Burgos Michelle, Neary Joseph T, González Fernando A
Department of Biochemistry, Medical-Sciences Campus, University of Puerto Rico, San Juan, Puerto Rico.
J Neurochem. 2007 Dec;103(5):1785-800. doi: 10.1111/j.1471-4159.2007.04872.x. Epub 2007 Sep 14.
Nucleotides as well as other neurotransmitters are known to be released to the extracellular space upon injury. To determine whether nucleotides acting on P2Y(2) nucleotide receptors promote protective or degenerative events after trauma in astrocytic cells, a well-established model of in vitro brain trauma was applied to 1321N1 cells expressing recombinant P2Y(2) nucleotide receptors (P2Y(2)R-1321N1). Cellular death was examined by measuring DNA fragmentation and caspase activation. Fragmented DNA was observed 48 h post-injury in 1321N1 cells, while P2Y(2) nucleotide receptor expressing cells did not show DNA fragmentation. A laddering pattern of fragmented DNA following injury was observed upon inhibition of P2Y(2) nucleotide receptors with suramin. Time-dependent increases of cleaved caspase-9, a mitochondrial-associated caspase, correlated with injury-induced cellular death. A decreased bax/bcl-2 gene expression ratio was observed in P2Y(2)R-1321N1 cells after traumatic injury, while untransfected 1321N1 cells showed a significant time-dependent increase of the bax/bcl-2 gene expression ratio. Activation of protein kinases was assessed to determine the signaling pathways involved in cell death and survival responses following traumatic injury. In P2Y(2)R-1321N1 and 1321N1 cells p38 phosphorylation was stimulated in a time-dependent manner but the phosphatidylinositol 3-kinase-dependent activation of extracellular signal-regulated kinase 1/2 and protein kinase B (PKB)/Akt was only observed in P2Y(2)R-1321N1 cells after injury. The stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) signaling pathway was not activated by traumatic injury in either astrocytic cell line. Inhibition of p38 kinase signaling pathway by treatment with PD1693, a MKK3/6 inhibitor, abolished the expression of cleaved caspase-9, the increase in the bax/bcl-2 gene expression ratio, as well as the fragmentation of DNA that followed injury of 1321N1 cells. Taken together, our results demonstrate a novel role for P2Y(2) nucleotide receptors and extracellular nucleotides in mediating survival responses to glial cells undergoing cellular death induced by trauma.
已知核苷酸以及其他神经递质在损伤后会释放到细胞外空间。为了确定作用于P2Y(2)核苷酸受体的核苷酸在星形胶质细胞创伤后是促进保护性事件还是退行性事件，将一种成熟的体外脑创伤模型应用于表达重组P2Y(2)核苷酸受体的1321N1细胞（P2Y(2)R - 1321N1）。通过测量DNA片段化和半胱天冬酶激活来检测细胞死亡。在1321N1细胞损伤后48小时观察到DNA片段化，而表达P2Y(2)核苷酸受体的细胞未显示DNA片段化。用苏拉明抑制P2Y(2)核苷酸受体后，观察到损伤后DNA片段化的梯状模式。裂解的半胱天冬酶 - 9（一种与线粒体相关的半胱天冬酶）的时间依赖性增加与损伤诱导的细胞死亡相关。创伤性损伤后，P2Y(2)R - 1321N1细胞中bax/bcl - 2基因表达比率降低，而未转染的1321N1细胞中bax/bcl - 2基因表达比率呈显著的时间依赖性增加。评估蛋白激酶的激活以确定创伤性损伤后参与细胞死亡和存活反应的信号通路。在P2Y(2)R - 1321N1和1321N1细胞中，p38磷酸化以时间依赖性方式被刺激，但细胞外信号调节激酶1/2和蛋白激酶B（PKB）/Akt的磷脂酰肌醇3 - 激酶依赖性激活仅在损伤后的P2Y(2)R - 1321N1细胞中观察到。在两种星形胶质细胞系中，创伤性损伤均未激活应激激活蛋白激酶/c - Jun NH2末端激酶（SAPK/JNK）信号通路。用MKK3/6抑制剂PD1693处理抑制p38激酶信号通路后，消除了裂解的半胱天冬酶 - 9的表达、bax/bcl - 2基因表达比率的增加以及1321N1细胞损伤后随之而来的DNA片段化。综上所述，我们的结果表明P2Y(2)核苷酸受体和细胞外核苷酸在介导对经历创伤诱导细胞死亡的神经胶质细胞的存活反应中具有新作用。
