Abstract
The dietary cis-polyunsaturated fatty acid, arachidonic acid, stimulates adhesion of metastatic human breast carcinoma cells (MDA-MB-435) to the extracellular matrix, but the molecular mechanisms by which fatty acids modify the behavior of these cells are unclear. Exposure to arachidonic acid activates multiple signaling pathways. Activation of p38 mitogen-activated protein kinase (p38 MAPK) is required for increased cell adhesion to type IV collagen, and this activation is sensitive to inhibitors of lipoxygenases, suggesting a requirement for arachidonic acid metabolism. The goals of the current study were to identify the one or more key metabolites of arachidonic acid that are responsible for activation of p38 MAPK and to elucidate the upstream kinases that lead to p38 MAPK activation. High performance liquid chromatographic analysis revealed that MDA-MB-435 cells metabolize exogenous arachidonic acid predominantly to 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE). Immunoblot analysis with antibodies specific to 15(S)-lipoxygenase-1 (LOX-1) and 15(S)-lipoxygenase-2 (LOX-2) demonstrated the expression of 15-LOX-2, but not 15-LOX-1, in these tumor cells. A LOX inhibitor, nordihydroguaiaretic acid, attenuated production of 15(S)-HETE and inhibited the phosphorylation of p38 MAPK following exposure to arachidonic acid. In contrast, overexpression of LOX-2 sensitized the cells to the addition of arachidonic acid, leading to increased activation of p38 MAPK. Addition of exogenous 15(S)-HETE to MDA-MB-435 cells stimulated cell adhesion to type IV collagen and activated the p38 MAPK pathway, including the upstream kinases transforming growth factor-beta1-activated protein kinase-1 (TAK1) and MAPK kinase 6. Transfection of these cells with a dominant negative form of TAK1 blocked arachidonic acid-stimulated p38 MAPK phosphorylation. These data demonstrate that 15(S)-LOX-2 generation of 15(S)-HETE activates specific growth factor receptor-related signaling pathways, thereby initiating signal transduction events leading to increased cell adhesion to the extracellular matrix.
Highlights
The cis-polyunsaturated fatty acid arachidonic acid and its many metabolites are important mediators of cell signaling with roles in inflammation, platelet aggregation, tissue development, and carcinogenesis [1,2,3,4,5]
We show for the first time the presence of 15(S)-LOX-2 in breast carcinoma cells and demonstrate the involvement of upstream kinases TAK1 and MKK6 in the activation of p38 MAPK by the arachidonic acid metabolite, 15(S)-hydroxyeicosatetraenoic acid (HETE)
We show that 15(S)-HETE is the primary metabolite of arachidonic acid exogenously added to these cells and that the addition of 15(S)HETE recapitulated the effects of arachidonic acid
Summary
The cis-polyunsaturated fatty acid arachidonic acid and its many metabolites are important mediators of cell signaling with roles in inflammation, platelet aggregation, tissue development, and carcinogenesis [1,2,3,4,5]. 15(S)-LOX-2, on the other hand, has a more limited tissue distribution (prostate, lung, skin, and cornea) and exclusively metabolizes arachidonic acid to 15(S)-HETE [14, 16] These two isozymes display important functional differences in their metabolism of arachidonic acid. The inhibition of LOX activity blocks the ability of arachidonic acid to promote cell adhesion These results suggested that arachidonic acid or its metabolites could affect metastasis through alteration of the adhesive properties of tumor cells and encouraged us to investigate the lipid-induced signaling pathways involved in this process. Nor is it clear which upstream kinases are responsible for the activation of the p38 MAPK in this system
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