Mutant Erk1 Overexpression Attenuates Growth Factor Induced Proliferation in Rabbit Corneal Epithelial Cells

Abstract

Abstract: : Purpose: The mitogenic response to epidermal growth factor (EGF) in many different cell types is dependent on activation of the ERK limb of the mitogen activated protein kinase (MAPK) cascade. However, this determination is based on the use of modulators whose selectivity may be open to question. Given this uncertainty, it is conceivable that other signaling pathways could also be involved in mediating this response. To help deal with this issue, we created a tetracycline-inducible (tet-on) system (Invitrogen) in SV40-immortalized rabbit corneal epithelial cells (tRCEC). Such a system enables selective increases in Erk1 mutant expression without alterations of other signaling pathways. Methods: tRCEC were cotransfected with pcDNA6/TR and a dominant negative cDNA Erk1 mutant construct (provided by M. Cobb). Results: They exhibited a low level of native Erk1 expression in the absence of tetracycline. Following exposure to (1 µg/ml) tetracycline for 24 h, the Erk1 mutant protein was greatly induced reaching a 10-fold induction. EGF (5ng/ml)-induced stimulation of Erk1 activity after 15 min was only 30% of the level measured in the absence of tetracycline. In untransfected cells, this concentration of EGF stimulated Erk1 activity in a time dependent manner. At 15 min, the increase was maximal reaching a level that was 3-fold above that measured in the absence of EGF. Without Erk1 induction, 5 ng/ml EGF maximally increased proliferation 2.5-fold. However, mutant Erk1 overexpression eliminated this response without having any cytotoxic effects. Conclusions: This study demonstrates that Erk1 expression and its activation by EGF are required for the control of cell cycle progression. Such a cell line will be beneficial in identifying interactions among signaling pathways mediating growth factor control of proliferation.

Keywords: cornea: epithelium • gene/expression • cell-cell communication