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Involvement of Phosphatidylinositol 3-Kinase and Mitogen-activated Protein Kinases in Glycine-extended Gastrin-induced Dissociation and Migration of Gastric Epithelial Cells*

Open AccessPublished:November 02, 2001DOI:https://doi.org/10.1074/jbc.M105090200
      The various molecular forms of gastrin can act as promoters of proliferation and differentiation in different regions of the gastrointestinal tract. We report a novel stimulatory effect of glycine-extended gastrin17 only on cell/cell dissociation and cell migration in a non-tumorigenic mouse gastric epithelial cell line (IMGE-5). In contrast, both amidated and glycine-extended gastrin17 stimulated proliferation of IMGE-5 cells via distinct receptors. Glycine-extended gastrin17-induced dissociation preceded migration and was blocked by selective inhibitors of phosphatidylinositol 3-kinase (PI3-kinase) but did not require mitogen-activated protein (MAP) kinase activation. Furthermore, glycine-extended gastrin17 induced a PI3-kinase-mediated tyrosine phosphorylation of the adherens junction protein β-catenin, partial dissociation of the complex between β-catenin and the transmembrane protein E-cadherin, and delocalization of β-catenin into the cytoplasm. Long lasting activation of MAP kinases by glycine-extended gastrin17 was specifically required for the migratory response, in contrast to the involvement of a rapid and transient MAP kinase activation in the proliferative response to both amidated and glycine-extended gastrin17. Therefore, the time course of MAP kinase activation appears to be a critical determinant of the biological effects mediated by this pathway. Together with the involvement of PI3-kinase in the dissociation of adherens junctions, long term activation of MAP kinases seems responsible for the selectivity of this novel effect of G17-Gly on the adhesion and migration of gastric epithelial cells.
      G17-NH2
      amidated gastrin 17
      CCK
      cholecystokinin
      MAP
      mitogen-activated protein
      PI3-kinase
      phosphatidylinositol 3-kinase
      FCS
      fetal calf serum
      PBS
      phosphate-buffered saline
      BrdUrd
      bromodeoxyuridine
      DMEM
      Dulbecco's modified Eagle's medium
      DTT
      dithiothreitol
      PCR
      polymerase chain reaction
      ANOVA
      analysis of variance
      G17-Gly
      glycine-extended gastrin 17
      MEK
      mitogen-activated protein kinase/extracellular signal-regulated kinase kinase
      Gastrin is an important hormone for the development and function of the gastrointestinal tract (for review see Ref.
      • Dockray G.J.
      ). Amidated gastrin (G17-NH2)1has been shown to activate various events such as gastric acid secretion, endocrine secretion of histamine and somatostatin, expression of epidermal growth factors (
      • Miyazaki Y.
      • Shinomura Y.
      • Tsutsui S.
      • Zushi S.
      • Higashimoto Y.
      • Kanayama S.
      • Higashiyama S.
      • Taniguchi N.
      • Matsuzawa Y.
      ), activation of early genes (
      • Todisco A.
      • Takeuchi Y.
      • Seva C.
      • Dickinson C.J.
      • Yamada T.
      ), and proliferation (
      • Baldwin G.S.
      ). Compelling evidence also demonstrates that alternative forms of progastrin processing, such as glycine-extended gastrin 17 (G17-Gly) (
      • Dockray G.J.
      ,
      • Seva C.
      • Dickinson C.
      • Yamada T.
      ,
      • Hollande F.
      • Imdhal A.
      • Mantamadiotis T.
      • Ciccotosto G.D.
      • Shulkes A.
      • Baldwin G.S.
      ) and progastrin-(6–80) (
      • Baldwin G.S.
      • Hollande F.
      • Yang Z.
      • Karelina Y.
      • Paterson A.
      • Strang R.
      • Fourmy D.
      • Neumann G.
      • Shulkes A.
      ), have a biological role in the stimulation of proliferation. Recently, G17-Gly has been shown to promote the invasiveness of the colon cancer cell line LoVo (
      • Kermorgant S.
      • Levy T.
      ), and various reports showed that gastrin-related peptides are capable of activating a set of focal adhesion proteins such as p125FAK, p130Cas, and paxillin, which participate in the regulation of various cell functions such as preservation of morphology and migration (see Ref.
      • Rozengurt E.G.
      • Walsh J.H.
      for review). However, direct effects of gastrins on epithelial cell adhesion have not yet been described.
      Binding of G17-NH2 to the gastrin/cholecystokinin B (gastrin/CCK-B) receptor has been shown to activate various intracellular transduction pathways depending on the cell type. In the rat pancreatic cell line AR4-2J, gastrin-induced cell proliferation is thought to be mediated by activation of MAP kinases, leading to subsequent expression of immediate early genes like c-fos and c-jun (
      • Todisco A.
      • Takeuchi Y.
      • Seva C.
      • Dickinson C.J.
      • Yamada T.
      ,
      • Stepan V.M.
      • Dickinson C.J.
      • Del Valle J.D.
      • Matsushima M.
      • Todisco A.
      ), whereas gastrin-promoted cell growth in the rat pituitary adenoma cell line GH3 is supported by a Ca2+-dependent mechanism (
      • Stepan V.M.
      • Tatewaki M.
      • Matsushima M.
      • Dickinson C.J.
      • del Valle J.
      • Todisco A.
      ). Furthermore, in a rat intestinal epithelial cell line (IEC-6) (
      • Singh P.
      • Narayan S.
      • Adiga R.B.
      ) and in Chinese hamster ovary cells expressing transfected gastrin/CCK-B receptors (
      • Daulhac L.
      • Kowalski-Chauvel A.
      • Pradayrol L.
      • Vaysse N.
      • Seva C.
      ), gastrin stimulates c-Src-like tyrosine kinases upstream of phosphatidylinositol 3-kinase (PI3-kinase) and MAP kinase.
      To date, G17-Gly-induced transduction pathways have been studied mostly in tumor cell lines. In AR4-2J cells (
      • Todisco A.
      • Takeuchi Y.
      • Seva C.
      • Dickinson C.J.
      • Yamada T.
      ), as well as in the human colon cancer cell lines HT29 and LoVo (
      • Stepan V.M.
      • Sawada M.
      • Todisco A.
      • Dickinson C.J.
      ), G17-Gly stimulates c-Jun amino-terminal kinase activation independently of the MAP kinase pathway. Binding studies strongly suggest that G17-Gly effects are mediated by a novel receptor that is insensitive to G17-NH2 and classical gastrin/CCK-B receptor antagonists (
      • Seva C.
      • Dickinson C.
      • Yamada T.
      ,
      • Hollande F.
      • Imdhal A.
      • Mantamadiotis T.
      • Ciccotosto G.D.
      • Shulkes A.
      • Baldwin G.S.
      ,
      • Stepan V.M.
      • Sawada M.
      • Todisco A.
      • Dickinson C.J.
      ), although a different receptor binding with a similar affinity to G17-Gly and G17-NH2 was also identified on Swiss 3T3 fibroblasts (
      • Singh P.
      • Owlia A.
      • Espeijo R.
      • Dai B.
      ). More studies are necessary in order to determine the signal transduction pathways activated by G17-Gly on other tumoral and non-tumoral cell lines and to correlate these processes with the biological roles of the peptide.
      For this work, we used a recently established gastric epithelial cell line (IMGE-5) (
      • Hollande F.
      • Blanc E.M.
      • Bali J.P.
      • Whitehead R.H.
      • Pelegrin A.
      • Baldwin G.S.
      • Choquet A.
      ) to compare the effects of amidated and glycine-extended gastrins on proliferation as well as cell adhesion and migration. The phenotype of these non-tumorigenic cells can be modulated in vitro by shifting them from a permissive temperature (33 °C) in the presence of γ-interferon to a non-permissive temperature (39 °C), allowing their differentiation toward an epithelial phenotype (
      • Hollande F.
      • Blanc E.M.
      • Bali J.P.
      • Whitehead R.H.
      • Pelegrin A.
      • Baldwin G.S.
      • Choquet A.
      ). The gastric origin of IMGE-5 cells together with their differentiated phenotype make them particularly suitable to study the cellular effects of gastrin-related peptides.
      In this paper, we report for the first time that, while both molecular forms of gastrin stimulate proliferation of IMGE-5 cells, dissociation and migration of these cells in a wound healing assay was induced only by G17-Gly. G17-Gly also induced tyrosine phosphorylation of the adherens junction protein β-catenin and dissociation of the complex between β-catenin and the transmembrane protein E-cadherin, followed by the partial disappearance of β-catenin from the cell membrane. We also show that a differential time course in the activation of MAP kinases by the two gastrin derivatives, as well as the involvement of PI3-kinase in the effect of G17-Gly on β-catenin, seem responsible for the selectivity of the effects of G17-Gly on the adhesion and migration of IMGE-5 cells.

      DISCUSSION

      The results presented in this paper demonstrate the exist-ence of similarities but also major differences in the biological effects of G17-Gly and G17-NH2 on a non-tumorigenic gastric epithelial cell line (IMGE-5). On the one hand, both G17-NH2 and G17-Gly stimulated IMGE-5 proliferation, as has been described previously in several other models (
      • Dockray G.J.
      ,
      • Baldwin G.S.
      ,
      • Seva C.
      • Dickinson C.
      • Yamada T.
      ,
      • Hollande F.
      • Imdhal A.
      • Mantamadiotis T.
      • Ciccotosto G.D.
      • Shulkes A.
      • Baldwin G.S.
      ). On the other hand, a major difference between the two gastrins is the novel effect of G17-Gly only on the dissociation and migration of gastric epithelial cells. Our results underline the existence of a biological role for G17-Gly that seems specific to this peptide and is not exhibited by G17-NH2. This finding contrasts with previous work on G17-Gly, which described biological effects also displayed by G17-NH2, such as cell proliferation (
      • Dockray G.J.
      ,
      • Seva C.
      • Dickinson C.
      • Yamada T.
      ,
      • Hollande F.
      • Imdhal A.
      • Mantamadiotis T.
      • Ciccotosto G.D.
      • Shulkes A.
      • Baldwin G.S.
      ) or stimulation of gastric acid secretion (
      • Chen D.
      • Zhao C.-M.
      • Dockray G.J.
      • Varro A.
      • Van Hoek A.
      • Sinclair N.F.
      • Wang T.C.
      • Koh T.J.
      ). However, it is in agreement with the recent description of a stimulatory effect of G17-Gly on the invasiveness of the human colon cancer cell line LoVo (
      • Kermorgant S.
      • Levy T.
      ). The enhancement of cell migration by G17-Gly reported herein may be profoundly important in the previously reported roles of progastrin-derived peptides during the differentiation of the gastrointestinal tract, as well as during cancer development and metastasis (see Refs.
      • Dockray G.J.
      and
      • Rozengurt E.G.
      • Walsh J.H.
      for review).
      Several reports have recently implicated gastrins in the activation of proteins involved in epithelial cell/matrix adhesion and morphology, such as p130Cas and paxillin (
      • Rozengurt E.G.
      • Walsh J.H.
      ). Recent results showing the phosphorylation and activation of p125FAK by G17-NH2 via a Src-related pathway are also compatible with a role in cell motility (
      • Daulhac L.
      • Kowalski-Chauvel A.
      • Pradayrol L.
      • Vaysse N.
      • Seva C.
      ), as the role of this non-receptor tyrosine kinase in cell migration is well documented (
      • Ilic D.
      • Damsky C.H.
      • Yamamoto T.
      ). It is worth noting, however, that G17-NH2 was shown to have an inhibitory effect on the spontaneous motility of glioblastoma cell lines (
      • De Hauwer C.
      • Camby I.
      • Darro F.
      • Migeotte I.
      • Decaestecker C.
      • Verbeek C.
      • Danguy A.
      • Pasteels J.-L.
      • Brotchi J.
      • Salmon I.
      • Van Ham P.
      • Kiss R.
      ). The results reported in this paper provide, to our knowledge, the first demonstration of a directstimulatory effect for a non-amidated progastrin-derived peptide on cell/cell adhesion between epithelial cells.
      This effect was detected at G17-Gly concentrations as low as 100 pm. Serum concentrations of G17-Gly are generally thought to be around 20–50 pm in the fasting state. However, gastrin precursor production is significantly increased in various physiological conditions such as birth and weaning (
      • Tornhage C.J.
      • Rehfeld J.F.
      ,
      • Hilsted L.
      • Bardram L.
      • Rehfeld J.F.
      ), as well as during pathological processes such as gastric adenocarcinoma (
      • Henwood M.
      • Clarke P.A.
      • Smith A.M.
      • Watson S.A.
      ) and gastrinoma (
      • Azuma T.
      • Magami Y.
      • Habu Y.
      • Kawai K.
      • Taggart R.T.
      • Walsh J.H.
      ,
      • Jais P.
      • Mignon M.
      • Rehfeld J.F.
      ). The migratory effect of G17-Gly was maximal around 5 nm, as compared with 1 nm for the maximal proliferative effect of the peptide. This slight difference in potency could be explained by differences in the sensitivity of the signaling pathways involved. Furthermore, previous in vitro investigations of G17-Gly effects on renal (
      • Stepan V.M.
      • Krametter D.F.
      • Matsushima M.
      • Todisco A.
      • Delvalle J.
      • Dickinson C.J.
      ), pancreatic (
      • Todisco A.
      • Takeuchi Y.
      • Seva C.
      • Dickinson C.J.
      • Yamada T.
      ,
      • Seva C.
      • Dickinson C.
      • Yamada T.
      ), colonic (
      • Hollande F.
      • Imdhal A.
      • Mantamadiotis T.
      • Ciccotosto G.D.
      • Shulkes A.
      • Baldwin G.S.
      ,
      • Stepan V.M.
      • Dickinson C.J.
      • Del Valle J.D.
      • Matsushima M.
      • Todisco A.
      ), or gastric cells (
      • Kaise M.
      • Muraoka A.
      • Seva C.
      • Takeda H.
      • Dickinson C.J.
      • Yamada T.
      ,
      • Iwase K.
      • Evers B.M.
      • Hellmich M.R.
      • Guo Y.-S.
      • Higashide S.
      • Kim H.J.
      • Townsend Jr., C.M.
      ) showed a maximal effective dose for G17-Gly varying between 0.1 (
      • Stepan V.M.
      • Sawada M.
      • Todisco A.
      • Dickinson C.J.
      ) and 10 nm (
      • Todisco A.
      • Takeuchi Y.
      • Seva C.
      • Dickinson C.J.
      • Yamada T.
      ), even when the apparent affinity of G17-Gly receptors seemed higher (see Refs.
      • Stepan V.M.
      • Krametter D.F.
      • Matsushima M.
      • Todisco A.
      • Delvalle J.
      • Dickinson C.J.
      ,
      • Kaise M.
      • Muraoka A.
      • Seva C.
      • Takeda H.
      • Dickinson C.J.
      • Yamada T.
      ,
      • Iwase K.
      • Evers B.M.
      • Hellmich M.R.
      • Guo Y.-S.
      • Higashide S.
      • Kim H.J.
      • Townsend Jr., C.M.
      and this work). This apparent discrepancy might indicate that the G17-Gly receptors could exist in various affinity states or that fractional occupancy only is necessary to activate the biological effects downstream of these receptors.
      The signal transduction pathways activated by G17-NH2 in IMGE-5 cells have been reported previously in some, but not all, cell types. The results obtained in this study indicate that MAP kinase activation is essential for the proliferative effect of G17-NH2 on IMGE-5 cells, and Stepan et al. (
      • Stepan V.M.
      • Dickinson C.J.
      • Del Valle J.D.
      • Matsushima M.
      • Todisco A.
      ) recently showed that G17-NH2 stimulated proliferation through a pathway involving MAP kinases in AR42J cells. In contrast, in GH3 cells, G17-NH2 did not activate this pathway but induced proliferation in a Ca2+-dependent manner. In previous studies, activation of G/CCK-B receptors by G17-NH2 has been shown to induce PI3-kinase activation in transfected Chinese hamster ovary cells (
      • Daulhac L.
      • Kowalski-Chauvel A.
      • Pradayrol L.
      • Vaysse N.
      • Seva C.
      ), probably via the prior formation of a p60Src-p125FAKcomplex (
      • Daulhac L.
      • Kowalski-Chauvel A.
      • Pradayrol L.
      • Vaysse N.
      • Seva C.
      ). Thus, there seems to be a certain degree of cell type specificity in the transduction pathways involved in the proliferative effects of G17-NH2.
      Much less is known about the signal transduction pathways activated by G17-Gly. Reports by Todisco et al. (
      • Todisco A.
      • Takeuchi Y.
      • Seva C.
      • Dickinson C.J.
      • Yamada T.
      ) on pancreatic carcinoma cells and Stepan et al. (
      • Stepan V.M.
      • Sawada M.
      • Todisco A.
      • Dickinson C.J.
      ) on colorectal carcinoma cells indicate that G17-Gly regulates the transcriptional activation of early genes, through the activation of enzymes such as c-Jun kinase. In these cell lines, the MAP kinase pathway is not involved in the proliferative effect of G17-Gly (
      • Todisco A.
      • Takeuchi Y.
      • Seva C.
      • Dickinson C.J.
      • Yamada T.
      ,
      • Daulhac L.
      • Kowalski-Chauvel A.
      • Pradayrol L.
      • Vaysse N.
      • Seva C.
      ). On the contrary, our results show that p42/p44 MAP kinase activation by G17-Gly as well as G17-NH2 was involved in their proliferative effect on IMGE-5 cells. However, PI3-kinase was involved selectively in the proliferative effect of G17-Gly only. Further studies on other cell lines will be necessary to assess whether there is also a cell type specificity in the transduction pathways triggered by G17-Gly, whether there are several subtypes of G17-Gly receptors, or whether the signal transduction coupled to these receptors is different in tumor cell lines.
      The novel effect of G17-Gly on dissociation and migration of gastric epithelial cells involved both PI3-kinase and MAP kinase pathways. Activation of PI3-kinase was essential at least for cell dissociation, whereas the MAP kinase pathway seemed to be involved only in the motility response of IMGE-5 cells, without affecting their dissociation. This result is interesting, as the role of these two pathways in the dissociating effect of growth factors on other cell types is still unclear. PI3-kinase activation has been shown to participate in the ligand-induced migration of several cell types including renal epithelial cells (
      • Royal I.
      • Fournier T.M.
      • Park M.
      ) and vascular smooth muscle cells (
      • Duan C.
      • Bauchat J.R.
      • Hsieh T.
      ). However, the specific involvement of these pathways in the successive steps of a migratory response, i.e. cell dissociation and motility, has not been clearly defined. A report by Potempa and Ridley (
      • Potempa S.
      • Ridley A.J.
      ) showed that both PI3-kinase and MAP kinase activation (by Ras) seemed essential to hepatocyte growth factor-induced adherens junction disassembly in Madin-Darby canine kidney cells. However, in the same cell line, Royal et al.(
      • Royal I.
      • Fournier T.M.
      • Park M.
      ) reported that hepatocyte growth factor-induced motility required PI3-kinase activation, whereas pathways downstream from Grb2 (including MAP kinases) were involved in branching tubulogenesis. Recent results on HepG2 human hepatoma cells also showed that PI3-kinase was involved in cell dissociation, whereas inhibition of MEK blocked the motility response to growth factors (
      • Sipeki S.
      • Bander E.
      • Buday L.
      • Farkas G.
      • Bacsy E.
      • Ways D.K.
      • Farago A.
      ). Our results also directly implicate the PI3-kinase pathway in the G17-Gly-induced tyrosine phosphorylation of β-catenin, as well as its dissociation from E-cadherin and delocalization from the adherens junctions. In contrast to HepG2 cells, the MAP kinase pathway does not appear to be involved in this G17-Gly-induced event in IMGE-5 cells.
      We also found in this study that the time course of MAP kinase activation by G17-Gly and G17-NH2was different, with the former triggering a rapid but short lived phosphorylation, whereas the latter induced a long term activation lasting for 3 h. Furthermore, we showed that the early activation of p42/44 MAP kinases is essential to the proliferative effect of G17-NH2 and G17-Gly, whereas a longer term activation seems necessary for the migratory response to G17-Gly. A previous study (
      • Sipeki S.
      • Bander E.
      • Buday L.
      • Farkas G.
      • Bacsy E.
      • Ways D.K.
      • Farago A.
      ) suggested a correlation between the lack of effect of epidermal growth factors on HepG2 cell motility and its ability to induce only a short term increase in the phosphorylation of p42/p44 MAP kinases, whereas factors inducing a scattering of these cells, like hepatocyte growth factor, stimulated MAP kinases for a longer time. However, to our knowledge, this is the first direct demonstration that long term activation of MAP kinases is essential to the migratory response to an exogenous factor.
      Interestingly, the stimulation induced by G17-Gly triggered a delayed increase in the activation of Akt/PKB, which was not detected after stimulation with G17-NH2. This result contradicts recent data (
      • Pece S.
      • Chiariello M.
      • Murga C.
      • Gutkind J.S.
      ) showing the activation of an Akt-dependent anti-apoptotic pathway by the formation of E-cadherin-mediated cell/cell contacts in Madin-Darby canine kidney cells. However, the existence of a similar mechanism has been recently demonstrated by Taupin et al. (
      • Taupin D.R.
      • Kinoshita K.
      • Podolsky D.K.
      ), who showed that the migratory effect of intestinal trefoil factor on intestinal cell lines is coupled to anti-apoptotic signals. It is possible that a balancing mechanism would induce activation of an anti-apoptotic pathway when cells are induced to migrate by an exogenous physiological activator. Such a mechanism would be necessary when cells need to migrate in vivo during the epithelial/mesenchymal transition or during processes leading to mucosal restitution and ulcer repair.
      Our data showed that IMGE-5 cells are sensitive to both amidated and glycine-extended forms of gastrin. To date the only cell line responding to both molecular forms of gastrin is the rat pancreatic carcinoma cell line AR42J (
      • Seva C.
      • Dickinson C.
      • Yamada T.
      ). The ligand selectivity of the G17-Gly receptor identified on IMGE-5 cells was similar to that described previously (
      • Hollande F.
      • Imdhal A.
      • Mantamadiotis T.
      • Ciccotosto G.D.
      • Shulkes A.
      • Baldwin G.S.
      ) both on AR42J and on YAMC cells. Furthermore, the expression of receptors for both G17-Gly and G17-NH2 was correlated to the differentiation status of IMGE-5 cells. Although IMGE-5 cells do not express G/CCK-B receptors under permissive conditions, they still express binding sites for G17-Gly, albeit at a lower density. IMGE-5 could therefore represent a unique tool for the parallel and independent study of biological effects and signal transduction pathways associated with G17-Gly and G17-NH2 activation.
      Finally, our results demonstrating an effect of G17-Gly on the migration of gastric epithelial cells could reflect a potential physiological role for this peptide during ontogeny, in gastroduodenal ulcer disease, or during the progression of carcinomas. Although the available results are still scarce, there seems to be a general tendency toward the expression of partially processed rather than mature forms of gastrin in the early stages of development (at a stage when migration of cells to form the gastric pits is maximal), as well as during carcinoma development (
      • Read M.A.
      • Chick P.
      • Hardy K.J.
      • Shulkes A.
      ,
      • Read M.
      • Shulkes A.
      ,
      • Ciccotosto G.D.
      • Shulkes A.
      ). The role of these partially processed forms in colonic proliferation is well established in vivo (
      • Dockray G.J.
      ,
      • Rozengurt E.G.
      • Walsh J.H.
      ,
      • Wang T.C.
      • Koh T.J.
      • Varro A.
      • Cahill R.J.
      • Dangler C.A.
      • Fox J.G.
      • Dockray G.J.
      ). Furthermore, a correlation could exist between serum concentrations of total progastrin products and the presence of liver metastasis in colorectal cancer (
      • Kameyama M.
      • Fukuda I.
      • Imakoa S.
      • Nakamori S.
      • Iwanaga T.
      ) as well as in patients affected by the rare Zollinger-Ellison syndrome (
      • Jais P.
      • Mignon M.
      • Rehfeld J.F.
      ). Furthermore, antibodies neutralizing both amidated and glycineextended forms of gastrin have been shown to inhibit the spontaneous metastasis of a human colorectal tumor when injected into immunodeficient mice (
      • Watson S.A.
      • Michaeli D.
      • Morris T.M.
      • Clarke P.
      • Varro A.
      • Griffin N.
      • Smith A.
      • Justin T.
      • Hardcastle J.D.
      ). Therefore, the potential role of progastrin-derived peptides on migration needs to be further investigated in other models, in order to assess to what extent the results presented in this paper extend beyond the gastric mucosa and represent a general regulatory mechanism in the gastrointestinal tract.

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