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Relationship between protein phosphatase type-2C activity and induction of apoptosis in cultured neuronal cells
Neurochemistry International 41 (2002) 251–259
Relationship between protein phosphatase type-2C activity and induction of apoptosis in cultured neuronal cells Susanne Klumpp a,∗ , Dagmar Selke a , Barbara Ahlemeyer b , Christine Schaper b , Josef Krieglstein b b
a Department of Biochemistry, Faculty of Pharmacy, Philipps-University, Marburg, Germany Faculty of Pharmacy, Institute for Pharmacology and Toxicology, Philipps-University, Marburg, Germany
Received 27 August 2001; accepted 7 January 2002
Abstract The cellular composition and concentration of fatty acids are crucial for proliferation and survival. We recently showed stimulation of protein phosphatase type-2C (PP2C) by unsaturated fatty acids. Here, we describe that treatment of cultured chick neurons with 100 M oleic acid for 24 h increased the percentage of damaged neurons to 61 ± 9% compared with 25 ± 4% in controls. Oleic acid-induced cell death showed features of apoptosis such as chromatin condensation, shrinkage of the nucleus, DNA fragmentation and caspase-3 activation. Extensive studies with a variety of fatty acids revealed a striking correlation between activation of PP2C and induction of apoptosis. Lipophilicity, oxidizability, and an acidic group were required for both effects. In addition, activation of PP2C and induction of apoptosis could discriminate between cis- and trans-conformation of the fatty acids. The results are in favor of PP2C playing an important, yet unidentified role in apoptosis. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Caspase-3; Chick neurons; Lipophilicity; Oleic acid; Phosphatases; Unsaturated fatty acids
1. Introductory statement The reversible phosphorylation of proteins, catalyzed by protein kinases and protein phosphatases, is a major mechanism for the regulation of almost all cellular functions from metabolism to signal transduction, differentiation, proliferation, or apoptosis. A lot has been learned about the contribution of kinases. Knowledge on phosphatases is less clear and has only recently received more investigative attention. Phosphatases that dephosphorylate serine and threonine residues are encoded by the PPP and PPM gene families, which are defined by distinct amino acid sequences and crystal structures (Barton et al., 1994; Cohen, 1994). The PPP family includes the signature phosphatases PP1, PP2A and PP2B (calcineurin), while the PPM family comprises phosphatase type-2C (PP2C) and pyruvate dehydrogenase phosphatase (Cohen, 1994). PP2C enzymes are characterized by a strict requirement of Mg2+ ions for activity (McGowan and Cohen, 1988) and they are insensitive ∗ Corresponding author. Present address: Pharmazeutische Chemie, Marbacher Weg 6, D-35032 Marburg, Germany. Tel.: +49-6421-2826645; fax: +49-6421-2826646. E-mail address: [email protected] (S. Klumpp).
to okadaic acid (Bialojan and Takai, 1988). The ␣- and -isozymes, monomers of 43–48 kDa, are ubiquitously expressed. We recently discovered that unsaturated fatty acids increase the activity of PP2C 10–15-fold (Klumpp et al., 1998a). The cellular lipid composition is crucial for cell viability. Treatment of neuronal cells with unsaturated fatty acids yielded contradictory results which are likely due to variations in the time of exposure, concentration applied, or the cell type used. For example, arachidonic and docosahexanoic acids have been shown to prevent (Lauritzen et al., 2000; Kim et al., 2001) as well as to induce (Surette et al., 1999; Garrido et al., 2001) neuronal apoptosis. In addition, polyunsaturated, but not saturated fatty acids prevented neuronal death in an animal model of transient global ischemia and also reduced kainic acid-induced death of cultured neurons (Lauritzen et al., 2000). In the present study, we have compared the ability of lipophilic compounds to stimulate the activity of recombinant PP2C with their effect on viability of cultured neuronal cells. We demonstrate that activation of PP2C correlates with the induction of apoptosis in neurons. These findings show a remarkable specificity for the chemistry of the activators as well as for the type of phosphatase. The data suggest an involvement of PP2C in apoptosis.
0197-0186/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 7 - 0 1 8 6 ( 0 2 ) 0 0 0 2 0 - 7
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2. Experimental procedures 2.1. Materials Dulbecco’s modified Eagle medium (DMEM), antibiotics and fetal bovine serum were purchased from Life Technologies, Germany. Staurosporine, poly-l-lysine, Hoechst 33258, bovine serum albumin (BSA), casein, alkaline phosphatase (P7923), acid phosphatase (P0157), cAMP-dependent protein kinase (P-4890), fatty acids and derivatives, fluorimetric caspase-3 assay kit and dimethylsulfoxide (DMSO) were from Sigma–Aldrich, Germany. Recombinant PP2C isozymes were obtained from cDNA clones encoding PP2C␣ and PP2C from bovine retina, and expressed in Escherichia coli BL21(DE3)pLysS. His-tag containing proteins were purified from the soluble extract by chromatography on Ni2+ -NTA agarose (Klumpp et al., 1998b). Protein phosphatases-2A (14-111) and -2B (14-104) were from Upstate Biotechnology, Germany, tyrosine phosphatase (539446) and protein phosphatase 1 (539555) from Calbiochem, Germany. [␥-32 P]ATP (110 TBq mmol−1 ) was purchased from Amersham, Germany. Apotag Kit from Oncor, USA, was used for terminal-transferase-mediated ddUTP-digoxigenin nick-end labeling (TUNEL) assay. The bicincolinic acid protein assay kit was from Pierce, USA. 2.2. Methods Primary neuronal cultures were derived from 7-day-old chick embryo telencephalons as previously described by Pettmann et al. (1979). Briefly, the tissue was mechanically dissociated through nylon meshes of 48 m mesh width and the resulting cell suspension was seeded at a density of 4 × 104 cells/cm2 onto poly-l-lysine-coated flasks or poly-l-lysine coated glass cover slips that were placed into petri dishes for TUNEL assay. Cultures were kept at 37 ◦ C and 5% CO2 in a humidified atmosphere in DMEM supplemented with 20% fetal bovine serum and antibiotics. The medium was changed every 2 days. Up to 7 days in culture, 98% of the cells were neurons as evaluated by immunohistochemical demonstration of neurofilament NF160 (Ahlemeyer et al., 2000). A total of 4 days after seeding, cultures were exposed to serum-free medium for 24 h. Thereafter, the cells were treated with fatty acids and derivatives or vehicle for the indicated time period. Fatty acids and derivatives were initially dissolved in pure DMSO and then diluted with culture medium to the final concentration of 0.016% DMSO in all experiments. Nuclear shape and chromosomal structure can be visualized by staining nuclear DNA with Hoechst 33258. Cultures were incubated for 10 min with 10 g/ml Hoechst 33258 in methanol and then washed with methanol and phosphate-buffered saline (PBS). Thereafter, nuclear morphology was observed under a fluorescence microscope. The number of neurons with shrunken nuclei and condensed
chromatin were counted in three areas in each of four different culture flasks and the results were expressed as the percent ratio of neurons with condensed chromatin and a shrunken nucleus versus the total number of cells. To detect DNA fragmentation, the TUNEL assay was performed using a commercial kit according to the manufacturer’s instructions. Briefly, cell monolayers were fixed in methanol at −20 ◦ C for 20 min and then incubated at 37 ◦ C for 1 h with digoxigenin-labelled ddUTP in the presence of terminal transferase. Thereafter, the reaction was stopped and anti-digoxigenin-antibodies coupled with fluorescein isothiocyanate were added for further 30 min. Fluorescence was observed under a confocal laser scanning microscope (LSM 510, Zeiss, Germany). Caspase-3 activity was measured using a fluorimetric assay kit. The assay based on the hydrolysis of the peptide substrate acetyl-Asp–Glu–Val–Asp–7-amido-4-methylcoumarin (Ac-DEVD–AMC) by caspase-3, resulting in the release of the fluorescent AMC moiety. Briefly, cells were harvested and pelleted by centrifugation at 600 × g for 5 min at 4 ◦ C. The supernatant was removed and the pellet was washed with PBS. Thereafter, the pellet was dissolved in lysis buffer containing 50 mM HEPES, pH 7.4, 5 mM 3-(3-cholamidopropyl)dimethylammonio-1propanesulfonate and 5 mM dithiothreitol and incubated for 20 min on ice, followed by a centrifugation at 14 000 × g for 15 min at 4 ◦ C. The supernatant was mixed with reaction buffer containing 10 mM Ac-DEVD-AMC, 20 mM HEPES, pH 7.4, 2 mM EDTA, 0.1% 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate and 5 mM dithiothreitol. Fluorescence of AMC was measured at 360 nm excitation wavelength and 460 nm emission wavelength. Results were calculated using an AMC standard curve and are expressed as nmol AMC/mg protein/min. Protein was measured using the bicincolinic acid protein assay kit and bovine serum albumin as a standard. Activities of PP2C and PP2A were assayed at 30 ◦ C for 10 min in 30 l containing 20 mM Tris–HCl pH 7.5, 0.01% 2-mercaptoethanol, 1.3 mg/ml BSA, and 1 M [32 P]casein (5 × 104 cpm) (McGowan and Cohen, 1988; Cohen, 1991). For determination of PP2C activity in context with fatty acids, 0.7 mM magnesium acetate was added. For detection of PP2C in neuronal cultures, 20 mM Mg2+ was present. Okadaic acid (100 nM) was used to inhibit PP2A activity. Reactions were terminated by the addition of 200 l 20% trichloroacetic acid. After centrifugation at 10 000 × g (5 min), 200 l of the supernatant were analyzed for [32 P]phosphate content. The activities of PP1, PP2B, tyrosine phosphatase, acid and alkaline phosphatase were monitored at 405 nm by formation of p-nitrophenol from 0.5 mg/ml p-nitrophenyl phosphate (Garcia-Rozas et al., 1982). Incubations for determination of acid and alkaline phosphatase activities were carried out in 1 ml at 37 ◦ C and stopped after sufficient color development by the addition of 250 l 13% K2 HPO4 . Alkaline phosphatase was assayed in the presence of 50 mM Tris–HCl pH 7.5 and 1 mM
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magnesium acetate; acid phosphatase in 50 mM sodium acetate pH 5.5. Activities of PP1, PP2B and tyrosine phosphatase were determined in 20 mM Tris–HCl pH 7.5, 1% glycerol, 0.1% 2-mercaptoethanol, and 5 mM MnCl2 (PP1) or 1 mg/ml BSA (tyrosine phosphatase) or 1 mM MnCl2 , 0.1 mM CaCl2 and 10 g/ml calmodulin (PP2B) in 100 l reactions, and terminated by 25 l 13% K2 HPO4 . Activity measurements were kept within the linear range of time and protein.
3. Results 3.1. Neuronal death induced by oleic acid When chick neurons were exposed to oleic acid, they underwent cell death in a dose-dependent manner (Fig. 1). Neuronal death induced by incubating the cultures with 100 M oleic acid for 24 h showed features of apoptosis such as a reduction in nuclear size and chromatin condensation detected by nuclear staining with Hoechst 33258 (Fig. 2, left column) and DNA fragmentation detected by TUNEL assay (Fig. 2, right column). Activation of caspases can be investigated before the relatively late morphological changes of apoptosis. As caspase-3 is recognized as an effector caspase,
Fig. 1. Oleic acid induces neuronal death in a dose-dependent manner. Chick neurons were incubated with different concentrations of oleic acid for 24 h. Thereafter, the percentage of neurons with a reduced nuclear size and chromatin condensation was determined by nuclear staining with Hoechst 33258. Values are given as means ± S.D. of four experiments.
Fig. 2. Oleic acid-induced neurotoxicity showed features of apoptosis. Chick neurons were treated with vehicle (controls) or 100 M oleic acid for 24 h. Thereafter, nuclear staining with Hoechst 33258 (left column) and TUNEL staining (right column) were performed. Please note a higher number of neurons with a reduced nuclear size and chromatin condensation as well as of TUNEL-positive neurons in cultures treated with oleic acid than in controls. Magnification: ×200.
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Fig. 3. Oleic acid activates caspase-3 in cultured chick neurons. Chick neurons were incubated with 100 M oleic acid and vehicle for different time periods. Thereafter, cell lysates were assayed for caspase-3 activity using a fluorimetric assay kit. Values are means±S.D. of six experiments.
we monitored caspase-3 activity in cell lysates using a fluorimetric substrate. Within 2 h after starting the exposure to 100 M oleic acid, a maximal caspase-3 activity was detected (Fig. 3). Oleic acid-induced caspase-3 activation was in the same range as found in cultures treated for 8 h with 200 nM staurosporine (data not shown) which has been previously shown to induce apoptosis in cultured chick neurons (Ahlemeyer and Krieglstein, 2000). Thus, neurons treated with oleic acid primarily underwent apoptosis as we detected an apoptotic nuclear morphology, DNA fragmentation as well as an activation of caspase-3. 3.2. Activation of PP2C correlates with the induction of apoptosis induced by fatty acids In previous studies, we have shown that unsaturated fatty acids activate PP2C (Klumpp et al., 1998a). To find out whether activation of PP2C was involved in the induction of neuronal apoptosis, we evaluated PP2C activity in our cell culture system and tried to find out whether there is a correlation between the activation of PP2C and induction of apoptosis by various fatty acids. The presence of PP2C in various brain areas has been suggested by mRNA analysis (Abe et al., 1992). However, evidence for PP2C in neurons in terms of enzyme activity was lacking. We were able to measure PP2C activity in cultured chick embryonic neurons. Specific activity of neuronal PP2C was in the range of 15–20 pmol Pi /min/mg which was comparable to the activity found in homogenates of other tissues. Identity of PP2C was verified by substrate specificity (dephosphorylation of casein, no dephosphorylation of p-nitrophenyl phosphate), insensitivity towards okadaic acid and an absolute requirement of Mg2+ ions.
Fig. 4. Effect of C-18 fatty acids on PP2C activity. Dephosphorylation of [32 P]casein was determined in the presence of 1.3 mg BSA with 3.3 ng PP2C␣ per incubation. Activity in the absence of fatty acids (100%) was 11 nmol Pi /min/mg. Fatty acids added were 18:0 (䊊), 18:1 cis-9 (䊉), 18:1 trans-9 (䉬), 18:1 cis-15 (), and 18:1 cis-9 methyl ester (䊏). Open squares show the activation by 18:1 cis-9 in the absence of BSA.
Recombinant PP2C was used for the subsequent in vitro enzyme assays because the amount of PP2C from neuronal cell extracts was limited. In addition, interference with endogenous lipids could be avoided. A dose–response curve showing the effect of oleic acid on the activity of PP2C␣ is presented in Fig. 4. Using standard assay conditions, maximal stimulation was observed at 500 M oleic acid. Absence of BSA upon the dephosphorylation reaction shifted the dose–response curve to significantly lower concentrations. The maximal rate of stimulation (14-fold) was not changed (Fig. 4). Oleic acid carries the double bond at C-9, cis-conformation. Other forms of 18:1 fatty acids were also tested for their ability to influence PP2C activity (Fig. 4). Octadecenoic acid with the double bond at C-15 (instead of C-9) did not stimulate PP2C activity. Similarly, elaidic acid, with trans-conformation at C-9 (instead of cis) failed to activate PP2C. The methyl ester derivative of oleic acid (instead of a free carboxy-group) also was not able to increase the activity of PP2C. Short chain unsaturated fatty acids were without effect (Fig. 5A). Polyunsaturated fatty acids like linolenic acid (18:3 cis-9,12,15 ) had about the same impact on PP2C stimulation as monounsaturated fatty acids (data not shown). The features required for the activation of PP2C are also prerequisites for the induction of apoptosis in neuronal cells (Fig. 5B). Even the discriminations between conformations (cis versus trans) and the position of the double bond (9 versus 15 ) were decisive criteria for the development
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Fig. 5. Activation of PP2C correlates with the induction of apoptosis in cultured neuronal cells. PP2C activity (A) was measured in vitro using [32 P]casein as a substrate in the presence of fatty acids (500 M) or DMSO. Induction of apoptosis was observed in primary cultures of chick embryonic neurons (B) treated with vehicle or 10 M ascorbic acid, palmitic acid and palmitoyl-ascorbic acid, or 100 M fatty acids as indicated for 24 h. The percentage of apoptotic neurons was determined by nuclear staining with Hoechst 33258.
of neuronal cell death. Compounds able to stimulate PP2C activity were found to induce apoptosis in neuronal cells. 3.3. Unsaturated fatty acids activate PP2C, but not other phosphatases Next, we addressed the question whether the lipophilic compounds stimulating PP2C acted specifically on that dephosphorylating enzyme. The activities of six phosphatases, representatives from various classes, were investigated: unspecific acid and alkaline phosphatases, serine/threonine protein phosphatases type-1, -2A, -2B, and a tyrosine phosphatase. Again, the members of the C-18 fatty acid series were used as test agents: saturated (stearic acid, 18:0), and unsaturated (oleic acid, 18:1 cis-9 ) fatty acids, and other forms of octadecenoic acid (18:1 cis-15 , trans-9 , and cis-9 methyl ester). None of the six phosphatases was
activated by oleic acid, and the other fatty acids tested either had no effect or were inhibitory (Fig. 6). This demonstrates that only a special phosphatase is activated by substances with specific properties (Fig. 4 versus Fig. 6).
4. Discussion Inhibitors specific for the various phosphatases are commonly used to investigate whether protein phosphatases play a role in cellular behavior and survival. Okadaic acid and tautomycin inhibit the activities of PP1 and PP2A in the nanomolar range (Hardie et al., 1991), PP2B activity is blocked by cyclosporin and FK506 (Antoni et al., 1993). Those inhibitors helped to establish the importance of protein phosphorylation and dephosphorylation in apoptosis (Yan et al., 1997; Nuydens et al., 1998). For instance,
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Fig. 6. Effect of C-18 fatty acids on serine/threonine protein phosphatase type-2C (A), type-1 (B), type-2A (C), type-2B (D), tyrosine protein phosphatase (E), acid phosphatase (F), alkaline phosphatase (G). Casein was used as a substrate for PP2A and PP2C. The other phosphatases were assayed upon dephosphorylation of p-nitrophenyl phosphate. Symbols for the fatty acids added are as in Fig. 4; 18:0 (䊊), 18:1 cis-9 (䊉), 18:1 trans-9 (䉬), 18:1 cis-15 (), and 18:1 cis-9 methyl ester (䊏).
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dephosphorylation of Bcl-2 is carried by PP2A (Deng et al., 1998; Ruvolo et al., 1999) and Bad is a substrate for PP2B (Wang et al., 1999). PP1 and PP2A are known to respond to ceramides by an increase in the dephosphorylation rate (Chalfant et al., 1999). PP2A and PP2B are substrates for caspase-3 and activated by proteolytic cleavage (Santoro et al., 1998; Mukerjee et al., 2000). PP2C is as ubiquitous and active as the phosphatases type-1, -2A and -2B (for review see Wera and Hemmings, 1995). Yet, PP2C has not been studied in context with cell death, mainly because there is no inhibitor available to specifically block the activity of PP2C. The data presented here, however, demonstrate that activation of PP2C by fatty acids and their derivatives correlates with the induction of neuronal apoptosis by these compounds. Apoptotic type of death caused by oleic acid is shown by different approaches such as nuclear staining with Hoechst, 33258 TUNEL assay and caspase-3 activity assay. Systematical analysis of a variety of fatty acids and derivatives revealed the chemical and structural features of compounds essential for activation of PP2C: (i) unsaturation; (ii) lipophilicity with a minimum chain length of 15 C-atoms; and (iii) a free negatively charged group. Only compounds that fulfilled all three requirements were capable to stimulate PP2C activity. This is best exemplified by ascorbic acid, palmitic acid, and the adduct of both (Fig. 5A). Ascorbic acid is not lipophilic, hence did not increase PP2C activity. Palmitic acid failed to stimulate PP2C because lack of oxidizability. Combination of both single molecules to palmitoyl-ascorbic acid, however, resulted in a compound strongly enhancing PP2C activity. The position and conformation of the double bond in fatty acids were crucial as well: 18:1 cis-9 caused enzyme activation, whereas 18:1 trans-9 and 18:1 cis-15 failed to activate PP2C. Our studies revealed that the lipophilic compounds containing the three structural elements necessary to activate PP2C were capable of inducing apoptosis in neuronal cells (Fig. 5). Furthermore, the differentiation between cis- and trans-conformation and the position of the double bond seen for PP2C was also obvious for apoptosis. The striking correlation observed between activation of PP2C in vitro and induction of apoptosis in cultured neuronal cells points to a hitherto unknown involvement of PP2C in cell death processes. The aforementioned correlation was strikingly specific for PP2C. Alkaline and acid phosphatases were not influenced by oleic acid. Furthermore, the serine/threonine phosphatases type-1, -2A and -2B, and tyrosine phosphatase activities were inhibited. The possibility that inhibition of the latter phosphatases by oleic acid was responsible for induction of apoptosis in cultured neuronal cells, instead of activation of PP2C, is unlikely: 18:1 trans-9 and 18:1 cis-15 inhibited PP1, PP2A, PP2B and tyrosine phosphatases similar to oleic acid, yet without leading to apoptosis. Maximal stimulation of PP2C activity in vitro by oleic acid was observed at 500 M, and induction of apoptosis in
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cultured neuronal cells also required at least 100 M. These concentrations seem to be beyond significance at first glance. The following reasons, however, are in favor of dealing with physiologically relevant effects: (i) activation of PP2C could be shifted to lower concentrations if the assays were carried out in the absence of BSA. (ii) Casein is known to chelate fatty acids; it most likely is not the substrate of PP2C in brain. (iii) With regard to cultured neurons, it is not clear to which extent long-chain fatty acids are taken up by diffusion through the lipid bilayer phase and/or get into cells via membrane transport proteins. Addition of [14 C]oleic acid to brain membrane preparations in vitro showed that 40% of the radioactivity was bound to membranes (Witt and Nielsen, 1994). (iv) Once in a cell, cytosolic fatty acid binding proteins with high affinity for oleic acid acts as carrier and buffer to fix the concentration of free oleic acid (Schnutgen et al., 1996). Numerous studies have shown that free fatty acids which are normally present in the brain in small concentrations (e.g. 0.5 M arachidonic acid) rapidly accumulate under pathological conditions including ischemia and hypoxia (Meves, 1994; Wright et al., 2000). Predominant among those free fatty acids is the unsaturated molecule oleate, followed by palmitate, stearate and arachidonate (Yasudo et al., 1985). Oleic acid has been described to regulate Mg2+ -dependent phosphatidylethanolamine N-methyltransferase activity in the retina; low concentrations of this fatty acid (40 M) inhibited, whereas high concentrations (200 M) activated the formation of phosphatidylcholine (Giusto et al., 1997). In the brain, unsaturated fatty acids, like oleic acid and arachidonic acid, modulate the activities of ion channels, protein kinase C and phospholipases. Furthermore, oleic and arachidonic acids have been shown to induce the release of neurotransmitters from synaptosomes and inhibited their reuptake (Rhoads et al., 1983; Troeger et al., 1984) as well as to increase GABA/benzodiazepine receptor ligand binding (Witt and Nielsen, 1994). Interestingly, the requirements of unsaturated fatty acids to enhance [3 H]diazepam binding were a minimum chain length of 15 C-atoms, a minimum of one unsaturated carbon–carbon bond, and cis-conformation. Esterified derivatives and trans isomers were inactive. This pattern is exactly the same as described in this paper for activation of PP2C in vitro and for induction of apoptosis in cultured neuronal cells. Benzodiazepines are known to protect neurons from degeneration after ischemia (Schwartz-Bloom et al., 2000) which, among others, can explain the discrepancy that although oleic acid-induced apoptosis in cultured neurons, it is harmless in brain under normal conditions. Polyunsaturated fatty acids differentially affect cell survival depending on the concentrations applied. In the presence of albumin, oleic acid stimulated cell proliferation by activation of phosphatidylinositol 3-kinase with a half maximal effective concentration of 5 M (Hardy et al., 2000) which is much lower than the concentrations to induce apoptosis in the absence of albumin used in this study. Similarly,
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polyunsaturated fatty acids like linolenic acid, docosahexanoic acid and arachidonic acid at low doses of 10 M have been shown to protect neurons from glutamate excitotoxicity (Lauritzen et al., 2000). Consistent with our findings, polyunsaturated fatty acids induced apoptosis when given at higher concentrations. Docosahexanoic acid (90 M) has been shown to activate caspase-3 and to cause DNA fragmentation as detected by TUNEL assay through an activation of PP1 and PP2C (Siddiqui et al., 2001). Linoleic acid was growth stimulatory at concentrations below 200 M, but induced apoptosis above 400 M (Phoon et al., 2001). In summary, our data suggest PP2C as additional player in the already complex apoptotic signal transduction cascades. These findings are independently supported by the fact that overexpression of PP2C is lethal (Wenk and Mieskes, 1995; Cheng et al., 1999). All this desperately calls for the identification of the physiological substrate(s) of PP2C in neuronal cells.
Acknowledgements The authors thank Sandra Engel and Michaela Stumpf for excellent technical assistance. Palmitoyl-ascorbic acid was a kind gift from K. Eger, Leipzig. Studies were supported by grants from the Deutsche Forschungsgemeinschaft to J.K. and to S.K. (KR-354/16-4).
References Abe, H., Tamura, S., Kondo, H., 1992. Localization of mRNA for protein phosphatase-2C in the brain of adult rats. Mol. Brain Res. 13, 283–288. Ahlemeyer, B., Fischer, D., Kissel, T., Krieglstein, J., 2000. Staurosporine-induced apoptosis in cultured chick embryonic neurons is reduced by polyethylenimine of low molecular weight used as a coating substrate. Neurosci. Res. 37, 245–253. Ahlemeyer, B., Krieglstein, J., 2000. Inhibition of glutathione depletion by retinoic acid and tocopherol protects cultured neurons from staurosporine-induced oxidative stress and apoptosis. Neurochem. Int. 36, 1–5. Antoni, F.A., Shipston, M.J., Smith, S.M., 1993. Inhibitory role for calcineurin in stimulus–secretion coupling revealed by FK506 and cyclosporin A in pituitary corticotrope tumor cells. Biochem. Biophys. Res. Commun. 194, 226–233. Barton, G.J., Cohen, P.T.W., Barford, D., 1994. Conservation analysis and structure prediction of the protein serine/threonine phoshatases. Sequence similarity with diadenosine tetraphosphatase from Escherichia coli suggests homology to the protein phosphatases. Eur. J. Biochem. 220, 225–237. Bialojan, C., Takai, A., 1988. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem. J. 256, 283–290. Chalfant, C.E., Kishikawa, K., Mumby, M.C., Kamibayashi, C., Bielawska, A., Hannun, Y.A., 1999. Long chain ceramides activate protein phosphatase-1 and protein phosphatase-2A. J. Biol. Chem. 274, 20313– 20317. Cheng, A., Ross, K.E., Kaldis, P., Solomon, M.J., 1999. Dephosphorylation of cyclin-dependent kinases by type-2C protein phosphatases. Genes Dev. 13, 2946–2957.
Cohen, P., 1991. Classification of protein-serine/threonine phosphatases: identification and quantitation in cell extracts. Meth. Enzymol. 201, 389–398. Cohen, P.T.W., 1994. Nomenclature and chromosomal localization of human protein serine/threonine phosphatase genes. Adv. Protein Phosphatases 8, 371–376. Deng, X., Ito, T., Carr, B., Mumby, M., May, W.S., 1998. Reversible phosphorylation of Bcl-2 following interleukin-3 or bryostatin-1 is mediated by direct interaction with protein phosphatase-2A. J. Biol. Chem. 273, 34157–34163. Garcia-Rozas, C., Plaza, A., Diaz-Espada, F., Kreisler, M., Martinez-Alonso, C., 1982. Alkaline phosphatase activity as a membrane marker for activated E cells. J. Immunol 129, 52–55. Garrido, R., Mattson, M.P., Hennig, B., Toborek, M., 2001. Nicotine protects against arachidonic-acid-induced caspase activation, cytochrome c release and apoptosis of cultured spinal cord neurons. J. Neurochem. 76, 1395–1403. Giusto, N.M., Castagnet, P.I., Ilincheta, M.G., Roque, M.E., Pasquare, S.J., 1997. Lipid metabolism in photoreceptor membranes: regulation and mechanism. Neurochem. Res. 22, 445–453. Hardie, D.G., Haystead, T.A., Sim, A.T., 1991. Use of okadaic acid to inhibit protein phosphatases in intact cells. Meth. Enzymol. 201, 469– 476. Hardy, S., Langelier, Y., Prentki, M., 2000. Oleate activates phosphatidylinositol 3-kinase and promotes proliferation and reduces apoptosis of MDA-MB-231 breast cancer cells, whereas palmitate has opposite effects. Cancer Res. 60, 6353–6358. Kim, H.Y., Akbar, M., Kim, K.Y., 2001. Inhibition of neuronal apoptosis by polyunsaturated fatty acids. J. Mol. Neurosci. 16, 223–227. Klumpp, S., Selke, D., Hermesmeier, J., 1998a. Protein phosphatase type-2C active at physiological Mg2+ : stimulation by unsaturated fatty acids. FEBS Lett. 437, 229–232. Klumpp, S., Selke, D., Fischer, D., Baumann, A., Müller, F., Thanos, S., 1998b. Protein phosphatase type-2C isozymes present in vertebrate retinae: purification, characterization and localization in photoreceptors. J. Neurosci. Res. 51, 328–338. Lauritzen, I., Blondeau, N., Heurteaux, C., Widmann, C., Romey, G., Lazdunski, M., 2000. Polyunsaturated fatty acids are potent neuroprotectors. Eur. Mol. Biol. Organisation J. 19, 1784–1793. McGowan, C.H., Cohen, P., 1988. Protein phosphatase-2C from rabbit skeletal muscle and liver: an Mg2+ -dependent enzyme. Meth. Enzymol. 159, 416–426. Meves, H., 1994. Modulation of ion channels by arachidonic acid. Progress Neurobiol. 43, 175–186. Mukerjee, N., McGinnis, K.M., Park, Y.H., Gnegy, M.E., Wang, K.K., 2000. Caspase-mediated proteolytic activation of calcineurin in thapsigargin-mediated apoptosis in SH-SY5Y neuroblastoma cells. Arch. Biochem. Biophys. 379, 337–343. Nuydens, R., de Jong, M., van den Kieboom, G., Heers, C., Dispersyn, G., Cornelissen, F., Nuyens, R., Borgers, M., Geerts, H., 1998. Okadaic acid-induced apoptosis in neuronal cells: evidence for an abortive mitotic attempt. J. Neurochem. 70, 1124–1133. Pettmann, B., Louis, J.C., Sensenbrenner, M.C.L., 1979. Morphological and biochemical maturation of neurones cultured in the absence of glial cells. Nature 281, 378–380. Phoon, M.C., Desbordes, C., Howe, J., Chow, V.T., 2001. Linoleic and linolelaidic acids differentially influence proliferation and apoptosis of molt-4 leukaemia cells. Cell Biol. Int. 25, 777–784. Rhoads, D.E., Osburn, L.D., Peterson, N.A., Raghupathy, E., 1983. Release of neurotransmitter amino acids from synaptosomes: enhancement of calcium-independent efflux by oleic and arachidonic acids. J. Neurochem. 41, 531–537. Ruvolo, P.P., Deng, X., Ito, T., Carr, B.K., May, W.S., 1999. Ceramide induces Bcl-2 dephosphorylation via a mechanism involving mitochondrial PP2A. J. Biol. Chem. 274, 20296–20300. Santoro, M.F., Annand, R.R., Robertson, M.M., Peng, Y.W., Brady, M.J., Mankovich, J.A., Hackett, M.C., Ghayur, T., Walter, G., Wong, W.W.,
S. Klumpp et al. / Neurochemistry International 41 (2002) 251–259 1998. Regulation of protein phosphatase-2A activity by caspase-3 during apoptosis. J. Biol. Chem. 273, 13119–13128. Schnutgen, F., Borchers, T., Müller, T., Spener, F., 1996. Heterologous expression and characterization of mouse brain fatty acid binding protein. Biol. Chem. Hoppe Seyler 377, 211– 215. Schwartz-Bloom, R.D., Miller, K.A., Evenson, D.A., Crain, B.J., Nadler, J.V., 2000. Benzodiazepines protect hippocampal neurons from degeneration after transient cerebral ischemia: an ultrastructural study. Neuroscience 98, 471–484. Siddiqui, R.A., Jenski, L.J., Neff, K., Harvy, K., Kovacs, R.J., Stillwell, W., 2001. Docosahexanoic acid induces apoptosis in Jurkat cells by a protein phosphatase-mediated process. Biochim. Biophys. Acta 1499, 265–275. Surette, M.E., Fonteh, A.N., Bernatchez, C., Chilton, F.H., 1999. Perturbations in the control of cellular arachidonic acid levels block cell growth and induce apoptosis in HL-60 cells. Carcinogenesis 20, 757–763. Troeger, M.B., Rafalowska, U., Erecinska, M., 1984. Effect of oleate on neurotransmitter transport and other plasma membrane functions in rat synaptosomes. J. Neurochem. 42, 1735–1742.
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Wang, H.G., Panthan, N., Ethell, I.M., Krajewski, S., Yamaguchi, Y., Shibasaki, F., McKeon, F., Bobo, T., Franke, T.F., Reed, J.C., 1999. Ca2+ -induced apoptosis through calcineurin dephosphorylation of Bad. Science 284, 339–343. Wenk, J., Mieskes, G., 1995. Cytosolic and nuclear localization of protein phosphatase-2C beta 1 in COS and BHK cells. Eur. J. Cell Biol. 68, 377–386. Wera, S., Hemmings, B.A., 1995. Serine/threonine protein phosphatases. Biochem. J. 311, 17–29. Witt, M.R., Nielsen, M., 1994. Characterization of the influence of unsaturated free fatty acids on brain GABA/benzodiazepine receptor binding in vitro. J. Neurochem. 62, 1432–1439. Wright, L.C., Groot, O.K.L., Delikatny, E.J., Santangelo, R.T., Sorell, T.C., 2000. The origin of 1 H NMR-visible triacylglycerol in human neutrophils. Eur. J. Biochem. 267, 68–78. Yan, Y., Shay, J.W., Wright, W.E., Mumby, M.C., 1997. Inhibition of protein phosphatase activity induces p53-dependent apoptosis in the absence of p53 transactivation. J. Biol. Chem. 272, 15220–15226. Yasudo, H., Kishiro, K., Izumi, N., Nakanishi, M., 1985. Biphasic liberation of arachidonic and stearic acids during cerebral ischemia. J. Neurochem. 45, 168–172.
Relationship between protein phosphatase type-2C activity and induction of apoptosis in cultured neuronal cells Susanne Klumpp a,∗ , Dagmar Selke a , Barbara Ahlemeyer b , Christine Schaper b , Josef Krieglstein b b
a Department of Biochemistry, Faculty of Pharmacy, Philipps-University, Marburg, Germany Faculty of Pharmacy, Institute for Pharmacology and Toxicology, Philipps-University, Marburg, Germany
Received 27 August 2001; accepted 7 January 2002
Abstract The cellular composition and concentration of fatty acids are crucial for proliferation and survival. We recently showed stimulation of protein phosphatase type-2C (PP2C) by unsaturated fatty acids. Here, we describe that treatment of cultured chick neurons with 100 M oleic acid for 24 h increased the percentage of damaged neurons to 61 ± 9% compared with 25 ± 4% in controls. Oleic acid-induced cell death showed features of apoptosis such as chromatin condensation, shrinkage of the nucleus, DNA fragmentation and caspase-3 activation. Extensive studies with a variety of fatty acids revealed a striking correlation between activation of PP2C and induction of apoptosis. Lipophilicity, oxidizability, and an acidic group were required for both effects. In addition, activation of PP2C and induction of apoptosis could discriminate between cis- and trans-conformation of the fatty acids. The results are in favor of PP2C playing an important, yet unidentified role in apoptosis. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Caspase-3; Chick neurons; Lipophilicity; Oleic acid; Phosphatases; Unsaturated fatty acids
1. Introductory statement The reversible phosphorylation of proteins, catalyzed by protein kinases and protein phosphatases, is a major mechanism for the regulation of almost all cellular functions from metabolism to signal transduction, differentiation, proliferation, or apoptosis. A lot has been learned about the contribution of kinases. Knowledge on phosphatases is less clear and has only recently received more investigative attention. Phosphatases that dephosphorylate serine and threonine residues are encoded by the PPP and PPM gene families, which are defined by distinct amino acid sequences and crystal structures (Barton et al., 1994; Cohen, 1994). The PPP family includes the signature phosphatases PP1, PP2A and PP2B (calcineurin), while the PPM family comprises phosphatase type-2C (PP2C) and pyruvate dehydrogenase phosphatase (Cohen, 1994). PP2C enzymes are characterized by a strict requirement of Mg2+ ions for activity (McGowan and Cohen, 1988) and they are insensitive ∗ Corresponding author. Present address: Pharmazeutische Chemie, Marbacher Weg 6, D-35032 Marburg, Germany. Tel.: +49-6421-2826645; fax: +49-6421-2826646. E-mail address: [email protected] (S. Klumpp).
to okadaic acid (Bialojan and Takai, 1988). The ␣- and -isozymes, monomers of 43–48 kDa, are ubiquitously expressed. We recently discovered that unsaturated fatty acids increase the activity of PP2C 10–15-fold (Klumpp et al., 1998a). The cellular lipid composition is crucial for cell viability. Treatment of neuronal cells with unsaturated fatty acids yielded contradictory results which are likely due to variations in the time of exposure, concentration applied, or the cell type used. For example, arachidonic and docosahexanoic acids have been shown to prevent (Lauritzen et al., 2000; Kim et al., 2001) as well as to induce (Surette et al., 1999; Garrido et al., 2001) neuronal apoptosis. In addition, polyunsaturated, but not saturated fatty acids prevented neuronal death in an animal model of transient global ischemia and also reduced kainic acid-induced death of cultured neurons (Lauritzen et al., 2000). In the present study, we have compared the ability of lipophilic compounds to stimulate the activity of recombinant PP2C with their effect on viability of cultured neuronal cells. We demonstrate that activation of PP2C correlates with the induction of apoptosis in neurons. These findings show a remarkable specificity for the chemistry of the activators as well as for the type of phosphatase. The data suggest an involvement of PP2C in apoptosis.
0197-0186/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 7 - 0 1 8 6 ( 0 2 ) 0 0 0 2 0 - 7
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2. Experimental procedures 2.1. Materials Dulbecco’s modified Eagle medium (DMEM), antibiotics and fetal bovine serum were purchased from Life Technologies, Germany. Staurosporine, poly-l-lysine, Hoechst 33258, bovine serum albumin (BSA), casein, alkaline phosphatase (P7923), acid phosphatase (P0157), cAMP-dependent protein kinase (P-4890), fatty acids and derivatives, fluorimetric caspase-3 assay kit and dimethylsulfoxide (DMSO) were from Sigma–Aldrich, Germany. Recombinant PP2C isozymes were obtained from cDNA clones encoding PP2C␣ and PP2C from bovine retina, and expressed in Escherichia coli BL21(DE3)pLysS. His-tag containing proteins were purified from the soluble extract by chromatography on Ni2+ -NTA agarose (Klumpp et al., 1998b). Protein phosphatases-2A (14-111) and -2B (14-104) were from Upstate Biotechnology, Germany, tyrosine phosphatase (539446) and protein phosphatase 1 (539555) from Calbiochem, Germany. [␥-32 P]ATP (110 TBq mmol−1 ) was purchased from Amersham, Germany. Apotag Kit from Oncor, USA, was used for terminal-transferase-mediated ddUTP-digoxigenin nick-end labeling (TUNEL) assay. The bicincolinic acid protein assay kit was from Pierce, USA. 2.2. Methods Primary neuronal cultures were derived from 7-day-old chick embryo telencephalons as previously described by Pettmann et al. (1979). Briefly, the tissue was mechanically dissociated through nylon meshes of 48 m mesh width and the resulting cell suspension was seeded at a density of 4 × 104 cells/cm2 onto poly-l-lysine-coated flasks or poly-l-lysine coated glass cover slips that were placed into petri dishes for TUNEL assay. Cultures were kept at 37 ◦ C and 5% CO2 in a humidified atmosphere in DMEM supplemented with 20% fetal bovine serum and antibiotics. The medium was changed every 2 days. Up to 7 days in culture, 98% of the cells were neurons as evaluated by immunohistochemical demonstration of neurofilament NF160 (Ahlemeyer et al., 2000). A total of 4 days after seeding, cultures were exposed to serum-free medium for 24 h. Thereafter, the cells were treated with fatty acids and derivatives or vehicle for the indicated time period. Fatty acids and derivatives were initially dissolved in pure DMSO and then diluted with culture medium to the final concentration of 0.016% DMSO in all experiments. Nuclear shape and chromosomal structure can be visualized by staining nuclear DNA with Hoechst 33258. Cultures were incubated for 10 min with 10 g/ml Hoechst 33258 in methanol and then washed with methanol and phosphate-buffered saline (PBS). Thereafter, nuclear morphology was observed under a fluorescence microscope. The number of neurons with shrunken nuclei and condensed
chromatin were counted in three areas in each of four different culture flasks and the results were expressed as the percent ratio of neurons with condensed chromatin and a shrunken nucleus versus the total number of cells. To detect DNA fragmentation, the TUNEL assay was performed using a commercial kit according to the manufacturer’s instructions. Briefly, cell monolayers were fixed in methanol at −20 ◦ C for 20 min and then incubated at 37 ◦ C for 1 h with digoxigenin-labelled ddUTP in the presence of terminal transferase. Thereafter, the reaction was stopped and anti-digoxigenin-antibodies coupled with fluorescein isothiocyanate were added for further 30 min. Fluorescence was observed under a confocal laser scanning microscope (LSM 510, Zeiss, Germany). Caspase-3 activity was measured using a fluorimetric assay kit. The assay based on the hydrolysis of the peptide substrate acetyl-Asp–Glu–Val–Asp–7-amido-4-methylcoumarin (Ac-DEVD–AMC) by caspase-3, resulting in the release of the fluorescent AMC moiety. Briefly, cells were harvested and pelleted by centrifugation at 600 × g for 5 min at 4 ◦ C. The supernatant was removed and the pellet was washed with PBS. Thereafter, the pellet was dissolved in lysis buffer containing 50 mM HEPES, pH 7.4, 5 mM 3-(3-cholamidopropyl)dimethylammonio-1propanesulfonate and 5 mM dithiothreitol and incubated for 20 min on ice, followed by a centrifugation at 14 000 × g for 15 min at 4 ◦ C. The supernatant was mixed with reaction buffer containing 10 mM Ac-DEVD-AMC, 20 mM HEPES, pH 7.4, 2 mM EDTA, 0.1% 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate and 5 mM dithiothreitol. Fluorescence of AMC was measured at 360 nm excitation wavelength and 460 nm emission wavelength. Results were calculated using an AMC standard curve and are expressed as nmol AMC/mg protein/min. Protein was measured using the bicincolinic acid protein assay kit and bovine serum albumin as a standard. Activities of PP2C and PP2A were assayed at 30 ◦ C for 10 min in 30 l containing 20 mM Tris–HCl pH 7.5, 0.01% 2-mercaptoethanol, 1.3 mg/ml BSA, and 1 M [32 P]casein (5 × 104 cpm) (McGowan and Cohen, 1988; Cohen, 1991). For determination of PP2C activity in context with fatty acids, 0.7 mM magnesium acetate was added. For detection of PP2C in neuronal cultures, 20 mM Mg2+ was present. Okadaic acid (100 nM) was used to inhibit PP2A activity. Reactions were terminated by the addition of 200 l 20% trichloroacetic acid. After centrifugation at 10 000 × g (5 min), 200 l of the supernatant were analyzed for [32 P]phosphate content. The activities of PP1, PP2B, tyrosine phosphatase, acid and alkaline phosphatase were monitored at 405 nm by formation of p-nitrophenol from 0.5 mg/ml p-nitrophenyl phosphate (Garcia-Rozas et al., 1982). Incubations for determination of acid and alkaline phosphatase activities were carried out in 1 ml at 37 ◦ C and stopped after sufficient color development by the addition of 250 l 13% K2 HPO4 . Alkaline phosphatase was assayed in the presence of 50 mM Tris–HCl pH 7.5 and 1 mM
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magnesium acetate; acid phosphatase in 50 mM sodium acetate pH 5.5. Activities of PP1, PP2B and tyrosine phosphatase were determined in 20 mM Tris–HCl pH 7.5, 1% glycerol, 0.1% 2-mercaptoethanol, and 5 mM MnCl2 (PP1) or 1 mg/ml BSA (tyrosine phosphatase) or 1 mM MnCl2 , 0.1 mM CaCl2 and 10 g/ml calmodulin (PP2B) in 100 l reactions, and terminated by 25 l 13% K2 HPO4 . Activity measurements were kept within the linear range of time and protein.
3. Results 3.1. Neuronal death induced by oleic acid When chick neurons were exposed to oleic acid, they underwent cell death in a dose-dependent manner (Fig. 1). Neuronal death induced by incubating the cultures with 100 M oleic acid for 24 h showed features of apoptosis such as a reduction in nuclear size and chromatin condensation detected by nuclear staining with Hoechst 33258 (Fig. 2, left column) and DNA fragmentation detected by TUNEL assay (Fig. 2, right column). Activation of caspases can be investigated before the relatively late morphological changes of apoptosis. As caspase-3 is recognized as an effector caspase,
Fig. 1. Oleic acid induces neuronal death in a dose-dependent manner. Chick neurons were incubated with different concentrations of oleic acid for 24 h. Thereafter, the percentage of neurons with a reduced nuclear size and chromatin condensation was determined by nuclear staining with Hoechst 33258. Values are given as means ± S.D. of four experiments.
Fig. 2. Oleic acid-induced neurotoxicity showed features of apoptosis. Chick neurons were treated with vehicle (controls) or 100 M oleic acid for 24 h. Thereafter, nuclear staining with Hoechst 33258 (left column) and TUNEL staining (right column) were performed. Please note a higher number of neurons with a reduced nuclear size and chromatin condensation as well as of TUNEL-positive neurons in cultures treated with oleic acid than in controls. Magnification: ×200.
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Fig. 3. Oleic acid activates caspase-3 in cultured chick neurons. Chick neurons were incubated with 100 M oleic acid and vehicle for different time periods. Thereafter, cell lysates were assayed for caspase-3 activity using a fluorimetric assay kit. Values are means±S.D. of six experiments.
we monitored caspase-3 activity in cell lysates using a fluorimetric substrate. Within 2 h after starting the exposure to 100 M oleic acid, a maximal caspase-3 activity was detected (Fig. 3). Oleic acid-induced caspase-3 activation was in the same range as found in cultures treated for 8 h with 200 nM staurosporine (data not shown) which has been previously shown to induce apoptosis in cultured chick neurons (Ahlemeyer and Krieglstein, 2000). Thus, neurons treated with oleic acid primarily underwent apoptosis as we detected an apoptotic nuclear morphology, DNA fragmentation as well as an activation of caspase-3. 3.2. Activation of PP2C correlates with the induction of apoptosis induced by fatty acids In previous studies, we have shown that unsaturated fatty acids activate PP2C (Klumpp et al., 1998a). To find out whether activation of PP2C was involved in the induction of neuronal apoptosis, we evaluated PP2C activity in our cell culture system and tried to find out whether there is a correlation between the activation of PP2C and induction of apoptosis by various fatty acids. The presence of PP2C in various brain areas has been suggested by mRNA analysis (Abe et al., 1992). However, evidence for PP2C in neurons in terms of enzyme activity was lacking. We were able to measure PP2C activity in cultured chick embryonic neurons. Specific activity of neuronal PP2C was in the range of 15–20 pmol Pi /min/mg which was comparable to the activity found in homogenates of other tissues. Identity of PP2C was verified by substrate specificity (dephosphorylation of casein, no dephosphorylation of p-nitrophenyl phosphate), insensitivity towards okadaic acid and an absolute requirement of Mg2+ ions.
Fig. 4. Effect of C-18 fatty acids on PP2C activity. Dephosphorylation of [32 P]casein was determined in the presence of 1.3 mg BSA with 3.3 ng PP2C␣ per incubation. Activity in the absence of fatty acids (100%) was 11 nmol Pi /min/mg. Fatty acids added were 18:0 (䊊), 18:1 cis-9 (䊉), 18:1 trans-9 (䉬), 18:1 cis-15 (), and 18:1 cis-9 methyl ester (䊏). Open squares show the activation by 18:1 cis-9 in the absence of BSA.
Recombinant PP2C was used for the subsequent in vitro enzyme assays because the amount of PP2C from neuronal cell extracts was limited. In addition, interference with endogenous lipids could be avoided. A dose–response curve showing the effect of oleic acid on the activity of PP2C␣ is presented in Fig. 4. Using standard assay conditions, maximal stimulation was observed at 500 M oleic acid. Absence of BSA upon the dephosphorylation reaction shifted the dose–response curve to significantly lower concentrations. The maximal rate of stimulation (14-fold) was not changed (Fig. 4). Oleic acid carries the double bond at C-9, cis-conformation. Other forms of 18:1 fatty acids were also tested for their ability to influence PP2C activity (Fig. 4). Octadecenoic acid with the double bond at C-15 (instead of C-9) did not stimulate PP2C activity. Similarly, elaidic acid, with trans-conformation at C-9 (instead of cis) failed to activate PP2C. The methyl ester derivative of oleic acid (instead of a free carboxy-group) also was not able to increase the activity of PP2C. Short chain unsaturated fatty acids were without effect (Fig. 5A). Polyunsaturated fatty acids like linolenic acid (18:3 cis-9,12,15 ) had about the same impact on PP2C stimulation as monounsaturated fatty acids (data not shown). The features required for the activation of PP2C are also prerequisites for the induction of apoptosis in neuronal cells (Fig. 5B). Even the discriminations between conformations (cis versus trans) and the position of the double bond (9 versus 15 ) were decisive criteria for the development
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Fig. 5. Activation of PP2C correlates with the induction of apoptosis in cultured neuronal cells. PP2C activity (A) was measured in vitro using [32 P]casein as a substrate in the presence of fatty acids (500 M) or DMSO. Induction of apoptosis was observed in primary cultures of chick embryonic neurons (B) treated with vehicle or 10 M ascorbic acid, palmitic acid and palmitoyl-ascorbic acid, or 100 M fatty acids as indicated for 24 h. The percentage of apoptotic neurons was determined by nuclear staining with Hoechst 33258.
of neuronal cell death. Compounds able to stimulate PP2C activity were found to induce apoptosis in neuronal cells. 3.3. Unsaturated fatty acids activate PP2C, but not other phosphatases Next, we addressed the question whether the lipophilic compounds stimulating PP2C acted specifically on that dephosphorylating enzyme. The activities of six phosphatases, representatives from various classes, were investigated: unspecific acid and alkaline phosphatases, serine/threonine protein phosphatases type-1, -2A, -2B, and a tyrosine phosphatase. Again, the members of the C-18 fatty acid series were used as test agents: saturated (stearic acid, 18:0), and unsaturated (oleic acid, 18:1 cis-9 ) fatty acids, and other forms of octadecenoic acid (18:1 cis-15 , trans-9 , and cis-9 methyl ester). None of the six phosphatases was
activated by oleic acid, and the other fatty acids tested either had no effect or were inhibitory (Fig. 6). This demonstrates that only a special phosphatase is activated by substances with specific properties (Fig. 4 versus Fig. 6).
4. Discussion Inhibitors specific for the various phosphatases are commonly used to investigate whether protein phosphatases play a role in cellular behavior and survival. Okadaic acid and tautomycin inhibit the activities of PP1 and PP2A in the nanomolar range (Hardie et al., 1991), PP2B activity is blocked by cyclosporin and FK506 (Antoni et al., 1993). Those inhibitors helped to establish the importance of protein phosphorylation and dephosphorylation in apoptosis (Yan et al., 1997; Nuydens et al., 1998). For instance,
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Fig. 6. Effect of C-18 fatty acids on serine/threonine protein phosphatase type-2C (A), type-1 (B), type-2A (C), type-2B (D), tyrosine protein phosphatase (E), acid phosphatase (F), alkaline phosphatase (G). Casein was used as a substrate for PP2A and PP2C. The other phosphatases were assayed upon dephosphorylation of p-nitrophenyl phosphate. Symbols for the fatty acids added are as in Fig. 4; 18:0 (䊊), 18:1 cis-9 (䊉), 18:1 trans-9 (䉬), 18:1 cis-15 (), and 18:1 cis-9 methyl ester (䊏).
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dephosphorylation of Bcl-2 is carried by PP2A (Deng et al., 1998; Ruvolo et al., 1999) and Bad is a substrate for PP2B (Wang et al., 1999). PP1 and PP2A are known to respond to ceramides by an increase in the dephosphorylation rate (Chalfant et al., 1999). PP2A and PP2B are substrates for caspase-3 and activated by proteolytic cleavage (Santoro et al., 1998; Mukerjee et al., 2000). PP2C is as ubiquitous and active as the phosphatases type-1, -2A and -2B (for review see Wera and Hemmings, 1995). Yet, PP2C has not been studied in context with cell death, mainly because there is no inhibitor available to specifically block the activity of PP2C. The data presented here, however, demonstrate that activation of PP2C by fatty acids and their derivatives correlates with the induction of neuronal apoptosis by these compounds. Apoptotic type of death caused by oleic acid is shown by different approaches such as nuclear staining with Hoechst, 33258 TUNEL assay and caspase-3 activity assay. Systematical analysis of a variety of fatty acids and derivatives revealed the chemical and structural features of compounds essential for activation of PP2C: (i) unsaturation; (ii) lipophilicity with a minimum chain length of 15 C-atoms; and (iii) a free negatively charged group. Only compounds that fulfilled all three requirements were capable to stimulate PP2C activity. This is best exemplified by ascorbic acid, palmitic acid, and the adduct of both (Fig. 5A). Ascorbic acid is not lipophilic, hence did not increase PP2C activity. Palmitic acid failed to stimulate PP2C because lack of oxidizability. Combination of both single molecules to palmitoyl-ascorbic acid, however, resulted in a compound strongly enhancing PP2C activity. The position and conformation of the double bond in fatty acids were crucial as well: 18:1 cis-9 caused enzyme activation, whereas 18:1 trans-9 and 18:1 cis-15 failed to activate PP2C. Our studies revealed that the lipophilic compounds containing the three structural elements necessary to activate PP2C were capable of inducing apoptosis in neuronal cells (Fig. 5). Furthermore, the differentiation between cis- and trans-conformation and the position of the double bond seen for PP2C was also obvious for apoptosis. The striking correlation observed between activation of PP2C in vitro and induction of apoptosis in cultured neuronal cells points to a hitherto unknown involvement of PP2C in cell death processes. The aforementioned correlation was strikingly specific for PP2C. Alkaline and acid phosphatases were not influenced by oleic acid. Furthermore, the serine/threonine phosphatases type-1, -2A and -2B, and tyrosine phosphatase activities were inhibited. The possibility that inhibition of the latter phosphatases by oleic acid was responsible for induction of apoptosis in cultured neuronal cells, instead of activation of PP2C, is unlikely: 18:1 trans-9 and 18:1 cis-15 inhibited PP1, PP2A, PP2B and tyrosine phosphatases similar to oleic acid, yet without leading to apoptosis. Maximal stimulation of PP2C activity in vitro by oleic acid was observed at 500 M, and induction of apoptosis in
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cultured neuronal cells also required at least 100 M. These concentrations seem to be beyond significance at first glance. The following reasons, however, are in favor of dealing with physiologically relevant effects: (i) activation of PP2C could be shifted to lower concentrations if the assays were carried out in the absence of BSA. (ii) Casein is known to chelate fatty acids; it most likely is not the substrate of PP2C in brain. (iii) With regard to cultured neurons, it is not clear to which extent long-chain fatty acids are taken up by diffusion through the lipid bilayer phase and/or get into cells via membrane transport proteins. Addition of [14 C]oleic acid to brain membrane preparations in vitro showed that 40% of the radioactivity was bound to membranes (Witt and Nielsen, 1994). (iv) Once in a cell, cytosolic fatty acid binding proteins with high affinity for oleic acid acts as carrier and buffer to fix the concentration of free oleic acid (Schnutgen et al., 1996). Numerous studies have shown that free fatty acids which are normally present in the brain in small concentrations (e.g. 0.5 M arachidonic acid) rapidly accumulate under pathological conditions including ischemia and hypoxia (Meves, 1994; Wright et al., 2000). Predominant among those free fatty acids is the unsaturated molecule oleate, followed by palmitate, stearate and arachidonate (Yasudo et al., 1985). Oleic acid has been described to regulate Mg2+ -dependent phosphatidylethanolamine N-methyltransferase activity in the retina; low concentrations of this fatty acid (40 M) inhibited, whereas high concentrations (200 M) activated the formation of phosphatidylcholine (Giusto et al., 1997). In the brain, unsaturated fatty acids, like oleic acid and arachidonic acid, modulate the activities of ion channels, protein kinase C and phospholipases. Furthermore, oleic and arachidonic acids have been shown to induce the release of neurotransmitters from synaptosomes and inhibited their reuptake (Rhoads et al., 1983; Troeger et al., 1984) as well as to increase GABA/benzodiazepine receptor ligand binding (Witt and Nielsen, 1994). Interestingly, the requirements of unsaturated fatty acids to enhance [3 H]diazepam binding were a minimum chain length of 15 C-atoms, a minimum of one unsaturated carbon–carbon bond, and cis-conformation. Esterified derivatives and trans isomers were inactive. This pattern is exactly the same as described in this paper for activation of PP2C in vitro and for induction of apoptosis in cultured neuronal cells. Benzodiazepines are known to protect neurons from degeneration after ischemia (Schwartz-Bloom et al., 2000) which, among others, can explain the discrepancy that although oleic acid-induced apoptosis in cultured neurons, it is harmless in brain under normal conditions. Polyunsaturated fatty acids differentially affect cell survival depending on the concentrations applied. In the presence of albumin, oleic acid stimulated cell proliferation by activation of phosphatidylinositol 3-kinase with a half maximal effective concentration of 5 M (Hardy et al., 2000) which is much lower than the concentrations to induce apoptosis in the absence of albumin used in this study. Similarly,
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polyunsaturated fatty acids like linolenic acid, docosahexanoic acid and arachidonic acid at low doses of 10 M have been shown to protect neurons from glutamate excitotoxicity (Lauritzen et al., 2000). Consistent with our findings, polyunsaturated fatty acids induced apoptosis when given at higher concentrations. Docosahexanoic acid (90 M) has been shown to activate caspase-3 and to cause DNA fragmentation as detected by TUNEL assay through an activation of PP1 and PP2C (Siddiqui et al., 2001). Linoleic acid was growth stimulatory at concentrations below 200 M, but induced apoptosis above 400 M (Phoon et al., 2001). In summary, our data suggest PP2C as additional player in the already complex apoptotic signal transduction cascades. These findings are independently supported by the fact that overexpression of PP2C is lethal (Wenk and Mieskes, 1995; Cheng et al., 1999). All this desperately calls for the identification of the physiological substrate(s) of PP2C in neuronal cells.
Acknowledgements The authors thank Sandra Engel and Michaela Stumpf for excellent technical assistance. Palmitoyl-ascorbic acid was a kind gift from K. Eger, Leipzig. Studies were supported by grants from the Deutsche Forschungsgemeinschaft to J.K. and to S.K. (KR-354/16-4).
References Abe, H., Tamura, S., Kondo, H., 1992. Localization of mRNA for protein phosphatase-2C in the brain of adult rats. Mol. Brain Res. 13, 283–288. Ahlemeyer, B., Fischer, D., Kissel, T., Krieglstein, J., 2000. Staurosporine-induced apoptosis in cultured chick embryonic neurons is reduced by polyethylenimine of low molecular weight used as a coating substrate. Neurosci. Res. 37, 245–253. Ahlemeyer, B., Krieglstein, J., 2000. Inhibition of glutathione depletion by retinoic acid and tocopherol protects cultured neurons from staurosporine-induced oxidative stress and apoptosis. Neurochem. Int. 36, 1–5. Antoni, F.A., Shipston, M.J., Smith, S.M., 1993. Inhibitory role for calcineurin in stimulus–secretion coupling revealed by FK506 and cyclosporin A in pituitary corticotrope tumor cells. Biochem. Biophys. Res. Commun. 194, 226–233. Barton, G.J., Cohen, P.T.W., Barford, D., 1994. Conservation analysis and structure prediction of the protein serine/threonine phoshatases. Sequence similarity with diadenosine tetraphosphatase from Escherichia coli suggests homology to the protein phosphatases. Eur. J. Biochem. 220, 225–237. Bialojan, C., Takai, A., 1988. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem. J. 256, 283–290. Chalfant, C.E., Kishikawa, K., Mumby, M.C., Kamibayashi, C., Bielawska, A., Hannun, Y.A., 1999. Long chain ceramides activate protein phosphatase-1 and protein phosphatase-2A. J. Biol. Chem. 274, 20313– 20317. Cheng, A., Ross, K.E., Kaldis, P., Solomon, M.J., 1999. Dephosphorylation of cyclin-dependent kinases by type-2C protein phosphatases. Genes Dev. 13, 2946–2957.
Cohen, P., 1991. Classification of protein-serine/threonine phosphatases: identification and quantitation in cell extracts. Meth. Enzymol. 201, 389–398. Cohen, P.T.W., 1994. Nomenclature and chromosomal localization of human protein serine/threonine phosphatase genes. Adv. Protein Phosphatases 8, 371–376. Deng, X., Ito, T., Carr, B., Mumby, M., May, W.S., 1998. Reversible phosphorylation of Bcl-2 following interleukin-3 or bryostatin-1 is mediated by direct interaction with protein phosphatase-2A. J. Biol. Chem. 273, 34157–34163. Garcia-Rozas, C., Plaza, A., Diaz-Espada, F., Kreisler, M., Martinez-Alonso, C., 1982. Alkaline phosphatase activity as a membrane marker for activated E cells. J. Immunol 129, 52–55. Garrido, R., Mattson, M.P., Hennig, B., Toborek, M., 2001. Nicotine protects against arachidonic-acid-induced caspase activation, cytochrome c release and apoptosis of cultured spinal cord neurons. J. Neurochem. 76, 1395–1403. Giusto, N.M., Castagnet, P.I., Ilincheta, M.G., Roque, M.E., Pasquare, S.J., 1997. Lipid metabolism in photoreceptor membranes: regulation and mechanism. Neurochem. Res. 22, 445–453. Hardie, D.G., Haystead, T.A., Sim, A.T., 1991. Use of okadaic acid to inhibit protein phosphatases in intact cells. Meth. Enzymol. 201, 469– 476. Hardy, S., Langelier, Y., Prentki, M., 2000. Oleate activates phosphatidylinositol 3-kinase and promotes proliferation and reduces apoptosis of MDA-MB-231 breast cancer cells, whereas palmitate has opposite effects. Cancer Res. 60, 6353–6358. Kim, H.Y., Akbar, M., Kim, K.Y., 2001. Inhibition of neuronal apoptosis by polyunsaturated fatty acids. J. Mol. Neurosci. 16, 223–227. Klumpp, S., Selke, D., Hermesmeier, J., 1998a. Protein phosphatase type-2C active at physiological Mg2+ : stimulation by unsaturated fatty acids. FEBS Lett. 437, 229–232. Klumpp, S., Selke, D., Fischer, D., Baumann, A., Müller, F., Thanos, S., 1998b. Protein phosphatase type-2C isozymes present in vertebrate retinae: purification, characterization and localization in photoreceptors. J. Neurosci. Res. 51, 328–338. Lauritzen, I., Blondeau, N., Heurteaux, C., Widmann, C., Romey, G., Lazdunski, M., 2000. Polyunsaturated fatty acids are potent neuroprotectors. Eur. Mol. Biol. Organisation J. 19, 1784–1793. McGowan, C.H., Cohen, P., 1988. Protein phosphatase-2C from rabbit skeletal muscle and liver: an Mg2+ -dependent enzyme. Meth. Enzymol. 159, 416–426. Meves, H., 1994. Modulation of ion channels by arachidonic acid. Progress Neurobiol. 43, 175–186. Mukerjee, N., McGinnis, K.M., Park, Y.H., Gnegy, M.E., Wang, K.K., 2000. Caspase-mediated proteolytic activation of calcineurin in thapsigargin-mediated apoptosis in SH-SY5Y neuroblastoma cells. Arch. Biochem. Biophys. 379, 337–343. Nuydens, R., de Jong, M., van den Kieboom, G., Heers, C., Dispersyn, G., Cornelissen, F., Nuyens, R., Borgers, M., Geerts, H., 1998. Okadaic acid-induced apoptosis in neuronal cells: evidence for an abortive mitotic attempt. J. Neurochem. 70, 1124–1133. Pettmann, B., Louis, J.C., Sensenbrenner, M.C.L., 1979. Morphological and biochemical maturation of neurones cultured in the absence of glial cells. Nature 281, 378–380. Phoon, M.C., Desbordes, C., Howe, J., Chow, V.T., 2001. Linoleic and linolelaidic acids differentially influence proliferation and apoptosis of molt-4 leukaemia cells. Cell Biol. Int. 25, 777–784. Rhoads, D.E., Osburn, L.D., Peterson, N.A., Raghupathy, E., 1983. Release of neurotransmitter amino acids from synaptosomes: enhancement of calcium-independent efflux by oleic and arachidonic acids. J. Neurochem. 41, 531–537. Ruvolo, P.P., Deng, X., Ito, T., Carr, B.K., May, W.S., 1999. Ceramide induces Bcl-2 dephosphorylation via a mechanism involving mitochondrial PP2A. J. Biol. Chem. 274, 20296–20300. Santoro, M.F., Annand, R.R., Robertson, M.M., Peng, Y.W., Brady, M.J., Mankovich, J.A., Hackett, M.C., Ghayur, T., Walter, G., Wong, W.W.,
S. Klumpp et al. / Neurochemistry International 41 (2002) 251–259 1998. Regulation of protein phosphatase-2A activity by caspase-3 during apoptosis. J. Biol. Chem. 273, 13119–13128. Schnutgen, F., Borchers, T., Müller, T., Spener, F., 1996. Heterologous expression and characterization of mouse brain fatty acid binding protein. Biol. Chem. Hoppe Seyler 377, 211– 215. Schwartz-Bloom, R.D., Miller, K.A., Evenson, D.A., Crain, B.J., Nadler, J.V., 2000. Benzodiazepines protect hippocampal neurons from degeneration after transient cerebral ischemia: an ultrastructural study. Neuroscience 98, 471–484. Siddiqui, R.A., Jenski, L.J., Neff, K., Harvy, K., Kovacs, R.J., Stillwell, W., 2001. Docosahexanoic acid induces apoptosis in Jurkat cells by a protein phosphatase-mediated process. Biochim. Biophys. Acta 1499, 265–275. Surette, M.E., Fonteh, A.N., Bernatchez, C., Chilton, F.H., 1999. Perturbations in the control of cellular arachidonic acid levels block cell growth and induce apoptosis in HL-60 cells. Carcinogenesis 20, 757–763. Troeger, M.B., Rafalowska, U., Erecinska, M., 1984. Effect of oleate on neurotransmitter transport and other plasma membrane functions in rat synaptosomes. J. Neurochem. 42, 1735–1742.
259
Wang, H.G., Panthan, N., Ethell, I.M., Krajewski, S., Yamaguchi, Y., Shibasaki, F., McKeon, F., Bobo, T., Franke, T.F., Reed, J.C., 1999. Ca2+ -induced apoptosis through calcineurin dephosphorylation of Bad. Science 284, 339–343. Wenk, J., Mieskes, G., 1995. Cytosolic and nuclear localization of protein phosphatase-2C beta 1 in COS and BHK cells. Eur. J. Cell Biol. 68, 377–386. Wera, S., Hemmings, B.A., 1995. Serine/threonine protein phosphatases. Biochem. J. 311, 17–29. Witt, M.R., Nielsen, M., 1994. Characterization of the influence of unsaturated free fatty acids on brain GABA/benzodiazepine receptor binding in vitro. J. Neurochem. 62, 1432–1439. Wright, L.C., Groot, O.K.L., Delikatny, E.J., Santangelo, R.T., Sorell, T.C., 2000. The origin of 1 H NMR-visible triacylglycerol in human neutrophils. Eur. J. Biochem. 267, 68–78. Yan, Y., Shay, J.W., Wright, W.E., Mumby, M.C., 1997. Inhibition of protein phosphatase activity induces p53-dependent apoptosis in the absence of p53 transactivation. J. Biol. Chem. 272, 15220–15226. Yasudo, H., Kishiro, K., Izumi, N., Nakanishi, M., 1985. Biphasic liberation of arachidonic and stearic acids during cerebral ischemia. J. Neurochem. 45, 168–172.