Up-regulation of cyclooxygenase-2 by cobalt chloride-induced hypoxia is mediated by phospholipase D isozymes in human astroglioma cells

Cyclooxygenase-2 (COX-2) is an isoform of prostaglandin H synthase induced by hypoxia and has been implicated in the growth and progression of a variety of human cancers. In the present study, we investigated the role of phospholipase D (PLD) isozymes in cobalt chloride (CoCl₂)-induced hypoxia-driven COX-2 expression in U87 MG human astroglioma cells. CoCl₂ stimulated PLD activity and synthesis of COX-2 protein in a dose and time-dependent manner. Moreover, elevated expression of PLD1 and PLD2 increased hypoxia-induced COX-2 expression and prostaglandin E2 (PGE₂) production. Pretreatment of cells with 1-butanol, but not 3-butanol, suppressed CoCl₂-induced COX-2 expression and PGE₂ formation. In addition, evidence that PLD activity was involved in the stimulation of COX-2 expression was provided by the observations that overexpression of wild type PLD isozymes, but not catalytically inactive PLD isozymes, stimulated CoCl₂-induced COX-2 expression and PGE₂ production. PLD1 enhanced COX-2 expression by CoCl₂ via reactive oxygen species (ROS), p38 MAPK kinase, PKC-δ, and PKA, but not ERK, whereas PLD2 enhanced CoCl₂-induced COX-2 expression via ROS and p38 MAPK, but not ERK, PKC-δ, and PKA. Differential regulation of COX-2 expression mediated through PLD isozymes was comparable with that of CoCl₂-induced PLD activity in these two PLD isozymes. Taken together, our results demonstrate for the first time that PLD1 and PLD2 isozymes enhance CoCl₂-induced COX-2 expression through differential signaling pathways in astroglioma cells.