The role of mPGES-1 for prostaglandin E2 production in primary human synovial cells

The effectiveness of nonsteroidal anti-inflammatory drugs and specific cyclooxygenase (COX)-2 inhibitors for treatment of arthritis provides clinical evidence that increased local prostaglandin (PG) production in joint tissues contributes to symptoms of pain, swelling, and stiffness. Despite improved gastrointestinal safety of specific COX-2 inhibitors, unwanted effects associated with inhibition of COX-2 continue to occur in patients treated with these agents. Since production of stable PGs requires synthase enzymes that function downstream of COX, there are other potential targets for therapeutic intervention in arthritis patients. PGE2 is the most abundant prostanoid in synovial fluid and tissues, andits biosynthesis is catalyzed by the coordinate action of COX enzymes and PGE synthases (PGES). There are constitutive forms (COX-1, microsomal PGES-2 [mPGES-2] and cytosolic PGES) and inducible forms (COX-2 and mPGES-1) of both biosynthetic enzymes, all of which are expressed in human fibroblast-like synoviocytes (FLS). For this reason, FLS are ideal for the study of PGE2 biosynthetic pathways in a clinically relevant cell. Both COX-2 and mPGES-1 are increased by the proinflammatory cytokines IL-1 and tumor necrosis factor. However, we have shown that the time frame of their stimulated expression is distinctly different. mPGES-1 mRNA expression lags COX-2 by 2 hours and is sustained up to 48 hours, while mPGES-1 protein lags COX-2 by 4 hours and is sustained to 72 hours. Upregulation of mPGES-1 protein is required for high-level PGE2 production as measured by enzyme immunoassay and HPLC, and PGE2 biosynthetic capacity remains robust at 48 hours. At the 48 hour time point, PGE2 production is blocked by the selective COX-1 inhibitor, suggesting that mPGES-1 is capable of efficiently generating PGE2 from COX-1-derived substrate. In contrast to COX-2, IL-induced transcription of mPGES-1 is blocked by the protein synthesis inhibitor cyclohexamide, suggesting a requirement for new protein synthesis. It was recently reported that the transcription factor early growth response gene 1 (Egr-1) binds to the murine mPGES-1 promoter and regulates transcription. Total and nuclear Egr-1 expression is upregulated by IL-1 in FLS. This expression is blocked by the inhibitors of MAPK, as is upregulation of mPGES-1. Two tandem GC boxes are present in the mPGES-1 promoter region, with the proximal GC box overlapping a potential Egr-1 binding site. Under basal conditions, at least three complexes (CI, CII and CIII) bind to this region of the mPGES-1 promoter (-125 to -98) by electrophoretic mobility shift assay. We identified the induced CII complex as Egr-1 by supershift assay. Egr-1 binds to the proximal GC box as determined by competition assays using mutated oligonucleotides. CI and CIII contain Sp3 by supershift assay. It is not yet clear that Sp3 is displaced by Egr-1. We confirmed the functional significance of Egr-1 binding for induced endogenous mPGES-1 expression using plasmids containing wild-type and mutant Egr-1. In summary, mPGES-1 is a cytokine-inducible enzyme required for production of PGE2 in FLS. mPGES-1 deficiency in null mice completely blocks PGE2 production and susceptibility to collagen-induced arthritis. mPGES-1 represents a novel therapeutic target for treatment of the inflammation associated with arthritis.