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MAP Kinase Activation by Cyclosporine A
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
236, 599–603 (1997)
RC977017
MAP Kinase Activation by Cyclosporine A Liliana Paslaru,* Sylviane Trigon,† Martin Kuhlmann,‡ and Michel Morange†,1 †Groupe de Biologie Mole´culaire du Stress, Unite´ de Ge´ne´tique mole´culaire, Ecole Normale Supe´rieure, 46 rue d’Ulm, 75230 Paris, Cedex 05, France; *University of Medicine and Pharmacy Carol Davila, Post-Graduate Department of Biochemistry, Fundeni Hospital, Sos Fundeni No. 258, Bucharest, Romania; and ‡Schwerpunkt Nephrologie, Innere Medizin IV, Med. Universita¨tsklinik, D-66421 Homburg/Saar, Germany
Received June 10, 1997
Short treatment of HeLa cells with cyclosporine A led to the activation in the crude cell extracts of a MAP kinase-like activity. Fractionation by chromatography on a Mono Q column allowed the separation of two activities co-eluting with the MAP kinases ERK1 and ERK2. The activation of these two MAP kinases was demonstrated in Western Blotting by the appearance, after CsA treatment, of two new slowly migrating forms on SDS electrophoretic gels. A similar activation was also obtained in renal epithelial BSC-1 cells and 3T3 fibroblasts. MAP kinase activation might result from a perturbation of calcium homeostasis induced by CsA treatment. q 1997 Academic Press
Cyclosporine A (CsA) is an immunosuppressive drug whose use has resulted in a dramatic increase in the efficiency of organ transplantation in recent years (1,2). The major cellular target of the immunosuppressive action of CsA is the T lymphocyte (3). The drug interacts with cyclophilins and the resulting complexes inhibit a calmodulin-dependent protein phosphatase, called PP2B or calcineurin (4,5). This inhibition of calcineurin leads to a blockade in the activation of the transcription factor NFAT which is involved in cytokine production and in the subsequent activation of T lymphocytes (6,7). Many studies have demonstrated the pleiotropic action of CsA. This drug blocks the direct interaction of the YY1 transcription factor with cyclophilin A (8). CsA also leads to an alteration in the efficiency of a subset of general transcription factors in addition to NFAT (9). It has also a destabilizing effect on some mRNAs (10). In organs such as kidney, prolonged treatment with CsA has toxic effects (11). As seen from the action of CsA on YY1 factor, it is highly probable that some of these effects do not result from the well documented 1
Corresponding author. Fax: 33 1 44 32 39 41.
inhibitory action of CsA on calcineurin phosphatase activity. In fact, as far as the different metabolites or analogs of CsA are concerned, their nephrotoxicity does not correlate with their immunosuppressive action (12,13). In a previous paper, we described the stress-like effects of CsA on different cellular systems. CsA inhibits general protein synthesis in parallel with the specific stimulation of BiP (Grp78) synthesis and increases HSP 70 and HSP 90 synthesis after heat-shock (14). These observations suggested to us that CsA might be included in the family of chemical agents that induce the stress response. To confirm this hypothesis, we sought other stress effects of CsA. Recently, it was shown that MAP kinases are induced by stress (15,16). Here we demonstrate that two members of the MAP kinase family, ERK1 and ERK2, are activated by short treatments with CsA in the three cell systems we used. The characterization of these new intracellular targets of CsA action might help to explain the pleiotropic effects of this drug. MATERIALS AND METHODS Cell culture, CsA treatment, and crude cell extract preparation. HeLa (MRL2 clone), 3T3 fibroblasts or renal proximal tubular (BSC1) monkey cells were cultured at 377C in a 10% CO2 water-saturated atmosphere in Dulbecco’s modified Eagle medium containing 10% heat-inactivated fetal calf serum, 2 mM glutamine, 50 mg/ml streptomycin and 50 units/ml penicillin. The physiological properties of the BSC-1 cells have been described previously (17). They were obtained from American Type Culture Collection (ATCC), Rockville MD, USA. 3T3 cells were a kind gift of M. Yaniv’s laboratory (Institut Pasteur). Serum induction was carried out 24 h after replacing the normal medium with serum-free medium, by addition of a 20% serum-containing medium for 15 min. Cells were plated at low density and incubated with CsA during the exponential growth phase. CsA (or analogs) were prediluted in culture medium and incubated with cells for the indicated times. Crude extracts were prepared as previously described (16). Briefly, cells were washed in Buffer A (50 mM Glycerophosphate, 10 mM MgCl2 , 1 mM EGTA, 10% glycerol pH 7.3) and lysed in the same buffer supplemented with 1% (v/v) Nonidet P-40, 1 mg/ml pepstatin,
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1 mg/ml aprotinin, 0.5 mM phenyl methyl sulfonyl fluoride, 1 mM sodium orthovanadate, 50 mM NaF and 0.01% (v/v) b-mercaptoethanol. Lysates were centrifuged at 10,000g for 10 min and supernatants collected (about 1.5 mg/ml). CsA was kindly provided by Mrs Annick Roche (Sandoz France). It was diluted in cremophor/ethanol (2:1, v/v) at 10 mg/ml. Two analogs of CsA, MeAla-6 (CsA1), which is a non-immunosuppressive analog but an efficient PPIase inhibitor, and MeBm2t (CsA2), which is immunosuppressive but devoid of any effect on PPIase activity, were kindly provided by Dr. Philippe Durette (Merck, Sharp and Dohme Research Laboratories) (13). Chromatography on Mono Q. Prior to chromatography, the lysates were clarified by centrifugation at 100,000 1 g for 15 min at 47C. Supernatants were applied to an HR 5/5 Mono Q column (Pharmacia) previously equilibrated in Buffer A (18). After washing (10 ml), proteins were eluted with a 30 ml linear 0 to 300 mM NaCl gradient in the same buffer. The flow rate was 0.5 ml/min during loading and 1.0 ml/min during washing and elution. Fractions of 1.0 ml were collected. Hepta-4 kinase assay. The reaction mixture (24 ml) contained (final concentrations): 60 mM glycerophosphate, 10 mM EGTA, 10 mM MgCl2 , 7.5% glycerol, 1 mM sodium orthovanadate, 50 mM NaF, g32P ATP (0.2 mM, 1 mCi), 0.1% b-mercaptoethanol, hepta-4 (SPTSPSY)4 (0.4 mg/ml). Hepta-4 peptide corresponds to a four-fold repeat of a motif highly repeated in the C-terminal part of RNA polymerase II. It was synthesized by Dr. Odile Siffert in the Organic Chemical Laboratory of Institut Pasteur. Two microliters of crude cell extracts or 4 microliters of the different fractions from the Mono Q column were added and in vitro reactions were performed in initial rate conditions at 307C for 30 min. Reactions were stopped by the addition of 1 volume of Laemmli sample buffer containing 5% b-mercaptoethanol, and samples were analyzed by 15% SDS polyacrylamide gel electrophoresis performed according to Laemmli (19). Gels were fixed in ethanol/acetic acid/trichloracetic acid/water (3:1:1:5, v/v), dried, and submitted to autoradiography with intensifying screens at 0707C. Signals were quantified with a BAS READER (FUJI) and measurements were made with PC BAS. Western blot analysis. After electrophoresis on a 10% polyacrylamide gel, proteins were electrotransferred on nitrocellulose. Western Blots were performed as described in the ECL kit (Amersham Corp.). An anti ERK1-ERK2 antibody was used (Ab 956/837 from Santa Cruz Biotechnology).
RESULTS Induction of a MAP Kinase-Like Activity in HeLa Cells by Short Treatments with CsA For the MAP kinase assay, we used a short peptide of 28 amino acids (hepta-4) bearing a four-fold repeat
FIG. 1. Activation of a MAP kinase-like activity by CsA treatment. Cell treatment with 100 mM CsA for 30 min, 1, 2, 3, 4 and 5 hours. Crude extract preparation and assays were performed as described in Materials and Methods.
FIG. 2. Dependence of MAP kinase activity on the concentration of CsA added during the pretreatment. Same experimental conditions as in Figure 1. The duration of CsA treatment was 3 hours. The activity in control cells was considered as 100%.
of the consensus sequence of MAP kinases, which was demonstrated in previous studies to be a good in vitro substrate for MAP kinases, in particular for ERK1 and ERK2 (16). The activation of hepta-4 kinase activity by CsA treatment was time and concentration dependent. The increase in enzymatic activity in crude cell extracts was already detectable between 30-60 min after the addition of 100 mM CsA and was roughly proportional to the incubation time between 1 and 5 hours (Figure 1). This kinase activity was dependent on CsA concentration, being maximal at high concentrations of CsA, but already detectable at low concentrations (10 mM) (Figure 2). The Increased Hepta-4 Kinase Activity after CsA Treatment Results from Activation of the Major MAP Kinases ERK1 and ERK2 The hepta-4 peptide is a good in vitro substrate for MAP kinases, and the amount of hepta-4 peptide phosphorylated by crude cell extracts increases dramatically when MAP kinases are activated by the addition of mitogenic agents (16). However, this peptide can be also efficiently phosphorylated by protein kinases unrelated to MAP kinases, such as the dsDNA dependent protein kinase (Trigon, submitted). To confirm that the increase in hepta-4 protein kinase activity observed after CsA treatment in crude extracts resulted from activation of MAP kinases, these extracts were fractionated by chromatography on a Mono Q column. In a preliminary control experiment (Figure 3A), extracts from untreated or serum activated HeLa cells (see Materials and Methods for the experimental condi-
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FIG. 3. MAP kinases ERK1 and ERK2 are activated by serum addition and CsA treatment. Crude extracts from untreated, serum or CsA-treated cells were fractionated on a Mono Q column and the different fractions assayed for hepta-4 kinase activity or for the presence of MAP kinases by Western blotting with anti ERK1-ERK2. A) Hepta-4 kinase activity induction by serum addition. B) Detection of ERK1 and ERK2 in the fractions of Experiment A containing hepta-4 kinase activity (a: without serum addition; b: after serum addition). C) Same experiment as in A, but the cells were treated with 100 mM CsA for 3 hours, instead of being treated with serum. D) Characterization of ERK1 and ERK2 in the fractions of column C containing hepta-4 kinase activity (a: without CsA; b: after CsA addition). Dotted line: NaCl gradient. After quantification, the activities are expressed in arbitrary units, but the values can be directly compared between experiments 3A and 3C.
tions used for serum activation) were fractionated on the same column. Each fraction was assayed for hepta4 kinase activity. Two peaks of hepta-4 kinase activity were obtained, eluting at 0.175 and 0.2 M NaCl. The fractions containing hepta-4 kinase activity after serum addition and the corresponding fractions from the extracts of untreated HeLa cells were analyzed by western blotting with antibodies specific for ERK1 and ERK2 (Figure 3B). The two proteins were present in the fractions containing hepta-4 kinase activity. In the extracts from serum treated cells (b), two forms of ERK2 (p42) migrating differently on SDS gels and two forms of ERK1 (p44) eluting at different positions on the Mono Q column were clearly detectable, whereas only one form of each ERK enzyme was present in extracts from untreated cells (a). The slowly migrating form of ERK2 and the retarded species of ERK1 coincide exactly with the first and second peaks of hepta4 kinase activity, respectively, and correspond to the phosphorylated, activated forms of ERK2 and ERK1. When the same experiment was repeated with extracts from CsA-treated cells (Figure 3C; 3 hours of pretreatment with 100 mM CsA), two peaks of hepta-4 kinase activity were increased by CsA treatment, eluting at the same positions as the protein kinase activi-
ties stimulated by serum. However the maximal activity levels were higher after serum addition than after CsA treatment. The activation of MAP kinases by CsA was confirmed by western blotting of the corresponding fractions. Figure 3D shows that the fractions in which hepta-4 kinase activity was found correspond to the fractions containing the slowly migrating form of ERK2 and the retarded species of ERK1 (in this experiment, we were able to separate the active and inactive forms of ERK1 on SDS gels). These activated forms of ERK1 and ERK2 were only detected in extracts from CsA-treated (b), but not untreated HeLa cells (a). However the relative amount of the activated forms was lower than that obtained after serum addition, in full agreement with the hepta-4 kinase activity measurements. Activation of Hepta-4 Kinase Activity Can Be Also Obtained with CsA Analogs and in Different Cell Types Since the kidney is one of the organs most sensitive to CsA treatment, it was interesting to see whether MAP kinase induction by CsA would also be observed in kidney epithelial cells. As shown in Figure 4, this
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FIG. 4. Activation of a MAP kinase activity by CsA pretreatment in kidney BSC-1 cells. Same experimental conditions as in Figure 1, except that CsA pretreatment was 2 hours.
was indeed the case in BSC-1 cells in the same range of CsA concentrations as those which increased hepta4 kinase activity in HeLa cells: the stimulatory effect was already detected at 1 mM CsA after two hours of treatment, but was higher with 100 mM CsA. The activation of hepta-4 kinase activity was not limited to epithelial cells but was also obtained after CsA treatment of 3T3 fibroblasts (data not shown). We tested the action of two analogs of CsA. CsA1 (MeAla-6) binds efficiently to cyclophilins and inhibits their PPIase activity, but has no immunosuppressive action. CsA2 (MeBm2t) is immunosuppressive but has no inhibitory effect on the PPIase activity of cyclophilins. Figure 5 shows that both analogs could stimulate hepta-4 kinase activity, though to a lower extent than CsA itself. DISCUSSION Our experiments demonstrated that CsA treatment induces the activation of the MAP kinases, ERK1 and ERK2. Activation by CsA was rapid: detectable after 1 hour, it peaked at about 5 hours and disappeared after longer treatments (data not shown). High concentrations of CsA induced stronger MAP kinase activation, but low, ‘‘physiological’’, therapeutic doses of CsA (1 mM) were sufficient to induce the phenomenon. Activation was not limited to HeLa cells, since CsA stimulation of MAP kinases was similar in kidney epithelial and 3T3 cells, nor was it specific to CsA, since two analogs of this drug, with different potential immunosuppressive actions, had a nearly identical effect. In T cells, MAP kinases are essential components of the activation pathway resulting from the recognition of antigens presented by the MHC complex and leading to the synthesis of the growth factor IL2. Reciprocally, it has recently been shown that the functional unresponsiveness of T cells, called anergy, is parallel to a blockade in the activation of these protein kinases (20, 21). Therefore, it may appear surprising that components of a signalling pathway involved in mitogenesis and activation are stimulated by CsA, whereas in lym-
phocytes this drug has an inhibitory effect. Our observation thus raises two questions: the mechanism by which MAP kinases are activated and the physiological significance of this activation. An increase in intracellular calcium is one of the numerous stimuli known to activate MAP kinases (22) maybe through the intermediary action of PYK2 tyrosine kinase (23). In a previous report, we showed that CsA, like calcium ionophores, can induce GRP78 synthesis inside the endoplasmic reticulum. Since GRP78 synthesis can be induced by a decrease in the calcium concentration inside the endoplasmic reticulum (24), a possible link between the earlier and present observations would be that CsA treatment results in a perturbation of calcium homeostasis, with a decrease in calcium concentrations inside the endoplasmic reticulum and a concomitant increase in cytosolic calcium. The former would lead to the stimulation of GRP78 synthesis and the latter to the activation of MAP kinases. In fact, modifications in calcium homeostasis by CsA have been already observed in different systems. These modifications are complex, involving both a mitochondrial calcium overload (25) and variations in the level of intracytosolic calcium. The latter have been observed with CsA alone, or when CsA was added to other hormonal agents which themselves modify calcium homeostasis (26, 27). Use of CsA analogs supported the hypothesis that activation of MAP kinases by CsA is not dependent on inhibition of calcineurin phosphatase, but most probably results from variations in intracellular calcium levels. The two analogs which were tested have different immunosuppressive actions (and effects on calcineurin) (13) but equally induce MAP kinase activity. As noted previously (27), similar modifications in intracellular calcium concentrations are induced by different CsA analogs, irrespective of their immunosuppressive action. How might CsA alter calcium homeostasis? Apart from its specific action on the calcium channels of the
FIG. 5. Activation of a MAP kinase activity by two analogs of CsA. HeLa cells were preincubated with 100 mM of CsA or the two analogs (CsA1 MeAla-6; CsA2 MeBm2t) for 3 hours. Extracts and assays were performed as described in Materials and Methods.
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inner mitochondrial membrane, CsA is able to modify the properties and fluidity of biological membranes by inserting into these membranes (28-30). Such modifications might result in altered transmembrane calcium flux. MAP kinases have pleiotropic effects, due to the large number of proteins which have been demonstrated to be their phosphorylation targets, both in vivo and in vitro (31). One of the roles proposed for MAP kinases is activation by phosphorylation of transcription factors such as Elk-1 or SAP-1a. These factors, when complexed with the SRF, are responsible for serum activation of the genes containing a Serum Responsive Element in their promoter (32, 33). Protein synthesis might also be altered by the stimulation of MAP kinases. Non-phosphorylated PHAS-1 binds to initiation factor 4E (eIF-4E) and inhibits protein synthesis. When PHAS-1 is phosphorylated by the MAP kinases, it no longer interacts with eIF-4E and translation can be initiated (34, 35). Other nuclear and cytoplasmic proteins are potential substrates for MAP kinases. Their modifications might result in other cellular effects, such as cytoskeletal alterations. The diversity of MAP kinase targets is similar to the diversity of CsA effects on cells. Moreover, by modifying the level of intracellular calcium, CsA might alter the action of numerous hormones which use calcium as second messenger and play fundamental roles in the control of physiological processes. ACKNOWLEDGMENTS We are very indebted to Rosemary Sousa-Yeh for her critical reading of the manuscript and to Miss Murielle Rallu for her help in the experiments. L.P. was an invited Professor of the ENS (PAST program). CsA was provided to us by Mrs. Annick Roche (Sandoz France) and the two analogs by Dr. Philippe Durette (Merck, Sharp and Dohme Research Laboratory).
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7. Park, J., Yaseen, N. R., Hogan, P. G., Rao, A., and Sharma, S. (1995) J. Biol. Chem. 270, 20653–20659. 8. Yang, W. M., Inouye, C. J., and Seto, E. (1995) J. Biol. Chem. 270, 15187–15193. 9. Mahajan, P. B., and Thompson, E. A. (1993) J. Biol. Chem. 268, 16693–16698. 10. Nair, A. P. K., Hahn, S., Banholzer, R., Hirsch, H. H., and Moroni, C. (1994) Nature 369, 239–242. 11. Morris, S. M., Kepka-Lenhart, D., McGill, R. L., Curthoys, N. P., and Adler, S. (1992) J. Biol. Chem. 267, 13768–13771. 12. Bowers, L. D. (1990) Transplant. Proc. 22, 1135–1136. 13. Sigal, N. H., Dumont, F., Durette, P., Siekierka, J. J., Peterson, L., Rich, D. H., Dunlap, B. E., Staruch, M. J., Melino, M. R., Koprak, S. L., Williams, D., Witzel, M. R., and Pisano, J. M. (1991) J. Exp. Med. 173, 619–628. 14. Paslaru, L., Pinto, M., and Morange, M. (1994) FEBS Lett. 350, 304–308. 15. Dubois, M., and Bensaude, O. (1993) FEBS Lett. 324, 191–195. 16. Trigon, S., and Morange, M. (1995) J. Biol. Chem. 270, 13091– 13098. 17. Hopps, H. E., Bernheim, B., Nisalak, A., Tjio, J. H., and Smadel, J. E. (1963) J. Immunol. 91, 416–424. 18. Northwood, I. C., Gonzalez, F. A., Wartmann, M., Raden, D. L., and Davis, R. J. (1991) J. Biol. Chem. 266, 15266–15276. 19. Laemmli, U. K. (1970) Nature 227, 680–685. 20. Li, W., Whaley, C. D., Mondino, A., and Mueller, D. L. (1996) Science 271, 1272–1276. 21. Fields, P. E., Gajewski, T. F., and Fitch, F. W. (1996) Science 271, 1276–1278. 22. Chao, T.-S. O., Byron, K. L., Lee, K.-M., Villereal, M., and Rosner, M. R. (1992) J. Biol. Chem. 267, 19876–19883. 23. Lev, S., Moreno, H., Martinez, R., Canoll, P., Peles, E., Musacchio, J. M., Plowman, G. D., Rudy, B., and Schlessinger, J. (1995) Nature 376, 737–745. 24. Li, W. W., Alexandre, S., Cao, X., and Lee, A. S. (1993) J. Biol. Chem. 268, 12003–12009. 25. Jiang, T., and Acosta, D., Jr. (1995) Toxicology 95, 155–166. 26. Nicchitta, C. V., Kamoun, M., and Williamson, J. R. (1985) J. Biol. Chem. 260, 13613–13618. 27. Goldberg, H. J., Wong, P. Y., Cole, E. H., Levy, G. A., and Skorecki, K. L. (1989) Transplant. 47, 731–733. 28. LeGrue, S. J., Friedman, A. W., and Kahan, B. D. (1983) J. Immunol. 131, 712–718. 29. O’Leary, T. J., Ross, P. D., Lieber, M. R., and Levin, I. W. (1986) Biophysical J. 49, 795–801. 30. Skorecki, K. L., Rutledge, W. P., and Schrier, R. W. (1992) Kidney Intern. 42, 1–10. 31. Davis, R. J. (1993) J. Biol. Chem. 268, 14553–14556. 32. Treisman, R. (1994) Curr. Opin. Genet. Dev. 4, 96–101. 33. Janknecht, R., Ernst, W. H., and Nordheim, A. (1995) Oncogene 10, 1209–1216. 34. Lin, T.-A., Kong, X., Haystead, T. A. J., Pause, A., Belsham, G., Sonenberg, N., and Lawrence, J.-C., Jr. (1994) Science 266, 653– 656. 35. Haystead, T. A. J., Haystead, C. M. M., Hu, C., Lin, T.-A., and Lawrence, J. C. (1994) J. Biol. Chem. 269, 23185–23191.
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236, 599–603 (1997)
RC977017
MAP Kinase Activation by Cyclosporine A Liliana Paslaru,* Sylviane Trigon,† Martin Kuhlmann,‡ and Michel Morange†,1 †Groupe de Biologie Mole´culaire du Stress, Unite´ de Ge´ne´tique mole´culaire, Ecole Normale Supe´rieure, 46 rue d’Ulm, 75230 Paris, Cedex 05, France; *University of Medicine and Pharmacy Carol Davila, Post-Graduate Department of Biochemistry, Fundeni Hospital, Sos Fundeni No. 258, Bucharest, Romania; and ‡Schwerpunkt Nephrologie, Innere Medizin IV, Med. Universita¨tsklinik, D-66421 Homburg/Saar, Germany
Received June 10, 1997
Short treatment of HeLa cells with cyclosporine A led to the activation in the crude cell extracts of a MAP kinase-like activity. Fractionation by chromatography on a Mono Q column allowed the separation of two activities co-eluting with the MAP kinases ERK1 and ERK2. The activation of these two MAP kinases was demonstrated in Western Blotting by the appearance, after CsA treatment, of two new slowly migrating forms on SDS electrophoretic gels. A similar activation was also obtained in renal epithelial BSC-1 cells and 3T3 fibroblasts. MAP kinase activation might result from a perturbation of calcium homeostasis induced by CsA treatment. q 1997 Academic Press
Cyclosporine A (CsA) is an immunosuppressive drug whose use has resulted in a dramatic increase in the efficiency of organ transplantation in recent years (1,2). The major cellular target of the immunosuppressive action of CsA is the T lymphocyte (3). The drug interacts with cyclophilins and the resulting complexes inhibit a calmodulin-dependent protein phosphatase, called PP2B or calcineurin (4,5). This inhibition of calcineurin leads to a blockade in the activation of the transcription factor NFAT which is involved in cytokine production and in the subsequent activation of T lymphocytes (6,7). Many studies have demonstrated the pleiotropic action of CsA. This drug blocks the direct interaction of the YY1 transcription factor with cyclophilin A (8). CsA also leads to an alteration in the efficiency of a subset of general transcription factors in addition to NFAT (9). It has also a destabilizing effect on some mRNAs (10). In organs such as kidney, prolonged treatment with CsA has toxic effects (11). As seen from the action of CsA on YY1 factor, it is highly probable that some of these effects do not result from the well documented 1
Corresponding author. Fax: 33 1 44 32 39 41.
inhibitory action of CsA on calcineurin phosphatase activity. In fact, as far as the different metabolites or analogs of CsA are concerned, their nephrotoxicity does not correlate with their immunosuppressive action (12,13). In a previous paper, we described the stress-like effects of CsA on different cellular systems. CsA inhibits general protein synthesis in parallel with the specific stimulation of BiP (Grp78) synthesis and increases HSP 70 and HSP 90 synthesis after heat-shock (14). These observations suggested to us that CsA might be included in the family of chemical agents that induce the stress response. To confirm this hypothesis, we sought other stress effects of CsA. Recently, it was shown that MAP kinases are induced by stress (15,16). Here we demonstrate that two members of the MAP kinase family, ERK1 and ERK2, are activated by short treatments with CsA in the three cell systems we used. The characterization of these new intracellular targets of CsA action might help to explain the pleiotropic effects of this drug. MATERIALS AND METHODS Cell culture, CsA treatment, and crude cell extract preparation. HeLa (MRL2 clone), 3T3 fibroblasts or renal proximal tubular (BSC1) monkey cells were cultured at 377C in a 10% CO2 water-saturated atmosphere in Dulbecco’s modified Eagle medium containing 10% heat-inactivated fetal calf serum, 2 mM glutamine, 50 mg/ml streptomycin and 50 units/ml penicillin. The physiological properties of the BSC-1 cells have been described previously (17). They were obtained from American Type Culture Collection (ATCC), Rockville MD, USA. 3T3 cells were a kind gift of M. Yaniv’s laboratory (Institut Pasteur). Serum induction was carried out 24 h after replacing the normal medium with serum-free medium, by addition of a 20% serum-containing medium for 15 min. Cells were plated at low density and incubated with CsA during the exponential growth phase. CsA (or analogs) were prediluted in culture medium and incubated with cells for the indicated times. Crude extracts were prepared as previously described (16). Briefly, cells were washed in Buffer A (50 mM Glycerophosphate, 10 mM MgCl2 , 1 mM EGTA, 10% glycerol pH 7.3) and lysed in the same buffer supplemented with 1% (v/v) Nonidet P-40, 1 mg/ml pepstatin,
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1 mg/ml aprotinin, 0.5 mM phenyl methyl sulfonyl fluoride, 1 mM sodium orthovanadate, 50 mM NaF and 0.01% (v/v) b-mercaptoethanol. Lysates were centrifuged at 10,000g for 10 min and supernatants collected (about 1.5 mg/ml). CsA was kindly provided by Mrs Annick Roche (Sandoz France). It was diluted in cremophor/ethanol (2:1, v/v) at 10 mg/ml. Two analogs of CsA, MeAla-6 (CsA1), which is a non-immunosuppressive analog but an efficient PPIase inhibitor, and MeBm2t (CsA2), which is immunosuppressive but devoid of any effect on PPIase activity, were kindly provided by Dr. Philippe Durette (Merck, Sharp and Dohme Research Laboratories) (13). Chromatography on Mono Q. Prior to chromatography, the lysates were clarified by centrifugation at 100,000 1 g for 15 min at 47C. Supernatants were applied to an HR 5/5 Mono Q column (Pharmacia) previously equilibrated in Buffer A (18). After washing (10 ml), proteins were eluted with a 30 ml linear 0 to 300 mM NaCl gradient in the same buffer. The flow rate was 0.5 ml/min during loading and 1.0 ml/min during washing and elution. Fractions of 1.0 ml were collected. Hepta-4 kinase assay. The reaction mixture (24 ml) contained (final concentrations): 60 mM glycerophosphate, 10 mM EGTA, 10 mM MgCl2 , 7.5% glycerol, 1 mM sodium orthovanadate, 50 mM NaF, g32P ATP (0.2 mM, 1 mCi), 0.1% b-mercaptoethanol, hepta-4 (SPTSPSY)4 (0.4 mg/ml). Hepta-4 peptide corresponds to a four-fold repeat of a motif highly repeated in the C-terminal part of RNA polymerase II. It was synthesized by Dr. Odile Siffert in the Organic Chemical Laboratory of Institut Pasteur. Two microliters of crude cell extracts or 4 microliters of the different fractions from the Mono Q column were added and in vitro reactions were performed in initial rate conditions at 307C for 30 min. Reactions were stopped by the addition of 1 volume of Laemmli sample buffer containing 5% b-mercaptoethanol, and samples were analyzed by 15% SDS polyacrylamide gel electrophoresis performed according to Laemmli (19). Gels were fixed in ethanol/acetic acid/trichloracetic acid/water (3:1:1:5, v/v), dried, and submitted to autoradiography with intensifying screens at 0707C. Signals were quantified with a BAS READER (FUJI) and measurements were made with PC BAS. Western blot analysis. After electrophoresis on a 10% polyacrylamide gel, proteins were electrotransferred on nitrocellulose. Western Blots were performed as described in the ECL kit (Amersham Corp.). An anti ERK1-ERK2 antibody was used (Ab 956/837 from Santa Cruz Biotechnology).
RESULTS Induction of a MAP Kinase-Like Activity in HeLa Cells by Short Treatments with CsA For the MAP kinase assay, we used a short peptide of 28 amino acids (hepta-4) bearing a four-fold repeat
FIG. 1. Activation of a MAP kinase-like activity by CsA treatment. Cell treatment with 100 mM CsA for 30 min, 1, 2, 3, 4 and 5 hours. Crude extract preparation and assays were performed as described in Materials and Methods.
FIG. 2. Dependence of MAP kinase activity on the concentration of CsA added during the pretreatment. Same experimental conditions as in Figure 1. The duration of CsA treatment was 3 hours. The activity in control cells was considered as 100%.
of the consensus sequence of MAP kinases, which was demonstrated in previous studies to be a good in vitro substrate for MAP kinases, in particular for ERK1 and ERK2 (16). The activation of hepta-4 kinase activity by CsA treatment was time and concentration dependent. The increase in enzymatic activity in crude cell extracts was already detectable between 30-60 min after the addition of 100 mM CsA and was roughly proportional to the incubation time between 1 and 5 hours (Figure 1). This kinase activity was dependent on CsA concentration, being maximal at high concentrations of CsA, but already detectable at low concentrations (10 mM) (Figure 2). The Increased Hepta-4 Kinase Activity after CsA Treatment Results from Activation of the Major MAP Kinases ERK1 and ERK2 The hepta-4 peptide is a good in vitro substrate for MAP kinases, and the amount of hepta-4 peptide phosphorylated by crude cell extracts increases dramatically when MAP kinases are activated by the addition of mitogenic agents (16). However, this peptide can be also efficiently phosphorylated by protein kinases unrelated to MAP kinases, such as the dsDNA dependent protein kinase (Trigon, submitted). To confirm that the increase in hepta-4 protein kinase activity observed after CsA treatment in crude extracts resulted from activation of MAP kinases, these extracts were fractionated by chromatography on a Mono Q column. In a preliminary control experiment (Figure 3A), extracts from untreated or serum activated HeLa cells (see Materials and Methods for the experimental condi-
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FIG. 3. MAP kinases ERK1 and ERK2 are activated by serum addition and CsA treatment. Crude extracts from untreated, serum or CsA-treated cells were fractionated on a Mono Q column and the different fractions assayed for hepta-4 kinase activity or for the presence of MAP kinases by Western blotting with anti ERK1-ERK2. A) Hepta-4 kinase activity induction by serum addition. B) Detection of ERK1 and ERK2 in the fractions of Experiment A containing hepta-4 kinase activity (a: without serum addition; b: after serum addition). C) Same experiment as in A, but the cells were treated with 100 mM CsA for 3 hours, instead of being treated with serum. D) Characterization of ERK1 and ERK2 in the fractions of column C containing hepta-4 kinase activity (a: without CsA; b: after CsA addition). Dotted line: NaCl gradient. After quantification, the activities are expressed in arbitrary units, but the values can be directly compared between experiments 3A and 3C.
tions used for serum activation) were fractionated on the same column. Each fraction was assayed for hepta4 kinase activity. Two peaks of hepta-4 kinase activity were obtained, eluting at 0.175 and 0.2 M NaCl. The fractions containing hepta-4 kinase activity after serum addition and the corresponding fractions from the extracts of untreated HeLa cells were analyzed by western blotting with antibodies specific for ERK1 and ERK2 (Figure 3B). The two proteins were present in the fractions containing hepta-4 kinase activity. In the extracts from serum treated cells (b), two forms of ERK2 (p42) migrating differently on SDS gels and two forms of ERK1 (p44) eluting at different positions on the Mono Q column were clearly detectable, whereas only one form of each ERK enzyme was present in extracts from untreated cells (a). The slowly migrating form of ERK2 and the retarded species of ERK1 coincide exactly with the first and second peaks of hepta4 kinase activity, respectively, and correspond to the phosphorylated, activated forms of ERK2 and ERK1. When the same experiment was repeated with extracts from CsA-treated cells (Figure 3C; 3 hours of pretreatment with 100 mM CsA), two peaks of hepta-4 kinase activity were increased by CsA treatment, eluting at the same positions as the protein kinase activi-
ties stimulated by serum. However the maximal activity levels were higher after serum addition than after CsA treatment. The activation of MAP kinases by CsA was confirmed by western blotting of the corresponding fractions. Figure 3D shows that the fractions in which hepta-4 kinase activity was found correspond to the fractions containing the slowly migrating form of ERK2 and the retarded species of ERK1 (in this experiment, we were able to separate the active and inactive forms of ERK1 on SDS gels). These activated forms of ERK1 and ERK2 were only detected in extracts from CsA-treated (b), but not untreated HeLa cells (a). However the relative amount of the activated forms was lower than that obtained after serum addition, in full agreement with the hepta-4 kinase activity measurements. Activation of Hepta-4 Kinase Activity Can Be Also Obtained with CsA Analogs and in Different Cell Types Since the kidney is one of the organs most sensitive to CsA treatment, it was interesting to see whether MAP kinase induction by CsA would also be observed in kidney epithelial cells. As shown in Figure 4, this
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FIG. 4. Activation of a MAP kinase activity by CsA pretreatment in kidney BSC-1 cells. Same experimental conditions as in Figure 1, except that CsA pretreatment was 2 hours.
was indeed the case in BSC-1 cells in the same range of CsA concentrations as those which increased hepta4 kinase activity in HeLa cells: the stimulatory effect was already detected at 1 mM CsA after two hours of treatment, but was higher with 100 mM CsA. The activation of hepta-4 kinase activity was not limited to epithelial cells but was also obtained after CsA treatment of 3T3 fibroblasts (data not shown). We tested the action of two analogs of CsA. CsA1 (MeAla-6) binds efficiently to cyclophilins and inhibits their PPIase activity, but has no immunosuppressive action. CsA2 (MeBm2t) is immunosuppressive but has no inhibitory effect on the PPIase activity of cyclophilins. Figure 5 shows that both analogs could stimulate hepta-4 kinase activity, though to a lower extent than CsA itself. DISCUSSION Our experiments demonstrated that CsA treatment induces the activation of the MAP kinases, ERK1 and ERK2. Activation by CsA was rapid: detectable after 1 hour, it peaked at about 5 hours and disappeared after longer treatments (data not shown). High concentrations of CsA induced stronger MAP kinase activation, but low, ‘‘physiological’’, therapeutic doses of CsA (1 mM) were sufficient to induce the phenomenon. Activation was not limited to HeLa cells, since CsA stimulation of MAP kinases was similar in kidney epithelial and 3T3 cells, nor was it specific to CsA, since two analogs of this drug, with different potential immunosuppressive actions, had a nearly identical effect. In T cells, MAP kinases are essential components of the activation pathway resulting from the recognition of antigens presented by the MHC complex and leading to the synthesis of the growth factor IL2. Reciprocally, it has recently been shown that the functional unresponsiveness of T cells, called anergy, is parallel to a blockade in the activation of these protein kinases (20, 21). Therefore, it may appear surprising that components of a signalling pathway involved in mitogenesis and activation are stimulated by CsA, whereas in lym-
phocytes this drug has an inhibitory effect. Our observation thus raises two questions: the mechanism by which MAP kinases are activated and the physiological significance of this activation. An increase in intracellular calcium is one of the numerous stimuli known to activate MAP kinases (22) maybe through the intermediary action of PYK2 tyrosine kinase (23). In a previous report, we showed that CsA, like calcium ionophores, can induce GRP78 synthesis inside the endoplasmic reticulum. Since GRP78 synthesis can be induced by a decrease in the calcium concentration inside the endoplasmic reticulum (24), a possible link between the earlier and present observations would be that CsA treatment results in a perturbation of calcium homeostasis, with a decrease in calcium concentrations inside the endoplasmic reticulum and a concomitant increase in cytosolic calcium. The former would lead to the stimulation of GRP78 synthesis and the latter to the activation of MAP kinases. In fact, modifications in calcium homeostasis by CsA have been already observed in different systems. These modifications are complex, involving both a mitochondrial calcium overload (25) and variations in the level of intracytosolic calcium. The latter have been observed with CsA alone, or when CsA was added to other hormonal agents which themselves modify calcium homeostasis (26, 27). Use of CsA analogs supported the hypothesis that activation of MAP kinases by CsA is not dependent on inhibition of calcineurin phosphatase, but most probably results from variations in intracellular calcium levels. The two analogs which were tested have different immunosuppressive actions (and effects on calcineurin) (13) but equally induce MAP kinase activity. As noted previously (27), similar modifications in intracellular calcium concentrations are induced by different CsA analogs, irrespective of their immunosuppressive action. How might CsA alter calcium homeostasis? Apart from its specific action on the calcium channels of the
FIG. 5. Activation of a MAP kinase activity by two analogs of CsA. HeLa cells were preincubated with 100 mM of CsA or the two analogs (CsA1 MeAla-6; CsA2 MeBm2t) for 3 hours. Extracts and assays were performed as described in Materials and Methods.
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inner mitochondrial membrane, CsA is able to modify the properties and fluidity of biological membranes by inserting into these membranes (28-30). Such modifications might result in altered transmembrane calcium flux. MAP kinases have pleiotropic effects, due to the large number of proteins which have been demonstrated to be their phosphorylation targets, both in vivo and in vitro (31). One of the roles proposed for MAP kinases is activation by phosphorylation of transcription factors such as Elk-1 or SAP-1a. These factors, when complexed with the SRF, are responsible for serum activation of the genes containing a Serum Responsive Element in their promoter (32, 33). Protein synthesis might also be altered by the stimulation of MAP kinases. Non-phosphorylated PHAS-1 binds to initiation factor 4E (eIF-4E) and inhibits protein synthesis. When PHAS-1 is phosphorylated by the MAP kinases, it no longer interacts with eIF-4E and translation can be initiated (34, 35). Other nuclear and cytoplasmic proteins are potential substrates for MAP kinases. Their modifications might result in other cellular effects, such as cytoskeletal alterations. The diversity of MAP kinase targets is similar to the diversity of CsA effects on cells. Moreover, by modifying the level of intracellular calcium, CsA might alter the action of numerous hormones which use calcium as second messenger and play fundamental roles in the control of physiological processes. ACKNOWLEDGMENTS We are very indebted to Rosemary Sousa-Yeh for her critical reading of the manuscript and to Miss Murielle Rallu for her help in the experiments. L.P. was an invited Professor of the ENS (PAST program). CsA was provided to us by Mrs. Annick Roche (Sandoz France) and the two analogs by Dr. Philippe Durette (Merck, Sharp and Dohme Research Laboratory).
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