The lipoxygenase metabolite, 12(S)-HETE, induces a protein kinase C-dependent cytoskeletal rearrangement and retraction of microvascular endothelial cells
Tang, D. G., Timar, J., Grossi, I., Renaud, C., Kimler, V. A., Diglio, C. A., ... Honn, K. V. (1993). The lipoxygenase metabolite, 12(S)-HETE, induces a protein kinase C-dependent cytoskeletal rearrangement and retraction of microvascular endothelial cells. Experimental Cell Research, 207(2), 361-375.
We previously reported that a lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], induced large vessel endothelial cell (EC) retraction and increased tumor cell adhesion to exposed extracellular matrix (Honn et al., FASEB J. 3, 2285-2293, 1989). Here, we present evidence that 12(S)-HETE induces the retraction of microvascular ECs in a time- and concentration-dependent manner. The EC retraction was observed 15 min after 12(S)-HETE treatment and reached a peak level between 1 and 2 h. The monolayer reformed by 24 h. Silver staining and 'gap-FRAP' experiments suggest that 12(S)-HETE altered the normally apposed cell junctions and impaired gap junction-mediated cell-cell communication. It appears that the 12(S)-HETE effect was mediated by cytoskeletal alteration. The first observed alteration in EC cytoskeleton following 12(S)-HETE stimulation is vimentin bundling, followed by the rearrangement and disruption of vinculin-containing adhesion plaques and/or simultaneous redistribution of alpha-actinin and disruption of spectrin. These changes are accompanied by progressive microfilament dissolution. During the same time interval, alpha-actinin is mobilized to the cell periphery at cell 'ruffles.' However, 12(S)-HETE showed little or no effects on actin-binding proteins filamin and tropomyosin or on microtubules. 12(S)-HETE effects on these cytoskeletal elements were fully reversible by 24 h and appeared to be mediated through enhancing protein phosphorylation. Following 12(S)-HETE (0.1 microM) treatment increased phosphorylation of proteins that comigrated with myosin light chain (20 kDa), actin (42 kDa), and vimentin (57 kDa) were observed. The enhanced phosphorylation of these cytoskeletal proteins was confirmed by 2D gel analysis. The phosphorylation-promoting effect of 12(S)-HETE on cytoskeletal proteins could be totally abolished by calphostin C, partially inhibited by staurosporine, but was not influenced by N-[2-(methylamine)ethyl]-5-isoquinolinesilfonamide dihydrochloride (HS), suggesting that the 12(S)-HETE effect was mediated via protein kinase C. This was further substantiated by quantitative experiments demonstrating that calphostin C, but not H8, inhibited 12(S)-HETE-induced EC retraction