title: Protein kinase C-dependent effects of 12(S)-HETE on endothelial cell vitronectin receptor and fibronectin receptor.
abstract: 12(S)-HETE, a lipoxygenase metabolite of arachidonic acid induced a nondestructive and reversible endothelial cell (EC) retraction. 12(S)-HETE induced EC retraction was inhibited by protein kinase C inhibitors calphostin C and staurosporine but not by the protein kinase A inhibitor H8. The role of EC integrins alpha v beta 3 and alpha 5 beta 1 in 12(S)-HETE induced EC retraction was investigated. In confluent EC cultures, alpha v beta 3 is localized to focal adhesions at both the cell body and cell-cell borders and is colocalized with vinculin-containing focal adhesions. In contrast, alpha 5 beta 1 is primarily enriched at the cell-cell borders, demonstrating codistribution with cell cortical microfilaments and extracellular fibronectin. Both receptors were functional in mediating cell-cell or cell-matrix interactions based on the observations that specific antibodies inhibited EC adhesion to intact subendothelial matrix and disrupted the monolayer integrity. 12(S)-HETE induced a multistep, temporally defined redistribution of the alpha v beta 3-containing focal adhesions, leading to an eventual decrease in alpha v beta 3 plaques in the retracted ECs. This effect of 12(S)-HETE was inhibited by calphostin C but not by H8. The alterations of alpha v beta 3-containing focal adhesions preceded the development of EC retraction. 12(S)-HETE also enhanced EC alpha v beta 3 surface expression as revealed by immunofluorescence, flow cytometry, and digitized image analysis. 12(S)-HETE-induced alpha v beta 3 rearrangement (i.e., decreased focal adhesion localization and enhanced surface expression) did not result from altered mRNA transcription (as revealed by semi-quantitative RT-PCR analysis) or protein translation (as revealed by Western blotting). In contrast to its effect on alpha v beta 3, 12(S)-HETE did not demonstrate a temporally related, well-defined effect on the distribution pattern and the surface expression of alpha 5 beta 1, although the cell-cell border staining pattern of alpha 5 beta 1 was disrupted due to EC retraction. It is concluded that 12(S)-HETE-induced decrease of alpha v beta 3 localization to focal adhesions may contribute to the development of EC retraction and that 12(S)-HETE induced increase in alpha v beta 3 surface expression may promote adhesion of inflammatory leukocytes as well as tumor cells to endothelium.
authors.affiliation: Department of Radiation Oncology, Wayne State University, Detroit, Michigan 48202.
authors.forename: D G
authors.initials: DG
authors.lastname: Tang
authors.forename: Y Q
authors.initials: YQ
authors.lastname: Chen
authors.forename: C A
authors.initials: CA
authors.lastname: Diglio
authors.forename: K V
authors.initials: KV
authors.lastname: Honn
mesh_terms: D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D001011:Aorta; D001483:Base Sequence; D002448:Cell Adhesion / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects; D004730:Endothelium, Vascular / Q000187:drug effects*; D006893:Hydroxyeicosatetraenoic Acids / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D051379:Mice; D008833:Microcirculation / Q000187:drug effects; D008969:Molecular Sequence Data; D009281:Naphthalenes; D011083:Polycyclic Compounds / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats; D017207:Rats, Sprague-Dawley; D016024:Receptors, Cytoadhesin / Q000187:drug effects* / Q000378:metabolism; D017469:Receptors, Fibronectin / Q000187:drug effects* / Q000378:metabolism; D019030:Receptors, Vitronectin; D019311:Staurosporine; D016596:Vinculin
chemical_list: D000470:Alkaloids; D006893:Hydroxyeicosatetraenoic Acids; D009281:Naphthalenes; D011083:Polycyclic Compounds; D016024:Receptors, Cytoadhesin; D017469:Receptors, Fibronectin; D019030:Receptors, Vitronectin; D016596:Vinculin; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D011493:Protein Kinase C; D019311:Staurosporine; C058819:calphostin C