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The antimicrobial peptide human β-defensin-3 (hBD-3) is produced by epidermal keratinocytes and protects the skin from infections. This peptide induces the release of a lipid mediator, prostaglandin D2 from dermal mast cells. Prostaglandin D2 binds to cell-surface G protein-coupled receptors, D prostanoid receptor, and chemoattractant receptor-homologous molecule expressed on T helper cell type 2 (CRTH2). Both receptors are detected on epidermal keratinocytes. It is reported that prostaglandin D2 is involved in cutaneous allergy, however, its role in antimicrobial defense is unknown. We examined the in vitro effects of prostaglandin D2 on hBD-3 production in normal human keratinocytes. Prostaglandin D2 enhanced hBD-3 secretion and mRNA expression in human keratinocytes. Prostaglandin D2-induced hBD-3 production was suppressed by the CRTH2 antagonist ramatroban and by antisense oligonucleotides against c-Jun and c-Fos, components of a transcription factor, activator protein-1 (AP-1). Prostaglandin D2 enhanced the transcriptional activity and DNA binding of AP-1, expression, phosphorylation, and DNA binding of c-Fos proteins in keratinocytes. Prostaglandin D2-induced hBD-3 production, AP-1 activity, and c-Fos expression and phosphorylation were suppressed by U0126, PP2, and pertussis toxin, which are inhibitors of mitogen-activated protein kinase kinase (MEK), src, and Gi proteins, respectively. The phosphorylation of extracellular signal-regulated kinase (ERK), downstream kinase of MEK, was induced by prostaglandin D2, and suppressed by ramatroban, pertussis toxin, PP2, and U0126. These results suggest that prostaglandin D2 induces hBD-3 production in human keratinocytes by activating AP-1 through the expression and phosphorylation of c-Fos via the CRTH2/Gi/src/MEK/ERK pathway. Prostaglandin D2 may promote cutaneous antimicrobial activity via hBD-3.