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054 CONSTITUTIVE ACTIVATION OF ERK1B/MAP KINASE induces TRANSFORMATION OF INTESTINAL EPITHELIAL CRYPT CELLS INDEPENDENTLY OF PHOSPHATIDYLINOSITOL 3-KINASE/AKT, COX-2 AND EGF RECEPTOR ACTIVATION V Durand, E Lemieux, N Rivard Strong evidences exist for the critical involvement of Ras/Raf/MEK/ERK cascade in the regulation of intestinal epithelial cell proliferation. K-rasV12G is the most frequently mutated oncogene in colorectal cancer and targeted expression of K-rasV12G in the intestinal epithelium causes activation of the MEK/ERK pathway and tumorigenesis in mice. Moreover, blockade of the MEK/ERK pathway suppresses growth of colon tumors in vivo. However, the mechanisms by which the MEK/ERK cascade induces intestinal epithelial cell tumorigenesis remain to be clarified.
Department of Anatomy and Cell Biology, University of Sherbrooke, Sherbrooke, Quebec
METHODS: Retrovirus encoding the HA-tagged MEK1 wild type (wtMEK) or a constitutive active mutant of MEK1 (MEK1-S218D/S222D, caMEK) were used to infect normal intestinal epithelial crypt cells IEC-6. The effects of pharmacological inhibitors of MEK1/2 (UO126, 10 uM), PI-3Kinase (LY294002, 10 uM), COX-2 (NS-398, 10 uM) and EGF receptor (PD153035, 10 uM) were examined on cell growth and morphology. Activation profiles of ERKs and Akt as well as COX-1/2 protein expression were examined by Western blotting.
RESULTS: 1) Stable expression of caMEK, but not wtMEK, in IEC-6 cells induced growth factor relaxation for DNA synthesis, enhanced migration (Boyden chambers), induced deregulation of contact inhibition cell growth, promoted morphological transformation and growth in soft agar. In addition, we found a significant up-regulation of c-myc, phosphorylated (T58,S62) c-myc and cyclin D1 expression levels. 2) Intriguingly, expression and activity of ERK1/2 and Akt remained comparable in wtMEK- and caMEK-expressing IEC-6 cells; however, the alternatively spliced isoform of ERK1, ERK1b, is constitutively and markedly activated in caMEK-expressing IEC-6 cells. 3) Interestingly, expression levels of COX1/2 were not enhanced in caMEK- versus wtMEK-expressing IEC-6 cells. 4) Pharmacological inhibition of PI-3Kinase, COX-2 or EGF receptor tyrosine kinase did not influence the transformed phenotype of caMEK-expressing IEC-6 cells. 5) By contrast, treatment of caMEK-expressing IEC-6 cells with UO126 inhibitor reduced ERK1/2 phosphorylation levels, abolished ERK1b phosphorylation levels and resulted in the reversion of the transformed phenotype of these cells.
CONCLUSION: Our results suggest that ERK1b, an alternatively spliced isoform of ERK1, contributes to the induction of transformation of intestinal epithelial cells by MEK, independently of PI-3Kinase/Akt, COX-2 and EGF receptor activation.