12(S)-hydroxyeicosatetraenoic acid and 13(S)-hydroxyoctadecadienoic acid regulation of protein kinase C-alpha in melanoma cells: role of receptor-mediated hydrolysis of inositol phospholipids.

Abstract

Protein kinase C (PKC) isoenzymes are essential components of cell signaling. In this study, we investigated the regulation of PKC-a in murine B16 amelanotic melanoma (B16a) Inhibitor studies revealed that PKC was involved in basal and 12(S)-HETE-regulated tumor cell and endothelial cell integrin expression (5), adhesion (6, 7), spreading (5), and experimental metastasis (7). However, the involvement of specific PKC isoform(s) and particularly the related mechanism(s) of action for 12(S)-HETE and 13(S)-HODE have not been explored.Previously, exploration of mechanisms of action for monoHETEs placed emphasis on investigating their fate after uptake by cells and their intracellular targets. For example, extracellular mono-HETEs are esterified into phospholipids, possibly affecting certain membrane functions (8). In vitro kinase assays revealed that 15-HETE can increase PKC activity (9), and 12-HETE can activate PKC-y (10). Finally, incorporated 12(S)-HETE has been shown to bind to an intracellular protein (11). Although receptors have been identified for some eicosanoids (12-18), studies on possible cell membrane receptor-mediated signaling processes for 12(S)-HETE and/or 13(S)-HODE are lacking. In this study, we propose that 12(S)-HETE and 13(S)-HODE interact with a cell surface receptor(s) whose activation leads to generation of second messengers and further downstream activation of PKC.Protein kinase C (PKC) consists of a family of protein-serine/ threonine kinases that play important roles in mediating cell growth, differentiation, and tumor promotion (1). Binding of extracellular stimulators (e.g., hormones, growth factors, cytokines, and neurotransmitters) to their cell surface receptors triggers the phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol bisphosphate, generating the second messengers diacylglycerol (DAG) and D-myo-inositol 1,4,5-trisphosphate (IP3). While DAG directly activates PKC, IP3 releases Ca2+ from intracellular stores (1). Tumor-promoting phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), mimic DAG and directly activate PKC.Metastasis is a multistep process involving tumor cell detachment from the primary tumor, invasion through basement membrane, and intravasation (2). Once blood borne, the tumor cell must adhere to endothelium, induce endothelial cell retraction, and again invade through basement membrane to form a successful secondary tumor. Previously, we have observed that the ability of tumor cells to accomplish many of these steps in the metastatic cascade is enhanced by 12(S)- MATERIALS AND METHODSMaterials. Polyclonal antibodies against PKC-a, -P3I, -,BII, -ry, -8, -a, or -(were prepared as described (19). PMA, pertussis toxin, and cholera toxin were obtained from Calbiochem. Genistein was purchased from Sigma. 12(S)-[3H]HETE (215.3 Ci/mmol; 1 Ci = 37 GBq) was from NEN. Murine B16 amelanotic melanoma (B16a) cells were maintained as described (5). Subcellular Fractionation, PKC Activity Assay, and Western Blotting. Cytosol and membrane PKC wer...

The Oxidized Lipid and Lipoxygenase Product 12(S)-Hydroxyeicosatetraenoic Acid Induces Hypertrophy and Fibronectin Transcription in Vascular Smooth Muscle Cells via p38 MAPK and cAMP Response Element-binding Protein Activation

“…In contrast, the stereoisomer 12(R)-HETE, which is not an LO product, did not have these stimulatory effects, indicating specificity for the LO pathway. Although there is evidence in other cell types for a 12(S)-HETE receptor (37,38), it is not clear from this study whether the effects of 12(S)-HETE on Ras-MAPK activation are mediated by a specific receptor. Because 12(S)-HETE did not directly induce oxidant stress in these cells, 2 additional unknown mechanisms for MAPK activation may be operative.…”

“…Lipoxygenase products can modulate calcium currents in VSMCs (47) and also induce oxidant stress and inflammatory gene expression (48,49). In cancer cells, 12(S)-HETE has been shown to induce multiple signaling events through a potential receptormediated mechanism (37). Our initial unpublished experiments 2 did not support the presence of specific 12(S)-HETE receptors on VSMCs.…”

Eicosanoid Activation of Protein Kinase C ϵ

“…As mentioned, previous reports suggest that the effects of 12(S)-HETE on live cells were the result of receptor-mediated stimulation of phospholipase C, the release of DAG and free Ca 2ϩ , and subsequent kinase activation (33,34). Although such a complex pathway may exist in some systems, it is unlikely to function in growth cones.…”

“…A number of reports also implicate AA and/or its metabolites in PKC stimulation (27)(28)(29)(30)(31)(32), but they do not provide evidence for a direct mechanism of activation. Instead, some of them suggest kinase activation via intermediate steps such as the intracellular release of DAG and Ca 2ϩ (33,34). Therefore, the issue has remained unclear.…”

Arachidonic acid activates Jun N-terminal kinase in vascular smooth muscle cells

“…Earlier studies have demonstrated that cis-polyunsaturated fatty acids and their metabolites such as HETEs activate PKC isozymes of conventional type (a, b and g isozymes) in several cell types (Liu et al, 1995;Kikkawa et al, 1988;Hansson et al, 1986;McPhail et al, 1984). To understand the mechanism by which arachidonic acid and 12-HpETE induce JNK1 activity, the role of PKC on arachidonic acid and 12-HpETE activation of JNK1 was tested.…”