BACTERIAL PHOSPHOLIPASE-C UP-REGULATES MATRIX METALLOPROTEINASE EXPRESSION BY CULTURED EPITHELIAL-CELLS

Phospholipase C (PLC) is a putative virulence factor of several pathog enic bacteria. We studied if exogenous PLC would perturb epithelial be havior in infected tissues, Gelatin and casein zymography of cell cult ure medium indicated that the broad-spectrum PLC of Bacillus cereus in duced matrix metalloproteinase (MMP) production in epithelial cells of human skin (NHEK), human gingiva (HGE), and porcine periodontal ligam ent (PLE). In all three cell types, the strongest increase (ninefold) at 0.1 U/ml was seen in the MMP-9 (92-kDa gelatinase) activity, and th e effect was dose dependent in the range of 0.1 to 1.0 U/ml, A relativ ely weaker increase (twofold) in MMP-2 (72-kDa gelatinase) was also ob served in each cell type. PLC induction of MMP-3 (48-kDa stromelysin) was also seen in NHEK and HGE on gelatin and more sensitively for PLE by casein zymography (fivefold), Total gelatinolytic activity as measu red by degradation of C-14-labeled denatured type I collagen increased by about Id-fold (NHEK), 12-fold (HGE), and 14-fold (PLE), Northern a nalysis showed a clear increase in the MMP-9, and a minor increase in MMP-3 mRNA levels but no significant increase in MMP-2 mRNA levels, Fu rther studies with PLE revealed that MMP-9 induction by PLC progressiv ely increased with the length of cell culture time in the absence of s erum. PLC induction of MMPs was polar, with MMP-9 and MMP-3 secreted p rimarily in the apical direction and MMP-2 secreted mainly in the basa l direction, The PLC effect was blocked by neomycin, an inhibitor of t he phosphoinositol signal pathway, No significant effects were observe d in MMP expression with the calcium ionophore A23187 or phospholipase A(2). Morphologically, PLC treatment resulted in reduced contacts bet ween the cultured cells and loss of the cell surface microvilli, These results suggest that PLC secreted by bacterial pathogens may disrupt epithelium of infected tissue and increase the subepithelial tissue de struction through induction of MMPs.