Ras activated ERK and PI3K pathways differentially affect directional movement of cultured fibroblasts.

Background: Cell migration is essential in physiological and pathological processes, such as wound healing and metastasis formation. Ras involvement in these processes has been extensively demonstrated. This work attempts to characterize Ras regulation of the phenomena determining directional cell migration by separately analyzing the role of its principal effector pathways, MAPK and PI3K. Methods: NIH3T3 and NIHRasV12 fibroblasts were followed in wound healing assays to study, in time and under a directional stimulus, cell migration both under standard conditions and in presence of MAPK and PI3K inhibitors. Several parameters, descriptive of specific aspects of cell motion, were evaluated by coupling dynamic microscopy with quantitative and statistical methods. Quantitative Western Blots coupled with immunofluorescence stainings, were used to evaluate ERK activation. Results: Constitutive RasV12 activation confers to NIH3T3 the ability to close the wound faster. Neither increased cell proliferation nor higher speed explains the accelerated healing, but the increased directional migration drives the wound closure. Inhibition of ERK activation, which occurs immediately after wound, greatly blocks the directional migration, while inhibition of PI3K pathway reduces cell speed but does not prevent wound closure. Conclusion: Ras is greatly involved in determining and regulating directionality, ERK is its key effector for starting, driving and regulating directional movement