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Inhibition of Glucocorticoid-Induced Apoptosis with 5-Aminoimidazole-4-carboxamide Ribonucleoside, a Cell-Permeable Activator of AMP-Activated Protein Kinase
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
243, 821–826 (1998)
RC988154
Inhibition of Glucocorticoid-Induced Apoptosis with 5-Aminoimidazole-4-carboxamide Ribonucleoside, a Cell-Permeable Activator of AMP-Activated Protein Kinase Claudio Stefanelli,1 Ivana Stanic’, Francesca Bonavita, Flavio Flamigni, Carla Pignatti, Carlo Guarnieri, and Claudio M. Caldarera Department of Biochemistry ‘‘G. Moruzzi,’’ University of Bologna, Via Irnerio, 48, I40126 Bologna, Italy
Received January 5, 1998
The AMP-activated protein kinase (AMPK) is related to a growing family of protein kinases that are believed to protect cells against environmental and nutritional stress. In the present study the hypothesis of a protective role for AMPK against thymocyte apoptosis has been tested. It is shown that AMPK is expressed in rat thymocytes that contain the transcript for the a1 isoform of the AMPK catalytic subunit and can be activated by treatment with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a wellestablished activator of AMPK. AICAR is not toxic and prevents glucocorticoid-induced apoptosis in the same concentration range used to activate AMPK. At concentrations higher than 1 mM, AICAR fully restores cell viability and inhibits DNA laddering in dexamethasone-treated thymocytes. Furthermore, AICAR blocks the dexamethasone-induced activation of caspase 3-like enzymes, which are believed to play a pivotal role in apoptotic cell death. Activation of AMPK by oligomycin, which depletes thymocytes of ATP, is also correlated to inhibition of caspase 3-like activity in dexamethasone-treated cells. However, AICAR and oligomycin do not exert any protective action when apoptosis is induced by staurosporine. These results indicate that AICAR is a powerful inhibitor of glucocorticoid-induced apoptosis and suggest that AMPK activation may interfere with a step in the apoptotic cascade triggered by dexamethasone. q 1998 Academic Press
1 To whom correspondence should be addressed. Fax: /39-51351224. E-mail: [email protected]. Abbreviations used: AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; AMC, 7-amino-4-methyl-coumarin; AMPK, AMP- activated protein kinase; AMPKK, AMPK kinase; bp, base pairs; DEVDAMC, acetyl-asp-glu-val-asp-AMC; DEVDase, enzymatic activity cleaving the peptide DEVD-AMC; RT-PCR, reverse transcriptionpolymerase chain reaction; SAPK, stress-activated protein kinase; ZMP, AICAR monophosphate; ZTP, AICAR triphosphate.
Cells respond to changes in environmental conditions by adapting their metabolism and recruiting specific signal transduction pathways. ATP depletion is a cellular stress which can be caused by hypoxia and several eukaryotic cells have evolved capabilities for surviving relatively long periods with limited supplies of oxygen (1-2). Thymic lymphocytes not only survive hypoxia (3), but in these cells ATP depletion may also inhibit apoptotic cell death induced by the synthetic glucocorticoid dexamethasone (4), suggesting that the adaptation to low ATP of these hypoxia-tolerant cells includes a step which interferes with apoptosis. The programmed cell death cascade can be divided in several stages (5). Death-triggering agents activate multiple signal pathways leading to the execution phase, when the activation of caspases occurs. Activation of the proteolytic cascade of caspases in turn leads to characteristic apoptotic structural lesions accompanying cell death (6). Extracellular stress elicits the activation of protein kinase cascades involving the stress activated protein kinases (SAPKs) (7,8); however, SAPK activation does not occur during anoxia, but only during the repletion of cellular ATP pool (9,10). On the contrary, ATP depletion activates a specific pathway: the AMP-activated protein kinase (AMPK) cascade (reviewed in ref. 11). AMPK consists of a family of isoenzymes (12), thought to act as ‘‘low-fuel warning system’’, being switched on by depletion of ATP with consequent increase of AMP (11). This kinase is activated by high levels of AMP via a complex mechanism which involves phosphorylation and allosteric regulation (11,13). AMP activates the AMPK by direct allosteric activation of the enzyme, activation of an upstream kinase (AMPKK), and inhibition of AMPK dephosphorylation. AMPK substrates include biosynthetic enzymes of lipid metabolism, which are inhibited upon phosphorylation by AMPK (11). These targets have suggested that the function of
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AMPK might be to inhibit fatty acid and sterol synthesis under conditions where the cellular energy status is compromised. However, studies of the yeast omologue of AMPK, SNF1, have suggested that AMPK could be more generally involved in gene regulation in response to metabolic stress (11,14,15). In agreement with this hypothesis, Raf-1 kinase has recently been identified as a substrate of AMPK (16). To date, no specific inhibitors of AMPK are available to test the involvement of AMPK in the antiapoptotic effect of ATP depletion in thymic lymphocytes, but AICAR represents an useful activator (17), extensively used to show the involvement of AMPK activation in specific cellular processes (17-20). AICAR is a cell-permeable precursor of its monophosphate nucleotide ZMP (21), an AMP analogue that mimics the effects of AMP on the allosteric activation of AMPK (22,23). In this study it is shown that thymus cells express AMPK and that its activation by AICAR correlates to inhibition of dexamethasone-induced apoptosis. MATERIALS AND METHODS Materials and cells. AICAR, DEVD-AMC and other biochemicals were products of Sigma. [g-33P]ATP and L-[1-14C]leucine were purchased from Amersham. The SAMS peptide was custom synthesized by Neosystem Laboratoire (Strasbourg, France). rTth and Taq DNA polymerases were from Perkin Elmer. Oligonucleotides were synthesized with an ABI 391 DNA synthesizer and purified by HPLC. Thymocytes were obtained from immature male Wistar rats. The isolated cells were suspended at the indicated concentration in RPMI 1640 medium containing 10 mM Hepes and 100 U/ml penicillin and streptomycin. The treatments were prepared in the culture medium or dimethylsulphoxide if water-insoluble. Each experimental point was derived from two wells each assayed in duplicate. AMPK activity. Tissues were rapidly homogenized in homogenization buffer HB (12), while thymocyte suspensions (2 1 108 cells/4 ml of medium) were collected at the end of the incubations, rapidly washed in ice-cold phosphate-buffered saline, homogenized in 1 ml of HB in a glass/teflon homogenizer and centrifuged 5 min at 3,000g. The supernatants were then centrifuged at 100,000g for 40 min and a 35% saturated ammonium sulphate fraction was prepared for each sample. The resultant pellet was resuspended in HB and diluted to a protein concentration of 0.2 mg/ml. The activity of AMPK was assayed by the phosphorylation of the SAMS peptide (24). Aliquots containing 1 mg of protein were incubated 10 min at 30 7C in a final volume of 25 ml containing 0.1 M Hepes pH 7, 0.2 M NaCl, 5 mM MgCl2 , 20% glycerol, 200 mM SAMS peptide and 200 mM [g-33P]ATP (220 cpm/pmol). Reactions were performed in duplicate { 5*-AMP (200 mM) with a minus peptide substrate control. The reaction was stopped in ice with 5 ml of 30% phosphoric acid and 10 ml aliquots were removed and spotted on phosphocellulose paper dishes that were washed and dried (24) before radioactivity counting. In preliminary experiments it was confirmed the linearity of the assay with respect to the incubation time and the amount of assayed protein. Detection of AMPK expression by RT-PCR. Total RNA was isolated from 107 thymocytes by the single-step guanidine thiocyanate method with the TriPure reagent (Boehringer Mannheim) according to the manufacturer’s instruction. The RNA preparation was digested with DNase and then 0.5 mg aliquots were reverse-transcribed, amplified for 40 cycles and analysed by agar gel electrophoresis as previously described (25). Specific primers were designed on the basis of published sequences as follows: a1 AMPK (12) forward
5*-GTCATCAGTACACCGTCTGA-3*, reverse 5*-CAAGCAGGACGTTTTCAGGT-3*; a2 AMPK (14) forward 5*-GTCATCTCAGGAAGGCTGTA-3*, reverse 5*-GTGGCAATAGAACGGTTGAG-3*; b-actin (26) forward 5*-TCTTCCAGCCTTCCTTCCT-3*, reverse 5*-GGAGCAATGATCTTGATCTTC-3*. The expected size of the amplification products were: 199 bp for a1 AMPK (cDNA nt 244-442); 179 bp for a2 AMPK (cDNA nt 550-728); 214 bp for b-actin (gene nt 25763001 with intervening intron). The mRNA for b-actin was determined as positive control for RNA extraction and RT-PCR. Since the intron/ exon structure of the AMPK genes is not known, in order to avoid the possible amplification of contaminating DNA, firstly the extracted RNA was extensively digested with DNase, furthermore, for each sample control aliquots were amplified for 40 cycles with Taq polymerase omitting the reverse transcription phase. Under these conditions DNA contamination was never found. Determination of caspase 3-like (DEVDase) activity. Caspase 3like activity was measured by the cleavage of the fluorogenic peptide substrate DEVD-AMC (27). Thymocytes (21107 cells) were incubated in one ml of medium. At the end of the experiment the cells were collected, washed in phosphate-buffered saline, suspended in 150 ml of lysis buffer (20 mM Hepes pH 7, 5 mM dithiothreitol, 2 mM EDTA, 0.1% CHAPS, 0.1% Triton X-100, 1 mM phenylmethylsulphonyl fluoride, 1 mg/ml each of aprotinin, pepstatin and leupeptin), vortexed and left 10 min in ice. The lysates were centrifuged 1 min at 28,000g and the supernatant was used as enzyme source. Ten microliters of this extract (containing about 10 mg of protein), were combined with 20 ml of assay buffer containing 100 mM Hepes pH 7, 5 mM dithiothreitol, 0.1% CHAPS, 10% sucrose and 0.15 mM DEVD-AMC and incubated 15 min at 37 7C. The reaction was stopped in ice by adding 0.1 ml of 2% sodium acetate in 0.2 M acetic acid. The samples were diluted with 2.5 ml of water and the specific cleavage of the fluorogenic peptide DEVD-AMC was monitored by AMC liberation using 370 nm excitation and 455 nm emission wavelengths. One unit is defined as the amount of enzyme activity cleaving 1.0 nmol of substrate per minute in the standard conditions described. Other assays. Cell death was evaluated by trypan blue exclusion. Internucleosomal DNA fragmentation was visualized by standard agarose gel electrophoresis and ethidium bromide staining (2 mg of DNA/lane) after DNA extraction from 107 cells. The rate of protein synthesis was measured in 2 x 107 cells by incorporation of L-[114 C]leucine in the acid-insoluble fraction during a 4 h incubation, as previously described (4).
RESULTS Thymocytes contain an activable AMPK. To explore the hypothesis that AMPK activation could be correlated to inhibition of apoptosis in ATP-depleted thymus cells, the presence of AMPK in rat thymocytes was tested. Previous studies on the tissue distribution of AMPK activity and expression revealed that AMPK is widely distributed (12,24). However, the presence of a kinase activable by AMP in thymus cells has not yet been reported. AMPK activity was assayed in the 0-35% saturated ammonium sulphate fraction, in which AMPK is almost totally recovered whereas inhibitors are removed (24). Fig. 1A shows that an AMP-activable kinase phosphorylating the SAMS peptide, a specific substrate of AMPK (24), is present in the rat thymus. The basal activity is low with respect to the activity measured in liver extracts, however, the activation by saturating (200 mM) AMP was more pronounced for the thymus
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bation of thymocytes with AICAR for 1 h activated cellular AMPK in a dose-dependent manner (Fig. 2A). AICAR, at the concentration of 2 mM, increased 3-fold AMPK activity measured in the presence of 200 mM AMP. While activating AMPK, AICAR did not affect energy-requiring processes like protein synthesis nor elicit significant toxic effect. Staurosporine is a powerful trigger of apoptosis virtually in all cell types. AICAR did not influence cell death induced by staurosporine; however, incubation of thymocytes with AICAR together with dexamethasone, produced a dose-dependent inhibition of dexamethasone-induced cell death (Fig. 2B). At dosages higher than 1 mM, AICAR fully preserved the viability of thymocytes incubated 20 h in the presence of dexamethasone. Accordingly, AICAR prevented DNA laddering in dexamethasone-treated cells (Fig. 2C). These data show that activation of AMPK by AICAR correlates with inhibition of glucocorticoid-induced apoptosis.
FIG. 1. Thymus cells express AMPK. (A) Phosphorylation of the SAMS peptide (AMPK activity) was measured in the absence or presence of 200 mM AMP in 0-35% ammonium sulphate fraction of rat liver or thymus extracts. Results are the mean of two separate experiments, whose range is shown. (B) SAMS phosphorylation in the presence of 200 mM AMP was measured in samples obtained from untreated thymocytes (control) or from cells incubated 1 h in the presence of 100 ng/ml oligomycin or 1 mM AICAR. The data are means of two experiments and the range is indicated. (C) Analysis of a1 and a2 AMPK transcripts by RT-PCR. Aliquots of 0.5 mg of total RNA were reverse-transcribed and then amplified for 40 cycles with primers specific for AMPK isoforms. b-Actin mRNA was used as a positive control. The expected size of amplification products is indicated.
enzyme (4-fold) with respect to the liver enzyme, that was activated about twice. As shown for other cell types, AMPK was activable in intact thymocytes (Fig. 1B). In fact, the activity of AMPK was further increased in cells depleted of ATP by treatment with 100 ng/ml of oligomycin (4), and in cells incubated 1 h with 1 mM AICAR, a cell permeable activator of AMPK (17). Since AMPK activation was evident in samples assayed in the presence of saturating AMP, the activation appears to be sustained by phosphorylation of AMPK by AMPKK (11,17,18). AMPK consists of a family of isoenzymes and two isoforms of the catalytic subunit, a1 and a2 , have been described to date (12). To determine the identity of thymic AMPK, thymocyte RNA was analyzed by RT-PCR using isoform-specific primers (Fig. 1C). Only the product corresponding to amplification of the a1 transcript was found. Owing to the sensitivity of RT-PCR, it seems likely that a1 is the only isoform expressed in thymus cells. Activation of AMPK by AICAR correlates to inhibition of dexamethasone-induced thymocyte apoptosis. Incu-
Effect of AICAR on caspase 3-like activity. The activation of caspase 3-like proteases is believed to play a pivotal role in the proteolytic cascade of apoptosis (6,28). As shown in Fig. 3A, caspase 3-like activity cleaving the fluorogenic substrate DEVD-AMC (DEVDase activity) gradually increased following dexamethasone, and after 4-6 h was about threefold the control’s level. The activation of caspase 3-like activity was strongly inhibited by AICAR (Fig. 3B). On the other hand, AICAR did not inhibit DEVDase activation induced by staurosporine (Fig. 3C). These results are fully consistent with the effects of AICAR on cell death reported on Fig. 2. Similarly to AICAR, oligomycin, while activating AMPK (Fig. 1), inhibited caspase 3like activity triggered by dexamethasone, but not by staurosporine. DISCUSSION Glucocorticoids kill immature lymphocytes as well as some leukemic cells by switching on the cell death program leading to apoptosis (29). Glucocorticoid-induced death of rodent thymocytes is one of the early model of apoptosis studied (30) and represents a prototypic model system for research in the field of programmed cell death. The molecular mechanism by which glucocorticoids induce apoptosis, however, is still poorly understood. Depending of the cell model it has been proposed that glucocorticoid-induced apoptosis is due to induction of ‘‘lysis genes’’ or to repression of ‘‘survival genes’’ (31). Whatever is the upstream mechanism, glucocorticoids finally cause the activation of caspase 3-like activity in both tumor cells (32) and thymocytes (Fig. 3). Thymocyte apoptosis caused by the synthetic glucocorticoid dexamethasone, but not by staurosporine, is inhibited in ATP-depleted thymocytes (4), suggesting
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FIG. 2. AICAR inhibits thymocyte apoptosis induced by dexamethasone. (A) Effect of different concentrations of AICAR on AMPK activity and the rate of protein synthesis in isolated thymocytes. AMPK activity was measured by SAMS phosphorylation in the presence of 200 mM AMP in extracts obtained from cells incubated with AICAR for 1 h. In control cells the activity was 356 { 27 cpm/min/ mg of protein (n Å 4). Protein synthesis was measured by leucine incorporation in acid-insoluble material following a 4 h incubation. Control thymocytes incorporated 206 { 19 cpm/106 cells (n Å 4). The reported data were obtained in a typical experiment representative of three. (B) Cell suspensions were incubated with different concentrations of AICAR in the presence of 1 mM dexamethasone (dex), 1 mM staurosporine (stauro), or 1 ml/ml of dimethylsulphoxide (control). After 16 h, cell survival was measured by trypan blue exclusion.
that the adaptive response to this cellular stress interferes with a specific pathway leading to apoptosis. It is worth noting that rat thymocytes well survive ATP depletion (3,4), differently from most cell types that are early damaged by treatments depleting cellular ATP. Cellular stress activates protein kinase cascades (7,8) and, at least in some cells, these kinases may have a protective role against apoptosis (8). On the other hand, ATP depletion does not recruit the SAPK pathway (9,10 and data not shown), but activates AMPK (11). In the examination of AMPK’s involvement in the antiapoptotic effect of ATP depletion in thymocytes, it must be born in mind that treatments depleting cellular ATP are likely to have non-specific side-effects, since ATP depletion can influence several cellular processes. However, a few years ago it has been reported that ZMP, a normal intermediate in the biosynthesis of purine nucleotides, can mimic the effect of AMP on allosteric activation of AMPK (22,23). Administration of AICAR to intact cells causes ZMP to accumulate inside the cell (17,21) and this has been demonstrated to be a rather specific method for activating AMPK (17), frequently used to demonstrate the involvement of AMPK in cellular processes (17-20). In the present study it is described for the first time a correlation between AMPK and apoptosis. AMPK is expressed in rat thymocytes, that contain the transcript for the widely distributed a1 isoform of the catalytic subunit, and can be activated by AICAR as well as by ATP depletion that follows oligomycin, a specific inhibitor of ATP synthase (Fig. 1). While activating AMPK, these treatments also block the death of dexamethasone-treated thymocytes (ref. 4 and Fig. 2). In particular, AICAR, in the same concentration range used to activate AMPK, inhibits internucleosomal DNA fragmentation and activation of caspase 3-like activity, which is considered the ‘‘point of no return’’ in apoptosis (6). It is worth noting that both AICAR and oligomycin do not affect caspase activation caused by staurosporine, suggesting a specific interference of AMPK in the death-pathway induced by glucocorticoids. AMPK phosphorylates and inactivates a number of key enzymes of lipid metabolism, including 3-hydroxy-3methylglutaryl-CoA reductase, catalyzing the first step of isoprenoid biosynthesis, acetyl-CoA carboxylase, the first enzyme of fatty acid biosynthesis, and hormonesensitive lipase (reviewed in ref. 11). These targets could suggest an interference of AMPK with some lipid molecule implicated in glucocorticoid-induced
The figure reports data from one experiment representative of three. (C) Electrophoretic fractionation of DNA (2 mg/lane) extracted from thymocytes incubated 6 h without any treatment (control) or with 1 mM dexamethasone (dex) together with the indicated concentration of AICAR.
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FIG. 3. AICAR inhibits caspase 3-like activity induced by dexamethasone. (A) Caspase 3-like activity cleaving the fluorogenic substrate DEVD-AMC (DEVDase activity) was measured at various times in control thymocytes and in cells treated with 1 mM dexamethasone (dex) to obtain a time course. (B) Control and dexamethasone-treated (dex) thymocytes were incubated 4 h in the presence of the indicated concentration of AICAR. Afterward DEVDase activity was measured. (C) Thymocytes were treated with 1 mM dexamethasone (dex) or 1 mM staurosporine (stauro) or none (control); each group was then incubated 5 h without any further treatment or with 2 mM AICAR or 100 ng/ml oligomycin. The panels report the data obtained in a typical experiment representative of several (2 to 5).
apoptosis since AMPK activation largely affects lipid metabolism. Recently, Raf-1 has been identified as a substrate of AMPK, which phosphorylates it at Ser621 (16). The role of this phosphorylation is not known, but interestingly, the Raf-1 kinase has been implicated in the regulation of apoptosis (33). However, it is evident that any step from dexamethasone binding to the activation of caspases may represent a site where AMPK could interfere with the progression of the death program, and several evidences indicate that a number of novel AMPK targets remain to be identified (11). Like all pharmacological approaches, results of experiments using AICAR should be interpreted with caution. In fact, even if AICAR represents the most specific tool for activation of AMPK in intact cells available to date (17), secondary effects cannot be ruled out. In some cell types, for example, a prolonged exposition to AICAR may alter purine and pyrimidine nucleotide levels and cause ZTP to accumulate (17,21). The supplementation of 100 mM uridine (34) does not hamper the inhibition of DEVDase activity by AICAR (data not shown), suggesting that a decrease of pyrimidine nucleotide level is not involved in the antiapoptotic power of AICAR. Furthermore, AICAR does not significantly affect protein synthesis (Fig. 2), which is necessary for glucocorticoid-induced apoptosis (35). On the other hand, the consequences of ZTP elevation are not known, but it could competitively interfere with some ATP or GTP-dependent processes. Unfortunately, AMPK inhibitors are not available to date. However, the strict correlation between ATP depletion and inhibition of dexamethasone-induced apoptosis (4), together with the similar antiapoptotic effect of AICAR and oligomycin, two structurally unrelated molecules both activating AMPK in different ways, support an apoptosis-inhibiting effect of AMPK in thymocytes. The discovery of the step affected by AICAR will contribute
to provide insight into the molecular mechanism triggered by glucocorticoids to induce thymocyte suicide. ACKNOWLEDGMENTS This work was supported by grants from the National Research Council (CNR) and Italian MURST (fondi 60%).
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14. Carling, D., Aguan, K., Woods, A., Verhoeven, A. J. M., Beri, R. K., Brennan, C. H., Sidebottom, C., Davison, M. D., and Scott, J. (1994) J. Biol. Chem. 269, 11442–11448. 15. Mitchelhill, K. I., Stapleton, D., Gao, G., House, C. M., Michell, B., Frosa, Katsis, Witters, L. A., and Kemp, B. E. (1994) J. Biol. Chem. 269, 2361–2364. 16. Sprenkle, A. B., Davies, S. P., Carling, D., Hardie, D. G., and Sturgill, T. W. (1997) FEBS Lett. 403, 254–258. 17. Corton, J. M., Gillespie, J. G., Hawley, S. A., and Hardie, D. G. (1995) Eur. J. Biochem. 229, 558–565. 18. Sullivan, J. E., Brocklehurst, K. J., Marley, A. E., Carey, F., Carling, D., and Beri, R. K. (1994) FEBS Lett. 353, 33–36. 19. Hemin, N., Vincent, M. F., Gruber, H. E., and van den Berghe, G. (1995) FASEB J. 9, 541–546. 20. Velasco, G., Geelen, M. J. H., and Guzman, M. (1997) Arch. Biochem. Biophys. 337, 169–175. 21. Sabina, R. L., Patterson, D., and Holmes, E. W. (1985) J. Biol. Chem. 260, 6107–6114. 22. van den Berghe, G. H., and Gruber, H. (1993) International Patent WO93/03734. 23. Sullivan, J. E., Carey, F., Carling, D., and Beri, R. K. (1994) Biochem. Biophys. Res. Commun. 200, 1551–1556. 24. Davies, S. P., Carling, D., and Hardie, D. G. (1989) Eur. J. Biochem. 186, 123–128.
25. Flamigni, F., Faenza, I., Marmiroli, S., Stanic, I., Giaccari, A., Muscari, C., Stefanelli, C., and Rossoni, C. (1997) Biochem. J. 324, 783–789. 26. Nudel, U., Zakut, R., Shani, M., Neuman, S., Levy, Z., and Yaffe, D. (1983) Nucleic Acids Res. 11, 1759–1771. 27. Nicholson, D. W., Ali, A., Thornberry, N. A., Vaillancourt, J. P., Ding, C. K., Gallant, M., Gareau, Y., Griffin, P. R., Labelle, M., Lazebnik, Y. A., Munday, N. A., Raju, S. M., Smulson, M. E., Yamin, T-T., Yu, V. L., and Miller, D. K. (1995) Nature 376, 37– 43. 28. Patel, T., Gores, G. J., and Kaufmann, S. H. (1996) FASEB J. 10, 587–597. 29. Bansal, N., Houle, A., and Melnykovych, G. (1991) FASEB J. 5, 211–216. 30. Wyllie, A. H. (1980) Nature 343, 76–79. 31. Helmberg, A., Auphan, N., Caelles, C., and Karin, M. (1995) EMBO J. 14, 452–460. 32. Miyashita, T., U. M., Inoue, T., Reed, J. C., and Yamada, M. (1997) Biochem. Biophys. Res. Commun. 233, 781–787. 33. Wang, H.-G., Takayaka, S., Rapp, V. R., and Reed, J. C. (1996) Proc. Natl. Acad. Sci. USA 93, 7063–7068. 34. Thomas, C. B., Meade, J. C., and Holmes, E. W. (1981) J. Cell. Physiol. 107, 335–344. 35. Wyllie, A. H., Morris, R. G., Smith, A. L., and Dunlop, D. (1984) J. Pathol. 142, 67–77.
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Inhibition of Glucocorticoid-Induced Apoptosis with 5-Aminoimidazole-4-carboxamide Ribonucleoside, a Cell-Permeable Activator of AMP-Activated Protein Kinase Claudio Stefanelli,1 Ivana Stanic’, Francesca Bonavita, Flavio Flamigni, Carla Pignatti, Carlo Guarnieri, and Claudio M. Caldarera Department of Biochemistry ‘‘G. Moruzzi,’’ University of Bologna, Via Irnerio, 48, I40126 Bologna, Italy
Received January 5, 1998
The AMP-activated protein kinase (AMPK) is related to a growing family of protein kinases that are believed to protect cells against environmental and nutritional stress. In the present study the hypothesis of a protective role for AMPK against thymocyte apoptosis has been tested. It is shown that AMPK is expressed in rat thymocytes that contain the transcript for the a1 isoform of the AMPK catalytic subunit and can be activated by treatment with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a wellestablished activator of AMPK. AICAR is not toxic and prevents glucocorticoid-induced apoptosis in the same concentration range used to activate AMPK. At concentrations higher than 1 mM, AICAR fully restores cell viability and inhibits DNA laddering in dexamethasone-treated thymocytes. Furthermore, AICAR blocks the dexamethasone-induced activation of caspase 3-like enzymes, which are believed to play a pivotal role in apoptotic cell death. Activation of AMPK by oligomycin, which depletes thymocytes of ATP, is also correlated to inhibition of caspase 3-like activity in dexamethasone-treated cells. However, AICAR and oligomycin do not exert any protective action when apoptosis is induced by staurosporine. These results indicate that AICAR is a powerful inhibitor of glucocorticoid-induced apoptosis and suggest that AMPK activation may interfere with a step in the apoptotic cascade triggered by dexamethasone. q 1998 Academic Press
1 To whom correspondence should be addressed. Fax: /39-51351224. E-mail: [email protected]. Abbreviations used: AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; AMC, 7-amino-4-methyl-coumarin; AMPK, AMP- activated protein kinase; AMPKK, AMPK kinase; bp, base pairs; DEVDAMC, acetyl-asp-glu-val-asp-AMC; DEVDase, enzymatic activity cleaving the peptide DEVD-AMC; RT-PCR, reverse transcriptionpolymerase chain reaction; SAPK, stress-activated protein kinase; ZMP, AICAR monophosphate; ZTP, AICAR triphosphate.
Cells respond to changes in environmental conditions by adapting their metabolism and recruiting specific signal transduction pathways. ATP depletion is a cellular stress which can be caused by hypoxia and several eukaryotic cells have evolved capabilities for surviving relatively long periods with limited supplies of oxygen (1-2). Thymic lymphocytes not only survive hypoxia (3), but in these cells ATP depletion may also inhibit apoptotic cell death induced by the synthetic glucocorticoid dexamethasone (4), suggesting that the adaptation to low ATP of these hypoxia-tolerant cells includes a step which interferes with apoptosis. The programmed cell death cascade can be divided in several stages (5). Death-triggering agents activate multiple signal pathways leading to the execution phase, when the activation of caspases occurs. Activation of the proteolytic cascade of caspases in turn leads to characteristic apoptotic structural lesions accompanying cell death (6). Extracellular stress elicits the activation of protein kinase cascades involving the stress activated protein kinases (SAPKs) (7,8); however, SAPK activation does not occur during anoxia, but only during the repletion of cellular ATP pool (9,10). On the contrary, ATP depletion activates a specific pathway: the AMP-activated protein kinase (AMPK) cascade (reviewed in ref. 11). AMPK consists of a family of isoenzymes (12), thought to act as ‘‘low-fuel warning system’’, being switched on by depletion of ATP with consequent increase of AMP (11). This kinase is activated by high levels of AMP via a complex mechanism which involves phosphorylation and allosteric regulation (11,13). AMP activates the AMPK by direct allosteric activation of the enzyme, activation of an upstream kinase (AMPKK), and inhibition of AMPK dephosphorylation. AMPK substrates include biosynthetic enzymes of lipid metabolism, which are inhibited upon phosphorylation by AMPK (11). These targets have suggested that the function of
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AMPK might be to inhibit fatty acid and sterol synthesis under conditions where the cellular energy status is compromised. However, studies of the yeast omologue of AMPK, SNF1, have suggested that AMPK could be more generally involved in gene regulation in response to metabolic stress (11,14,15). In agreement with this hypothesis, Raf-1 kinase has recently been identified as a substrate of AMPK (16). To date, no specific inhibitors of AMPK are available to test the involvement of AMPK in the antiapoptotic effect of ATP depletion in thymic lymphocytes, but AICAR represents an useful activator (17), extensively used to show the involvement of AMPK activation in specific cellular processes (17-20). AICAR is a cell-permeable precursor of its monophosphate nucleotide ZMP (21), an AMP analogue that mimics the effects of AMP on the allosteric activation of AMPK (22,23). In this study it is shown that thymus cells express AMPK and that its activation by AICAR correlates to inhibition of dexamethasone-induced apoptosis. MATERIALS AND METHODS Materials and cells. AICAR, DEVD-AMC and other biochemicals were products of Sigma. [g-33P]ATP and L-[1-14C]leucine were purchased from Amersham. The SAMS peptide was custom synthesized by Neosystem Laboratoire (Strasbourg, France). rTth and Taq DNA polymerases were from Perkin Elmer. Oligonucleotides were synthesized with an ABI 391 DNA synthesizer and purified by HPLC. Thymocytes were obtained from immature male Wistar rats. The isolated cells were suspended at the indicated concentration in RPMI 1640 medium containing 10 mM Hepes and 100 U/ml penicillin and streptomycin. The treatments were prepared in the culture medium or dimethylsulphoxide if water-insoluble. Each experimental point was derived from two wells each assayed in duplicate. AMPK activity. Tissues were rapidly homogenized in homogenization buffer HB (12), while thymocyte suspensions (2 1 108 cells/4 ml of medium) were collected at the end of the incubations, rapidly washed in ice-cold phosphate-buffered saline, homogenized in 1 ml of HB in a glass/teflon homogenizer and centrifuged 5 min at 3,000g. The supernatants were then centrifuged at 100,000g for 40 min and a 35% saturated ammonium sulphate fraction was prepared for each sample. The resultant pellet was resuspended in HB and diluted to a protein concentration of 0.2 mg/ml. The activity of AMPK was assayed by the phosphorylation of the SAMS peptide (24). Aliquots containing 1 mg of protein were incubated 10 min at 30 7C in a final volume of 25 ml containing 0.1 M Hepes pH 7, 0.2 M NaCl, 5 mM MgCl2 , 20% glycerol, 200 mM SAMS peptide and 200 mM [g-33P]ATP (220 cpm/pmol). Reactions were performed in duplicate { 5*-AMP (200 mM) with a minus peptide substrate control. The reaction was stopped in ice with 5 ml of 30% phosphoric acid and 10 ml aliquots were removed and spotted on phosphocellulose paper dishes that were washed and dried (24) before radioactivity counting. In preliminary experiments it was confirmed the linearity of the assay with respect to the incubation time and the amount of assayed protein. Detection of AMPK expression by RT-PCR. Total RNA was isolated from 107 thymocytes by the single-step guanidine thiocyanate method with the TriPure reagent (Boehringer Mannheim) according to the manufacturer’s instruction. The RNA preparation was digested with DNase and then 0.5 mg aliquots were reverse-transcribed, amplified for 40 cycles and analysed by agar gel electrophoresis as previously described (25). Specific primers were designed on the basis of published sequences as follows: a1 AMPK (12) forward
5*-GTCATCAGTACACCGTCTGA-3*, reverse 5*-CAAGCAGGACGTTTTCAGGT-3*; a2 AMPK (14) forward 5*-GTCATCTCAGGAAGGCTGTA-3*, reverse 5*-GTGGCAATAGAACGGTTGAG-3*; b-actin (26) forward 5*-TCTTCCAGCCTTCCTTCCT-3*, reverse 5*-GGAGCAATGATCTTGATCTTC-3*. The expected size of the amplification products were: 199 bp for a1 AMPK (cDNA nt 244-442); 179 bp for a2 AMPK (cDNA nt 550-728); 214 bp for b-actin (gene nt 25763001 with intervening intron). The mRNA for b-actin was determined as positive control for RNA extraction and RT-PCR. Since the intron/ exon structure of the AMPK genes is not known, in order to avoid the possible amplification of contaminating DNA, firstly the extracted RNA was extensively digested with DNase, furthermore, for each sample control aliquots were amplified for 40 cycles with Taq polymerase omitting the reverse transcription phase. Under these conditions DNA contamination was never found. Determination of caspase 3-like (DEVDase) activity. Caspase 3like activity was measured by the cleavage of the fluorogenic peptide substrate DEVD-AMC (27). Thymocytes (21107 cells) were incubated in one ml of medium. At the end of the experiment the cells were collected, washed in phosphate-buffered saline, suspended in 150 ml of lysis buffer (20 mM Hepes pH 7, 5 mM dithiothreitol, 2 mM EDTA, 0.1% CHAPS, 0.1% Triton X-100, 1 mM phenylmethylsulphonyl fluoride, 1 mg/ml each of aprotinin, pepstatin and leupeptin), vortexed and left 10 min in ice. The lysates were centrifuged 1 min at 28,000g and the supernatant was used as enzyme source. Ten microliters of this extract (containing about 10 mg of protein), were combined with 20 ml of assay buffer containing 100 mM Hepes pH 7, 5 mM dithiothreitol, 0.1% CHAPS, 10% sucrose and 0.15 mM DEVD-AMC and incubated 15 min at 37 7C. The reaction was stopped in ice by adding 0.1 ml of 2% sodium acetate in 0.2 M acetic acid. The samples were diluted with 2.5 ml of water and the specific cleavage of the fluorogenic peptide DEVD-AMC was monitored by AMC liberation using 370 nm excitation and 455 nm emission wavelengths. One unit is defined as the amount of enzyme activity cleaving 1.0 nmol of substrate per minute in the standard conditions described. Other assays. Cell death was evaluated by trypan blue exclusion. Internucleosomal DNA fragmentation was visualized by standard agarose gel electrophoresis and ethidium bromide staining (2 mg of DNA/lane) after DNA extraction from 107 cells. The rate of protein synthesis was measured in 2 x 107 cells by incorporation of L-[114 C]leucine in the acid-insoluble fraction during a 4 h incubation, as previously described (4).
RESULTS Thymocytes contain an activable AMPK. To explore the hypothesis that AMPK activation could be correlated to inhibition of apoptosis in ATP-depleted thymus cells, the presence of AMPK in rat thymocytes was tested. Previous studies on the tissue distribution of AMPK activity and expression revealed that AMPK is widely distributed (12,24). However, the presence of a kinase activable by AMP in thymus cells has not yet been reported. AMPK activity was assayed in the 0-35% saturated ammonium sulphate fraction, in which AMPK is almost totally recovered whereas inhibitors are removed (24). Fig. 1A shows that an AMP-activable kinase phosphorylating the SAMS peptide, a specific substrate of AMPK (24), is present in the rat thymus. The basal activity is low with respect to the activity measured in liver extracts, however, the activation by saturating (200 mM) AMP was more pronounced for the thymus
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bation of thymocytes with AICAR for 1 h activated cellular AMPK in a dose-dependent manner (Fig. 2A). AICAR, at the concentration of 2 mM, increased 3-fold AMPK activity measured in the presence of 200 mM AMP. While activating AMPK, AICAR did not affect energy-requiring processes like protein synthesis nor elicit significant toxic effect. Staurosporine is a powerful trigger of apoptosis virtually in all cell types. AICAR did not influence cell death induced by staurosporine; however, incubation of thymocytes with AICAR together with dexamethasone, produced a dose-dependent inhibition of dexamethasone-induced cell death (Fig. 2B). At dosages higher than 1 mM, AICAR fully preserved the viability of thymocytes incubated 20 h in the presence of dexamethasone. Accordingly, AICAR prevented DNA laddering in dexamethasone-treated cells (Fig. 2C). These data show that activation of AMPK by AICAR correlates with inhibition of glucocorticoid-induced apoptosis.
FIG. 1. Thymus cells express AMPK. (A) Phosphorylation of the SAMS peptide (AMPK activity) was measured in the absence or presence of 200 mM AMP in 0-35% ammonium sulphate fraction of rat liver or thymus extracts. Results are the mean of two separate experiments, whose range is shown. (B) SAMS phosphorylation in the presence of 200 mM AMP was measured in samples obtained from untreated thymocytes (control) or from cells incubated 1 h in the presence of 100 ng/ml oligomycin or 1 mM AICAR. The data are means of two experiments and the range is indicated. (C) Analysis of a1 and a2 AMPK transcripts by RT-PCR. Aliquots of 0.5 mg of total RNA were reverse-transcribed and then amplified for 40 cycles with primers specific for AMPK isoforms. b-Actin mRNA was used as a positive control. The expected size of amplification products is indicated.
enzyme (4-fold) with respect to the liver enzyme, that was activated about twice. As shown for other cell types, AMPK was activable in intact thymocytes (Fig. 1B). In fact, the activity of AMPK was further increased in cells depleted of ATP by treatment with 100 ng/ml of oligomycin (4), and in cells incubated 1 h with 1 mM AICAR, a cell permeable activator of AMPK (17). Since AMPK activation was evident in samples assayed in the presence of saturating AMP, the activation appears to be sustained by phosphorylation of AMPK by AMPKK (11,17,18). AMPK consists of a family of isoenzymes and two isoforms of the catalytic subunit, a1 and a2 , have been described to date (12). To determine the identity of thymic AMPK, thymocyte RNA was analyzed by RT-PCR using isoform-specific primers (Fig. 1C). Only the product corresponding to amplification of the a1 transcript was found. Owing to the sensitivity of RT-PCR, it seems likely that a1 is the only isoform expressed in thymus cells. Activation of AMPK by AICAR correlates to inhibition of dexamethasone-induced thymocyte apoptosis. Incu-
Effect of AICAR on caspase 3-like activity. The activation of caspase 3-like proteases is believed to play a pivotal role in the proteolytic cascade of apoptosis (6,28). As shown in Fig. 3A, caspase 3-like activity cleaving the fluorogenic substrate DEVD-AMC (DEVDase activity) gradually increased following dexamethasone, and after 4-6 h was about threefold the control’s level. The activation of caspase 3-like activity was strongly inhibited by AICAR (Fig. 3B). On the other hand, AICAR did not inhibit DEVDase activation induced by staurosporine (Fig. 3C). These results are fully consistent with the effects of AICAR on cell death reported on Fig. 2. Similarly to AICAR, oligomycin, while activating AMPK (Fig. 1), inhibited caspase 3like activity triggered by dexamethasone, but not by staurosporine. DISCUSSION Glucocorticoids kill immature lymphocytes as well as some leukemic cells by switching on the cell death program leading to apoptosis (29). Glucocorticoid-induced death of rodent thymocytes is one of the early model of apoptosis studied (30) and represents a prototypic model system for research in the field of programmed cell death. The molecular mechanism by which glucocorticoids induce apoptosis, however, is still poorly understood. Depending of the cell model it has been proposed that glucocorticoid-induced apoptosis is due to induction of ‘‘lysis genes’’ or to repression of ‘‘survival genes’’ (31). Whatever is the upstream mechanism, glucocorticoids finally cause the activation of caspase 3-like activity in both tumor cells (32) and thymocytes (Fig. 3). Thymocyte apoptosis caused by the synthetic glucocorticoid dexamethasone, but not by staurosporine, is inhibited in ATP-depleted thymocytes (4), suggesting
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FIG. 2. AICAR inhibits thymocyte apoptosis induced by dexamethasone. (A) Effect of different concentrations of AICAR on AMPK activity and the rate of protein synthesis in isolated thymocytes. AMPK activity was measured by SAMS phosphorylation in the presence of 200 mM AMP in extracts obtained from cells incubated with AICAR for 1 h. In control cells the activity was 356 { 27 cpm/min/ mg of protein (n Å 4). Protein synthesis was measured by leucine incorporation in acid-insoluble material following a 4 h incubation. Control thymocytes incorporated 206 { 19 cpm/106 cells (n Å 4). The reported data were obtained in a typical experiment representative of three. (B) Cell suspensions were incubated with different concentrations of AICAR in the presence of 1 mM dexamethasone (dex), 1 mM staurosporine (stauro), or 1 ml/ml of dimethylsulphoxide (control). After 16 h, cell survival was measured by trypan blue exclusion.
that the adaptive response to this cellular stress interferes with a specific pathway leading to apoptosis. It is worth noting that rat thymocytes well survive ATP depletion (3,4), differently from most cell types that are early damaged by treatments depleting cellular ATP. Cellular stress activates protein kinase cascades (7,8) and, at least in some cells, these kinases may have a protective role against apoptosis (8). On the other hand, ATP depletion does not recruit the SAPK pathway (9,10 and data not shown), but activates AMPK (11). In the examination of AMPK’s involvement in the antiapoptotic effect of ATP depletion in thymocytes, it must be born in mind that treatments depleting cellular ATP are likely to have non-specific side-effects, since ATP depletion can influence several cellular processes. However, a few years ago it has been reported that ZMP, a normal intermediate in the biosynthesis of purine nucleotides, can mimic the effect of AMP on allosteric activation of AMPK (22,23). Administration of AICAR to intact cells causes ZMP to accumulate inside the cell (17,21) and this has been demonstrated to be a rather specific method for activating AMPK (17), frequently used to demonstrate the involvement of AMPK in cellular processes (17-20). In the present study it is described for the first time a correlation between AMPK and apoptosis. AMPK is expressed in rat thymocytes, that contain the transcript for the widely distributed a1 isoform of the catalytic subunit, and can be activated by AICAR as well as by ATP depletion that follows oligomycin, a specific inhibitor of ATP synthase (Fig. 1). While activating AMPK, these treatments also block the death of dexamethasone-treated thymocytes (ref. 4 and Fig. 2). In particular, AICAR, in the same concentration range used to activate AMPK, inhibits internucleosomal DNA fragmentation and activation of caspase 3-like activity, which is considered the ‘‘point of no return’’ in apoptosis (6). It is worth noting that both AICAR and oligomycin do not affect caspase activation caused by staurosporine, suggesting a specific interference of AMPK in the death-pathway induced by glucocorticoids. AMPK phosphorylates and inactivates a number of key enzymes of lipid metabolism, including 3-hydroxy-3methylglutaryl-CoA reductase, catalyzing the first step of isoprenoid biosynthesis, acetyl-CoA carboxylase, the first enzyme of fatty acid biosynthesis, and hormonesensitive lipase (reviewed in ref. 11). These targets could suggest an interference of AMPK with some lipid molecule implicated in glucocorticoid-induced
The figure reports data from one experiment representative of three. (C) Electrophoretic fractionation of DNA (2 mg/lane) extracted from thymocytes incubated 6 h without any treatment (control) or with 1 mM dexamethasone (dex) together with the indicated concentration of AICAR.
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FIG. 3. AICAR inhibits caspase 3-like activity induced by dexamethasone. (A) Caspase 3-like activity cleaving the fluorogenic substrate DEVD-AMC (DEVDase activity) was measured at various times in control thymocytes and in cells treated with 1 mM dexamethasone (dex) to obtain a time course. (B) Control and dexamethasone-treated (dex) thymocytes were incubated 4 h in the presence of the indicated concentration of AICAR. Afterward DEVDase activity was measured. (C) Thymocytes were treated with 1 mM dexamethasone (dex) or 1 mM staurosporine (stauro) or none (control); each group was then incubated 5 h without any further treatment or with 2 mM AICAR or 100 ng/ml oligomycin. The panels report the data obtained in a typical experiment representative of several (2 to 5).
apoptosis since AMPK activation largely affects lipid metabolism. Recently, Raf-1 has been identified as a substrate of AMPK, which phosphorylates it at Ser621 (16). The role of this phosphorylation is not known, but interestingly, the Raf-1 kinase has been implicated in the regulation of apoptosis (33). However, it is evident that any step from dexamethasone binding to the activation of caspases may represent a site where AMPK could interfere with the progression of the death program, and several evidences indicate that a number of novel AMPK targets remain to be identified (11). Like all pharmacological approaches, results of experiments using AICAR should be interpreted with caution. In fact, even if AICAR represents the most specific tool for activation of AMPK in intact cells available to date (17), secondary effects cannot be ruled out. In some cell types, for example, a prolonged exposition to AICAR may alter purine and pyrimidine nucleotide levels and cause ZTP to accumulate (17,21). The supplementation of 100 mM uridine (34) does not hamper the inhibition of DEVDase activity by AICAR (data not shown), suggesting that a decrease of pyrimidine nucleotide level is not involved in the antiapoptotic power of AICAR. Furthermore, AICAR does not significantly affect protein synthesis (Fig. 2), which is necessary for glucocorticoid-induced apoptosis (35). On the other hand, the consequences of ZTP elevation are not known, but it could competitively interfere with some ATP or GTP-dependent processes. Unfortunately, AMPK inhibitors are not available to date. However, the strict correlation between ATP depletion and inhibition of dexamethasone-induced apoptosis (4), together with the similar antiapoptotic effect of AICAR and oligomycin, two structurally unrelated molecules both activating AMPK in different ways, support an apoptosis-inhibiting effect of AMPK in thymocytes. The discovery of the step affected by AICAR will contribute
to provide insight into the molecular mechanism triggered by glucocorticoids to induce thymocyte suicide. ACKNOWLEDGMENTS This work was supported by grants from the National Research Council (CNR) and Italian MURST (fondi 60%).
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