Prostaglandin E₂is an enhancer for IL-1β-induced expression of membrane-associated prostaglandin E synthase in rheumatoid synovial fibroblasts

Introduction

Membrane-associated prostaglandin E synthase (mPGES) is a recently identified terminal enzyme of arachidonic acid cascade that catalyzes prostaglandin (PG)H₂ to PGE₂. We recently reported that expression of mPGES in rheumatoid synovial fibroblasts (RSF) was upregulated by IL-1β, and that the time-course of its expression was delayed and prolonged when compared with that of cyclooxygenase-2 (COX-2).

Objective

To identify the regulating factors of mPGES expression in RSF.

Methods

RSF from patients with rheumatoid arthritis (RA) on surgery, who gave written consent to the use of tissues for this research, were treated with IL-1β, rofecoxib, NS-398, and meloxicam (selective COX-2 inhibitors). The effects of PGE₂ and selective EP1, EP2, EP3, and EP4 receptor agonists (DI-004, AE1-259-01, AE-243, and AE1-329, respectively; Ono Pharmaceutical Co., Osaka, Japan) were also studied in various conditions. Expression of the mRNA and protein of mPGES and COX-2 were analyzed by northern and Western blot analyses, respectively. Expression of PGE₂ receptor mRNA in RSF was determined by RT-PCR. PGE₂ and cAMP production was measured by an ELISA.

Results

Enhanced expression of mPGES mRNA and protein in IL-1β-stimulated RSF was attenuated by rofecoxib, NS-398, or meloxicam. This attenuation was restored by addition of PGE₂. Exogenous PGE₂ only (without IL-1β stimulation) did not induce expression of mPGES in RSF, indicating that PGE₂ was an enhancer of mPGES expression in the IL-1β-stimulated condition.

We then examined which PGE₂ receptor was important in the enhancing action of PGE₂ in RSF. EP2 and EP4 receptor mRNA was detected in RSF; however, EP1 and EP3 were negative. Addition of AE1-259-01 (EP2 agonist) or AE1-329 (EP4 agonist) as well as PGE₂ restored the inhibitory effects of mPGES expression by rofecoxib. In addition, the restoration by PGE₂ was mimicked by forskolin, a direct activator of adenylate cyclase. Intracellular cAMP was increased by IL-1β and it was inhibited by rofecoxib.

Conclusions

The enhancing property of PGE₂ via EP2/EP4 receptors on mPGES expression may play an important role in the cytokine-stimulated articular inflammation in RA patients. It also seems that COX-2 inhibitors effectively decrease PGE₂ production not only by COX-2 inhibition, but also by reduction of mPGES expression.