Cytosolic Phospholipase A₂ and Cyclooxygenase-2 Mediate Release and Metabolism of Arachidonic Acid in Tumor Necrosis Factor-α-Primed Cultured Intestinal Epithelial Cells (INT 407)

Abstract

Background: We have recently reported that tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine that has been suggested to play a role in the pathogenesis of inflammatory bowel disease, potentiates phospholipase A₂ (PLA₂)-stimulated arachidonic acid (AA) release and prostaglandin E₂(PGE₂) formation in cultured intestinal epithelial cells (INT 407). The aim of the present study was to investigate which particular isoforms of PLA₂ and cyclooxygenase (COX) are involved in these processes. Methods: Cells were labeled with ¹⁴C-AA or ¹⁴C-oleic acid, and the amounts of released fatty acid and PGE₂ were analyzed by thin-layer chromatography. mRNA was analyzed by reverse transcription and polymerase chain reaction. Results: The cells contained mainly mRNA for cytosolic PLA₂ (cPLA₂) and only trace amounts of mRNA for group I and II PLA₂. TNF-α potentiated the release of ¹⁴C-AA but not of ¹⁴C-oleic acid. The TNF-α-potentiated PGE₂ release was reduced after inhibition of cellular COX activity or mRNA synthesis. TNF-α increased the amounts of mRNA for COX-2 but not for COX-1. Conclusions: The results point to the possibility that TNF-α may modulate the intestinal mucosal content of biologically active AA metabolites by priming cPLA₂- and COX-2-mediated processes in the epithelial cells.

Key Words:

• Arachidonic acid
• cyclooxygenase activation
• cyclooxygenase gene expression
• intestinal epithelial cells
• phospholipase A₂ activation
• phospholipase A₂ gene expression
• prostaglandin E₂
• tumor necrosis factor-α