Inducible expression of a MAP kinase phosphatase-3-GFP chimera specifically blunts fibroblast growth and ras-dependent tumor formation in nude mice

Abstract

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 U0126, 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 de-phosphorylation 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-terminal 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.