Signaling mechanisms involved in the activation of arachidonic acid metabolism in human astrocytoma cells by tumor necrosis factor-alpha: phosphorylation of cytosolic phospholipase A2 and transactivation of cyclooxygenase-2.
Tumor necrosis factor-alpha (TNF-alpha) is a cytokine that elicits cell responses by activating the mitogen-activated protein kinase (MAP kinase) cascade and transcription factors such as nuclear factor-kappaB (NF-kappaB). As these elements play a central role in the mechanisms of signaling involved in the activation of cytosolic phospholipase A2 (cPLA2) and cyclooxygenase-2 (COX-2), the effect of TNF-alpha on arachidonate (AA) metabolism in 1321N1 astrocytoma cells was assayed. TNF-alpha produced a phosphorylation of cPLA2, which was preceded by an activation of both c-Jun N-terminal kinase (JNK) and p38-MAP kinase, and this was associated with the release of [3H]AA. In contrast, TNF-alpha did not activate the extracellular signal-regulated kinase (MAP kinase) p42, nor did it elicit a mitogenic response. Analysis of [3H]AA metabolites by reverse-phase HPLC showed that all of the [3H]AA released during the first hour after TNF-alpha addition eluted as authentic AA, whereas in samples obtained at 24 h after addition of TNF-alpha, 25% of the [3H]AA had been converted into COX products as compared with only 9% in control cells. In keeping with these findings, TNF-alpha produced an increase of COX-2 expression, as judged from both RT-PCR studies and immunoblot of COX-2 protein, and a long-lasting activation of NF-kappaB. These data show that TNF-alpha produces in astrocytoma cells an early activation of both p38-MAP kinase and JNK, which is followed by the phosphorylation of cPLA2 and the release of AA. On the other hand, the activation of NF-kappaB may explain the induction of the expression of COX-2 and the delayed generation of prostanoids.
Hernández
M
M
Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas, Facultad de Medicina, Valladolid, Spain.
Bayón
Y
Y
Sánchez Crespo
M
M
Nieto
M L
ML
D016718:Arachidonic Acid / Q000378:metabolism*; D001254:Astrocytoma; D001932:Brain Neoplasms; D051546:Cyclooxygenase 2; D003600:Cytosol / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D007700:Kinetics; D008565:Membrane Proteins; D016328:NF-kappa B / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010766:Phosphorylation; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D007527:Isoenzymes; D008565:Membrane Proteins; D016328:NF-kappa B; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D016718:Arachidonic Acid; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D010741:Phospholipases A; D054467:Phospholipases A2