Inducible expression of a MAP kinase phosphatase-3-GFP chimera specifically blunts fibroblast growth and ras-dependent tumor formation in nude mice
Inducible expression of a MAP kinase phosphatase-3-GFP chimera specifically blunts fibroblast growth and ras-dependent tumor formation in nude mice
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DOI：
10.1002/jcp.10465
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发表时间：
2004-06-01
影响因子：
5.6
通讯作者：
Pagès, G
中科院分区：
文献类型：
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作者：
Marchetti, S;Gimond, C;Pagès, G
p42/p44丝裂原活化蛋白激酶（MAPK）途径参与广泛的细胞程序，包括增殖、迁移、分化和存活。特异性药理学抑制剂，如PD 98059和U 01 26，通常用于抑制p42/p44 MAPK信号传导。然而，这些抑制剂不适合研究这些激酶在整个生物体中的功能。因此，我们开发了一种诱导系统，旨在通过表达对这两种激酶特异的磷酸酶MAPK磷酸酶3（MKP-3）来抑制p42/p44 MAPK活性。建立了成纤维细胞系，其中MKP-3表达受四环素控制。四环素诱导的MKP-3导致血清刺激的细胞中p42/p44 MAPK的部分去磷酸化。然而，当MKP-3的C末端与绿色荧光蛋白（GFP）融合时，我们可以提高MKP-3的稳定性，从而提高MAPK去磷酸化的速率。重要的是，GFP与MKP-3的融合没有改变磷酸酶对其MAPK底物的特异性。我们进一步表明，MKP-3-GFP在该成纤维细胞系中的条件性表达导致抑制：（a）p42/p44 MAPK底物Elk 1和HIF-1 α的磷酸化，（B）响应MAPK通路激活的血管内皮生长因子（VEGF）、细胞周期蛋白D1和c-fos基因转录，以及（c）细胞增殖。最后，用Ha-ras转化MKP-3-GFP诱导细胞系并注射到裸鼠体内。与未处理的对照组相比，用四环素类似物多西环素处理小鼠导致肿瘤出现和生长的大幅延迟，表明MKP-3-GFP活性在体内得以维持。总之，这些结果表明，MKP-3-GFP的诱导型表达构成了一个有价值的工具，研究p42/p44 MAPK在培养的细胞和动物模型中的各种细胞反应中的作用，一个工具，也可以用来阻止不需要的细胞生长的病理条件。J.细胞。199：441-450，2004。(C)2004 Wiley-Liss，Inc.
The p42/p44 mitogen activated protein kinase (MAPK) pathway participates in a wide range of cellular programs including proliferation, migration, differentiation, and survival. Specific pharmacological inhibitors, like PD98059 and U01 26, are often used to inhibit p42/p44 MAPK signaling. However, these inhibitors are not appropriate to study the function of these kinases in whole organisms. We thus developed an inducible system designed to inhibit p42/p44 MAPK activity through the expression of a phosphatase specific for these two kinases, the MAPK phosphatase 3 (MKP-3). A fibroblast cell line was established in which MKP-3 expression is controlled by tetracycline. Tetracycline-induced MKP-3 resulted in partial dephosphorylation of p42/p44 MAPKs in serum-stimulated cells. However, we could improve MKP-3 stability and thereby the rate of MAPK de-phosphorylation, when the C-tenninal end of MKP-3 was fused to the green fluorescent protein (GFP). Importantly, the fusion of GFP to MKP-3 did not alter the specificity of the phosphatase towards its MAPK substrates. We further show that conditional expression of MKP-3-GFP in this fibroblast cell line results in the inhibition of: (a) the phosphorylation of the p42/p44 MAPK substrates Elk1 and HIF-1alpha, (b) vascular endothelial growth factor (VEGF), cyclin D1, and c-fos gene transcription in response to MAPK pathway activation, and (c) cell proliferation. Finally, the MKP-3-GFP inducible cell line was transformed by Ha-ras and injected into nude mice. Treatment of mice with the tetracycline analog doxycycline resulted in a large delay in tumor emergence and growth as compared to the untreated control group, indicating that MKP-3-GFP activity is maintained in vivo. Altogether, these results show that inducible expression of MKP-3-GFP constitutes a valuable tool to study the role of p42/p44 MAPKs in various cellular responses in both cultured cell and animal models, a tool that may also be used to block unwanted cell growth in pathological conditions. J. Cell. Physiol. 199: 441-450, 2004. (C) 2004 Wiley-Liss, Inc.