Involvement of Cytosolic Phospholipase A₂ in Human β-defensin-2 mRNA Up-regulation

Intracellular calcium ion and p38 mitogen-activated protein kinase, two critical signaling molecules in regulating human β-defensin-2 (hBD-2) expression, were previously shown to activate cytosolic phospholipase A₂ (cPLA₂), a key enzyme for the generation of various lipid inflammatory mediators. Objectives: We tested the hypothesis whether hBD-2 mRNA up-regulation in human gingival epithelial cells (HGECs) was mediated by cPLA₂ activation. Methods: HGECs were stimulated with either 10 μg/ml of Fusobacterium nucleatum cell wall extract or 10 ng/ml of phorbol 12-myristate 13-acetate (PMA) in the absence or presence of aristolochic acid (ARA) or 4-bromophenacyl bromide (4-BPB), two PLA₂ inhibitors. Total RNA was harvested and analyzed for cPLA₂ and hBD-2 expression by RT-PCR. Whole cell lysates were extracted and analyzed for the presence of phosphorylated cPLA₂ and cPLA₂ by Western blot. Results: cPLA₂ mRNA was constitutively expressed in HGECs, yet PMA inhibited its mRNA expression in a dose-dependent manner, suggesting a negative feedback mechanism. The kinetics study showed that this inhibition was remarkable after HGECs were stimulated with PMA for 12 h. Interestingly, activation of cPLA₂ by phosphorylation at serine 505 residue was observed from 30 min to 3 h and 1 to 12 h for HGECs stimulated with Fusobacterium nucleatum and PMA, respectively. HBD-2 mRNA up-regulation by Fusobacterium nucleatum was inhibited by pretreatment with 10 μM ARA and 20 μM 4-BPB by 4.6 and 2.3 fold, respectively, suggesting involvement of cPLA₂ in regulating hBD-2 mRNA expression. Conclusion: These results demonstrate the involvement of cPLA₂ in hBD-2 mRNA up-regulation by Fusobacterium nucleatum in addition to phospholipase D, an upstream signaling molecule, as previously reported. This study was supported by the Thailand Research Fund, Grant no. RMU5080035, and the Center for Innovation in Chemistry: Postgraduate Education and Research Program in Chemistry.