12(S)-HETE-induced microvascular endothelial cell retraction results from PKC-dependent rearrangement of cytoskeletal elements and alpha V beta 3 integrins.
12(S)-HETE, a lipoxygenase metabolite of arachidonic acid, has been demonstrated to induce a reversible retraction of vascular endothelial cells (EC). 12(S)-HETE-induced microvascular EC retraction was blocked by a selective protein kinase C inhibitor, calphostin C, but not by the protein kinase A inhibitor, H8. EC exposed to 12(S)-HETE demonstrated a gradual dissolution of actin microfilaments and a decrease of vinculin-containing focal adhesions. The intermediate filaments, vimentin, also underwent extensive reorganization (i.e., filament bundling and enrichment to the cell filapodia) following 12(S)-HETE treatment. In vivo phosphorylation studies revealed that 12(S)-HETE induced a hyperphosphorylation of several major cytoskeletal proteins including myosin light chain, actin, and vimentin. The increased phosphorylation of these cytoskeletal proteins following 12(S)-HETE stimulation was abolished by calphostin C but not by H8. Confluent EC express alpha v beta 3 in focal adhesions at both the cell body and the cell-cell borders. 12(S)-HETE induced a sequential rearrangement of the alpha v beta 3-containing focal adhesions, resulting in a general decrease in alpha v beta 3 integrin receptors, especially in those retracted EC. 12(S)-HETE-induced rearrangement of alpha v beta 3 was inhibited by calphostin C but not by H8. In contrast to alpha v beta 3, confluent EC enrich alpha 5 beta 1 integrin receptors primarily at the cell-cell borders, colocalizing with extracellular fibronectin and cell cortical microfilaments. 12(S)-HETE treatment also disrupted the cell-border distribution pattern of alpha 5 beta 1 as EC retracted, but no distinct alterations (such as time-related redistribution and quantitative differences) in alpha 5 beta 1 were observed.
Tang
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DG
Department of Radiation Oncology, Wayne State University, Detroit, MI 48202.
Diglio
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CA
Honn
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KV
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D000199:Actins / Q000187:drug effects; D002448:Cell Adhesion / Q000187:drug effects; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000187:drug effects* / Q000648:ultrastructure; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects*; D006893:Hydroxyeicosatetraenoic Acids / Q000494:pharmacology*; D016023:Integrins; D007382:Intermediate Filaments / Q000187:drug effects; D007546:Isoquinolines / Q000494:pharmacology; D008833:Microcirculation / Q000502:physiology; D009281:Naphthalenes; D011083:Polycyclic Compounds / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D047428:Protein Kinase Inhibitors; D016024:Receptors, Cytoadhesin / Q000187:drug effects*; D019030:Receptors, Vitronectin; D014746:Vimentin / Q000187:drug effects; D016596:Vinculin / Q000032:analysis
D000199:Actins; D006893:Hydroxyeicosatetraenoic Acids; D016023:Integrins; D007546:Isoquinolines; D009281:Naphthalenes; D011083:Polycyclic Compounds; D047428:Protein Kinase Inhibitors; D016024:Receptors, Cytoadhesin; D019030:Receptors, Vitronectin; D014746:Vimentin; C061465:calphostin complex; D016596:Vinculin; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; C053177:N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide; D011493:Protein Kinase C