Alheim K, McDowell T L, Symons J A, Duff G W, Bartfai T
Department of Neurochemistry and Neurotoxicology, Stockholm University, Sweden.
Neurochem Int. 1996 Nov;29(5):487-96. doi: 10.1016/0197-0186(96)00017-4.
The nerve growth factor which induces phenotypic changes in PC12 pheochromocytoma cells also induces the expression of the proinflammatory cytokine interleukin 1 alpha in these cells. We have studied the signal transduction and transcriptional mechanisms involved in this induction of interleukin 1 alpha by nerve growth factor. The nerve growth factor induction of interleukin 1 alpha transcription in PC12 cells is exerted via the TrkA receptor, as demonstrated by inhibition of the nerve growth factor stimulated increases in the interleukin 1 alpha mRNA levels by the TrkA specific alkaloid K-252a. The promoter region(s) involved in induction of interleukin 1 alpha expression by nerve growth factor in PC12 pheochromocytoma cells were studied by deletion mutagenesis in a part of the 5' regulatory region of the human interleukin 1 alpha gene (bases -163 to +64). This promoter region was inserted into the promoterless pBLCAT3 plasmid, using the interleukin 1 alpha 5' fragment as the promoter to drive nerve growth factor inducible expression of the CAT (chloramphenicol acetyl transferase) reporter gene. Four mutants, with deletions of 9-15 bases in the 5' regulatory region of the human interleukin 1 alpha gene, were constructed: three deleted stretches correspond to regions with high sequence similarity to regions in other genes, coding for nerve growth factor-induced proteins, e.g. NGFI-A, NGFI-B, NGFI-C, ERK2 and VGF gene. These deletions, of which some reduced the basal, non-nerve growth factor stimulated expression of the CAT reporter protein, do not prevent the two- to threefold induction by nerve growth factor. The deletion which eliminated a putative AP-1 binding site, immediately upstream of the transcription start site in the interleukin 1 alpha promoter, almost completely prevented the nerve growth factor mediated induction of CAT reporter gene expression, suggesting that in PC12 cells the major site of nerve growth factor regulation of interleukin 1 alpha expression is at this AP-1 site.
诱导PC12嗜铬细胞瘤细胞发生表型变化的神经生长因子，也能诱导这些细胞中促炎细胞因子白细胞介素1α的表达。我们研究了神经生长因子诱导白细胞介素1α表达所涉及的信号转导和转录机制。神经生长因子对PC12细胞中白细胞介素1α转录的诱导作用是通过TrkA受体实现的，这一点可通过TrkA特异性生物碱K-252a抑制神经生长因子刺激的白细胞介素1α mRNA水平升高得到证明。通过对人白细胞介素1α基因5'调控区（碱基-163至+64）的一部分进行缺失诱变，研究了PC12嗜铬细胞瘤细胞中神经生长因子诱导白细胞介素1α表达所涉及的启动子区域。将该启动子区域插入无启动子的pBLCAT3质粒中，使用白细胞介素1α 5'片段作为启动子来驱动氯霉素乙酰转移酶（CAT）报告基因的神经生长因子诱导表达。构建了四个人白细胞介素1α基因5'调控区缺失9-15个碱基的突变体：三个缺失片段对应于与其他基因区域具有高度序列相似性的区域，这些基因编码神经生长因子诱导蛋白，如NGFI-A、NGFI-B、NGFI-C、ERK2和VGF基因。这些缺失，其中一些降低了CAT报告蛋白的基础的、非神经生长因子刺激的表达，但并不妨碍神经生长因子诱导的两到三倍的表达。消除白细胞介素1α启动子转录起始位点上游一个假定的AP-1结合位点的缺失，几乎完全阻止了神经生长因子介导的CAT报告基因表达的诱导，这表明在PC12细胞中，神经生长因子调节白细胞介素1α表达的主要位点就在这个AP-1位点。
