PKC mediates 12(S)‐HETE‐induced cytoskeletal rearrangement in B16A melanoma cells

Abstract

The fatty acid 12(S)-HETE may be a new second messenger capable of activating PKC. In tumor cells 12(S)-HETE stimulates cytoskeleton-dependent cellular responses such as adhesion and spreading. Analysis of 12(S)-HETE effects on B16a melanoma cell cytoskeleton revealed reversible rearrangement of microtubules, microfilaments, the actin-binding proteins, vinculin, myosin heavy (MHC) and light chains (MLC), as well as bundling of vimentin intermediate filaments. The alterations in microfilaments and intermediate filaments occurred very rapidly, i.e., 5 min after exposure of tumor cells to 12(S)-HETE. The 12(S)-HETE-induced cytoskeletal alterations were accompanied by centrifugal organelle-translocation. Interestingly, MLC exhibited clear association with the cytoplasmic organelles. Biochemical analysis of the 12(S)-HETE effect indicated a PKC-mediated reversible hyperphosphorylation of MLC, vimentin, and a 130 kD cytoskeletal-associated protein. Optimal effects were obtained after 5 min treatment with 12(S)-HETE at 0.1 microM concentration. 12(S)-HETE pretreatment induced tumor cell spreading on a fibronectin matrix which required the intactness of all three major cytoskeletal components. The spreading process was dependent upon the activity of PKC. Our data suggest that 12(S)-HETE is a physiological stimulant of PKC. Further, it induces rearrangement of the cytoskeleton of tumor cells in interphase resulting in the stimulation of cytoskeleton-dependent cell activity such as spreading.

Modulation of Cell-Substrate Adhesion by Arachidonic Acid: Lipoxygenase Regulates Cell Spreading and ERK1/2-inducible Cyclooxygenase Regulates Cell Migration in NIH-3T3 Fibroblasts

“…In previous work, LOX has been found to facilitate cell spreading by activation of protein kinase C⑀ (PKC⑀), resulting in the induction of actin polymerization (Chun and Jacobson, 1993;Timar et al, 1993;Chun et al, 1997). In the work presented herein, the marginal inhibition of migration (Ͻ20%) when LOX activity was inhibited after the cells were allowed to spread could reflect the need for turnover between monomeric and polymeric actin during the process of migration.…”

Cell-cell interactions during transendothelial migration of tumor cells

“…These gap junctions are thought to permit the transfer of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), a lipoxygenase metabolite of arachidonic acid, from tumor cells to endothelial cells, eventually leading to the retraction of endothelial cells. 12(S)-HETE is known to affect the redistribution of PECAM-1 and ␣ v ␤ 3 integrin (Tang and Honn, 1994a,b;Tang et al, 1993a,b), while triggering a protein kinase C-dependent rearrangement of the actin cytoskeleton (Tang et al, 1993c(Tang et al, , 1995Timar et al, 1993). Interestingly, lipoxygenase metabolites have also been shown to facilitate the transendothelial migration of monocytes (Sultana et al, 1996).…”

Regulation of melanoma‐cell motility by the lipoxygenase metabolite 12‐(S)‐hete

“…We therefore propose the existence of a putative "autocrine motility cycle" (Fig. 8) involving: (a) AMF binding and phosphorylation of gp78 Watanabe et al, 1991a), (b) receptor-ligand internalization (Watanabe et aZ., 1991b;Nabi et al, 1992), (c) G-protein-dependent signal transduction (Stracke et al, 1987;Watanabe et al, 1991b), (d) activation of inositol metabolism (Kohn et al, 1990) which is followed or paralleled by (el AMF or 12-(S)-HETE-induced activation of PKC (this study); (f) gp78-mediated activation of 12-lipoxygenase (this study); (g) cytoskeletal and morphological alterations Timar et al, 1993) including (h) gp78 phosphorylation and up-regulation at the cell surface (this study); and finally (i) enhanced tumor-cell motility.…”