Cytosolic Phospholipase A2 Is Required for Cytokine-induced Expression of Type IIA Secretory Phospholipase A2 That Mediates Optimal Cyclooxygenase-2-dependent Delayed Prostaglandin E2 Generation in Rat 3Y1 Fibroblasts

Abstract

Activation of rat fibroblastic 3Y1 cells with interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha) induced delayed prostaglandin (PG) E2 generation over 6-48 h, which occurred in parallel with de novo induction of type IIA secretory phospholipase A2 (sPLA2) and cyclooxygenase (COX)-2, without accompanied by changes in the constitutive expression of type IV cytosolic PLA2 (cPLA2) and COX-1. Types V and IIC sPLA2s were barely detectable in these cells. Studies using an anti-type IIA sPLA2 antibody, sPLA2 inhibitors, and a type IIA sPLA2-specific antisense oligonucleotide revealed that IL-1 beta/TNF alpha-induced delayed PGE2 generation by these cells was largely dependent on inducible type IIA sPLA2, which was functionally linked to inducible COX-2. Delayed PGE2 generation was also suppressed markedly by the cPLA2 inhibitor arachidonoyl trifluoromethyl ketone (AACOCF3), which attenuated induction of type IIA sPLA2, but not COX-2, expression. AACOCF3 inhibited the initial phase of cytokine-stimulated arachidonic acid release, and supplementing AACOCF3-treated cells with exogenous arachidonic acid partially restored type IIA sPLA2 expression. These results suggest that certain metabolites produced by the cPLA2-dependent pathway are crucial for the subsequent induction of type IIA sPLA2 expression and attendant delayed PGE2 generation. Some lipoxygenase-derived products might be involved in this event, since IL-1 beta/TNF alpha-induced type IIA sPLA2 induction and PGE2 generation were reduced markedly by lipoxygenase, but not COX, inhibitors. In contrast, Ca2+ ionophore-stimulated immediate PGE2 generation was regulated predominantly by the constitutive enzymes cPLA2 and COX-1, even when type IIA sPLA2 and COX-2 were maximally induced after IL-1 beta/TNF alpha treatment, revealing functional segregation of the constitutive and inducible PG biosynthetic enzymes.