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Neurosci Bull. 2008 August; 24(4): 231–243.
Published online 2008 August 2. doi:  10.1007/s12264-008-0430-x
PMCID: PMC5552591

Language: English | Chinese

Effects of P2Y1 receptor on glial fibrillary acidic protein and glial cell line-derived neurotrophic factor production of astrocytes under ischemic condition and the related signaling pathways

P2Y1受体对缺血状态下星形胶质细胞产生胶质原纤维酸性蛋白与胶质细胞源性神经营养因子的影响及相关信号通路

Abstract

Objective

The present study aimed to explore the role of P2Y1 receptor in glial fibrillary acidic protein (GFAP) production and glial cell line-derived neurotrophic factor (GDNF) secretion of astrocytes under ischemic insult and the related signaling pathways.

Methods

Using transient right middle cerebral artery occlusion (tMCAO) and oxygen-glucose-serum deprivation for 2 h as the model of ischemic injury in vivo and in vitro, immunofluorescence, quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, enzyme linked immunosorbent assay (ELISA) were used to investigate location of P2Y1 receptor and GDNF, the expression of GFAP and GDNF, and the changes of signaling molecules.

Results

Blockage of P2Y1 receptor with the selective antagonist N6-methyl-2′-deoxyadenosine 3′,5′-bisphosphate diammonium (MRS2179) reduced GFAP production and increased GDNF production in the antagonist group as compared with simple ischemic group both in vivo and in vitro. Oxygen-glucose-serum deprivation and blockage of P2Y1 receptor caused elevation of phosphorylated Akt and cAMP response element binding protein (CREB), and reduction of phosphorylated Janus kinase2 (JAK2) and signal transducer and activator of transcription3 (STAT3, Ser727). After blockage of P2Y1 receptor and deprivation of oxygen-glucose-serum, AG490 (inhibitor of JAK2) reduced phosphorylation of STAT3 (Ser727) as well as expression of GFAP; LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), decreased phosphorylation of Akt and CREB; the inhibitor of mitogen-activated protein kinase kinase1/2 (MEK1/2) U0126, an important molecule of Ras/extracellular signal-regulated kinase (ERK) signaling pathway, decreased the phosphorylation of JAK2, STAT3 (Ser727), Akt and CREB.

Conclusion

These results suggest that P2Y1 receptor plays a role in the production of GFAP and GDNF in astrocytes under transient ischemic condition and the related signaling pathways may be JAK2/STAT3 and PI3-K/Akt/CREB, respectively, and that crosstalk probably exists between them.

Keywords: P2Y1 receptor, gliosis, glial fibrillary acidic protein, glial cell line-derived neurotrophic factor, PI3-K/Akt/CREB, JAK2/STAT3, Ras/ERK

摘要

目的

研究P2Y1受体对缺血时星形胶质细胞产生胶质原纤维酸性蛋白(glial fibrillary acidic protein, GFAP)及胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor, GDNF)的影响及其相关信号通路。

方法

分别利用右侧大脑中动脉线拴阻塞及培养细胞缺氧无营养后恢复正常培养, 造成体内、 外缺血再灌注模型。 用免疫荧光标记、 实时定量RT-PCR、 Western blotting、 酶联免疫吸附试验观察P2Y1受体、 GDNF定位, 检测GFAP、 GDNF及信号分子的表达变化。

结果

与单纯性缺血组比较, 用选择性拮抗剂MRS2179阻断P2Y1受体后, 可使体内、 外星形胶质细胞产生的GFAP减少, 同时使其产生GDNF增加。 体外缺氧无营养并阻断P2Y1受体后: 可使磷酸化蛋白激酶B(Akt)及cAMP反应元件结合蛋白(cAMP response element binding protein, CREB)升高, 而使磷酸化JAK2及STAT3(Ser727)降低; JAK2的抑制剂AG490在降低磷酸化STAT3(Ser727)的同时也降低GFAP表达水平; PI3-K的抑制剂LY294002可降低磷酸化的Akt及CREB; MEK1/2抑制剂U0126可同时降低磷酸化的JAK2、STAT3 (Ser727)、 Akt及CREB。

结论

P2Y1受体参与短时性缺血时星形胶质细胞GFAP及GDNF的产生过程, 相关信号途径分别为JAK2/STAT3和PI3-K/AKT/CREB, 并且两条途径存在串话。

关键词: P2Y1受体, 胶质化, 胶质原纤维酸性蛋白, 胶质细胞源性神经营养因子, PI3-K/Akt/CREB, JAK2/STAT3, Ras/ERK

References

[1] Norenberg M.D. Astrocyte responses to CNS injury. J Neuropathol Exp Neurol. 1994;53:213–220. doi: 10.1097/00005072-199405000-00001. [PubMed] [Cross Ref]
[2] Hwang I.K., Yoo K.Y., Kim D.W., Lee B.H., Kang T.C., Choi S.Y., et al. Ischemia-related changes of glial-derived neurotrophic factor and phosphatidylinositol 3-kinase in the hippocampus: Their possible correlation in astrocytes. Brain Res. 2006;1072:215–223. doi: 10.1016/j.brainres.2005.12.012. [PubMed] [Cross Ref]
[3] Airaksinen M.S., Saarma M. The GDNF family: signaling, biological functions and therapeutic value. Nat Rev Neurosci. 2002;3:383–394. doi: 10.1038/nrn812. [PubMed] [Cross Ref]
[4] Burnstock G. Purinergic nerves. Pharmacol Rev. 1972;24:509–581. [PubMed]
[5] Burnstock G. Do some nerve cells release more than one transmitter? Neuroscience. 1976;1:239–248. doi: 10.1016/0306-4522(76)90054-3. [PubMed] [Cross Ref]
[6] Burnstock G. A basis for distinguishing two types of purinergic receptor. In: Bolis L., Straub R.W., editors. Cell Membrane Receptors for Drugs and Hormones. New York: Raven Press; 1978. pp. 107–118.
[7] Washburn K.B., Neary J.T. P2 purinergic receptors signal to stat3 in astrocytes: difference in STAT3 responses to P2Y and P2X receptor activation. Neuroscience. 2006;142:411–423. doi: 10.1016/j.neuroscience.2006.06.034. [PubMed] [Cross Ref]
[8] Koyama Y., Egawa H., Osakada M., Baba A., Matsuda T. Increased by FK960, a novel cognitive enhancer, in glial cell line-derived neurotrophic factor production in cultured rat astrocytes. Biochem Pharmacol. 2004;68:275–282. doi: 10.1016/j.bcp.2004.03.023. [PubMed] [Cross Ref]
[9] Porter N.M., Thibault O., Thibault V., Chen K.C., Landfield P.W. Calcium channel density and hippocampal cell death with age in long-term culture. J Neurosci. 1997;17:5629–5639. [PubMed]
[10] Liu Y., Lu J.B., Chen Q., Ye Z.R. Involvement of MAPK/ERK kinase-ERK pathway in exogenous bFGF induced Egr-1 binding activity enhancement in anoxia-reoxygenation injured astrocytes. Neurosci Bull. 2007;23:221–228. doi: 10.1007/s12264-007-0033-y. [PMC free article] [PubMed] [Cross Ref]
[11] Paxinos G., Watson C. The Rat Brain in Stereotactic Coordinates. New York: Academic Press; 1982.
[12] Longa E.Z., Weinstein P.R., Carlson S., Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke. 1989;20:84–91. [PubMed]
[13] Sriram K., Benkovic S.A., Hebert M.A., Miller D.B., O’Callaghan J.P. Induction of gp130-related cytokines and activation of JAK2/STAT3 pathway in astrocytes precedes up-regulation of glial fibrillary acidic protein in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of neurodegeneration. J Biol Chem. 2004;279:19936–19947. doi: 10.1074/jbc.M309304200. [PubMed] [Cross Ref]
[14] Rudolphi K.A., Schubert P. Adenosine and brain ischemia. In: Belardinelli L., Pelleg A., editors. Adenosine and Adenine Nucleotides: from Molecular Biology to Integrative Physiology. Norwell: Kluwer Academic Publishers; 1995. pp. 391–397.
[15] Ralevic V., Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev. 1998;50:413–492. [PubMed]
[16] Barnard E.A., Simon J., Webb T.E. Nucleotide receptors in the nervous system. An abundant component using diverse transduction mechanisms. Mol Neurobiol. 1997;15:103–129. doi: 10.1007/BF02740631. [PubMed] [Cross Ref]
[17] Webb T.E., Simon J., Krishek B.J., Bateson A.N., Smart T.G., King B.F., et al. Cloning and functional expression of a brain G-proteincoupled ATP receptor. FEBS Lett. 1993;324:219–225. doi: 10.1016/0014-5793(93)81397-I. [PubMed] [Cross Ref]
[18] Tokuyama Y., Hara M., Jones E.M., Fan Z., Bell G.I. Cloning of rat and mouse P2Y purinoceptors. Biochem Biophys Res Commun. 1995;211:211–218. doi: 10.1006/bbrc.1995.1798. [PubMed] [Cross Ref]
[19] Ayyanathan K., Webb T.E., Sandhu A.K., Athwal R.S., Banard E.A., Kunapuli S.P. Cloning and chromosomal localization of the human P2Y1 purinoceptor. Biochem Biophys Res Commun. 1996;218:783–788. doi: 10.1006/bbrc.1996.0139. [PubMed] [Cross Ref]
[20] Raivich G., Bohatschek M., Kloss C.U., Werner A., Jones L.L., Kreutzberg G.W. Neuroglial activation repertoire in the injured brain: graded response, molecular mechanisms and cues to physiological function. Brain Res Rev. 1999;30:77–105. doi: 10.1016/S0165-0173(99)00007-7. [PubMed] [Cross Ref]
[21] Eddleston M., Mucke L. Molecular profile of reactive astrocytes—implications for their role in neurologic disease. Nuroscience. 1993;54:15–36. doi: 10.1016/0306-4522(93)90380-X. [PubMed] [Cross Ref]
[22] McKeon R.J., Jurynec M.J., Buck C.R. The chondroitin sulfate proteoglycans neurocan and phosphacan are expressed by reactive astrocytes in the chronic CNS glial scar. J Neurosci. 1999;19:10778–10788. [PubMed]
[23] Franke H., Krügel U., Schmidt R., Grosche J., Reichenbach A., Illes P. P2 receptor-types involved in astrogliosis in vivo. Br J Pharmacol. 2001;134:1180–1189. doi: 10.1038/sj.bjp.0704353. [PMC free article] [PubMed] [Cross Ref]
[24] Franke H., Krügel U., Grosche J., Heine C., Härtig W., Allgaier C., et al. P2Y receptor expression on astrocytes in the nucleus accumbens of rats. Neuroscience. 2004;127:431–441. doi: 10.1016/j.neuroscience.2004.05.003. [PubMed] [Cross Ref]
[25] Neary J.T., Kang Y., Willoughby K.A., Ellis E.F. Activation of extracellular signal-regulated kinase by stretch-induced injury in astrocytes involves extracellular ATP and P2 purinergic receptors. J Neurosci. 2003;23:2348–2356. [PubMed]
[26] Czajkowski R., Banachewicz W., Ilnytska O., Drobot L.B., Barañska J. Differential effects of P2Y1 and P2Y12 nucleotide receptors on ERK1/ERK2 and phosphatidylinositol 3-kinase signalling and cell proliferation in serum-deprived and nonstarved glioma C6 cells. Br J Pharmacol. 2004;141:497–507. doi: 10.1038/sj.bjp.0705639. [PMC free article] [PubMed] [Cross Ref]
[27] Justicia C., Gabriel C., Planas A.M. Activation of the JAK/STAT pathway following transient focal cerebral ischemia: signaling through Jak1 and Stat3 in astrocytes. Glia. 2000;30:253–270. doi: 10.1002/(SICI)1098-1136(200005)30:3<253::AID-GLIA5>3.0.CO;2-O. [PubMed] [Cross Ref]
[28] Wang Y., Smith S.B., Ogilvie J.M., McCool D.J., Sarthy V. Ciliary neurotrophic factor induces glial fibrillary acidic protein in retinal Müller cells through the JAK/STAT signal transduction pathway. Curr Eye Res. 2002;24:305–312. doi: 10.1076/ceyr.24.4.305.8408. [PubMed] [Cross Ref]
[29] Na Y.J., Jin J.K., Kim J.I., Choi E.K., Carp R.I., Kim Y.S. JAK-STAT signaling pathway mediates astrogliosis in brains of scrapie-infected mice. J Neurochem. 2007;103:637–649. doi: 10.1111/j.1471-4159.2007.04769.x. [PubMed] [Cross Ref]
[30] Lin L.F., Doherty D.H., Lile J.D., Bektesh S., Collins F. GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science. 1993;260:1130–1132. doi: 10.1126/science.8493557. [PubMed] [Cross Ref]
[31] Lin L.F., Zhang T.J., Collins F., Armes L.G. Purification and initial characterization of rat B49 glial cell line-derived neurotrophic factor. J Neurochem. 1994;63:758–768. doi: 10.1046/j.1471-4159.1994.63020758.x. [PubMed] [Cross Ref]
[32] Parsadanian A., Pan Y., Li W., Myckatyn T.M., Brakefield D. Astrocyte-derived transgene GDNF promotes complete and long-term survival of adult facial motoneurons following avulsion and differentially regulates the expression of transcription factors of AP-1 and ATF/CREB families. Exp Neurol. 2006;200:26–37. [PubMed]
[33] Movaghar V.R., Yan H.Q., Li Y., Ma X., Akbarian F., Dixon C.E. Increased expression of glial cell line-derived neurotrophic factor in rat brain after traumatic brain injury. Acta Medica iranica. 2005;43:7–10.
[34] Miyazaki H., Nagashima K., Okuma Y., Nomura Y. Expression of glial cell line-derived neurotrophic factor induced by transient forebrain ischemia in rats. Brain Res. 2001;922:165–117. doi: 10.1016/S0006-8993(01)03013-X. [PubMed] [Cross Ref]
[35] Hayashi H., Ichihara M., Iwashita T., Murakami H., Shimono Y., Kawai K., et al. Characterization of intracellular signals via tyrosine 1062 in RET activated by glial cell line-derived neurotrophic factor. Oncogene. 2000;19:4469–4475. doi: 10.1038/sj.onc.1203799. [PubMed] [Cross Ref]
[36] Takahashi M. The GDNF/RET signaling pathway and human diseases. Cytokine Growth Factor Rev. 2001;12:361–373. doi: 10.1016/S1359-6101(01)00012-0. [PubMed] [Cross Ref]
[37] Ichihara M., Murakumo Y., Takahashi M. RET and neuroendocrine tumors. Cancer Lett. 2004;204:197–211. doi: 10.1016/S0304-3835(03)00456-7. [PubMed] [Cross Ref]
[38] Cen X., Nitta A., Ohya S., Zhao Y., Ozawa N., Mouri A., et al. An analog of a dipeptide-like structure of FK506 increases glial cell line-derived neurotrophic factor expression through cAMP response element-binding protein activated by heat shock protein 90/Akt signaling pathway. J Neurosci. 2006;26:3335–3344. doi: 10.1523/JNEUROSCI.5010-05.2006. [PubMed] [Cross Ref]
[39] Yu H., Oh-Hashi K., Tanaka T., Sai A., Inoue M., Hirata Y., et al. Rehmannia glutinosa induces glial cell line-derived neurotrophic factor gene expression in astroglial cells via cPKC and ERK1/2 pathways independently. Pharmacol Res. 2006;54:39–45. doi: 10.1016/j.phrs.2006.01.014. [PubMed] [Cross Ref]

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