotic actions of NO in trophic factor-deprived neuronal apoptosis have been shown to be associated with cGMP production (19
, 20
, 23)
. To investigate the role of cGMP in the anti-apoptotic signal cascade induced by NO, we examined DNA fragmentation, cytochrome c release, and caspase activation in the presence of the specific inhibitor of soluble guanylyl cyclase, ODQ, after treatment of cells with 6-OHDA and SNAP. SNAP suppressed 6-OHDA-induced apoptotic cell death and DNA fragmentation, and this suppression was markedly reduced by ODQ (Fig. 3
A, B). Western blots revealed that ODQ also suppressed the inhibitory effect of SNAP on mitochondrial cytochrome c release and caspase-3 activation in 6-OHDA-treated PC12 cells (Fig. 3C
). Moreover, SNAP suppressed the DEVDase activity increased by 6-OHDA, and this suppression was markedly reversed by ODQ (Fig. 3D
). These findings indicate that the anti-apoptotic effect of NO on 6-OHDA-induced apoptosis is associated with cGMP production, which inhibits DEVDase activation through the suppression of mitochondrial cytochrome c release.
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nNOS gene transfer inhibits apoptosis via PKG-dependent inhibition of cytochrome c release
To determine whether endogenous NO inhibits PC12 cell apoptosis, we stably transfected PC12 cells with pCMV-nNOS and examined the effect of nNOS-derived NO on 6-OHDA-mediated cell death. PC12 cells transfected with nNOS gene expressed nNOS protein, as determined by Western blot analysis, and produced 6 µM of NO2-/106 cells/24 h compared with 1 µM of NO2-/106 cells/24 h in control transfectant. nNOS-expressing PC12 cells decreased apoptotic cell death induced by 6-OHDA treatment (Fig. 4
A) and cytochrome c was not detected in their cytosols (Fig. 4B
). Control cells transfected with LacZ were not as protected as nNOS-transfected cells. 6-OHDA elevated DEVDase activity in LacZ-transfected PC12 cells but not in nNOS-transfected cells (Fig. 4C
). These anti-apoptotic effects in nNOS-expressing PC12 cells were reversed by the NOS inhibitor N-monomethyl-L-arginine (NMA), the guanylyl cyclase inhibitor ODQ, and the PKG inhibitor KT5823 (Fig. 4A-C
). These results suggest that endogenous NO derived from nNOS also prevents PC12 cell apoptosis, probably by cGMP and in a PKG-dependent manner.
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cGMP inhibits 6-OHDA-induced apoptosis through PKG activation
We and others have shown that cGMP can suppress apoptosis in hepatocytes (32)
and neuronal cells (19
, 20)
. To confirm the anti-apoptotic function of cGMP in PC12 cells exposed to 6-OHDA, we first examined the effects of the membrane-permeable cGMP analog 8-Br-cGMP on PC12 cell apoptosis induced by 6-OHDA. Exogenous 8-Br-cGMP inhibited 6-OHDA-induced cell death in a dose-dependent manner (Fig. 5
A). Maximal inhibition was observed at 100 µM, and this inhibition was significantly reversed by the PKG inhibitor KT5823. Other cell-permeable analogs of cGMP, 8-pCTP-cGMP, and dibutyryl-cGMP showed similar protective effects (data not shown). We further examined whether 8-Br-cGMP regulates mitochondrial cytochrome c release, caspase-3 and -9 activation/activity, and cleavage of the endogenous DEVDase substrate PARP in the cells exposed to 6-OHDA. 6-OHDA increased the release of mitochondrial cytochrome c to cytosol but KT5823 alone did not. However, treatment with 6-OHDA and 8-Br-cGMP resulted in a significant inhibition of mitochondrial cytochrome c release, and this inhibition was blocked by KT5823 (Fig. 5B
). Moreover, 8-Br-cGMP suppressed the activation and/or activity of caspase-9 and -3 in the lysates of the cells treated with 6-OHDA, as determined by Western blotting and colorimetric assays, and these suppressive effects were inhibited by KT5823 (Fig. 5C, D
). One of the established endogenous substrates for caspase-3 protease is PARP, which is cleaved from 116 kDa intact protein into 85 and 31 kDa fragments during apoptosis (36)
. 8-Br-cGMP inhibited PARP cleavage detected in the lysate of 6-OHDA-treated PC12 cells, and this inhibition was blocked by KT5823 (Fig. 5C
). These results suggest that cGMP prevented 6-OHDA-induced apoptosis by the suppression of mitochondrial cytochrome c release upstream of caspase-3 and -9 via a mechanism that involved protein kinase G.
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NO donor activates PI3K-dependent Akt activation
Activation of PI3K and its downstream effector Akt has been shown to suppress apoptosis and promote cell survival (26
, 37
, 38)
. To explore whether NO may suppress apoptosis and apoptotic signaling via the Akt survival pathway, we examined the effects of NO on endogenous Akt activation. Since phosphorylation of Akt at Ser473 is required for its full activation (39
, 40)
, we first examined the phosphorylation status of endogenous Akt using an antibody that specifically recognizes Akt phosphorylated at Ser473. Incubation of cells with different concentration of SNAP resulted in activation of endogenous Akt in a concentration-dependent manner, and this activation was correlated with the production of NO and cGMP (Fig. 6
A). We further examined whether the upstream target of Akt, PI3K, is involved in SNAP-induced Akt activation. It was found that the PI3K inhibitor LY294002 or Wortmannin markedly abolished the subsequent Akt activation by SNAP (Fig. 6B
). In addition, SNAP increased Akt activity about threefold, as measured by phosphorylation of GSK3, its biological substrate, and this increase was blocked by LY294002 or Wortmannin (Fig. 6C
). As activation of the PI3K/Akt survival pathway has been shown to suppress apoptosis induced by a variety of stimuli in other systems, we examined the involvement of the PI3K/Akt survival signal in the anti-apoptotic action of NO. The inhibitory effect of SNAP on the 6-OHDA-induced increase in DEVDase activity was reversed by LY294002 or Wortmannin (Fig. 6D
). Furthermore, inhibition of Akt activation reduced SNAP-mediated protection of PC12 cell apoptosis (Fig. 6E
). This demonstrates that the PI3K/Akt pathway may play an important role in the anti-apoptotic effects of NO in dopaminergic PC12 cells.
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cGMP is a critical mediator for the activation of PI3K/Akt by NO
To investigate the role of cGMP in the activation of PI3K/Akt by NO, we examined the effect of ODQ on SNAP-mediated Akt phosphorylation. As shown in Fig. 7
A, ODQ largely inhibited Akt activation by SNAP, indicating the involvement of cGMP in SNAP-mediated Akt activation. Moreover, incubation of cells with 8-Br-cGMP activated Akt, and this activation was suppressed either by the PKG inhibitor KT5823 or the PI3K inhibitor LY294002 (Fig. 7B
). One possible mechanism by which Akt signaling suppresses apoptosis is associated with the inhibition of cytochrome c release (41)
by phosphorylating proapoptotic Bad and blocking its translocation into mitochondria. We therefore examined the effects of cGMP or SNAP on Bad phosphorylation, its intracellular localization, and cytochrome c release by Western blot analysis. 8-Br-cGMP increased Bad phosphorylation compared with control cells, and this phosphorylation was blocked by KT5823 or LY294002 (Fig. 7C
). Bad predominantly localized in the cytosol from control cells, while 6-OHDA increased the translocation of Bad to mitochondria (Fig. 7D
). This translocation was suppressed by SNAP, and the suppressive effect of SNAP was reversed by KT5823 or LY294002. Moreover, the inhibitory effect of 8-Br-cGMP on 6-OHDA-induced cytochrome c release was decreased by treatment with KT5823 or LY294002 (Fig. 7E
). These results indicate that NO inhibits 6-OHDA-induced PC12 cell death by suppressing cytochrome c release through PKG-dependent activation of the PI3K/Akt pathway and subsequent Bad phosphorylation.
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NO protects differentiated PC12 cells from 6-OHDA-induced apoptosis
To investigate the possible role of NO in Akt activation and cell survival of postmitotic PC12 neurons, we differentiated PC12 cells by treatment with NGF for 7 days and then examined the effect of SNAP on apoptosis induced by 6-OHDA. SNAP induced Akt activation, and this activation was inhibited by the soluble guanylyl cyclase inhibitor ODQ or the PI3K inhibitor LY294002 (Fig. 8
A). SNAP prevented the differentiated PC12 cell apoptosis induced by 6-OHDA, and the protective effect of SNAP was inhibited by ODQ and LY294002 (Fig. 8B
). Moreover, SNAP suppressed the increased DEVDase activity in differentiated PC12 cells by treatment of 6-OHDA, and this suppression was reversed by ODQ and LY294002 (Fig. 8C
). These data suggest that NO/cGMP/Akt pathway may be involved in neuronal cell survival in development as well as pathological conditions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, motor and sympathetic neurons (19
, 23)
, and cultured hippocampal neurons (42)
. However, the suppressive mechanism involved remained elusive. This study was undertaken to characterize the mechanism by which NO suppresses 6-OHDA-induced apoptosis. We demonstrated here that concentrations of NO, adequate to activate soluble guanylyl cyclase, inhibited 6-OHDA-induced undifferentiated and differentiated PC12 cell apoptosis by a PKG-dependent activation of the PI3K/Akt pathway, resulting in Bad phosphorylation and subsequent suppression of cytochrome c release and caspase-3 activation. This mechanism was further supported by the observations that suppression of the apoptotic signal cascade by cGMP analog 8-Br-cGMP was blocked both by PKG inhibitor and PI3K inhibitors. We concluded that the NO/cGMP pathway suppresses 6-OHDA-induced PC12 cell apoptosis by Bad phosphorylation and inhibition of cytochrome c release and caspase-3 activation through a PKG-dependent activation of the PI3K/Akt pathway.
6-OHDA is a neurotoxin that generates reactive oxygen species (13)
and induces neuronal cell apoptosis in in vitro and in vivo experimental models for the study of Parkinson’s disease (1
, 14
, 15)
. It has been demonstrated that 6-OHDA induces apoptosis through mitochondrial cytochrome c release in cerebellar granule neurons and PC12 cells, which precedes caspase-3 activation (15
, 16)
. Antioxidants such as N-acetyl cysteine and a mixture of catalase and superoxide dismutase protected PC12 cells from 6-OHDA-induced cell death (data not shown), indicating that the major cytotoxic action of 6-OHDA is associated with reactive oxygen generation. Superoxide rapidly reacts with NO and forms peroxynitrite, which is considered to be toxic in cultured motor neuron and PC12 cells (43
, 44)
. It is also possible that this reaction inhibits the antiapoptotic effect of NO, probably by decreasing NO bioavailability and cGMP production (45)
. In addition, peroxynitrite is unstable and rapidly decomposed to nontoxic nitrate (46)
, suggesting that NO prevents superoxide-induced apoptotic cell death. We observed that 6-OHDA did not significantly suppressed cGMP production from PC12 cells treated with SNAP (data not shown). Moreover, we showed that NO produced either from nNOS gene delivery or SNAP and 8-Br-cGMP suppressed the 6-OHDA-induced apoptotic phenomena, such as mitochondrial cytochrome c release, caspase-3 activation, and DNA fragmentation. These results suggest that the NO
cGMP pathway may protect neuronal cells from apoptosis induced by 6-OHDA or other reactive oxygen-generating genotoxic drugs.
We previously demonstrated that NO prevents apoptosis by inhibiting caspase activity and proteolytic activation of downstream caspase proteases through direct S-nitrosylation of their redox-sensitive catalytic thiol (32)
. We went on to show that this process was efficient in iron-rich hepatocytes and iron-preloaded RAW264.7 cells, but not in control RAW274.7 (47)
. The catalytic activity of S-nitrosylated caspases was restored by preincubating with the reducing agent dithiothreitol (32)
. However, DEVDase activity of the cytosol from PC12 cells cotreated with 6-OHDA and SNAP was partially, but not significantly, increased by preincubation with dithiothreitol (data not shown). These results indicate that NO does not modify the catalytic thiol of the caspases in PC12 cells, probably due to low cellular non-heme iron concentrations.
NO is known to activate soluble guanylyl cyclase, resulting in accumulation of intracellular cGMP. Cyclic GMP can prevent apoptotic cell death in PC12 cells (20
, 23)
, sympathetic neurons (19)
, cultured hippocampal neurons (42)
, and hepatocytes (32)
by suppressing cytochrome c release and/or caspase activation. The anti-apoptotic effect of cGMP may be associated with PKG activation in trophic factor-deprived PC12 cells (20)
and hepatocytes (32)
. Consistent with these previous reports, we found that SNAP inhibited cytochrome c release as well as the activation of caspase-9 and -3 via cGMP production. Moreover, the membrane-permeable cGMP analog 8-Br-cGMP showed the same effect as NO in preventing PC12 cell apoptosis induced by 6-OHDA, and this protective effect was required for PKG activation. These results suggest that NO suppressed the apoptotic signal cascade by inhibiting mitochondrial cytochrome c release, upstream of caspase-9 and -3 through a cGMP-dependent PKG activation.
To explore the mechanism by which NO/cGMP pathway modulates the apoptosis signaling cascade, we tested the possibility that NO or cGMP may modulate or activate cellular survival pathways that antagonize the apoptosis signal. Activation of Akt by growth factors and cytokines promotes cell survival by intervening the apoptosis signal cascade via phosphorylation and inactivation of the proapoptotic proteins, such as Bad (26)
and procaspase-9 (27)
. It has also been shown that Akt activation blocks the apoptosis signal cascade by activation of NF-B and subsequent increases in anti-apoptotic gene expression (29)
. Therefore, we examined whether NO/cGMP has any effect on Akt activation in PC12 cells and whether this activation accounts for the protective function of NO/cGMP. We found that NO donor and 8-Br-cGMP activated endogenous Akt in undifferentiated and differentiated PC12 cells. As shown in previous reports (48
, 49)
, 8-Br-cAMP (100 µM) strongly protected PC12 cells from 6-OHDA-induced apoptosis and this protection was inhibited by the cAMP-dependent protein kinase inhibitor KT5720 (data not shown). However, we have previously shown that membrane-permeable cGMP analogs activate Akt through a PI3K-dependent pathway without elevation of the intracellular cAMP level in hepatocytes (48)
, indicating that cAMP is not involved in NO/cGMP-dependent Akt activation. Furthermore, we found that two structurally unrelated PI3K-specific inhibitors, LY294002 and Wortmannin, completely inhibited SNAP- and 8-Br-cGMP-mediated Akt activation in PC12 cells. These results suggest that NO/cGMP stimulated PI3K-dependent Akt activation in PC12 cells. However, SNAP-induced Akt activation was significantly, but not completely, inhibited in PC12 cells by the soluble guanylyl cyclase inhibitor ODQ (Fig. 7A
), indicating that NO may activate the PI3K/Akt pathway, mostly in a cGMP-dependent manner and partially by a cGMP-independent pathway. This result is consistent with the previous report showing that NO directly activates p21(Ras) by S-nitrosylation of redox-sensitive Cys118 and activates PI3K in the Jurkat T cell line (50)
. However, it is still unclear how other proteins upstream of PI3K are involved in the activation of the PI3K/Akt pathway. Thus, further study is required to elucidate the molecular mechanism for NO/cGMP-mediated Akt activation in cells.
Activated Akt has been shown to be required and sufficient for the prevention of apoptosis or promotion of cell survival. This is apparently accomplished through Akt-mediated phosphorylation of several proteins including proapoptotic Bad (26)
. Serine phosphorylation of Bad results in its binding to cytosolic protein 14–3-3 rather than to mitochondrial Bcl-2 or Bcl-XL and alters the subcellular distribution of Bad to prevent interaction with Bcl-2 or Bcl-XL at mitochondrial membranes, thus blocking the proapoptotic function of Bad. Increases in the functional activity of these anti-apoptotic Bcl-2 family proteins prevent apoptosis by interrupting mitochondrial apoptotic signaling (14)
. To explore target molecules downstream of Akt, we examined whether NO/cGMP phosphorylates proapoptotic Bad. We found that SNAP and 8-Br-cGMP increased Bad phosphorylation in 6-OHDA-treated PC12 cells, suppressing the translocation of cytosolic Bad to mitochondria. This event inhibited PC12 cell apoptosis induced by 6-OHDA through the suppression of mitochondrial cytochrome c release and caspase activation. Bad phosphorylation by SNAP and 8-Br-cGMP was blocked by PKG or PI3K inhibitors. This result indicates that activation of PI3K/Akt by NO/cGMP prevents apoptosis by inhibiting cytochrome c release and caspase activation via Bad phosphorylation. Our proposed mechanism for this regulation is shown schematically in Fig. 9
. It thus appears that, in addition to serving as a neurotransmitter, endogenous production of NO may function as a regulator of neuronal apoptosis in pathological conditions.
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ACKNOWLEDGMENTS |
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Received for publication July 31, 2002. Accepted for publication February 14, 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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