091 HELICOBACTER PYLORI UPREGULATES MMP-7 IN EPITHELIAL CELLS IN A CAG-DEPENDENT MANNER

J.R. Bebb¹, D.P. Letley¹, R.J. Thomas¹, H.M. Collins², S.A. Watson², J.C. Atherton¹.Division of Gastroenterology and Institute of Infections and Immunity, Academic Unit of Cancer Studies, University Hospital, Nottingham, UK

Background: MMP-7 (matrilysin) is a member of the metalloproteinase family of enzymes, which are important in normal and pathological remodelling of epithelial-matrix interactions. Several studies have shown increased expression of MMP-7 in gastric cancer tissue compared to non-cancer tissue. More virulent strains of H.pylori possess the cagPAI (encoding a Type IV secretion system) and an active form of VacA, a toxin that induces vacuolation in vitro. This study examines the effect of H.pylori on MMP-7 expression in HT29 cells.

Methods:H.pylori strains 60190 (cagPAI+, vacA s1/m1), Tx30a (cagPAI-, vacA s2/m2) and the VacA and CagE isogenic mutants of 60190 were co-cultured with HT29 cells for 24 hours. Cell pellets were used for RNA extraction and reverse transcription, and cDNA levels assessed for MMP-7 levels (and GAPDH) by Real Time PCR. HT29 supernatants were assessed for MMP-7 expression by western blot and casein zymography and for other metalloproteinase activity by gelatin zymography. Experiments were performed at least three times.

Results:H.pylori pathogenic strain 60190 increased MMP-7 RNA levels (13-fold vs untreated, p=0.06, unpaired t-test) more than non-pathogenic strain Tx30a. Use of isogenic mutants showed this effect to be VacA-independent but CagE-dependent. Western blot gave a 29kDa band for 60190 and its VacA mutant. Casein zymography revealed a 29kDa band of activity for all samples but reduced intensity for strain Tx30a and the 60190 cagE mutant. This band corresponds to the predicted size for pro-MMP-7. Gelatin zymography showed no differences between treatments.

Conclusion: In HT29 cells H.pylori co-culture leads to upregulation of MMP-7 at both RNA …