AbstractElevated levels of prostaglandins such as PGE2 in inflamed gingiva play a significant role in the tissue destruction caused by periodontitis, partly by targeting local fibroblasts. Only very few studies have shown that PGE2 inhibits the proliferation of a gingival fibroblast (GF) cell line, and we expanded this research by using primary human GFs (hGFs) and looking into the mechanisms of the PGE2 effect. GFs derived from healthy human gingiva were treated with PGE2 and proliferation was assessed by measuring cell number and DNA synthesis and potential signaling pathways were investigated using selective activators or inhibitors. PGE2 inhibited the proliferation of hGFs dose‐dependently. The effect was mimicked by forskolin (adenylate cyclase stimulator) and augmented by IBMX (a cAMP‐breakdown inhibitor), pointing to involvement of cAMP. Indeed, PGE2 and forskolin induced cAMP generation in these cells. Using selective EP receptor agonists we found that the anti‐proliferative effect of PGE2 is mediated via the EP2 receptor (which is coupled to adenylate cyclase activation). We also found that the effect of PGE2 involved activation of Epac (exchange protein directly activated by cAMP), an intracellular cAMP sensor, and not PKA. While serum increased the amount of phospho‐ERK in hGFs by ∼300%, PGE2 decreased it by ∼50%. Finally, the PGE2 effect does not require endogenous production of prostaglandins since it was not abrogated by two COX‐inhibitors. In conclusion, in human gingival fibroblasts PGE2 activates the EP2—cAMP—Epac pathway, reducing ERK phosphorylation and inhibiting proliferation. This effect could hamper periodontal healing and provide further insights into the pathogenesis of inflammatory periodontal disease. J. Cell. Biochem. 108: 207–215, 2009. © 2009 Wiley‐Liss, Inc.