Human beta-defensin-3 up-regulates cyclooxygenase-2 expression and prostaglandin E2 synthesis in human gingival fibroblasts.

Oral epithelial cells express three antimicrobial peptide human beta-defensins (hBDs) that have previously been demonstrated to exert proinflammatory effects on various immune cells. We wanted to examine whether hBDs could induce cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) synthesis in non-immune cells, such as human gingival fibroblasts. Cultured fibroblasts were treated with different concentrations of hBD-1, -2, -3 or interleukin-1 beta, as a positive control, for various times, in the presence or absence of NS-398, a specific COX-2 inhibitor. The levels of COX-1 and COX-2 mRNA expression were analyzed using RT-PCR and real-time PCR. Whole cell lysates were analyzed for COX-1 and COX-2 protein expression by western blotting. Cell-free culture supernatants were assayed for PGE(2) levels by ELISA. The lactate dehydrogenase assay was performed to determine the cytotoxicity of hBDs. Ten and 40 microg/mL of hBD-3 up-regulated COX-2 mRNA and protein expression, consistent with COX-2 up-regulation by interleukin-1 beta, whereas hBD-1 and hBD-2 did not. However, COX-1 mRNA and protein were constitutively expressed. The time-course study revealed that hBD-3 up-regulated COX-2 mRNA and protein expression at 6 and 12 h, respectively. Consistent with COX-2 up-regulation, 10 and 40 microg/mL of hBD-3 significantly increased PGE(2) levels in cell-free culture supernatants (p < 0.05), and this was inhibited by NS-398 in a dose-dependent manner. Neither of the hBD concentrations tested in this study was toxic to the cells. These findings indicate that epithelial human beta-defensin-3 functions as a proinflammatory mediator in controlling arachidonic acid metabolism in underlying fibroblasts.