Gastrin stimulates MMP-1 expression in gastric epithelial cells: putative role in gastric epithelial cell migration

Abstract

The pyloric antral hormone gastrin plays a role in remodeling of the gastric epithelium, but the specific targets of gastrin that mediate these effects are poorly understood. Glandular epithelial cells of the gastric corpus express matrix metalloproteinase (MMP)-1, which is a potential determinant of tissue remodeling; some of these cells express the CCK-2 receptor at which gastrin acts. We have now examined the hypothesis that gastrin stimulates expression of MMP-1 in the stomach. We determined MMP-1 transcript abundance in gastric mucosal biopsies from Helicobacter pylori negative human subjects with normal gastric mucosal histology, who had a range of serum gastrin concentrations due in part to treatment with proton pump inhibitors (PPI). The effects of gastrin were studied on gastric epithelial AGS-GR cells using Western blot and migration assays. In human subjects with increased serum gastrin due to PPI usage, MMP-1 transcript abundance was increased 2-fold; there was also increased MMP-7 transcript abundance but not MMP-3. In Western blots, gastrin increased proMMP-1 abundance, as well that of a minor band corresponding to active MMP-1, in the media of AGS-GR cells, and the response was mediated by protein kinase C and p42/44 MAP kinase. There was also increased MMP-1 enzyme activity. Gastrin-stimulated AGS-GR cell migration in both scratch wound and Boyden chamber assays was inhibited by MMP-1 immunoneutralization. We conclude that MMP-1 expression is a target of gastrin implicated in mucosal remodeling.

Keywords: MMP-1; gastric epithelium; gastrin.

Fig. 1.

Association between serum gastrin and gastric matrix metalloproteinase (MMP)-1 transcript abundance. A: serum gastrin concentrations (left y-axis, pM) and abundance of MMP-1, MMP-3, and MMP-7 transcripts relative to GAPDH (right y-axis) in subjects on proton pump inhibitors (PPIs) (gray bars, n = 85) normalized to control (Cntrl, black bars, n = 77). B: similar data for subjects on PPIs divided into two groups on the basis of serum gastrin below (n = 27) or above (n = 58) the upper limit of the reference range (30 pM). C: similar data for those control (n = 65) and PPI-treated subjects (n = 27) in whom serum gastrin was below the upper limit of the reference range. D: MMP-1 transcript abundance relative to GAPDH expressed for each quartile of serum gastrin concentration and the mean gastrin concentration within each quartile. Data are means ± SE. *P < 0.05.

Fig. 2.

Immunohistochemical localization of MMP-1 in gastric epithelial cells. A and B: subject treated with PPIs and with normal serum gastrin. C and D: control subject with normal serum gastrin. E and F: two different subjects with elevated serum gastrin (>200 pM), both on PPIs. Note staining in glandular cells (arrows), but light staining in stroma, mucus neck, and surface epithelial cells. A and C: ×10; other panels: ×20.

Fig. 3.

Increased proMMP-1 in corpus biopsies from hypergastrinemic humans and mice, detected by Western blot. A: Western blot showing proMMP-1 in gastric corpus biopsies from four subjects with serum gastrin < 30 pm (Hi-gas, −) and four subjects with hypergastrinemia (Hi-gas, +). B: means ± SE of serum gastrin and abundance of MMP-1 estimated by densitometry of Western blots of the data shown in A. C: Western blot showing proMMP-1 in gastric corpus biopsies from four wild-type mice and four INS-Gas hypergastrinemic mice. D: means ± SE of serum gastrin and abundance of MMP-1 estimated by densitometry of Western blots of data shown in C.

Fig. 4.

Gastrin stimulates proMMP-1 expression by AGS-G_R cells revealed by Western blot. A: Western blots of HGT1-G_R, RMG1-G_R, and AGS-G_R cells reveal stimulation of proMMP-1 by hG17ns (10 nM) in media (top) and in cell extracts (middle) while there is no difference in cellular GAPDH (bottom). B: concentration-dependent increases in proMMP-1 (52 kDa) in response to hG17ns (1–10 nM). C: stimulation of proMMP-1 by hG17ns (10 nM) is inhibited by the CCK-2 receptor antagonist L740,093 (0.2 μg/ml), while responses to PMA (100 nM) are not; the changes in proMMP-1 can be seen both in media and cell extracts but there is no change in GAPDH abundance in cell extracts. D: hG17ns and PMA increase proMMP-1 abundance in AGS-G_R media, but only PMA also stimulates AGS cells which lack the CCK-2 receptor. E: the effects of both gastrin and PMA on proMMP-1 in media are inhibited by a PKC inhibitor, Ro320432 (2.0 μM), and an inhibitor of activation of p42/44 MAP kinase, U0126 (10 μM).

Fig. 5.

Brefeldin A (BFA) inhibits proMMP-1 secretion. A: BFA (10 μg/ml) inhibits the increase in proMMP-1 in media of AGS-G_R cells in response to hG17ns treatment for 16 h by arresting secretion and causing accumulation in the cells; when BFA is added to cells already treated with hG17ns for 16 h the pattern of bands in media after a further 8 h (i.e., at 24 h) is similar to that at 16 h suggesting a low capacity for proMMP-1 activation or degradation. B: enzyme activity of MMP-1 determined using a specific substrate [DNP-Pro-Cha-Abu-Cys(Me)-His-Ala-Lys(N-Me-Abz)-NH₂ (Cha, β-cyclohexylalanyl; Abu, l-α-aminobutyryl; Abz, 2-aminobenzoyl)] is increased in hG17ns stimulated AGS-G_R media (16 h) and is inhibited by treatment with BFA; no further increase in MMP-1 activity was detected in AGS-G_R media, after treatment with hG17ns and addition of BFA for a further 8 h. Data are means ± SE, n = 3; horizontal arrows, P < 0.05.

Fig. 6.

Gastrin increases AGS-G_R cell migration in part via MMP-1. A: images of a scratch wound assay showing control AGS-G_R cells, and cells treated with MMP-1 antibody (MMP-1 Ab, 2.5 μg/ml), gastrin (hG17ns, 10 nM, 8 h) and the combination of hG17ns and MMP-1 Ab. Gastrin stimulated AGS-G_R cell migration is inhibited by neutralizing antibody to MMP-1. B: mean data ± SE, for three scratch wound assays. C: Boyden chamber migration assays using AGS-G_R cells and treatments with gastrin, MMP-1 antibody alone, and in combination with gastrin. Data are means ± SE, n = 3; horizontal bars, P < 0.05.