Matrix Metalloproteinase-9 Induction via Cytosolic Phospholipase A₂ in Epithelial Cells

Regulation of matrix metalloproteinase-9 (MMP-9), one of the key enzymes in the pathogenesis of oral inflammatory disorders, is involved with the pathway of arachidonic acid metabolism in various cell lines. Objectives: We wanted to examine the regulation of MMP-9 expression in primary gingival epithelial cells (GECs). Methods: GECs were stimulated with either 1, 10, 100 ng/ml of phorbol 12-myristate 13-acetate (PMA) or 3, 10, 30 μg/ml of Fusobacterium nucleatum cell wall extract in the absence or presence of aristolochic acid (ARA) or 4-bromophenacyl bromide (4-BPB), two phospholipase A₂ (PLA₂) inhibitors. Total RNA was analyzed for cytosolic PLA₂ (cPLA₂) and MMP-9 expression by RT-PCR and real-time PCR. Whole cell lysates were analyzed for expression of phosphorylated cPLA₂ and cPLA₂ by western blot. Culture medium was analyzed for MMP-9 expression and activity by western blot and gelatin-zymography, respectively. Results: MMP-9 mRNA expression was induced by Fusobacterium nucleatum cell wall extract in a dose-dependent manner, while PMA already induced MMP-9 mRNA expression as low as 1 ng/ml and the induction reached the maximal level at 10 ng/ml. The time-course study demonstrated that MMP-9 mRNA was up-regulated at 3 h, whereas the MMP-9 secretion was detected at 12 h after stimulation. cPLA₂ mRNA expression was inhibited by PMA, yet its protein expression remained unchanged. Interestingly, transient activation of cPLA₂ by phosphorylation was observed in GECs stimulated with both stimulants. Induction of MMP-9 expression and activity was inhibited by ARA and 4-BPB in a dose-dependent fashion, suggesting involvement of cPLA₂ in regulating MMP-9. Conclusion: These findings demonstrate the involvement of cPLA₂ in regulating MMP-9 expression and activity in primary cells. 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.