Welsh N
Department of Medical Cell Biology, Uppsala University, Biomedicum, P. O. Box 571, S-751 23 Uppsala, Sweden.
J Biol Chem. 1996 Apr 5;271(14):8307-12. doi: 10.1074/jbc.271.14.8307.
The aim of this investigation was to study the putative involvement of lipid second messengers, protein kinases, and transcription factors in interleukin-1 beta (IL-1beta)-induced signal transduction in insulin-producing cells. For this purpose, insulin-producing RINm5F cells were exposed to IL-1beta (25 units/ml), and the ceramide, ceramide 1-phosphate, sphingomyelin, diacylglycerol, and phosphatidic acid contents of the cells were subsequently determined. It was found that IL-1beta induced a transient increase (2-5 min) in ceramide and diacylglycerol, which was not paralleled by an increase in ceramide 1-phosphate and phosphatidic acid. A rapid decrease in the sphingomyelin content of the cells was, however, observed. The cell-permeable ceramide analogue N-acetylsphingosine and the phorbol ester phorbol 12-myristate 13-acetate (PMA) both induced the phosphorylation and increased the activities of the protein kinase JNK1 and the transcription factor ATF2. These effects were, however, not as pronounced as those induced by IL-1beta. The DNA binding activity of transcription factors in nuclear extracts was determined using the electrophoretic mobility shift assay method. Transcription factor binding to the ATF/cAMP-responsive element consensus sequence was increased 4-5-fold by acetylsphingosine, PMA, or IL-1beta, whereas binding to the CCAAT/enhancer-binding protein and AP-1 elements was found to be only slightly stimulated by these three agents. Binding to the NF-kappaB element was strongly induced by IL-1beta, but not by acetylsphingosine or PMA. Finally, acetylsphingosine and PMA did not mimic the nitric oxide-inducing effects of IL-1beta. It is concluded that IL-1beta-stimulated formation of ceramide and diacylglycerol may contribute to JNK1 and ATF2 transcription factor activation, which may be a necessary (but not sufficient) step in beta-cell nitric-oxide synthase induction.
本研究的目的是探讨脂质第二信使、蛋白激酶和转录因子在白细胞介素-1β（IL-1β）诱导的胰岛素生成细胞信号转导中的潜在作用。为此，将胰岛素生成的RINm5F细胞暴露于IL-1β（25单位/毫升），随后测定细胞中神经酰胺、神经酰胺1-磷酸、鞘磷脂、二酰基甘油和磷脂酸的含量。结果发现，IL-1β诱导神经酰胺和二酰基甘油短暂增加（2-5分钟），而神经酰胺1-磷酸和磷脂酸并未随之增加。然而，观察到细胞中鞘磷脂含量迅速下降。细胞可渗透的神经酰胺类似物N-乙酰鞘氨醇和佛波酯佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）均诱导蛋白激酶JNK1和转录因子ATF2磷酸化并增加其活性。然而，这些作用不如IL-1β诱导的作用明显。使用电泳迁移率变动分析方法测定核提取物中转录因子的DNA结合活性。乙酰鞘氨醇、PMA或IL-1β使转录因子与ATF/cAMP反应元件共有序列的结合增加4-5倍，而这三种试剂对与CCAAT/增强子结合蛋白和AP-1元件的结合仅略有刺激。IL-1β强烈诱导与NF-κB元件的结合，但乙酰鞘氨醇或PMA则无此作用。最后，乙酰鞘氨醇和PMA不能模拟IL-1β诱导一氧化氮的作用。结论是，IL-1β刺激的神经酰胺和二酰基甘油形成可能有助于JNK1和ATF2转录因子激活，这可能是β细胞一氧化氮合酶诱导过程中的一个必要（但不充分）步骤。
