Carbachol-mediated activation of type M(3) muscarinic acetylcholine receptors induces the biosynthesis of the transcription factor Egr-1 in human SH-SY5Y neuroblastoma cells involving an activation of extracellular signal-regulated protein kinase. Carbachol triggered the phosphorylation of the ternary complex factor Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, and strikingly enhanced the transcriptional activation potential of Elk-1. Chromatin immunoprecipitation experiments revealed that Elk-1 binds in vivo to the 5'-upstream region of the Egr-1 gene in carbachol-stimulated neuroblastoma cells. Together, these data indicate that Elk-1 connects the intracellular signaling cascade elicited by activation of M(3) muscarinic acetylcholine receptors with the transcription of the Egr-1 gene. Lentiviral-mediated expression of either MAP kinase phosphatase-1 (MKP-1) or a constitutively active mutant of calcineurin A inhibited Egr-1 biosynthesis following carbachol stimulation, indicating that these phosphatases function as shut-off devices of muscarinic acetylcholine receptor signaling. Additionally, carbachol stimulation increased transcription of a chromatin-embedded collagenase promoter/reporter gene, showing that AP-1 activity is enhanced in carbachol-stimulated neuroblastoma. Expression experiments revealed that both MKP-1 and a constitutively active mutant of calcineurin A impaired carbachol-induced upregulation of AP-1 activity. The fact that carbachol stimulation of neuroblastoma cells activates the transcription factors Egr-1 and AP-1 suggests that changes in the gene expression pattern are an integral part of muscarinic acetylcholine receptor signaling.

译文

:Carbachol介导的M(3)型毒蕈碱乙酰胆碱受体的激活诱导人SH-SY5Y神经母细胞瘤细胞中转录因子Egr-1的生物合成,涉及细胞外信号调节蛋白激酶的激活。卡巴胆碱触发三元复合因子Elk-1的磷酸化,这是血清反应元件驱动的基因转录的关键转录调节因子,并显着增强了Elk-1的转录激活潜能。染色质免疫沉淀实验表明,Elk-1在体内与卡巴胆碱刺激的神经母细胞瘤细胞中Egr-1基因的5'上游区域结合。在一起,这些数据表明Elk-1连接通过Egr-1基因的转录激活M(3)毒蕈碱乙酰胆碱受体引起的细胞内信号传导级联。 MAP激酶磷酸酶-1(MKP-1)或钙调神经磷酸酶A的组成型活性突变体的慢病毒介导的表达在卡巴胆碱刺激后抑制了Egr-1的生物合成,表明这些磷酸酶起着毒蕈碱性乙酰胆碱受体信号转导的关闭装置的作用。此外,卡巴胆碱刺激增加了染色质嵌入的胶原酶启动子/报告基因的转录,表明AP-1活性在卡巴胆碱刺激的神经母细胞瘤中得到增强。表达实验表明,MKP-1和钙调神经磷酸酶A的组成型活性突变体均会破坏卡巴胆碱诱导的AP-1活性上调。卡巴胆碱刺激神经母细胞瘤细胞激活转录因子Egr-1和AP-1的事实表明,基因表达模式的改变是毒蕈碱型乙酰胆碱受体信号传导的组成部分。

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