- Email: [email protected]
Stimulation of P2Y-purinoceptors on astrocytes results in immediate early gene expression and potentiation of neuropeptide action
Pergamon
PII:
Neuroscience Vol. 85, No. 2, pp. 521–525, 1998 Copyright 1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00653-2
STIMULATION OF P2Y-PURINOCEPTORS ON ASTROCYTES RESULTS IN IMMEDIATE EARLY GENE EXPRESSION AND POTENTIATION OF NEUROPEPTIDE ACTION J. PRILLER,* M. REDDINGTON,* C. A. HAAS† and G. W. KREUTZBERG*‡ *Department of Neuromorphology, Max-Planck-Institute of Neurobiology, Martinsried, Germany †Institute of Anatomy I, University of Freiburg, Freiburg, Germany Abstract––The action of adenosine-5 -O-(2-thiodiphosphate), a non-hydrolysable purine analogue and potent P2Y1-purinoceptor agonist, was studied on immediate early gene expression in rat astrocyte cultures. A rapid and transient increase in c-fos, junB, c-jun and Tis11 messenger RNA was observed in cultured astrocytes after treatment with adenosine-5 -O-(2-thiodiphosphate). Maximal induction of immediate early gene expression was obtained within 30 min of stimulation and c-fos was the most sensitive indicator of P2Y-purinoceptor activation. Calcitonin gene-related peptide has also been shown to be a potent inducer of c-fos messenger RNA in cultured astroglial cells. The combined stimulation of astrocytes with calcitonin gene-related peptide and adenosine-5 -O-(2-thiodiphosphate) resulted in the potentiated expression of c-fos messenger RNA. The superinduction of immediate early gene expression by calcitonin gene-related peptide and extracellular ATP in cultured astrocytes might result from intracellular signal transduction cross-talk, since adenosine-5 -O-(2-thiodiphosphate) was found to increase calcitonin gene-related peptide-induced cyclic AMP accumulation by 35%. Phorbol 12-myristate 13-acetate also increased calcitonin gene-related peptide-evoked cyclic AMP accumulation and led to the induction of immediate early gene expression, suggesting that protein kinase C might be at least in part involved in purinergic cross-talk. Our results demonstrate synergistic roles for extracellular ATP and calcitonin gene-related peptide in the transcriptional activation of astroglial cells. 1998 IBRO. Published by Elsevier Science Ltd. Key words: neuroglia, adenine nucleotides, calcitonin gene-related peptide, transcription factors, purinergic receptors.
Cultured astrocytes are known to respond to stimulation with extracellular ATP. In recent receptor expression experiments, the two major P2purinoceptors expressed on astrocytes were identified as P2Y and P2U.12 The astroglial P2Y-receptor has been shown to be a G-protein-coupled receptor linked to phospholipases C and A2.4,24 Activation of protein kinase C, mobilization of intracellular calcium, eicosanoid release and stimulation of mitogen-activated protein kinase have been observed as a result of P2Y-purinoceptor activation.4,12,17,25 On the other hand, stimulation of the astroglial receptor for calcitonin gene-related peptide (CGRP), a 37-amino acid peptide generated by alternative RNA processing of the calcitonin gene transcript,23 results in the accumulation of cyclic AMP and increased expression of c-fos messenger RNA in cultured astrocytes.7,14 Since CGRP has been suggested to play a role in the activation of glial cells following motoneuron injury,22 and ATP is released in large quantities from
damaged cells,6 it was interesting to determine whether both substances could act synergistically in the mobilization of astroglial cells during CNS regeneration. For this purpose, we studied the effects of CGRP and adenosine-5 -O-(2-thiodiphosphate) (ADPâS), a hydrolysis-resistant purine analogue,29 on immediate early gene (IEG) expression in cultured astrocytes. Due to their intermediary role in gene expression,27 IEGs are a useful tool for the investigation of receptor-mediated transcriptional activation. The potential involvement of protein kinase C in the actions of ADPâS was studied by comparison with the effects of phorbol ester.
‡To whom correspondence should be addressed. Abbreviations: ADPâS, adenosine-5 -O-(2-thiodiphosphate); CGRP, calcitonin gene-related peptide; IEG, immediate early gene; PKC, protein kinase C; PMA, phorbol 12myristate 13-acetate. 521
EXPERIMENTAL PROCEDURES
Materials á-CGRP was obtained from Sigma Chemicals, Deisenhofen, Germany. ADPâS and anti-glial fibrillary acidic protein were from Boehringer–Mannheim, Mannheim, Germany. Phorbol 12-myristate 13-acetate (PMA) was obtained from Calbiochem, Bad Soden, Germany, and RO 20-1724 was from Research Biochemicals Inc., Natick, U.S.A. Cyclic AMP was determined using a radioimmunoassay kit from NEN-Du Pont, Bad Homburg, Germany. Wistar rats were obtained from the breeding facility, Max-Planck-Institute of Biochemistry, Martinsried.
522
J. Priller et al.
Cell culture Astrocyte cultures were obtained from neonatal rat brain according to the method of Lazar et al.14 Cells were harvested after 10–12 days in culture and replated in 6 cm Petri dishes or 12-well culture plates at a mean density of 200,000 cells/dish and 50,000 cells/well. Cultures were grown to confluency for four days before experiments were performed. A monoclonal anti-glial fibrillary acidic protein antibody (Boehringer–Mannheim) was used for immunocytochemical identification of astrocytes. Cyclic AMP determination For measurements of cyclic AMP, cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) containing 0.3 mM of the phosphodiesterase inhibitor RO 20-1724 for 30 min. Test compounds were added for another 25 min and cyclic AMP was extracted as described by Priller et al.21 Cyclic AMP was determined by radioimmunoassay using a commercially available kit (NEN-Du Pont). RNA extraction and northern blot analysis Preparation of total cellular RNA, northern transfer and hybridization were performed as described by Priller et al.21 32 P-labelled cDNA probes for junB, c-jun, Tis11 and 18S rRNA were obtained by random priming (Boehringer– Mannheim). A 32P-labelled c-fos cRNA probe was generated by in vitro transcription from a linearized pSP65 plasmid using SP6 RNA polymerase and á-[32P]-UTP. For the purpose of quantification, autoradiograms obtained from northern blots were scanned using laser densitometry. To allow correction for the recovery of RNA in each sample, IEG mRNA signals were normalized against 18S ribosomal RNA content by determining the ratio of respective optical densities. Statistical analysis An unpaired Student’s t-test was performed for statistical evaluation of the data. Significance was accepted for P<0.05. RESULTS
Induction of c-fos, junB, c-jun and Tis11 messenger RNA by adenosine-5 -O-(2-thiodiphosphate) and phorbol 12-myristate 13-acetate When astrocytes were treated with 1 µM ADPâS, a rapid and very transient increase in c-fos, junB, c-jun and Tis11 mRNA was observed (Fig. 1A). All IEG mRNAs were maximally induced within 30 min and returned to basal levels thereafter. However, the degree of stimulation by ADPâS varied for individual IEGs and was greatest for c-fos mRNA (mean increase five-fold over control), intermediate for junB and Tis11 mRNA (two-fold over control) and lowest for c-jun mRNA (1.5-fold over control). PMA, an activator of protein kinase C, also led to the rapid induction of c-fos, junB, c-jun and Tis11 mRNA in astrocytes (Fig. 1B). After 30 min incubation with 100 nM PMA, a dramatic increase in c-fos (22-fold over control), junB (nine-fold over control) and Tis11 (13-fold over control) mRNA was observed in astroglial cultures. While c-fos mRNA returned to basal levels within 2 h, the mRNAs of junB and Tis11 stayed elevated. In the case of c-jun mRNA, expression was maximal 2 h after addition of the phorbol ester (four-fold increase over control).
Superinduction of c-fos messenger RNA as a result of cross-talk by calcitonin gene-related peptide and adenosine-5 -O-(2-thiodiphosphate) CGRP has previously been shown to induce c-fos mRNA in cultured astrocytes.7 In this study, 1 µM CGRP doubled the amount of c-fos mRNA in astroglial cultures compared with control, while 1 µM ADPâS led to a 400% increase in c-fos mRNA expression (Fig. 2, Table 1). The simultaneous application of CGRP and ADPâS resulted in an 850% increase in c-fos mRNA (Fig. 2, Table 1), which is significantly higher than the merely additive effect of both agonists (i.e. a five-fold accumulation of c-fos mRNA over control). When astrocytes were treated with a combination of CGRP and phorbol ester, the induction of c-fos mRNA by PMA was generally so pronounced that cross-talk events with CGRP could not be discerned on the basis of IEG mRNA expression (data not shown). Effects of adenosine-5 -O-(2-thiodiphosphate) and phorbol 12-myristate 13-acetate on calcitonin generelated peptide-induced cyclic AMP accumulation In order to investigate further the signal cross-talk by CGRP and ADPâS in cultured astrocytes, we studied the effect of ADPâS on CGRP-evoked cyclic AMP accumulation. One-micromolar ADPâS had no effect of its own on cyclic AMP content, but increased CGRP-induced cyclic AMP accumulation by 35% (Fig. 3). This cross-talk seemed to require a certain threshold concentration of CGRP since 1 µM ADPâS failed to potentiate the effect of 100 nM CGRP on cyclic AMP accumulation. ATP was not used in these experiments since it stimulated adenylate cyclase activity, presumably via activation of A2-adenosine receptors following degradation of ATP to adenosine (data not shown). An even more pronounced potentiation of cyclic AMP accumulation was observed when CGRP was combined with 100 nM PMA (Fig. 3). While PMA slightly elevated basal cyclic AMP (increase of approximately 5 pmol/well), it potentiated CGRPevoked cyclic AMP accumulation by 25%, 30% and up to 80% when combined with 0.1 µM, 0.33 µM and 1.0 µM CGRP, respectively. DISCUSSION
Up to 100% of astrocytes within a given culture have been reported to respond to treatment with extracellular ATP by changes in intracellular calcium.20,24,26 The biological effects of adenine nucleotides on astroglial cells have been extensively investigated, but so far little is known about the role of transcription factors in the actions of ATP. In this study, stimulation of some P2Y-purinoceptors was shown to induce IEG expression in cultured astrocytes. Moreover, the purine analogue ADPâS was found to potentiate the effect of CGRP on IEG
ADPâS and CGRP effects on cultured astrocytes
523
Fig. 1. Time-course of immediate early gene induction by ADPßS (A) and PMA (B) in cultured astrocytes. Cells were treated with 1 µM ADPßS or 100 nM PMA for the times indicated and the RNA extracted was analysed as described under ‘‘Experimental Procedures’’. The autoradiograms shown represent one of 10 to 13 separate experiments performed in duplicate. The lower band of the c-fos mRNA signal represents the synthetic fos sense-transcript added as a recovery standard. ADPßS significantly induced c-fos (five-fold; P<0.001), junB (two-fold; P<0.001), c-jun (1.5-fold; P<0.05) and Tis11 (two-fold; P<0.05) mRNA. PMA also led to a significant increase in c-fos (22-fold; P<0.001), junB (nine-fold; P<0.001), c-jun (four-fold; P<0.05) and Tis11 (13-fold; P<0.001) mRNA.
mRNA expression in cultured astroglia. The IEGs Fos and Jun encode regulatory proteins that control the expression of target genes involved in adaptive responses in the nervous system.27 Tis genes, first identified in Swiss 3T3 cells following exposure to phorbol ester, also show characteristic features of IEGs in cultured astrocytes.1,15 A rapid and transient expression of the mRNAs of AP-1 transcription factor components (c-fos, junB, c-jun) and Tis11 was observed in astrocytes in response to stimulation with the non-hydrolysable P2Y1-agonist, ADPâS. The pattern of induction was identical to that observed in microglial cells,21 suggesting that neuroglia might show a rather uniform transcriptional response to extracellular ATP; P2-
purinoceptors have been described on the majority of glial cells, including Schwann cells, oligodendrocytes and enteric glia.2,11,13 Since the pattern of IEG induction by ADPâS in cultured astrocytes qualitatively resembled that of PMA, it is conceivable that protein kinase C (PKC) might play a role in purinergic stimulation of gene expression in astrocytes. PKC has also been found to mediate the uncoupling of astroglial gap junctions in response to P2Y-agonists.5 The fact that PMA led to a more dramatic and prolonged induction of IEG mRNA compared with ADPâS, might be explained by its direct, intracellular site of action as opposed to the receptor-mediated effects of ADPâS. Alternatively, phorbol esters might simply be more stable in cell culture than ATP
524
J. Priller et al.
Fig. 2. Effect of the combination of CGRP and ADPâS on c-fos mRNA expression in cultured astrocytes. Cells were treated with 1 µM CGRP, 1 µM ADPâS and CGRP plus ADPâS for 30 min. Immediate early gene expression was analysed as described under ‘‘Experimental Procedures’’. The autoradiogram shown is representative of three separate experiments performed in triplicate and two experiments performed in duplicate. The combination of CGRP and ADPâS led to an overadditive expression of c-fos mRNA. Table 1. Quantitative evaluation of the effect of agonist combination on c-fos messenger RNA expression Treatment 1 µM CGRP 1 µM ADPâS 1 µM CGRP+1 µM ADPâS
c-fos mRNA (fold stimulation) 2.090.17 5.020.63 9.531.27*
Values are meansS.E.M. (expressed as increase in c-fos mRNA relative to controls) of five separate experiments (see Fig. 2). The stimulation of astrocytes with CGRP plus ADPâS led to a potentiated expression of c-fos mRNA (combination vs additive effect of the agonists: *P<0.05).
analogues. In addition, ADPâS increases intracellular calcium in astrocytes19 which could potentially interfere with the expression of particular IEGs. In fact, Trejo and Brown have found that calcium represses c-jun expression through increased degradation of c-jun mRNA in astrocytoma cells.28 This study raises the possibility that neuropeptide receptors are co-expressed with P2-purinoceptors on cultured astrocytes since ADPâS and CGRP were found to have synergistic effects on second messenger activation and IEG expression in astroglial cultures. A similar synergism has been described for ADPâS and fibroblast growth factors with respect to DNA synthesis in astrocytes.18 The precise nature of the second messenger(s) mediating purinergic potentiation of CGRP effects in astroglia remains to be determined. The strong potentiation of CGRPevoked cyclic AMP accumulation by PMA suggests a role for PKC for this cross-talk. In fact, PKC has been shown to potentiate type II and V adenylate cyclase activities in the presence of ás or via direct phosphorylation.10,16 However, elevated intracellular calcium levels have also been reported to stimulate some types of adenylate cyclase,30 and metabotropic P2-purinoceptors are known to mobilize both PKC and intracellular calcium. Interestingly, pretreatment
Fig. 3. Synergistic actions of ADPâS and PMA on CGRPinduced cyclic AMP accumulation. Cells were incubated in 0.3 mM RO 20-1724 for 30 min, followed by the addition of increasing concentrations of CGRP in the absence or presence of 1 µM ADPâS or 0.1 µM PMA. After 25 min, cyclic AMP was extracted and determined by radioimmunoassay. The results are means of triplicate determinationsS.E.M. and the data are representative of those from three experiments. ADPâS increased CGRP-induced cyclic AMP accumulation by up to 35% (combination vs additive effect at 1 µM CGRP: P<0.05). PMA potentiated CGRP-evoked cyclic AMP accumulation by 80% when combined with 1 µM CGRP (P<0.01 compared to additive effect of the agonists). ADPâS did not affect basal cyclic AMP content, while PMA led to a slight increase.
of L-cells with ATP caused an enhancement of adenylate cyclase activity similar to that of PMA, but unaffected by prior depletion of PKC.9 It is clear from this study that astrocytes do not express the P2Y-purinoceptor subtype described by Boyer et al. on C6-2B rat glioma cells,3 which inhibits adenylate cyclase activity and has no effect on phospholipase C. CONCLUSION
The results suggest synergistic roles for extracellular ATP and CGRP in the transcriptional activation of cultured astroglial cells. It is not yet known what functional implications this might have. Preliminary experiments have indicated that IEGs other than c-fos (such as junB, c-jun and Tis11) do not respond to second messenger cross-talk by CGRP and ADPâS in astrocytes (Priller et al., unpublished observations). If the synergism of CGRP and extracellular ATP in astrocytes should lead to a selective increase in the synthesis of Fos relative to other members of the AP-1 transcription factor family, it is conceivable that the expression of target genes containing an AP-1 consensus sequence might be significantly enhanced, since Fos/Jun dimers are amongst the most potent transcriptional activators of these genes.8
ADPâS and CGRP effects on cultured astrocytes
525
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
Arenander A. T., de Vellis J. and Herschman H. R. (1989) Induction of c-fos and TIS genes in cultured rat astrocytes by neurotransmitters. J. Neurosci. Res. 24, 107–114. Berti-Mattera L. N., Wilkins P. L., Madhun Z. and Suchovsky D. (1996) P2-purinergic receptors regulate phospholipase C and adenylate cyclase activities in immortalized Schwann cells. Biochem. J. 314, 555–561. Boyer J. L., Lazarowski E. R., Chen X.-H. and Harden T. K. (1993) Identification of a P-2Y-purinergic receptor that inhibits adenylyl cyclase. J. Pharmac. exp. Ther. 267, 1140–1146. Bruner G. and Murphy S. (1993) Purinergic P2Y receptors on astrocytes are directly coupled to phospholipase A2. Glia 7, 219–224. Enkvist M. O. K. and McCarthy K. D. (1992) Activation of protein kinase C blocks astroglial gap junction communication and inhibits the spread of calcium waves. J. Neurochem. 59, 519–526. Gordon J. L. (1986) Extracellular ATP: effects, sources and fate. Biochem. J. 233, 309–319. Haas C. A., Reddington M. and Kreutzberg G. W. (1991) Calcitonin gene-related peptide stimulates the induction of c-fos gene expression in rat astrocyte cultures. Eur. J. Neurosci. 3, 708–712. Hughes P. and Dragunow M. (1995) Induction of immediate-early genes and the control of neurotransmitter-regulated gene expression within the nervous system. Pharmac. Rev. 47, 133–178. Johnson J. A., Friedman J., Halligan R. D., Birnbaumer M. and Clark R. B. (1991) Sensitization of adenylyl cyclase by P2 purinergic and M5 muscarinic receptor agonists in L cells. Molec. Pharmac. 40, 539–546. Kawabe J., Iwami G., Ebina T., Ohno S., Katada T., Ueda Y., Homcy C. J. and Ishikawa Y. (1994) Differential activation of adenylyl cyclase by protein kinase C isoenzymes. J. biol. Chem. 269, 16,554–16,558. Kimball B. C. and Mulholland M. W. (1996) Enteric glia exhibit P2U receptors that increase cytosolic calcium by a phospholipase C-dependent mechanism. J. Neurochem. 66, 604–612. King B. F., Neary J. T., Zhu Q., Wang S., Norenberg M. D. and Burnstock G. (1996) P2 purinoceptors in rat cortical astrocytes: expression, calcium-imaging and signalling studies. Neuroscience 74, 1187–1196. Kirischuk S., Scherer J., Kettenmann H. and Verkhratsky A. (1995) Activation of P2-purinoreceptors triggered Ca2+ release from InsP3-sensitive internal stores in mammalian oligodendrocytes. J. Physiol. 483, 41–57. Lazar P., Reddington M., Streit W., Raivich G. and Kreutzberg G. W. (1991) The action of calcitonin gene-related peptide on astrocyte morphology and cyclic AMP accumulation in astrocyte cultures from neonatal rat brain. Neurosci. Lett. 130, 99–102. Lim R. W., Varnum B. C. and Herschman H. (1987) Cloning of sequences induced as a primary response following mitogen treatment of density arrested Swiss 3T3 cells. Oncogene 1, 263–270. Lustig K. D., Conklin B. R., Herzmark P., Taussig R. and Bourne H. R. (1993) Type II adenylylcyclase integrates coincident signals from Gs, Gi and Gq. J. biol. Chem. 268, 13,900–13,905. Neary J. T. and Zhu Q. (1994) Signaling by ATP receptors in astrocytes. NeuroReport 5, 1617–1620. Neary J. T., Whittemore S. R., Zhu Q. and Norenberg M. D. (1994) Synergistic activation of DNA synthesis in astrocytes by fibroblast growth factors and extracellular ATP. J. Neurochem. 63, 490–494. Ogata T., Nakamura Y. and Schubert P. (1996) Potentiated cAMP rise in metabotropically stimulated rat cultured astrocytes by a Ca2+-related A1/A2 adenosine receptor cooperation. Eur. J. Neurosci. 8, 1124–1131. Pearce B. and Langley D. (1994) Purine- and pyrimidine-stimulated phosphoinositide breakdown and intracellular calcium mobilisation in astrocytes. Brain Res. 660, 329–332. Priller J., Haas C. A., Reddington M. and Kreutzberg G. W. (1995) Calcitonin gene-related peptide and ATP induce immediate early gene expression in cultured rat microglial cells. Glia 15, 447–457. Raivich G., Reddington M., Haas C. A. and Kreutzberg G. W. (1995) Peptides in motoneurons. In Neuropeptides in the Spinal Cord. (eds. Nyberg F., Sharma H. S. and Wiesenfeld-Hallin Z.), Progress in Brain Research. Vol. 104, pp. 3–20. Elsevier, Amsterdam. Rosenfeld M. G., Mermod J.-J., Amara S. G., Swanson L. W., Sawchenko P. E., Rivier J., Vale W. W. and Evans R. M. (1983) Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature 304, 129–135. Salter M. W. and Hicks J. L. (1995) ATP causes release of intracellular Ca2+ via the phospholipase Câ/IP3 pathway in astrocytes from the dorsal spinal cord. J. Neurosci. 15, 2961–2971. Sen R. P., Delicado E. G., Castro E. and Miras-Portugal M. T. (1993) Effect of P2Y agonists on adenosine transport in cultured chromaffin cells. J. Neurochem. 60, 613–619. Shao Y. and McCarthy K. D. (1993) Regulation of astroglial responsiveness to neuroligands in primary culture. Neuroscience 55, 991–1001. Sheng M. and Greenberg M. E. (1990) The regulation and function of c-fos and other immediate early genes in the nervous system. Neuron 4, 477–485. Trejo J. A. and Brown J. H. (1991) c-fos and c-jun are induced by muscarinic receptor activation of protein kinase C but are differentially regulated by intracellular calcium. J. biol. Chem. 266, 7876–7882. Welford L. A., Cusack N. J. and Hourani S. M. O. (1987) The structure–activity relationships of ectonucleotidases and of excitatory P2-purinoceptors: evidence that dephosphorylation of ATP analogues reduces pharmacological potency. Eur. J. Pharmac. 141, 123–130. Yoshimura M. and Cooper D. M. F. (1992) Cloning and expression of a Ca2+-inhibitable adenylyl cyclase from NCB-20 cells. Proc. natn. Acad. Sci. U.S.A. 89, 6716–6720. (Accepted 26 November 1997)
PII:
Neuroscience Vol. 85, No. 2, pp. 521–525, 1998 Copyright 1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00653-2
STIMULATION OF P2Y-PURINOCEPTORS ON ASTROCYTES RESULTS IN IMMEDIATE EARLY GENE EXPRESSION AND POTENTIATION OF NEUROPEPTIDE ACTION J. PRILLER,* M. REDDINGTON,* C. A. HAAS† and G. W. KREUTZBERG*‡ *Department of Neuromorphology, Max-Planck-Institute of Neurobiology, Martinsried, Germany †Institute of Anatomy I, University of Freiburg, Freiburg, Germany Abstract––The action of adenosine-5 -O-(2-thiodiphosphate), a non-hydrolysable purine analogue and potent P2Y1-purinoceptor agonist, was studied on immediate early gene expression in rat astrocyte cultures. A rapid and transient increase in c-fos, junB, c-jun and Tis11 messenger RNA was observed in cultured astrocytes after treatment with adenosine-5 -O-(2-thiodiphosphate). Maximal induction of immediate early gene expression was obtained within 30 min of stimulation and c-fos was the most sensitive indicator of P2Y-purinoceptor activation. Calcitonin gene-related peptide has also been shown to be a potent inducer of c-fos messenger RNA in cultured astroglial cells. The combined stimulation of astrocytes with calcitonin gene-related peptide and adenosine-5 -O-(2-thiodiphosphate) resulted in the potentiated expression of c-fos messenger RNA. The superinduction of immediate early gene expression by calcitonin gene-related peptide and extracellular ATP in cultured astrocytes might result from intracellular signal transduction cross-talk, since adenosine-5 -O-(2-thiodiphosphate) was found to increase calcitonin gene-related peptide-induced cyclic AMP accumulation by 35%. Phorbol 12-myristate 13-acetate also increased calcitonin gene-related peptide-evoked cyclic AMP accumulation and led to the induction of immediate early gene expression, suggesting that protein kinase C might be at least in part involved in purinergic cross-talk. Our results demonstrate synergistic roles for extracellular ATP and calcitonin gene-related peptide in the transcriptional activation of astroglial cells. 1998 IBRO. Published by Elsevier Science Ltd. Key words: neuroglia, adenine nucleotides, calcitonin gene-related peptide, transcription factors, purinergic receptors.
Cultured astrocytes are known to respond to stimulation with extracellular ATP. In recent receptor expression experiments, the two major P2purinoceptors expressed on astrocytes were identified as P2Y and P2U.12 The astroglial P2Y-receptor has been shown to be a G-protein-coupled receptor linked to phospholipases C and A2.4,24 Activation of protein kinase C, mobilization of intracellular calcium, eicosanoid release and stimulation of mitogen-activated protein kinase have been observed as a result of P2Y-purinoceptor activation.4,12,17,25 On the other hand, stimulation of the astroglial receptor for calcitonin gene-related peptide (CGRP), a 37-amino acid peptide generated by alternative RNA processing of the calcitonin gene transcript,23 results in the accumulation of cyclic AMP and increased expression of c-fos messenger RNA in cultured astrocytes.7,14 Since CGRP has been suggested to play a role in the activation of glial cells following motoneuron injury,22 and ATP is released in large quantities from
damaged cells,6 it was interesting to determine whether both substances could act synergistically in the mobilization of astroglial cells during CNS regeneration. For this purpose, we studied the effects of CGRP and adenosine-5 -O-(2-thiodiphosphate) (ADPâS), a hydrolysis-resistant purine analogue,29 on immediate early gene (IEG) expression in cultured astrocytes. Due to their intermediary role in gene expression,27 IEGs are a useful tool for the investigation of receptor-mediated transcriptional activation. The potential involvement of protein kinase C in the actions of ADPâS was studied by comparison with the effects of phorbol ester.
‡To whom correspondence should be addressed. Abbreviations: ADPâS, adenosine-5 -O-(2-thiodiphosphate); CGRP, calcitonin gene-related peptide; IEG, immediate early gene; PKC, protein kinase C; PMA, phorbol 12myristate 13-acetate. 521
EXPERIMENTAL PROCEDURES
Materials á-CGRP was obtained from Sigma Chemicals, Deisenhofen, Germany. ADPâS and anti-glial fibrillary acidic protein were from Boehringer–Mannheim, Mannheim, Germany. Phorbol 12-myristate 13-acetate (PMA) was obtained from Calbiochem, Bad Soden, Germany, and RO 20-1724 was from Research Biochemicals Inc., Natick, U.S.A. Cyclic AMP was determined using a radioimmunoassay kit from NEN-Du Pont, Bad Homburg, Germany. Wistar rats were obtained from the breeding facility, Max-Planck-Institute of Biochemistry, Martinsried.
522
J. Priller et al.
Cell culture Astrocyte cultures were obtained from neonatal rat brain according to the method of Lazar et al.14 Cells were harvested after 10–12 days in culture and replated in 6 cm Petri dishes or 12-well culture plates at a mean density of 200,000 cells/dish and 50,000 cells/well. Cultures were grown to confluency for four days before experiments were performed. A monoclonal anti-glial fibrillary acidic protein antibody (Boehringer–Mannheim) was used for immunocytochemical identification of astrocytes. Cyclic AMP determination For measurements of cyclic AMP, cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) containing 0.3 mM of the phosphodiesterase inhibitor RO 20-1724 for 30 min. Test compounds were added for another 25 min and cyclic AMP was extracted as described by Priller et al.21 Cyclic AMP was determined by radioimmunoassay using a commercially available kit (NEN-Du Pont). RNA extraction and northern blot analysis Preparation of total cellular RNA, northern transfer and hybridization were performed as described by Priller et al.21 32 P-labelled cDNA probes for junB, c-jun, Tis11 and 18S rRNA were obtained by random priming (Boehringer– Mannheim). A 32P-labelled c-fos cRNA probe was generated by in vitro transcription from a linearized pSP65 plasmid using SP6 RNA polymerase and á-[32P]-UTP. For the purpose of quantification, autoradiograms obtained from northern blots were scanned using laser densitometry. To allow correction for the recovery of RNA in each sample, IEG mRNA signals were normalized against 18S ribosomal RNA content by determining the ratio of respective optical densities. Statistical analysis An unpaired Student’s t-test was performed for statistical evaluation of the data. Significance was accepted for P<0.05. RESULTS
Induction of c-fos, junB, c-jun and Tis11 messenger RNA by adenosine-5 -O-(2-thiodiphosphate) and phorbol 12-myristate 13-acetate When astrocytes were treated with 1 µM ADPâS, a rapid and very transient increase in c-fos, junB, c-jun and Tis11 mRNA was observed (Fig. 1A). All IEG mRNAs were maximally induced within 30 min and returned to basal levels thereafter. However, the degree of stimulation by ADPâS varied for individual IEGs and was greatest for c-fos mRNA (mean increase five-fold over control), intermediate for junB and Tis11 mRNA (two-fold over control) and lowest for c-jun mRNA (1.5-fold over control). PMA, an activator of protein kinase C, also led to the rapid induction of c-fos, junB, c-jun and Tis11 mRNA in astrocytes (Fig. 1B). After 30 min incubation with 100 nM PMA, a dramatic increase in c-fos (22-fold over control), junB (nine-fold over control) and Tis11 (13-fold over control) mRNA was observed in astroglial cultures. While c-fos mRNA returned to basal levels within 2 h, the mRNAs of junB and Tis11 stayed elevated. In the case of c-jun mRNA, expression was maximal 2 h after addition of the phorbol ester (four-fold increase over control).
Superinduction of c-fos messenger RNA as a result of cross-talk by calcitonin gene-related peptide and adenosine-5 -O-(2-thiodiphosphate) CGRP has previously been shown to induce c-fos mRNA in cultured astrocytes.7 In this study, 1 µM CGRP doubled the amount of c-fos mRNA in astroglial cultures compared with control, while 1 µM ADPâS led to a 400% increase in c-fos mRNA expression (Fig. 2, Table 1). The simultaneous application of CGRP and ADPâS resulted in an 850% increase in c-fos mRNA (Fig. 2, Table 1), which is significantly higher than the merely additive effect of both agonists (i.e. a five-fold accumulation of c-fos mRNA over control). When astrocytes were treated with a combination of CGRP and phorbol ester, the induction of c-fos mRNA by PMA was generally so pronounced that cross-talk events with CGRP could not be discerned on the basis of IEG mRNA expression (data not shown). Effects of adenosine-5 -O-(2-thiodiphosphate) and phorbol 12-myristate 13-acetate on calcitonin generelated peptide-induced cyclic AMP accumulation In order to investigate further the signal cross-talk by CGRP and ADPâS in cultured astrocytes, we studied the effect of ADPâS on CGRP-evoked cyclic AMP accumulation. One-micromolar ADPâS had no effect of its own on cyclic AMP content, but increased CGRP-induced cyclic AMP accumulation by 35% (Fig. 3). This cross-talk seemed to require a certain threshold concentration of CGRP since 1 µM ADPâS failed to potentiate the effect of 100 nM CGRP on cyclic AMP accumulation. ATP was not used in these experiments since it stimulated adenylate cyclase activity, presumably via activation of A2-adenosine receptors following degradation of ATP to adenosine (data not shown). An even more pronounced potentiation of cyclic AMP accumulation was observed when CGRP was combined with 100 nM PMA (Fig. 3). While PMA slightly elevated basal cyclic AMP (increase of approximately 5 pmol/well), it potentiated CGRPevoked cyclic AMP accumulation by 25%, 30% and up to 80% when combined with 0.1 µM, 0.33 µM and 1.0 µM CGRP, respectively. DISCUSSION
Up to 100% of astrocytes within a given culture have been reported to respond to treatment with extracellular ATP by changes in intracellular calcium.20,24,26 The biological effects of adenine nucleotides on astroglial cells have been extensively investigated, but so far little is known about the role of transcription factors in the actions of ATP. In this study, stimulation of some P2Y-purinoceptors was shown to induce IEG expression in cultured astrocytes. Moreover, the purine analogue ADPâS was found to potentiate the effect of CGRP on IEG
ADPâS and CGRP effects on cultured astrocytes
523
Fig. 1. Time-course of immediate early gene induction by ADPßS (A) and PMA (B) in cultured astrocytes. Cells were treated with 1 µM ADPßS or 100 nM PMA for the times indicated and the RNA extracted was analysed as described under ‘‘Experimental Procedures’’. The autoradiograms shown represent one of 10 to 13 separate experiments performed in duplicate. The lower band of the c-fos mRNA signal represents the synthetic fos sense-transcript added as a recovery standard. ADPßS significantly induced c-fos (five-fold; P<0.001), junB (two-fold; P<0.001), c-jun (1.5-fold; P<0.05) and Tis11 (two-fold; P<0.05) mRNA. PMA also led to a significant increase in c-fos (22-fold; P<0.001), junB (nine-fold; P<0.001), c-jun (four-fold; P<0.05) and Tis11 (13-fold; P<0.001) mRNA.
mRNA expression in cultured astroglia. The IEGs Fos and Jun encode regulatory proteins that control the expression of target genes involved in adaptive responses in the nervous system.27 Tis genes, first identified in Swiss 3T3 cells following exposure to phorbol ester, also show characteristic features of IEGs in cultured astrocytes.1,15 A rapid and transient expression of the mRNAs of AP-1 transcription factor components (c-fos, junB, c-jun) and Tis11 was observed in astrocytes in response to stimulation with the non-hydrolysable P2Y1-agonist, ADPâS. The pattern of induction was identical to that observed in microglial cells,21 suggesting that neuroglia might show a rather uniform transcriptional response to extracellular ATP; P2-
purinoceptors have been described on the majority of glial cells, including Schwann cells, oligodendrocytes and enteric glia.2,11,13 Since the pattern of IEG induction by ADPâS in cultured astrocytes qualitatively resembled that of PMA, it is conceivable that protein kinase C (PKC) might play a role in purinergic stimulation of gene expression in astrocytes. PKC has also been found to mediate the uncoupling of astroglial gap junctions in response to P2Y-agonists.5 The fact that PMA led to a more dramatic and prolonged induction of IEG mRNA compared with ADPâS, might be explained by its direct, intracellular site of action as opposed to the receptor-mediated effects of ADPâS. Alternatively, phorbol esters might simply be more stable in cell culture than ATP
524
J. Priller et al.
Fig. 2. Effect of the combination of CGRP and ADPâS on c-fos mRNA expression in cultured astrocytes. Cells were treated with 1 µM CGRP, 1 µM ADPâS and CGRP plus ADPâS for 30 min. Immediate early gene expression was analysed as described under ‘‘Experimental Procedures’’. The autoradiogram shown is representative of three separate experiments performed in triplicate and two experiments performed in duplicate. The combination of CGRP and ADPâS led to an overadditive expression of c-fos mRNA. Table 1. Quantitative evaluation of the effect of agonist combination on c-fos messenger RNA expression Treatment 1 µM CGRP 1 µM ADPâS 1 µM CGRP+1 µM ADPâS
c-fos mRNA (fold stimulation) 2.090.17 5.020.63 9.531.27*
Values are meansS.E.M. (expressed as increase in c-fos mRNA relative to controls) of five separate experiments (see Fig. 2). The stimulation of astrocytes with CGRP plus ADPâS led to a potentiated expression of c-fos mRNA (combination vs additive effect of the agonists: *P<0.05).
analogues. In addition, ADPâS increases intracellular calcium in astrocytes19 which could potentially interfere with the expression of particular IEGs. In fact, Trejo and Brown have found that calcium represses c-jun expression through increased degradation of c-jun mRNA in astrocytoma cells.28 This study raises the possibility that neuropeptide receptors are co-expressed with P2-purinoceptors on cultured astrocytes since ADPâS and CGRP were found to have synergistic effects on second messenger activation and IEG expression in astroglial cultures. A similar synergism has been described for ADPâS and fibroblast growth factors with respect to DNA synthesis in astrocytes.18 The precise nature of the second messenger(s) mediating purinergic potentiation of CGRP effects in astroglia remains to be determined. The strong potentiation of CGRPevoked cyclic AMP accumulation by PMA suggests a role for PKC for this cross-talk. In fact, PKC has been shown to potentiate type II and V adenylate cyclase activities in the presence of ás or via direct phosphorylation.10,16 However, elevated intracellular calcium levels have also been reported to stimulate some types of adenylate cyclase,30 and metabotropic P2-purinoceptors are known to mobilize both PKC and intracellular calcium. Interestingly, pretreatment
Fig. 3. Synergistic actions of ADPâS and PMA on CGRPinduced cyclic AMP accumulation. Cells were incubated in 0.3 mM RO 20-1724 for 30 min, followed by the addition of increasing concentrations of CGRP in the absence or presence of 1 µM ADPâS or 0.1 µM PMA. After 25 min, cyclic AMP was extracted and determined by radioimmunoassay. The results are means of triplicate determinationsS.E.M. and the data are representative of those from three experiments. ADPâS increased CGRP-induced cyclic AMP accumulation by up to 35% (combination vs additive effect at 1 µM CGRP: P<0.05). PMA potentiated CGRP-evoked cyclic AMP accumulation by 80% when combined with 1 µM CGRP (P<0.01 compared to additive effect of the agonists). ADPâS did not affect basal cyclic AMP content, while PMA led to a slight increase.
of L-cells with ATP caused an enhancement of adenylate cyclase activity similar to that of PMA, but unaffected by prior depletion of PKC.9 It is clear from this study that astrocytes do not express the P2Y-purinoceptor subtype described by Boyer et al. on C6-2B rat glioma cells,3 which inhibits adenylate cyclase activity and has no effect on phospholipase C. CONCLUSION
The results suggest synergistic roles for extracellular ATP and CGRP in the transcriptional activation of cultured astroglial cells. It is not yet known what functional implications this might have. Preliminary experiments have indicated that IEGs other than c-fos (such as junB, c-jun and Tis11) do not respond to second messenger cross-talk by CGRP and ADPâS in astrocytes (Priller et al., unpublished observations). If the synergism of CGRP and extracellular ATP in astrocytes should lead to a selective increase in the synthesis of Fos relative to other members of the AP-1 transcription factor family, it is conceivable that the expression of target genes containing an AP-1 consensus sequence might be significantly enhanced, since Fos/Jun dimers are amongst the most potent transcriptional activators of these genes.8
ADPâS and CGRP effects on cultured astrocytes
525
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
Arenander A. T., de Vellis J. and Herschman H. R. (1989) Induction of c-fos and TIS genes in cultured rat astrocytes by neurotransmitters. J. Neurosci. Res. 24, 107–114. Berti-Mattera L. N., Wilkins P. L., Madhun Z. and Suchovsky D. (1996) P2-purinergic receptors regulate phospholipase C and adenylate cyclase activities in immortalized Schwann cells. Biochem. J. 314, 555–561. Boyer J. L., Lazarowski E. R., Chen X.-H. and Harden T. K. (1993) Identification of a P-2Y-purinergic receptor that inhibits adenylyl cyclase. J. Pharmac. exp. Ther. 267, 1140–1146. Bruner G. and Murphy S. (1993) Purinergic P2Y receptors on astrocytes are directly coupled to phospholipase A2. Glia 7, 219–224. Enkvist M. O. K. and McCarthy K. D. (1992) Activation of protein kinase C blocks astroglial gap junction communication and inhibits the spread of calcium waves. J. Neurochem. 59, 519–526. Gordon J. L. (1986) Extracellular ATP: effects, sources and fate. Biochem. J. 233, 309–319. Haas C. A., Reddington M. and Kreutzberg G. W. (1991) Calcitonin gene-related peptide stimulates the induction of c-fos gene expression in rat astrocyte cultures. Eur. J. Neurosci. 3, 708–712. Hughes P. and Dragunow M. (1995) Induction of immediate-early genes and the control of neurotransmitter-regulated gene expression within the nervous system. Pharmac. Rev. 47, 133–178. Johnson J. A., Friedman J., Halligan R. D., Birnbaumer M. and Clark R. B. (1991) Sensitization of adenylyl cyclase by P2 purinergic and M5 muscarinic receptor agonists in L cells. Molec. Pharmac. 40, 539–546. Kawabe J., Iwami G., Ebina T., Ohno S., Katada T., Ueda Y., Homcy C. J. and Ishikawa Y. (1994) Differential activation of adenylyl cyclase by protein kinase C isoenzymes. J. biol. Chem. 269, 16,554–16,558. Kimball B. C. and Mulholland M. W. (1996) Enteric glia exhibit P2U receptors that increase cytosolic calcium by a phospholipase C-dependent mechanism. J. Neurochem. 66, 604–612. King B. F., Neary J. T., Zhu Q., Wang S., Norenberg M. D. and Burnstock G. (1996) P2 purinoceptors in rat cortical astrocytes: expression, calcium-imaging and signalling studies. Neuroscience 74, 1187–1196. Kirischuk S., Scherer J., Kettenmann H. and Verkhratsky A. (1995) Activation of P2-purinoreceptors triggered Ca2+ release from InsP3-sensitive internal stores in mammalian oligodendrocytes. J. Physiol. 483, 41–57. Lazar P., Reddington M., Streit W., Raivich G. and Kreutzberg G. W. (1991) The action of calcitonin gene-related peptide on astrocyte morphology and cyclic AMP accumulation in astrocyte cultures from neonatal rat brain. Neurosci. Lett. 130, 99–102. Lim R. W., Varnum B. C. and Herschman H. (1987) Cloning of sequences induced as a primary response following mitogen treatment of density arrested Swiss 3T3 cells. Oncogene 1, 263–270. Lustig K. D., Conklin B. R., Herzmark P., Taussig R. and Bourne H. R. (1993) Type II adenylylcyclase integrates coincident signals from Gs, Gi and Gq. J. biol. Chem. 268, 13,900–13,905. Neary J. T. and Zhu Q. (1994) Signaling by ATP receptors in astrocytes. NeuroReport 5, 1617–1620. Neary J. T., Whittemore S. R., Zhu Q. and Norenberg M. D. (1994) Synergistic activation of DNA synthesis in astrocytes by fibroblast growth factors and extracellular ATP. J. Neurochem. 63, 490–494. Ogata T., Nakamura Y. and Schubert P. (1996) Potentiated cAMP rise in metabotropically stimulated rat cultured astrocytes by a Ca2+-related A1/A2 adenosine receptor cooperation. Eur. J. Neurosci. 8, 1124–1131. Pearce B. and Langley D. (1994) Purine- and pyrimidine-stimulated phosphoinositide breakdown and intracellular calcium mobilisation in astrocytes. Brain Res. 660, 329–332. Priller J., Haas C. A., Reddington M. and Kreutzberg G. W. (1995) Calcitonin gene-related peptide and ATP induce immediate early gene expression in cultured rat microglial cells. Glia 15, 447–457. Raivich G., Reddington M., Haas C. A. and Kreutzberg G. W. (1995) Peptides in motoneurons. In Neuropeptides in the Spinal Cord. (eds. Nyberg F., Sharma H. S. and Wiesenfeld-Hallin Z.), Progress in Brain Research. Vol. 104, pp. 3–20. Elsevier, Amsterdam. Rosenfeld M. G., Mermod J.-J., Amara S. G., Swanson L. W., Sawchenko P. E., Rivier J., Vale W. W. and Evans R. M. (1983) Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature 304, 129–135. Salter M. W. and Hicks J. L. (1995) ATP causes release of intracellular Ca2+ via the phospholipase Câ/IP3 pathway in astrocytes from the dorsal spinal cord. J. Neurosci. 15, 2961–2971. Sen R. P., Delicado E. G., Castro E. and Miras-Portugal M. T. (1993) Effect of P2Y agonists on adenosine transport in cultured chromaffin cells. J. Neurochem. 60, 613–619. Shao Y. and McCarthy K. D. (1993) Regulation of astroglial responsiveness to neuroligands in primary culture. Neuroscience 55, 991–1001. Sheng M. and Greenberg M. E. (1990) The regulation and function of c-fos and other immediate early genes in the nervous system. Neuron 4, 477–485. Trejo J. A. and Brown J. H. (1991) c-fos and c-jun are induced by muscarinic receptor activation of protein kinase C but are differentially regulated by intracellular calcium. J. biol. Chem. 266, 7876–7882. Welford L. A., Cusack N. J. and Hourani S. M. O. (1987) The structure–activity relationships of ectonucleotidases and of excitatory P2-purinoceptors: evidence that dephosphorylation of ATP analogues reduces pharmacological potency. Eur. J. Pharmac. 141, 123–130. Yoshimura M. and Cooper D. M. F. (1992) Cloning and expression of a Ca2+-inhibitable adenylyl cyclase from NCB-20 cells. Proc. natn. Acad. Sci. U.S.A. 89, 6716–6720. (Accepted 26 November 1997)