IL-1 induced shape changes mediated by ras

Abstract

IL-1, an inflammatory cytokine, is known to mediate a wide range of biological activities in a number of cell lines. IL-1 receptor binding causes modulation at focal adhesions and reorganisation of the actin cytoskeleton. Further, IL-1 induced signal transduction is regulated by integrin binding and fibronectin attachment is permissive in IL-1 mediated gene regulation. Members of the p21ras superfamily of GTP-binding proteins have been shown to play a central role in mediating coregulation of structural and growth factor/cytokine mediated responses. To determine the role of GTP-binding proteins in matrix regulation of IL-1 mediated responses we have made GTPase/EGFP constructs of human Ras and the dominant negative mutant Asn17-Ras. Confocal microscopy of live cells shows that IL-1 stimulation of Ha-Ras transfected cells plated on a fibronectin matrix show relocalisation of the Ras/EGFP fusion protein to the cell membrane, and subsequent rapid and pronounced shape changes, causing cell rounding to 50–70% of original section area. No such changes were seen upon stimulation with EGF, a known activator of Ras. The shape changes correlate with a transient activation of Ras in fibronectin attached cells, comparable with that of EGF. Transfection of cells with the dominant negative Ras construct (Asn17-Ras), or plating of Ras transfected cells on bare tissue culture plastic abrogates the observed IL-1 induced changes in cell morphology and activation. Automated screening of transfected cells at low magnification gave similar results, showing that IL-1 affects approximately 50% of the transfected cell population. These data indicate that IL-1 induced shape changes are due to activation of Ras, and that it is involved in regulating IL-1 mediated responses in a matrix dependent manner. The studies are supported by the STFUSH, the Medical Research Council and the Wellcome Trust.