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Background: IL-6 plays an important role in the pathogenesis of Graves' disease and its orbital component, thyroidassociated ophthalmopathy (TAO). Orbital tissues become inflamed in TAO, a process in which prostanoids have been implicated. Orbital fibroblasts both generate and respond to PGE2, underlying the inflammatory phenotype of these cells. Methodology/Principal Findings: Using cultured orbital and dermal fibroblasts, we characterized the effects of PGE2 on IL-6 expression. We found that the prostanoid provokes substantially greater cytokine synthesis in orbital fibroblasts, effects that are mediated through cell-surface EP2 receptors and increased steady-state IL-6 mRNA levels. The pre-translational upregulation of IL-6 results from increased gene promoter activity and can be reproduced with the PKA agonist, Sp-cAMP and blocked by interrupting the PKA pathway. PGE2-induced production of cAMP in orbital fibroblasts was far greater than that in dermal fibroblasts, resulting from higher levels of adenylate cyclase. PGE2 provokes CREB phosphorylation, increases the pCREB/CREB ratio, and initiates nuclear localization of the pCREB/CREB binding protein/p300 complex (CBP) preferentially in orbital fibroblasts. Transfection with siRNAs targeting either CREB or CBP blunts the induction of IL-6 gene expression. PGE2 promotes the binding of pCREB to its target DNA sequence which is substantially greater in orbital fibroblasts. Conclusion/Significance: These results identify the mechanism underlying the exaggerated induction of IL-6 in orbital fibroblasts and tie together two proinflammatory pathways involved in the pathogenesis of TAO. Moreover, they might therefore define an attractive therapeutic target for the treatment of TAO. [ABSTRACT FROM AUTHOR]