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doi: 10.1242/10.1242/jcs.00518

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Stimulation of MMP-7 (matrilysin) by Helicobacter pylori in human gastric epithelial cells: role in epithelial cell migration

¹ Physiological Laboratory, University of Liverpool, Liverpool L69 3BX, UK
² Department of Medicine, University of Liverpool, Liverpool L69 3BX, UK
³ Department of Medical Microbiology, University of Liverpool, Liverpool L69 3BX, UK
⁴ Department of Pathology, University of Liverpool, Liverpool L69 3BX, UK

View larger version (29K): [in a new window]   Fig. 1. Western blots of MMP-7 and ß-actin in gastric biopsies from control and H. pylori- infected subjects. Top panels, representative blots of MMP-7 and ß-actin from control subjects (-) and subjects infected with H. pylori (+) in biopsies from the gastric corpus (A) and antrum (B). Bottom panels, the relative abundance in corpus (C) and antrum (D) of MMP-7 and ß-actin. (C) Plasma gastrin concentrations are indicated in panel C, where MMP-7 and ß-actin data are also shown for the subset of H. pylori-positive patients in which plasma gastrin concentrations were in the normal range (i.e. <30 pM). Data for corpus based on n=11 control subjects (open bars) and 16 H. pylori-positive subjects; data for antrum based on n=10 control subjects and n=6 H. pylori-positive subjects. Means±s.e.m. *P<0.05.

View larger version (102K): [in a new window]   Fig. 2. Immunohistochemical localization of MMP-7 in human gastric epithelial cells. Immunoperoxidase labelling of MMP-7 and counter-staining with haematoxylin in antral biopsies from (A) H. pylori-negative and (B) H. pylori-positive subjects, and (C) in corpus biopsies from H. pylori-negative and (D) H. pylori-positive subjects (all x10). Higher magnification (x40) of the same samples (E–H, respectively) reveals cytosolic localization to a subnuclear region of surface mucus cells (arrow). (I) Immunofluorescence localization of MMP-7 to cells in a cultured antral gland from a H. pylori-positive subject (bar, 25 µm). The outline of the gland is indicated with a white broken line. (J) MMP-7 immunoreactivity in a cell at the periphery of a cultured antral gland from a control subject after H. pylori addition, demonstrating a lamellipodium with punctate MMP-7 immunoreactivity (bar, 50 µm). (K) Co-expression of MMP-7 (green) and mucin (red) in antral cells, demonstrating MMP-7 is not exclusively localized with mucin and showing the presence close to the plasma membrane of cells at the periphery of the cultured gland (bar, 10 µm). (L–N) Deconvolved images from a z-series through a single antral cell (outlined by a white broken line) showing that MMP-7 and mucin exhibit punctate staining in distinct subcellular compartments (bar, 10 µm).

View larger version (105K): [in a new window]   Fig. 3. Spreading of human gastric epithelial cells is stimulated by H. pylori by induction of MMP-7. (A–C) Frames taken at 17, 32 and 52 hours from the same human gastric gland (control subject) during time-lapse video-microscopy. (D–F) Similar frames taken from a gland from a H. pylori-positive subject. Subsequent staining of cell nuclei with Hoechst 33342 revealed 86 cells in A–C and 88 cells in D F. (G) The area of gastric colonies expressed relative to cell numbers at 17 and 40 hours indicates increased migration in H. pylori-positive (n=8; mean data from 4–15 glands per subject) compared with control subjects (n=12; mean data of 4–15 glands per subject). (H) Pretreatment with antisense (AS) oligonucleotides to MMP-7 (2 µM) inhibited the spreading of gastric glands at 40 hours from H. pylori-positive subjects (n=7), but had only a slight effect on control glands (n=7). Data expressed as means±s.e.m. *P<0.05. In A–F: bar, 200 µm.

View larger version (12K): [in a new window]   Fig. 4. H. pylori induction of MMP-7 in AGS cells is associated with stimulation of migration and invasion. (A) Western blots indicate that addition of H. pylori to cultured AGS cells with a multiplicity of infection [MOI] of 100 (centre lane) or 200 (right lane) produced a graded increase in the abundance of MMP-7; the latter co-migrated with the 19 kDa human recombinant MMP-7 (arrow). (B) H. pylori (hatched bars), increased migration of AGS cells through Transwell filters. The number of migrating cells per field are presented; mean±s.e.m., n=3. (C) H. pylori (hatched bars) increased invasion of AGS cells through Matrigel-coated Transwell filters. The sum of the invading cells in five fields is presented; mean±s.e.m., n=3. Neutralization of MMP-7 using an MMP-7 monoclonal antibody (4 µg ml-1) inhibited both migration and invasion.

View larger version (25K): [in a new window]   Fig. 5. Addition of H. pylori stimulates the expression of the 2.3 kb human MMP-7 promoter coupled to a luciferase reporter. (A) Stimulation of MMP-7-luc (1.0 µg well-1) by two different isolates of H. pylori, one vacA+ and cagA+ (E6), the other vacA- and cagA+ (E5). (B) Failure of H. pylori (60190) to stimulate MMP-7-luc when added to a filter over transfected AGS cells. (C) Stimulation of MMP-7-luc by the chemokine IL-8 (250 ng ml-1), proinflammatory cytokines TNF- (2.5 ng ml-1) and IL-6 (5 ng ml-1), and the growth factor EGF (100 ng ml-1). (D) However, the effect of H. pylori (60190) on MMP-7-luc was not mediated by soluble AGS cell factors, because application of H. pylori to AGS cells on filters cultured above MMP-7-luc transfected AGS cells had no effect on MMP-7-luc compared with direct application to AGS cells. Means±s.e.m., n=3.

View larger version (15K): [in a new window]   Fig. 6. H. pylori-increased p50, p65 and c-fos activity in AGS cells mediates MMP-7-luc expression. (A) In western blots of nuclear extracts, H. pylori increased NF-B p50 and p65 compatible with nuclear translocation. (B) In nuclear extracts of H. pylori-treated cells there was increased p65 and p50 binding to the NF-B consensus cis-element. (C) The inhibitor of IB degradation, BAY11-7082 (10 µM), depressed MMP-7-luc responses to H. pylori, as did the MEK inhibitor PD98059 (20 µM). (D) In nuclear extracts, there was also activation of c-fos, as indicated by binding to oligonucleotides with relevant consensus sequences. (E) Cotransfection with c-fos (0.6 µg well-1) increased expression of MMP-7-luc. Means±s.e.m., n=3.

View larger version (19K): [in a new window]   Fig. 7. Induction of MMP-7 by H. pylori is mediated by RhoA. (A) Expression of the MMP-7-luc plasmid (0.5 µg well-1) was increased in a time-dependent manner (hours after transfection) by cotransfection with plasmids encoding constitutively active forms of Rho (L63RhoA, 0.25 µg well-1) and, to lesser extent, Rac (L61Rac, 0.25 µg well-1); constitutively active cdc42 (L61cdc42, 0.25 µg well-1) had only a slight effect. (B) H. pylori-stimulated MMP-7-luc expression was inhibited by cotransfection with dominant-negative (DN) RhoA (N19RhoA, 0.5 µg well-1) and DN-Rac (N17Rac, 0.5 µg well-1). (C,D) L63RhoA-stimulated MMP-7-luc expression was inhibited by BAY11-7082 (10 µM) and by cotransfection with DN-jun vector (0.5 µg well-1). (E,F) L63Rac-stimulated MMP-7-luc expression was inhibited by BAY11-7082, but not by cotransfection with a DN-jun vector. Means±s.e.m., n=3.