Abstract
Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic proinflammatory cytokine that modulates a broad range of inflammatory and immunological processes. We have investigated the potential immunomodulatory properties of prostaglandin E2 (PGE2) by examining the molecular mechanism by which the eicosanoid suppresses T-cell-derived interleukin-17 (IL-17)-induced TNF-alpha mRNA expression and protein synthesis in human macrophages and rheumatoid arthritis-affected synovial fibroblasts. Initial studies confirmed that PGE2 induces egr-1 mRNA expression and protein synthesis by restricted SAPK2/p38 MAPK-dependent activating transcription factor-2 (ATF-2) dimer transactivation of the egr-1 promoter as judged by studies using wild-type (WT) and deletion mutant egr-1 promoter constructs, Northern and Western blotting, and standard and supershift electrophoretic mobility shift analyses. Using human leukemic monocytic THP-1 cells stably transfected with WT and dominant-negative mutant expression constructs of Egr-1, cotransfected or not with a WT pTNF-615SVOCAT construct, we observed that PGE2 inhibition of IL-17-stimulated TNF-alpha mRNA expression and promoter activity was dependent on Egr-1 expression, as mutants of Egr-1, alone or in combination, markedly abrogated any inhibitory effect of PGE2. Standard and supershift electrophoretic mobility shift analysis, signaling "decoy" overexpression studies, and pTNF-615SVOCAT promoter assays using WT and mutant promoter constructs revealed that IL-17-up-regulated promoter activity was largely dependent on ATF-2/c-Jun transactivation. PGE2 suppression of IL-17-induced ATF-2/c-Jun transactivation and DNA binding was dependent on Egr-1-mediated inhibition of induced c-Jun expression. We suggest that egr-1 is an immediate-early PGE2 target gene that may be a key regulatory factor in mediating eicosanoid control of genes involved in the immune and inflammatory responses.
Highlights
Tumor necrosis factor-␣ (TNF-␣) is a pleiotropic proinflammatory cytokine that modulates a broad range of inflammatory and immunological processes
We have investigated the potential immunomodulatory properties of prostaglandin E2 (PGE2) by examining the molecular mechanism by which the eicosanoid suppresses T-cell-derived interleukin-17 (IL-17)-induced TNF-␣ mRNA expression and protein synthesis in human macrophages and rheumatoid arthritis-affected synovial fibroblasts
Using human leukemic monocytic THP-1 cells stably transfected with WT and dominant-negative mutant expression constructs of Egr-1, cotransfected or not with a WT pTNF؊615SVOCAT construct, we observed that PGE2 inhibition of IL-17-stimulated TNF-␣ mRNA expression and promoter activity was dependent on Egr-1 expression, as mutants of Egr-1, alone or in combination, markedly abrogated any inhibitory effect of PGE2
Summary
Tumor necrosis factor-␣ (TNF-␣) is a pleiotropic proinflammatory cytokine that modulates a broad range of inflammatory and immunological processes. Type IV cytosolic phospholipase A2 translocates to the endoplasmic reticulum or outer nuclear membrane and releases arachidonic acid from the membrane lipids of most cell types [7, 8] The latter protein trafficking event is controlled by calcium fluxes and MAPK phosphorylation of cytosolic phospholipase A2 [8]. A co-localized, monotopic, integral membrane protein, prostaglandin H2 synthase ( referred to as cyclooxygenase (COX), with two isoforms, COX-1 and COX-2), catalyzes the first committed, rate-limiting step by converting arachidonic acid to prostaglandin H2 The latter intermediate is rapidly metabolized by cell- and tissue-specific synthases to one of several prostanoids (e.g. PGE2 by prostaglandin E synthase) [9, 10]. The C terminus of EP receptors harbors serine/threonine residues (anywhere from 1 to 9) that, when phosphorylated by activated protein kinase A, induce receptor desensitization and signal attenuation (reviewed in Ref. 13)
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