Signaling mechanisms involved in the activation of arachidonic acid metabolism in human astrocytoma cells by tumor necrosis factor-α:: 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-kappa B (NF-kappa B). As these elements play a central role in the mechanisms of signaling involved in the activation of cytosolic phospholipase A(2) (cPLA(2)) 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 cPLA(2), 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 [H-3]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 [H-3]AA metabolites by reverse-phase HPLC showed that all of the [H-3]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 [H-3]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-kappa B. 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 cPLA(2) and the release of AA. On the other hand, the activation of NF-kappa B may explain the induction of the expression of COX-2 and the delayed generation of prostanoids.