Cloning of bovine preproadrenomedullin and inhibition of its basal expression in vascular endothelial cells by staurosporine.
Cloning of bovine preproadrenomedullin and inhibition of its basal expression in vascular endothelial cells by staurosporine.
复制标题
牛前肾上腺髓质素原的克隆及其在血管内皮细胞中星形孢素的基础表达抑制。
DOI：
10.1016/s0024-3205(98)00079-4
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发表时间：
1998
期刊：
影响因子：
6.1
通讯作者：
R. Corder
中科院分区：
文献类型：
--
作者：
S. Barker;E. Wood;A. Clark;R. Corder
以牛主动脉内皮细胞（BAEC）总RNA为模板，采用逆转录聚合酶链反应（RT-PCR）和5'末端快速扩增技术，克隆了编码肾上腺髓质素前体（preproAM）的cDNA。牛preproAM cDNA与人、猪和大鼠preproAM具有高度的同源性。用牛特异性引物进行RT-PCR，研究该基因的调控。用非选择性蛋白激酶抑制剂staurosporine处理BAEC或人内皮细胞系（Ea.hy 926）导致preproAM mRNA水平显著降低。当Ea.hy 926细胞暴露于100 nM星形孢菌素2 h时，preproAM mRNA的减少似乎是绝对的。然而，用蛋白激酶A（PKA）抑制剂H-89或蛋白激酶C（PKC）抑制剂氯化白屈菜红碱和双吲哚马来酰亚胺I处理不能再现这种显著降低。这些观察结果表明，一种新的星形孢菌素敏感的蛋白激酶的激活是必要的preproAM基因在这些细胞中的基础表达。
The cDNA encoding preproadrenomedullin (preproAM) was cloned using reverse transcriptase polymerase chain reaction (RT-PCR) and 5' rapid amplification of cDNA ends from total RNA from bovine aortic endothelial cells (BAEC). Bovine preproAM cDNA shows high sequence homology with human, porcine and rat preproAM. Bovine-specific primers derived from this sequence were used in RT-PCR to study regulation of this gene. Treatment of BAEC or a human endothelial cell line (Ea.hy 926) with the non-selective protein kinase inhibitor staurosporine resulted in significantly reduced preproAM mRNA levels. The reduction in preproAM mRNA appeared to be absolute when Ea.hy 926 cells were exposed to 100 nM staurosporine for 2 h. However, this dramatic reduction could not be reproduced by treatment with the protein kinase A (PKA) inhibitor H-89, or the protein kinase C (PKC) inhibitors chelerythrine chloride and bisindolylmaleimide I. These observations suggest that activation of a novel staurosporine-sensitive protein kinase is necessary for basal expression of the preproAM gene in these cells.