Suzuki Tomohisa, Hide Izumi, Ido Katsutoshi, Kohsaka Shinichi, Inoue Kazuhide, Nakata Yoshihiro
Department of Pharmacology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan.
J Neurosci. 2004 Jan 7;24(1):1-7. doi: 10.1523/JNEUROSCI.3792-03.2004.
After a brain insult, ATP is released from injured cells and activates microglia. The microglia that are activated in this way then release a range of bioactive substances, one of which is tumor necrosis factor (TNF). The release of TNF appears to be dependent on the P2X7 receptor. The inhibitors 1,4-diamino-2,3-dicyano-1,4-bis[2-amino-phenylthio]butadiene (U0126), anthra[1,9-cd]pyrazol-6(2H)-one (SP600125), and 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)IH-imidazole (SB203580), which target MEK (mitogen-activated protein kinase kinase), JNK (c-Jun N-terminal kinase), and p38, respectively, all potently suppress the production of TNF in ATP-stimulated microglia, whereas the production of TNF mRNA is strongly inhibited by U0126 and SP600125. SB203580 did not affect the increased levels of TNF mRNA but did prevent TNF mRNA from accumulating in the cytoplasm. The ATP-provoked activation of JNK and p38 [but not extracellular signal-regulated kinase (ERK)] could be inhibited by brilliant blue G, a P2X7 receptor blocker, and by genistein and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, which are general and src-family-specific tyrosine kinase inhibitors, respectively. Most important, we found that treatment of the microglia in neuron-microglia cocultures with the P2X7 agonist 2'-3'-O-(benzoyl-benzoyl) ATP led to significant reductions in glutamate-induced neuronal cell death, and that either TNF-alpha converting enzyme inhibitor or anti-TNF readily suppressed the protective effect implied by this result. Together, these findings indicate that both ERK and JNK are involved in the regulation of TNF mRNA expression, that p38 is involved in the nucleocytoplasmic transport of TNF mRNA, and that a PTK (protein tyrosine kinase), possibly a member of the src family, acts downstream of the P2X7 receptor to activate JNK and p38. Finally, our data suggest that P2X7 receptor-activated microglia protect neurons against glutamate toxicity primarily because they are able to release TNF.
脑损伤后，三磷酸腺苷（ATP）从受损细胞中释放出来并激活小胶质细胞。以这种方式被激活的小胶质细胞随后释放一系列生物活性物质，其中之一是肿瘤坏死因子（TNF）。TNF的释放似乎依赖于P2X7受体。分别靶向丝裂原活化蛋白激酶激酶（MEK）、c-Jun氨基末端激酶（JNK）和p38的抑制剂1,4-二氨基-2,3-二氰基-1,4-双[2-氨基-苯硫基]丁二烯（U0126）、蒽[1,9-cd]吡唑-6(2H)-酮（SP600125）和4-(4-氟苯基)-2-(4-甲亚磺酰基苯基)-5-(4-吡啶基)1H-咪唑（SB203580），均能有效抑制ATP刺激的小胶质细胞中TNF的产生，而U0126和SP600125强烈抑制TNF mRNA的产生。SB203580不影响TNF mRNA水平的升高，但可阻止TNF mRNA在细胞质中积累。P2X7受体阻滞剂亮蓝G以及分别作为一般酪氨酸激酶抑制剂和src家族特异性酪氨酸激酶抑制剂的染料木黄酮和4-氨基-5-(4-氯苯基)-7-(叔丁基)吡唑并[3,4-d]嘧啶，可以抑制ATP引发的JNK和p38（而非细胞外信号调节激酶（ERK））的激活。最重要的是，我们发现用P2X7激动剂2'-3'-O-(苯甲酰-苯甲酰)ATP处理神经元-小胶质细胞共培养物中的小胶质细胞，可显著减少谷氨酸诱导的神经元细胞死亡，并且肿瘤坏死因子α转换酶抑制剂或抗TNF均可轻易抑制该结果所暗示的保护作用。总之，这些发现表明ERK和JNK均参与TNF mRNA表达的调节，p38参与TNF mRNA的核质转运，并且一种蛋白酪氨酸激酶（PTK），可能是src家族的成员，在P2X7受体下游发挥作用以激活JNK和p38。最后，我们的数据表明P2X7受体激活的小胶质细胞主要通过释放TNF来保护神经元免受谷氨酸毒性。
