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

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‐1α, (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. © 2004 Wiley‐Liss, Inc.