ACTIVATION OF MICROVASCULAR ENDOTHELIUM BY EICOSANOID 12(S)-HYDROXYEICOSATETRAENOIC ACID LEADS TO ENHANCED TUMOR-CELL ADHESION VIA UP-REGULATION OF SURFACE EXPRESSION OF ALPHA-V-BETA-3 INTEGRIN - A POSTTRANSCRIPTIONAL, PROTEIN-KINASE C-DEPENDENT AND CYTOSKELETON-DEPENDENT PROCESS

Tumor cell interaction with endothelial cells is a crucial step leadin g to organ-selective metastasis. Adhesion of murine B16 amelanotic mel anoma cells (B16a) to murine microvascular endothelial cells (CD3) was enhanced, in a dose- and time-dependent manner, by pretreating CD3 ce lls with 12(S)-hydroperoxyeicosatetraenoic acid [i.e., 12(S)-HETE], a 12-lipoxygenase metabolite of arachidonic acid. The metabolic precurso r of 12(S)-HETE, 12-HPETE, (12-hydroperoxyeicosatetraenoic acid) also enhanced B16a cell adhesion to CD3 monolayers, whereas other lipoxygen ase products, i.e., 5(S), 11(S), and 15(S)-HETEs were ineffective. 12( S)-HETE-enhanced tumor cell adhesion was blocked by treating endotheli al cells with antibodies against the alpha v beta 3 complex or against individual subunits but not with antibodies against alpha 5 beta 1. I n contrast, neither of these two integrins appeared to be involved in tumor cell adhesion to unstimulated endothelium. Flow cytometric analy sis, immunofluorescent labeling, and image analysis indicated that IZ( S)-HETE induced a time- and dose-dependent increase in the surface exp ression of alpha v beta 3 but not alpha 5 beta 1 on CD3 cells. The inc reased surface expression of alpha v beta 3 on endothelial cells did n ot result from an increased transcription or translation of alpha v be ta 3 message as confirmed by quantitative reverse transcription-polyme rase chain reaction, Northern blotting, and quantitative Western blott ing. Instead, subcellular fractionation studies revealed an increased translocation of alpha v beta 3 integrins from the cytosolic pool to t he membrane fractions. Pretreatment of endothelial cells with several cytoskeleton-disrupting agents (i.e., cycloheximide or acrylamide to d isrupt intermediate filament vimentin, cytochalasin D to disrupt micro filaments, colchicine or Nocodazole to disrupt microtubules) abolished the 12(S)-BETE-enhanced alpha v beta 3 surface expression as well as tumor cell adhesion to endothelial cells. Also, pretreatment of CD3 ce lls with protein kinase C inhibitor calphostin C, but not with protein kinase A inhibitor H8, blocked 12(S)-HETE-enhanced alpha v beta 3 sur face expression and tumor cell adhesion. Collectively, these results s uggest that eicosanoid 12(S)-HETE modulates tumor cell interaction wit h endothelium via protein kinase C- and cytoskeleton-dependent up-regu lation of the surface expression of alpha v beta 3 integrin.