12(S)-Hydroxyeicosatetraenoic acidand 13(S)-hydroxyoctadecadienoic acidregulation of protein kinaseC-ainmelanomacells: Roleof receptor-mediated hydrolysis ofinositol phospholipids

Protein kinase C (PKC)isoenzymes arees- sential components ofcell signaling. Inthis study, weinves- tigated theregulation ofPKC-ainmurineB16amelanotic melanoma (B16a) cells bythemonohydroxy fatty acids 12(S)- hydroxyeicosatetraenoic acid(12(S)-HETE) and13(S)- hydroxyoctadecadienoic acid(13(S)-HODE). 12(S)-HETE in- ducedatranslocation ofPKC-atotheplasma membrane and focal adhesion plaques, leading toenhanced adhesion ofB16a cells tothematrix protein fibronectin. However, 13(S)-HODE inhibited these 12(S)-HETE effects onPKC-a.A receptor- mediated mechanism ofaction for12(S)-HETE and13(S)- HODEissupported bythefollowing findings. First, 12(S)- HETEtriggered arapid increase incellular levels ofdiacyl- glycerol andinositol trisphosphate inB16acells. 13(S)-HODE blocked the12(S)-HETE-induced bursts ofbothsecond mes- sengers. Second, the12(S)-HETE-increased adhesion ofB16a cells tofibronectin wassensitive toinhibition byaphospho- lipase Cinhibitor andpertussis toxin. Finally, ahigh-affinity binding site (Kd= 1nM)for12(S)-HETE wasdetected inB16a cells, andbinding of12(S)-HETE toBl6acells waseffectively inhibited by13(S)-HODE (IC5o = 4nM).Insummary, our dataprovide evidence thatregulation ofPKC-aby12(S)- HETEand13(S)-HODE maybethrough aguanine nucleotide- binding protein-linked receptor-mediated hydrolysis ofino-