Signaling Mechanisms Involved in the Activation of Arachidonic Acid Metabolism in Human Astrocytoma Cells by Tumor Necrosis Factor‐α

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Abstract
: Tumor necrosis factor-α (TNF-α) is a cytokine that elicits cell responses by activating the mitogen-activated protein kinase (MAP kinase) cascade and transcription factors such as nuclear factor-kB (NF-kB). 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-α on arachidonate (AA) metabolism in 1321N1 astrocytoma cells was assayed. TNF-α 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-α 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-α addition eluted as authentic AA, whereas in samples obtained at 24 h after addition of TNF-α, 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-α 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-kB. These data show that TNF-α 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-kB may explain the induction of the expression of COX-2 and the delayed generation of prostanoids.