15S-Lipoxygenase-2 mediates arachidonic acid-stimulated adhesion of human breast carcinoma cells through the activation of TAK1, MKK6, and p38 MAPK.
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.
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Paul A
PA
Nony
Sarah B
SB
Kennett
Wayne C
WC
Glasgow
Kenneth
K
Olden
John D
JD
Roberts
D001093:Arachidonate 15-Lipoxygenase / Q000502:physiology*; D016718:Arachidonic Acid / Q000378:metabolism / Q000502:physiology*; D001943:Breast Neoplasms / Q000201:enzymology*; D002277:Carcinoma / Q000201:enzymology; D002448:Cell Adhesion / Q000502:physiology; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000502:physiology; D005260:Female; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D016859:Lipoxygenase Inhibitors; D048669:MAP Kinase Kinase 6 / Q000378:metabolism*; D048848:MAP Kinase Kinase Kinase 5 / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism*; D009637:Masoprocol / Q000494:pharmacology; D010766:Phosphorylation; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*; D000099151:MAP Kinase Kinase Kinase 7
D001093:Arachidonate 15-Lipoxygenase; D016718:Arachidonic Acid; D006893:Hydroxyeicosatetraenoic Acids; D016859:Lipoxygenase Inhibitors; D048669:MAP Kinase Kinase 6; D048848:MAP Kinase Kinase Kinase 5; D020930:MAP Kinase Kinase Kinases; D009637:Masoprocol; D048051:p38 Mitogen-Activated Protein Kinases; D000099151:MAP Kinase Kinase Kinase 7; C482858:MAP3K5 protein, human; C482566:MAP2K6 protein, human