Sale E M, Atkinson P G, Sale G J
Department of Biochemistry, University of Southampton, UK.
EMBO J. 1995 Feb 15;14(4):674-84. doi: 10.1002/j.1460-2075.1995.tb07046.x.
A phosphorothioate-oligonucleotide-based antisense strategy for depleting MAP kinase was developed. The 17mer antisense probe, EAS 1, caused a potent and concentration-dependent decrease in the steady state expression of p42 and p44 MAP kinase in 3T3 L1 fibroblasts and adipocytes with submicromolar concentrations effective. Antisense EAS 1 elicited a dose-dependent inhibition of insulin- and serum-stimulated DNA synthesis. Elimination of p42 MAP kinase by > 95% and p44 MAP kinase to levels undetected blocked the ability of serum in 3T3 L1 fibroblasts and insulin in 3T3 L1 adipocytes to stimulate DNA synthesis by 87-95%. The differentiation of 3T3 L1 fibroblasts into adipocytes was prevented by 1 microM antisense EAS 1. The corresponding sense, scrambled or sense plus antisense EAS 1 phosphorothioate oligonucleotides did not deplete the p42 or p44 MAP kinase from either cell type, did not inhibit stimulation of DNA synthesis and did not interfere with differentiation. Two kinases on different MAP kinase activation pathways were not depleted by antisense EAS 1 whereas the ability of insulin to activate p90 S6 kinase was > 90% eliminated in 3T3 L1 adipocytes by 4.5 microM antisense EAS 1. In conclusion these results show that MAP kinase is required for insulin and serum stimulation of DNA synthesis, for insulin stimulation of p90 S6 kinase activity and for differentiation of 3T3 L1 cells. Moreover, the development of the antisense probe EAS 1 against a target sequence of p42 MAP kinase that is conserved in p44 MAP kinase and across a range of species provides a molecular tool of general applicability for further dissecting the precise targets and roles of MAP kinase.
已开发出一种基于硫代磷酸酯寡核苷酸的反义策略来消耗丝裂原活化蛋白激酶（MAP激酶）。17聚体反义探针EAS 1能使3T3 L1成纤维细胞和脂肪细胞中p42和p44 MAP激酶的稳态表达产生有效且浓度依赖性的降低，亚微摩尔浓度即可起效。反义EAS 1对胰岛素和血清刺激的DNA合成产生剂量依赖性抑制。p42 MAP激酶消除95%以上且p44 MAP激酶降至检测不到的水平时，可使3T3 L1成纤维细胞中的血清以及3T3 L1脂肪细胞中的胰岛素刺激DNA合成的能力受阻87% - 95%。1 μM反义EAS 1可阻止3T3 L1成纤维细胞向脂肪细胞的分化。相应的正义链、随机序列或正义链加反义EAS 1硫代磷酸酯寡核苷酸均不会从任何一种细胞类型中消耗p42或p44 MAP激酶，不会抑制DNA合成的刺激，也不会干扰分化。反义EAS 1不会消耗不同MAP激酶激活途径上的两种激酶，而在3T3 L1脂肪细胞中，4.5 μM反义EAS 1可使胰岛素激活p90 S6激酶的能力消除90%以上。总之，这些结果表明，MAP激酶是胰岛素和血清刺激DNA合成、胰岛素刺激p90 S6激酶活性以及3T3 L1细胞分化所必需的。此外，针对p42 MAP激酶靶序列开发的反义探针EAS 1在p44 MAP激酶中保守且在一系列物种中都存在，这为进一步剖析MAP激酶的精确靶点和作用提供了一种具有普遍适用性的分子工具。
