Activation of Protein Kinase A and Atypical Protein Kinase C by A2A Adenosine Receptors Antagonizes Apoptosis Due to Serum Deprivation in PC12 Cells*

Nai-Kuei HuangDagger , Ya-Wen LinDagger , Chuen-Lin HuangDagger , Robert O. Messing§, and Yijuang ChernDagger

From the Dagger  Division of Neuroscience, Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, Republic of China, the § Department of Neurology, University of California, San Francisco, California 94608, and the Ernest Gallo Clinic and Research Center, Emeryville, California 94608

Received for publication, September 20, 2000, and in revised form, December 20, 2000




    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We found in the present study that stimulation of A2A adenosine receptors (A2A-R) prevents apoptosis in PC12 cells. This A2A-protective effect was blocked by protein kinase A (PKA) inhibitors and was not observed in a PKA-deficient PC12 variant. Stimulation of PKA also prevented apoptosis, suggesting that PKA is required for the protective effect of A2A-R. A general PKC inhibitor, but not down-regulation of conventional and novel PKCs, readily blocked the protective effect of A2A-R stimulation and PKA activation, suggesting that atypical PKCs (aPKCs) serve a critical role downstream of PKA. Consistent with this hypothesis, stimulation of A2A-R or PKA enhanced nuclear aPKC activity. In addition, the A2A-protective effect was blocked by a specific inhibitor of one aPKC, PKCzeta , whereas overexpression of a dominant-positive PKCzeta enhanced survival. In contrast, inhibitors of MAP kinase and phosphatidylinositol 3-kinase did not modulate the A2A-protective effect. Dominant-negative Akt also did not alter the A2A-protective effect, whereas it significantly reduced the protective action of nerve growth factor. Collectively, these data suggest that aPKCs can function downstream of PKA to mediate the A2A-R-promoted survival of PC12 cells. Furthermore, the results indicate that different extracellular stimuli can employ distinct signaling pathways to protect against apoptosis induced by the same insult.




    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Adenosine, which is released from metabolically active cells by facilitated diffusion or is generated extracellularly by degradation of released ATP, is a potent biological mediator (1). It is well known that adenosine modulates the activity of numerous cell types including various neuronal populations, platelets, neutrophils, and smooth muscle cells (1). To date, four adenosine receptors (A1, A2A, A2B, and A3) have been identified. These receptors all contain seven transmembrane domains and belong to the G protein-coupled receptor (GPCR)1 family (2). We previously cloned the cDNA and the gene for the rat A2A adenosine receptor (A2A-R; see Refs. 3 and 4). In the central nervous system, the rat A2A-R gene is heavily expressed by striatal neurons and colocalizes with the D2 dopamine receptor in GABAergic striopallidal neurons (5). Low level A2A-R expression is also observed in the cortex, hippocampus, cerebellum, and other areas of the brain (6). Importantly, A2A-R has been regarded as a potential therapeutic target in protecting against neurodegeneration (e.g. Parkinson's disease and Huntington's disease; see Refs. 7 and 8) and neuronal trauma (e.g. hypoxia/ischemia; see Ref. 9). Moreover, stimulation of A2A-R delays apoptosis in human neutrophils (10) and protects the hippocampus from excitotoxicity in a model of kainate-induced neuronal cell death (11). The molecular mechanisms underlying the protective effect of adenosine acting at A2A-R remain largely uncharacterized in neuronal cells.

The rat pheochromocytoma cell line PC12 displays phenotypic traits associated with both adrenal chromaffin cells and sympathetic neurons and is a useful model for studying the actions of neurotrophic factors and neurotransmitters. In the past decade, this cell line has also served as a popular model system for studying the functions of various survival factors, including nerve growth factor (NGF; see Ref. 12). We previously demonstrated that stimulation of A2A-R increases intracellular cAMP formation and activates novel protein kinase C isozymes in PC12 cells (13, 14). In the present study, we found that activation of A2A-R prevents apoptosis in serum-deprived PC12 cells. This protective mechanism involves transient enhancement of PKA activity and subsequent activation of atypical PKCs (aPKCs) and requires the activity of a serine-threonine protein phosphatase (PPase).


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents-- All reagents were purchased from Sigma except where specified. Forskolin, 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxyamidoadenosine (CGS 21680; CGS), 8-(3-chlorostyryl)caffeine (CSC), and okadaic acid (OKA) were purchased from Research Biochemical, Inc. (Natick, MA). DMEM, fetal bovine serum, and horse serum were purchased from Life Technologies, Inc. Bisindolylmaleimide I-HCl (BiM), calyculin A (Caly A), phorbol-12,13-didecanoate (PDD), and LY 294002 were purchased from Calbiochem-Novabiochem. H-89 and KT-5720 were from Biomol (Plymouth Meeting, PA). [gamma -32P]ATP was obtained from PerkinElmer Life Sciences. Anti-active c-Jun N-terminal kinase (JNK) and MAPK antibodies and TfxTM were purchased from Promega Co. (Madison, WI). Anti-phospho-Akt (Thr-308) and (Ser-473) antibodies were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY), and Cell Signaling (Beverly, MA), respectively. The Akt antibody that recognizes the total Akt protein was from Cell Signaling (Beverly, MA). The cell-permeable myristoylated PKCzeta pseudosubstrate (myr-SIYRRGARRWRKL) was obtained from Quality Controlled Biochemicals (Hopkinton, MA). NGF was obtained from Alomone (Jerusalem, Israel).

Cell Culture-- PC12 cells were maintained in DMEM supplemented with 10% v/v horse serum and 5% v/v fetal bovine serum. A123, a cAMP-dependent protein kinase (PKA)-deficient variant of PC12 cells (15), was kindly provided by Dr. J. A. Wagner (Cornell University Medical College, New York). A123 cells were maintained in DMEM supplemented with 5% v/v horse serum and 10% v/v fetal bovine serum. Novel PKC-dominant-negative PC12 variants (16) were maintained in DMEM supplemented with 10% v/v horse serum, 5% v/v fetal bovine serum, and G418 (50 µg/ml).

DNA Fragmentation-- Cells were plated at the density of 3 × 106 cells per 100-mm plate. After 24 h, cells were treated with the indicated reagent(s) for another 24 h and harvested by centrifugation, resuspended in 100 µl of lysis buffer (10 mM EDTA, 50 mM Tris-HCl, 0.5% Sarkosyl, and 0.5 mg/ml proteinase K), and incubated at 50 °C for 3 h. RNase (2 mg/ml) was added to the lysate for another 15 h. The lysate was extracted with 200 µl of phenol/chloroform and then centrifuged again for 5 min. DNA fragments present in the supernatant were separated using a 2% agarose gel.

MTT Assay-- Cells grown on 150-mm plates were washed twice with phosphate-buffered saline (PBS) and resuspended in DMEM. The resuspended cells were plated on 96-well plates (1.5 × 104 cells/well) and treated with the indicated reagent(s) for 24 h. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was then added to the medium (1 mg/ml), and cells were incubated at 37 °C for 3 h. Me2SO (100 µl) was then applied to the medium to dissolve the formazan crystal derived from mitochondrial cleavage of the tetrazolium ring of MTT. The absorbency at 570 nm in each well was measured on a micro-enzyme-linked immunosorbent assay plate reader. None of the reagents used in this study interfered with the MTT values.

PKC Activity Assay-- PKC activity was measured as described previously (14) with slight modifications. To measure atypical PKC activity, PC12 cells were first treated with PDD (1 µM) for 20 h to down-regulate conventional and novel PKCs. Cells were then washed with twice DMEM and incubated with the indicated reagents for the desired period of time. Different fractions of cells were then collected as described below. PKC activity was measured in a 40-µl reaction containing 136 mM NaCl, 5.4 mM KCl, 0.3 mM Na2HPO4, 0.3 mM KH2PO4, 10 mM Mg2SO4, 25 mM beta -glycerophosphate, 5 mM EGTA, 2.5 mM CaCl2, 1 mM glucose, 0.5% Triton X-100, and 25 mM HEPES, pH 7.2. Reactions were started by adding 150 µM of substrate (epsilon  peptide, Upstate Biotechnology Inc.) and 100 µM of [gamma -32P]ATP (2 Ci/mmol). After incubation for 15 min at 30 °C, the reaction was terminated by adding 10 µl of 25% (w/v) trichloroacetic acid. The samples were centrifuged at 7,500 × g for 10 min. The supernatants were then spotted on 2 × 2-cm phosphocellulose squares (Whatman P-81), washed three times using 75 mM phosphoric acid, and once using 75 mM sodium phosphate, pH 7.5. Radioactivity retained on the P-81 papers were measured by scintillation counting. PKCzeta activity was assayed as described above except that a PKCzeta -specific pseudosubstrate peptide (sequence 113-129; SIYRRGARRWRK-LYRAN) was added during the assay to block the PKCzeta activity. PKCzeta activity in PC12 cells was determined as the difference between the PKC activity assayed in the absence and in the presence of 300 µM PKCzeta -specific pseudosubstrate peptide (17). PKC activity increased linearly for up to 30 min using up to 30 µg of protein.

Isolation of Membrane, Cytosol, and Nuclear Fractions-- Membrane, cytosol, and nuclear fractions were isolated as described by Zhou et al. (18). Briefly, PC12 cells were collected by centrifugation (1000 × g, 2 min), resuspended, and incubated in 1 ml of PKC sonication buffer (2 mM Tris, pH 7.6, 50 mM 2-mercaptoethanol, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 100 µM leupeptin, 10 µM aprotinin, and 1 mM NaF) at room temperature for 2 min, and chilled on ice for 5 min. Nonidet P-40 was added to 1% (v/v) final concentration. Samples were forced once through a 20-gauge needle and then MgCl2 was added to 5 mM. The samples were centrifuged at 600 × g for 5 min to collect the nuclear fractions in the pellets. The supernatants were collected as the non-nuclear fraction or were further centrifuged at 100,000 × g for 45 min to separate the cytosol and membrane fractions. The pellets (i.e. the membrane fractions) were resuspended in 300 µl of PKC sonication buffer containing 0.1% Triton X-100. The nuclear fractions were resuspended in 150 µl of buffer C (20 mM HEPES, pH 8, 425 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 80 µg/ml PMSF, 1 mM NaVO4, 20 mM NaF, 100 nM okadaic acid, and 25% glycerol) and incubated for 30 min on ice. The nuclear samples were centrifuged at 3,000 rpm for 10 min at 4 °C to collect nuclear extracts in the supernatants. Protein concentrations were measured using the Bio-Rad Protein Assay Dye Reagent.

Western Blot Analysis-- PC12 cells were rinsed with ice-cold PBS and lysed in ice-cold lysis buffer (20 mM HEPES, 1 mM dithiothreitol, 20 mM EGTA, 10% glycerol, 50 mM beta -glycerophosphate, 10 mM NaF, 1% Triton X-100, 1 mM PMSF, 1 mM Na3VO4, 2 µM aprotinin, 100 µM leupeptin, 2 µM pepstatin, and 0.5 µM OKA). Cell debris was removed by centrifugation at 7,500 × g for 10 min. The supernatant was utilized for the Western blot analysis. Protein concentrations were determined using the Bio-Rad Protein Assay Dye Reagent. Equal amounts of sample were separated by SDS-polyacrylamide gel electrophoresis using 10% polyacrylamide gels. The resolved proteins were then electroblotted onto Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were blocked with 1% bovine serum albumin and incubated with the desired primary antibody for 1 h at room temperature, followed by the corresponding secondary antibody for 1 h at room temperature. Blots were then washed and immunoreactive bands were detected by enhanced chemiluminescence (Pierce) and recorded using Kodak XAR-5 film.

Transfection and Cell Viability Determinations-- All plasmids used in transient transfection experiments were prepared by CsCl purification. Cells were transfected using TfxTM (Promega), following the manufacturer's protocol, and then harvested between 48 and 72 h post-transfection. Transfection efficiency was typically between 10 and 15%. For survival analyses, cells were transiently transfected with a control vector or with vectors encoding the gene of interest along with one-seventh of the molar amount of an expression construct (pEGFP, CLONTECH; Palo Alto, CA) encoding green fluorescent protein (GFP), as indicated. Two days post-transfection, cells were subjected to serum deprivation for 24 h. Transfected cells were identified by GFP expression. Survival was determined as percentage of GFP-expressing cells by counting GFP-expressing cells in photomicrographs taken using a fluorescent microscope and normalized to the total number of cells counted from the corresponding photomicrographs of cells examined by phase contrast. An average of 1500 to 4000 total cells was counted for each experimental condition. Alternatively, GFP-expressing cells were quantified by flow cytometry as indicated below. These two methods produced similar results for the percentage of GFP-expressing cells. The survival index of 100% is designated as the percentage of GFP-expressing cells transfected with a control vector under the indicated treatment conditions.

Flow Cytometry-- PC12 cells were transiently transfected with the indicated plasmid construct plus one-seventh the amount of GFP vector. Forty eight hours post-transfection, serum was withdrawn for 24 h, and cells were analyzed for the expression of GFP by gently removing the cells from plates with PBS containing trypsin (0.15%) and EDTA (0.53 mM) and then analyzing cell samples by flow cytometry with a Becton Dickinson FACScan. The transfected cells were identified by the expression of GFP that was detected using the FL-1 channel (excitation, 488 nm; emission, 530/30 nm). For each transfected plasmid, 30,000 cells were analyzed.

Statistics-- Unless indicated otherwise, results were analyzed by one-way analyses of variance. Differences between means were assessed by the Student-Newman-Keuls method and were considered significant where p < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effects of an A2A-R-selective Agonist, CGS 21680, on Serum-deprived Apoptosis-- Serum deprivation for 24 h resulted in significant DNA fragmentation in PC12 cells (Fig. 1A). Serum deprivation also decreased cell survival, as measured by the MTT assay (Fig. 1C). Addition of an A2A-R-selective agonist, CGS21680 (CGS, 0.1 µM), reversed the DNA fragmentation and cell death induced by serum deprivation. Addition of CGS (0.1 µM) also significantly reduced phosphorylation of the stress-activated kinases JNK1 and JNK2 (Fig. 1B), which are implicated in apoptosis (12). Such protection by A2A-R required new protein synthesis, because a protein synthesis inhibitor blocked the prevention of apoptosis by activation of A2A-Rs in a dose-dependent manner (Fig. 1C).



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Fig. 1.   The A2A-R-selective agonist, CGS, inhibits serum-deprived apoptosis in PC12 cells. A, CGS prevented serum deprivation (24 h)-induced apoptosis as determined by a DNA fragmentation assay in PC12 cells. B, CGS prevented JNK1/2 phosphorylation after serum deprivation for 3 h. C, a protein synthesis inhibitor, cycloheximide, attenuated the protective effect of CGS in serum-deprived apoptosis in a dose-dependent manner. Cell viability is expressed as percentage of MTT metabolism observed in serum-treated cultures. Data points are mean ± S.E. values from three independent experiments. *, p < 0.05 compared with control (serum-free) cultures treated with the indicated concentration of cycloheximide.

The Role of PKA in the A2A-protective Effect in PC12 Cells-- Since activation of A2A-R led to a transient increase in cAMP in PC12 cells (13), we first examined whether PKA plays an important role in preventing apoptosis due to serum deprivation. As shown in Fig. 2A, two PKA inhibitors (H-89 and KT-5720) reduced the protective effect of CGS and forskolin (FK) in serum-deprived apoptosis. In addition, CGS and FK exerted no effect on serum-deprived apoptosis in a PKA-deficient PC12 variant (A123, Fig. 2B), further supporting our hypothesis that PKA is critical for the protective effect of A2A-R against apoptosis. Furthermore, the effect of CGS was markedly reduced by an A2A-R-selective antagonist, 8-(3-chlorostyryl)caffeine (CSC) (Fig. 2A). Thus, the effect of CGS is mediated by A2A-Rs.



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Fig. 2.   The protective effect of A2A-R stimulation against serum-deprived apoptosis is PKA-dependent. Cells (A, PC12 cells; B, A123 cells) were pretreated with the indicated reagent (KT-5720, 1 µM; H-89, 5 µM; or CSC, 10 µM) for 30 min before the addition of CGS (0.1 µM) or FK (5 µM) during serum deprivation. The indicated inhibitor remained present during serum deprivation. Data points represent mean ± S.E. values from three independent experiments. a, p < 0.05 compared with serum-deprived cells treated with CGS but not with CSC, KT-5720, or H-89. b, p < 0.05 compared with cells treated with FK but not with CSC, KT-5720, or H-89.

Phosphatases and PKC Mediate A2A-R-evoked Protection-- Because stimulation of A2A-R activates a serine/threonine PPase in neutrophils (19), we examined whether a PPase was involved in prevention of apoptosis by A2A-R activation. As shown in Fig. 3, two serine/threonine PPase inhibitors, okadaic acid (OKA) and calyculin A (Caly A), blocked the protective effect of A2A-R activation in a dose-dependent manner. Maximal inhibition of the A2A-protective effect by Caly A and OKA occurred at 1 and 10 nM, respectively. Caly A and OKA at these concentrations also markedly reduced the protective effect of FK (Fig. 3). Caly A (1 nM) or OKA (10 nM) alone did not markedly affect the survival of PC12 cells in the absence or presence of serum. Thus, a serine-threonine PPase appears to act downstream of PKA to facilitate survival of serum-starved cells upon A2A-R stimulation.



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Fig. 3.   The protective effect of A2A-R stimulation against serum-deprived apoptosis is PPase-dependent. Cells were pretreated with the indicated concentration of PPase inhibitor (Caly A or OKA) for 30 min before the addition of CGS (0.1 µM) or FK (5 µM) during serum deprivation. The indicated inhibitor remained present during serum deprivation. Data points represent mean ± S.E. values from three independent experiments. A, p < 0.05 compared with serum-deprived cells treated with CGS and not with Caly A or OKA. B, p < 0.05 compared with serum-deprived cells treated with FK and not with Caly A or OKA.

Activation of A2A-R has been shown to stimulate the ERK/MAPK pathway in several cell types, including PC12 cells (20). Therefore we examined whether this pathway is involved in the protection against apoptosis by A2A-R activation. As shown in Fig. 4A, treatment with CGS or FK increased phosphorylation of ERK1/ERK2 without altering protein levels. A MAPK kinase inhibitor (PD98059) blocked the FK- and CGS-mediated activation of ERK. However, PD98059 did not prevent the protective effect of CGS in serum-deprived cells (Fig. 4B). Therefore, activation of ERK is not required for A2A-R-mediated protection against apoptosis. This finding is consistent with the observation that ERK is not important for cAMP- or NGF-mediated survival of primary sympathetic neurons (21).



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Fig. 4.   Activation of MAPK is not important for the protective effect of A2A-R stimulation. A, PC12 cells were pretreated with PD98059 (20 µM) for 30 min before the addition of CGS (0.1 µM; 1 h) or FK (5 µM; 15 min). Phosphorylated MAPK and total MAPK were quantified by Western blot analysis. B, PC12 cells were pretreated with PD98059 (20 µM) for 30 min before serum deprivation in the presence or absence of CGS (0.1 µM). PD98059 of the indicated concentration remained present during serum deprivation. Data points are mean ± S.E. values from three independent experiments. *, p < 0.05 compared with serum-deprived control cells treated with or without PD98059.

We previously showed that stimulation of A2A-Rs activates novel PKCs (14). Therefore, we used a PKC inhibitor bisindolylmaleimide I-HCl (BiM) to examine whether PKC is involved in the protective effect of A2A-R. As shown in Table I, BiM markedly reduced the protective effect of CGS and FK. PKC therefore might be involved in A2A-R-mediated protection and exert its effect downstream of PKA.


                              
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Table I
A PKC inhibitor attenuates A2A-R-mediated protection against apoptosis in serum-deprived PC12 cells
Viability of PC12 cells was determined by MTT assay after 24 h of serum deprivation in the presence or absence of CGS (0.1 µM) or FK (5 µM). Cells were pretreated with the PKC inhibitor BiM (10 µM) for 30 min before serum deprivation. Where indicated, BiM remained present during serum deprivation. Results measured in cells treated with 10% fetal bovine serum and 5% horse serum without BiM, CGS, or FK were used to define 100% viability. Data points represent mean ± S.E. values from three independent experiments.

Atypical PKCs Mediate A2A-R Prevention of Serum-deprived Apoptosis-- PKC is a family of serine/threonine protein kinases that is composed of three subfamilies as follows: conventional, novel, and atypical. We previously demonstrated that two novel PKC isozymes (delta  and epsilon ) play significant roles in the desensitization of A2A-R-induced cAMP formation in PC12 cells (14). Moreover, two atypical PKC isozymes (aPKCs; lambda /iota and zeta ) and two conventional PKC isozymes (alpha  and gamma ) were also observed in our line of PC12 cells (Fig. 5, B and C). To identify the PKC isozymes involved in the protective effect of A2A-R, we treated PC12 cells with a PKC-stimulating phorbol ester, PDD (100 nM), for 20 h to induce proteolysis and down-regulation of conventional and novel PKCs. This treatment caused down-regulation of the conventional PKCs (alpha  and gamma ; Fig. 5C) and novel PKCs (delta  and epsilon ; see Ref. 14). Because aPKCs (lambda  and iota  and zeta ) are insensitive to diacylglycerols and phorbol esters, long term PDD treatment did not decrease levels of aPKCs in PC12 cells (Fig. 5B). Most interestingly, long term PDD treatment did not alter the response to A2A-R stimulation (Fig. 5A). Moreover, as shown in Table II, stimulation of A2A-R using CGS exerted a similar protective effect in PC12 variants expressing dominant-negative fragments of PKCepsilon or PKCdelta . Taken together, these data strongly suggest that conventional and novel PKCs are not involved in A2A-R-mediated protection against apoptosis in PC12 cells.



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Fig. 5.   Down-regulation of conventional and novel PKCs does not interfere with the protective effect of A2A-R stimulation. A, PC12 cells were pretreated with or without PDD (100 nM) for 20 h and then subjected to serum deprivation for 24 h in the presence of the indicated reagent. Data points are mean ± S.E. values from three independent experiments. *, p < 0.05 compared with serum-deprived cells cultured without CGS. Expression of atypical (B) and conventional (C) PKCs in PC12 cells after treatment with PDD for 20 h were analyzed using the corresponding antibody.


                              
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Table II
Stimulation of A2A-Rs promotes survival in PC12 cell lines that express dominant negative fragments of novel PKCs
Viability of the indicated PC12 cell line was determined by MTT assay after serum deprivation for 24 h in the presence or absence of CGS (0.1 µM). Results are expressed relative to values obtained for cells cultured in serum (10% fetal bovine serum and 5% horse serum). Data points are mean ± S.E. values from three independent experiments.

We next considered whether aPKCs are important for the A2-R-mediated protection by measuring aPKC activity after A2A-R stimulation in PC12 cells treated with PDD for 20 h. In these cells, CGS enhanced aPKC activity in both nuclear and non-nuclear fractions (Fig. 6, A and B). The increase in aPKC activity was much greater in the nuclear fraction as compared with the non-nuclear fraction. Western blot analysis showed that PKCzeta and PKClambda /iota immunoreactivities were markedly increased in the nuclear fraction following treatment with CGS (Fig. 6C). FK also enhanced aPKC activity in the nuclear fraction with a time course comparable to that observed with CGS (Fig. 7). These results suggest that PKA mediates increases in nuclear aPKCs during A2A-R stimulation.



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Fig. 6.   Stimulation of A2A-R increases nuclear aPKC activity and protein. PC12 cells were treated with or without CGS (0.1 µM) for the indicated time and then separated into nuclear (A) and non-nuclear fractions (B) for determination of PKC activities. Data points represent mean ± S.E. values from three independent experiments. *, p < 0.05 compared with untreated control samples at the same time point. C, Western analysis of PKCzeta and PKClambda /iota in the nuclear fractions after treatment with CGS for the indicated time. Data shown are representative of results from three independent experiments.



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Fig. 7.   Forskolin increases nuclear aPKC activity in PC12 cells. PC12 cells were treated with or without FK (5 µM) for the indicated time and then were collected to harvest nuclear fractions for determination of PKC activities. Data points represent mean ± S.E. values from three independent experiments. *, p < 0.05 compared with control samples incubated for the same time.

PKC has been implicated in survival following serum deprivation in PC12 cells (22). Therefore, we used a cell-permeable PKCzeta pseudosubstrate inhibitor to assess the role of PKC in the A2A-mediated protection. As shown in Fig. 8A, the PKCzeta -specific inhibitor blocked A2A-R-mediated protection against apoptosis. In addition, transient overexpression of a dominant-positive PKCzeta (PKCzeta +, see Ref. 23) enhanced the survival of serum-deprived PC12 cells by 39 ± 7% (Fig. 8B). Comparable results were obtained when control cells transfected with empty vector and the GFP-expressing construct were treated with CGS. Stimulation of A2A-R using CGS enhanced the number of GFP-expressing cells by 51 ± 20% (mean ± S.E.; p < 0.05, Student's t test, seven independent experiments). This observation is consistent with the protective effect of A2A-R assessed by the MTT assay (Fig. 1C). These findings indicate that PKCzeta specifically regulates A2A-R-mediated survival in PC12 cells.



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Fig. 8.   PKC mediates the protective effect of A2A-R stimulation. A, PC12 cells were pretreated with a cell-permeable PKCzeta -specific inhibitor (2.5 µM) for 30 min before the addition of CGS (0.1 µM) or NGF (100 ng/ml) as indicated. Data points represent mean ± S.E. values from three independent experiments. *, p < 0.05 compared with the corresponding serum-free-treated sample. #, p < 0.05 compared with the corresponding non-PKC inhibitor-treated sample. B, PC12 cells were transiently transfected with a control vector or with vectors encoding JNK1 or PKCzeta + along with one-seventh of the molar amount of a GFP vector as indicated. Two days post-transfection, cells were subjected to serum deprivation for 24 h. Transfected cells were identified by GFP expression, and the number of surviving GFP-expressing cells after 24 h serum deprivation was quantified directly by counting. The percentage of GFP-expressing cells transfected with the control vector was designated as a survival index of 100%. Data points are mean ± S.E. values from three independent experiments. A dominant-negative JNK1 (JNK1; see Ref. 48) was included here as a positive control since it was reported to increase survival upon serum deprivation in other cell types (12). *, p < 0.05 compared with cells transfected with the control vector. C, A123 cells were transiently transfected with a control vector or a vector encoding PKCzeta + along with one-seventh of the molar amount of a GFP vector as indicated. Two days post-transfection, cells were subjected to serum deprivation for 24 h. GFP-expressing cells were quantified by flow cytometry. The percentage of GFP-expressing cells transfected with the control vector was designated as a survival index of 100%. Data points are mean ± S.E. values from three independent experiments. *, p < 0.05 compared with cells transfected with the control vector.

To examine whether stimulation of A2A-Rs regulates PKCzeta , and whether this regulation is PKA-dependent, we next determined the activity of PKCzeta under our experimental conditions. We found that nuclear PKCzeta activity was increased by CGS (Fig. 6) and FK (Fig. 7). Moreover, transient overexpression of PKCzeta + enhanced the survival of serum-deprived A123 cells (a PKA-deficient PC12 variant) (Fig. 8C). Collectively, these results suggest that the protective effect of A2A-R stimulation in serum-deprived PC12 cells requires the sequential activation of PKA and PKCzeta .

The PI3K/Akt Pathway Is Not Involved in the A2A-R-mediated Protection against Apoptosis in Serum-deprived PC12 Cells-- Data from the above experiments suggest that PKCzeta mediates the A2A-protective effect in PC12 cells. Interestingly, the myristoylated PKCzeta pseudosubstrate inhibitor also partially suppressed the protective effect of NGF in serum-deprived PC12 cells (Fig. 8A). Thus, distinct anti-apoptotic signals may converge on PKCzeta in PC12 cells. Because phosphatidylinositol 3-kinase (PI3K) has been implicated in the NGF-induced translocation of PKCzeta to the nucleus (24) and in suppression of apoptosis (25), we next investigated if the PI3K pathway was involved in the protective effect of A2A-R stimulation. Although a PI3K inhibitor (LY294002) abolished the protective effect of NGF against apoptosis in a dose-dependent manner, it did not reduce CGS-mediated survival (Fig. 9A).



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Fig. 9.   The PI3K/Akt pathway is not required for the protective effect of A2A-R stimulation. A, cells were pretreated with the indicated concentration of LY 294002 for 60 min before the addition of CGS (0.1 µM) or NGF (100 ng/ml). Viability of PC12 cells was determined by MTT assay after 24 h of serum deprivation. Data points are mean ± S.E. values from three independent experiments. *p < 0.05 compared with cells not exposed to LY 294002. Akt phosphorylated on Ser-473 (B) or Thr-308 (C) and total Akt were detected by Western analysis in PC12 cells treated with CGS (0.1 µM) or NGF (100 ng/ml) for the indicated time. D, cells were transiently transfected with a control vector or dnAkt along with one-seventh of the molar amount of a GFP vector as indicated. Two days post-transfection, cells were subjected to serum deprivation in the presence of CGS (0.1 µM) or NGF (100 ng/ml) for 24 h. GFP-expressing cells were quantified by flow cytometry. The percentage of GFP-expressing cells transfected with a control vector in the presence of the indicated anti-apoptotic reagent (CGS or NGF) was used to define a survival index of 100%. Data points are mean ± S.E. values from three independent experiments. *, p < 0.05 compared with cells transfected with the control vector.

We next examined whether A2A-R stimulation activates Akt, one of the downstream targets of PI3K implicated in cell survival. We examined the phosphorylation levels of the two activating residues (26), threonine 308 and serine 473 of Akt, by using Western blot analysis (Fig. 9, B and C). Our data show that NGF markedly increases Akt phosphorylation, whereas stimulation of A2A-R does not. We next employed a kinase-dead Akt (dnAkt) that contains a point mutation in its catalytic domain (K179M; see Ref. 27) to determine whether Akt is involved in the A2A-R-protective effect. This K179M-Akt mutant is unable to transmit a signal downstream and effectively decreases upstream signals mediated by 3'-phosphorylated phosphoinositides and PDK1, which activate Akt (28). Overexpression of dnAkt reduced NGF-mediated cell survival during serum deprivation but did not alter CGS-mediated survival (Fig. 9D). Collectively, these results indicate that although the PI3K/Akt pathway plays a role in NGF-mediated survival, it is not activated by A2A-R stimulation nor is it required for the protective effect of A2A-R activation in serum-starved PC12 cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study, we found that A2A-R activation protects PC12 cells from apoptosis induced by serum deprivation. This protective effect requires PKA activation, since it is blocked by two different PKA inhibitors (H-89 and KT 5720), and is absent in PKA-deficient PC12 cells. In contrast to NGF-mediated survival, A2A-mediated protection does not require activation of PI3K or Akt. Although MAPK was activated by stimulation of A2A-R, blocking the MAPK pathway did not alter A2A-R-mediated protection against apoptosis. Compared with the well characterized mechanisms involving ERK/MAPKs and PI3K underlying NGF-mediated survival in PC12 cells, the results of the present study demonstrate that A2A-R utilizes a distinct set of signaling pathways to activate key downstream mediators (e.g. PKCzeta ) of the anti-apoptotic processes (Fig. 10).

In PC12 cells, cyclic AMP has been implicated in mitosis, apoptosis, and differentiation. Long lasting elevation of cellular cAMP, either by treatment with cAMP analogs or FK, rescues PC12 cells from cell death (29). Treatment of PC12 with cAMP analogs also eventually leads to neuronal differentiation, but this requires exposure to these agents for several days (30). Although activation of A2A-R increases cAMP, this response is transient (13) and does not stimulate neural differentiation of PC12 cells.2 Results in the present study suggest that transient activation of the cAMP/PKA pathway is sufficient to protect against cell death due to serum deprivation.

Our data also suggest that downstream of PKA, aPKCs mediate the protective effect of A2A-R against apoptosis in PC12 cells. This conclusion is based on the following evidence. First, the protective effect of A2A-R can be reversed by a general PKC inhibitor (BiM, Table I) and by a PKCzeta -specific inhibitor (Fig. 8A), but not by down-regulation of conventional and novel PKCs (Fig. 5 and Table II). BiM is widely used as a selective inhibitor of PKCs. Since BiM acts as a competitive inhibitor of ATP binding, it may also inhibit PKA at high concentrations (31). Although the in vitro Ki value of BiM for PKC is 10 nM, the effective concentration for inhibiting PKC in cultured cells appears to be higher. For example, in 3T3 fibroblasts, maximal inhibition of PKC-dependent phosphorylation by BiM occurs at 5 µM, which is a concentration that does not inhibit PKA-mediated phosphorylation in those cells (31). In addition, in rat basophilic leukemia (RBL-2H3) cells, 10 µM BiM blocks PKC- but not PKA-evoked phosphorylation of phospholipase C (32). BiM has therefore been routinely used to block PKC-mediated responses at concentrations ranging from 1 to 10 µM in studies employing various types of cultured cells (33, 34). Moreover, in our study, we demonstrated that in addition to BiM, a PKCzeta -specific pseudosubstrate peptide inhibitor also blocked the protective effect of A2A-R, thus confirming the involvement of PKCzeta in this process (Fig. 8A).

The second line of evidence supporting a role for aPKCs in A2AR-mediated survival comes from our observation that stimulation of A2A-R increased nuclear aPKC activity and the amount of nuclear PKCzeta and lambda /iota immunoreactivity in PC12 cells (Fig. 6). This process appears to be mediated by PKA, since FK also increases nuclear aPKC activity (Fig. 7). Increased nuclear aPKC may be important for antagonizing apoptosis since it has been observed following treatment with other mitogenic and differentiating factors that promote cell survival (24, 35). Finally, overexpression of a dominant-positive PKCzeta enhanced the survival of both wild-type and PKA-deficient PC12 cells (Fig. 8, B and C). These results indicate that aPKCs lie in a signal transduction pathway downstream of PKA that mediates that protective effect of A2A-R stimulation and that at least one aPKC, PKCzeta , is critical for the prevention of serum-deprived apoptosis in PC12 cells.

This protective effect of aPKCs is consistent with previous studies suggesting that aPKCs are potent anti-apoptotic kinases (36). For example, low concentrations of ceramide transiently activate PKCzeta in conjunction with NF-kappa B and promote survival of PC12 cells during serum deprivation (22). NGF, which promotes survival of PC12 cells, also increases the abundance of nuclear PKCzeta in PC12 cells (24). Results in the present study demonstrate that the myristoylated PKCzeta -selective inhibitor partially suppressed the protective effect of NGF (Fig. 9A), further suggesting that PKCzeta is an important anti-apoptotic factor in PC12 cells. Since PKCzeta has been implicated in the regulation of NF-kappa B (37), NF-kappa B might act downstream of PKCzeta to inhibit apoptosis due to serum deprivation. PKCiota has been shown to protect human leukemia cells against drug-induced apoptosis (38). We found that nuclear PKCiota is increased following A2A-R stimulation, suggesting that PKCiota may also contribute to important nuclear events that protect against apoptosis in PC12 cells. Further studies are required to clarify the role of PKClambda /iota in the protective effect of A2A-R stimulation.

The involvement of the PI3K/Akt pathway in preventing apoptosis has been well established (25). Various GPCRs activate Akt in either a PI3K-dependent or -independent manner (39, 40). In NGF-differentiated PC12 cells, PI3K plays a critical role in cell survival (41). We considered whether PI3K is important for A2A-mediated survival, since PKCzeta can be activated in a PI3K-dependent manner (42). However, we found that whereas a PI3K inhibitor blocked the ability of NGF to promote survival upon serum deprivation, the protective effect of A2A-R stimulation was PI3K-independent (Fig. 9A and Fig. 10). In addition, Akt, a kinase important for NGF-mediated survival (43), was not involved in the protective effect of A2A-R stimulation. These findings indicate that A2A-R agonists and NGF utilize different signaling pathways to prevent apoptosis. A similar situation exists in sympathetic neurons, where the PI3K/Akt pathway plays a critical role in depolarization-mediated survival, but not in survival mediated by the cAMP/PKA pathway (44). Our results also indicate that in PC12 cells these two pathways converge on at least one common anti-apoptotic factor, PKCzeta . The mechanism by which PKA activates PKCzeta independent of PI3K requires further study.



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Fig. 10.   Schematic representation of signaling pathways that mediate A2A-R-mediated survival.

Our findings also implicate serine/threonine protein phosphatases in the protective effect of A2A-R stimulation. Activation of A2A-R increases the activity of a serine/threonine PPase in neutrophils (19). In the present study, we found that two serine/threonine PPase inhibitors (Caly A and OKA) blocked the protective effect of CGS and FK at nM concentrations. Maximal inhibition was achieved using 1 nM of Caly A or 10 nM of OKA (Fig. 3). Caly A is a selective inhibitor of PP1 and PP2A (Ki = 0.5-2 nM). In contrast, OKA is a relatively specific inhibitor of PP2A with Ki values of 0.2 nM for PP2A and 20 nM for PP1. Because 10 nM of OKA completely blocked A2A-R-mediated protection against apoptosis (Fig. 3B), the PPase most likely involved in this process is a member of the PP2A family. Several important proteins involved in apoptosis, such as Ikappa B kinase, MAP kinases, and cell cycle regulators are substrates of PP2A (45). In a cell-free model of apoptosis, OKA suppresses caspase-3 activation and Akt cleavage, two key events in cell death (46). It remains to be determined whether stimulation of A2A-R activates a PP2A-like activity downstream of PKA that protects PC12 cells from apoptosis or whether basal PP2A activity is merely necessary for A2A-R-mediated survival.

A2A-Rs are expressed in many areas of the brain (6) and in various peripheral tissues (47). Previous work has suggested a role for A2A-Rs in protection against cell death. Kobayashi and Millhorn (9) reported that expression of A2A-Rs is increased by hypoxia. Stimulation of A2A-Rs in PC12 cells partially protects against cell death induced by hypoxia (9). In human neutrophils, stimulation of A2A-Rs delays apoptosis, presumably via a PKA-dependent mechanism (10). Our present study demonstrates that multiple mechanisms downstream of PKA underlie the action of A2A-Rs in preventing apoptosis of serum-deprived PC12 cells. Our findings provide the first clear evidence that PKCzeta is a key downstream component of a PKA-dependent, anti-apoptotic signaling pathway activated by a GPCR. Further knowledge about protective mechanisms evoked by A2A-R stimulation may help to facilitate the clinical application of A2A-R agonists in the treatment of neurodegeneration-associated nervous system trauma and neurological disease.


    ACKNOWLEDGEMENTS

We thank Dr. Peter Parker (Imperial Cancer Research Fund, London, UK) for the activated PKCzeta plasmid; Dr. Alex Toker (Beth Israel Deaconess Medical Center, Harvard Medical School, Boston) for the dominant-negative K179M-Akt mutant plasmid; and D. Platt for reading and editing the manuscript.


    FOOTNOTES

* This work was supported by National Science Council Grants NSC88-2316-B001-008-M46, NSC89-2316-B001-008-M46, and NSC90-2316-B001-005-M46) and by Academia Sinica, Taipei, Taiwan, Republic of China.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence and reprint requests should be addressed. Tel.: 886-2-26523913; Fax: 886-2-27829143; E-mail: bmychern@ibms.sinica.edu.tw.

Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M008589200

2 C. H. Chen and Y. Chern, unpublished observations.


    ABBREVIATIONS

The abbreviations used are: GPCR, G protein-coupled receptor; A2A-R, A2A adenosine receptor; CGS, CGS 21680; CSC, 8-(3-chlorostyryl)-caffeine; PKA, cAMP-dependent protein kinase; JNK, c-Jun N-terminal kinase; aPKCs, atypical PKCs; PP2A, protein phosphates 2A, PDD, phorbol-12,13-didecanoate; PBS, phosphate-buffered saline; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; FK, forskolin; PPase, protein phosphatase; OKA, okadaic acid; Caly A, calyculin A; BiM, Bisindolylmaleimide I-HCl; NGF, nerve growth factor; NF-kappa B, nuclear factor-kappa B; PI3K, phosphatidylinositol 3-kinase; PKC+, dominant-positive PKC; dnAkt, a dominant-negative Akt; GFP, green fluorescent protein; PMSF, phenylmethylsulfonyl fluoride; MAPK, mitogen-activated protein kinase; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular signal-regulated kinase.


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