Glycine-extended gastrin stimulates cell proliferation and migration through a Rho- and ROCK-dependent pathway, not a Rac/Cdc42-dependent pathway

Hong He, Julie Pannequin, John-Paul Tantiongco, Arthur Shulkes, and Graham S. Baldwin

Department of Surgery, University of Melbourne, Austin Health, Heidelberg, Victoria 3084, Australia

Submitted 28 January 2005 ; accepted in final form 14 April 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Both amidated gastrin (Gamide) and glycine-extended gastrin (Ggly) stimulate gastrointestinal cell proliferation and migration. Binding of Gamide to the cholecystokinin-2 receptor activates small GTP-binding proteins of the Rho family (Rho, Rac, and Cdc42), and dominant-negative mutants of Rho or Cdc42 block Gamide-stimulated cell proliferation and survival. In comparison, little is known about the Ggly signaling transduction pathway leading to cell proliferation and migration. The present study examined the roles of the small G proteins Rho, Rac, and Cdc42 in Ggly-induced proliferation and migration of the mouse gastric epithelial cell line IMGE-5. Ggly stimulated the activation of Rho and its downstream effector protein ROCK. The activation of Rho and ROCK mediated Ggly-induced cell proliferation and migration as inhibition of Rho by C3, or ROCK by Y-27632, completely blocked these effects of Ggly. Ggly also stimulated tyrosine phosphorylation of focal adhesion kinase, and stimulation was reversed by addition of C3 and Y-27632. In contrast to the effects of Rho and ROCK, inhibition of the Rac or Cdc42 pathways by expression of dominant-negative mutants of Rac or Cdc42 did not affect Ggly-induced cell proliferation and migration. These results demonstrate that Ggly stimulates IMGE-5 cell proliferation and migration through a Rho/ROCK-dependent pathway but not via Rac- or Cdc42-dependent pathways.

G proteins; cytoskeleton; focal adhesion kinase


RHO FAMILY PROTEINS, which include Rho, Rac, and Cdc42, are small monomeric G proteins that cycle between an inactive GDP-bound form and an active GTP-bound form. These proteins regulate the actin cytoskeleton and cell migration and proliferation (8, 23). Rho regulates actin polymerization, resulting in the formation of stress fibers and the assembly of focal adhesions. Rho exerts its distinct actions through interactions with specific target proteins. ROCK, a serine/threonine protein kinase, has been characterized as a downstream effector of Rho and has been implicated in the regulation of cell shape and the dynamic reorganization of cytoskeletal proteins (19, 20, 29) and in the control of transcription factors (57). Classical G protein-coupled receptors couple to Rho through either G{alpha}12 and/or G{alpha}13, which leads to tyrosine phosphorylation of focal adhesion kinase (FAK), Crk-associated substrate, and paxillin (7, 10, 27, 34, 39). Phosphorylation of these proteins is accompanied by changes in the actin cytoskeleton and the assembly of focal adhesions (7, 10, 27, 34), which are involved in cell adhesion, migration, and growth (17, 43, 47).

Rac and Cdc42 induce the formation of filopodia and lamellipodia, respectively, which contribute to the cytoskeletal rearrangements required for cell migration (28, 35, 41). Like Rho, Rac and Cdc42 also act through specific effector proteins such as p21-activated kinase (PAK), which is activated by binding to the GTP-bound form of Rac or Cdc42 (30, 32). Like ROCK, PAK plays important roles in regulating cytoskeleton organization and cell movement (31, 45, 60).

Gastrin, produced by G cells in the gastric antrum, is a circulating hormone that stimulates acid secretion from the gastric parietal cell (55). Gastrin also acts as a potent cell growth factor for normal and malignant gastrointestinal tissues (40, 48). The biological effects of amidated gastrin (Gamide) are mediated by the cholecystokinin (CCK)-2 receptor, a member of the G protein-coupled receptor family (21, 25, 56). Binding of Gamide to the CCK-2 receptor has been reported to activate various intracellular signaling molecules, including ERK, phosphatidylinositol 3-kinase (PI3-K), and c-Fos (5, 50). Recently, the involvement of small GTP-binding proteins including Rho, Rac, and Cdc42 in the growth-stimulating action of Gamide was demonstrated (49, 50). Furthermore, overexpression of the CCK-2 receptor in NIH/3T3 cells induces Rho-dependent actin remodeling (51), and Gamide stimulates plasminogen activator inhibitor-2 both directly and indirectly through Rho family GTPases (54).

Alternative products of progastrin processing, such as glycine-extended gastrin (Ggly) and progastrin itself, are able to induce the proliferation of various cell lines, including the gastric epithelial cell line IMGE-5 (16, 37, 46). Ggly has also been shown to stimulate invasion by the colon cancer cell line LoVo (24) and migration of the gastric epithelial cell line IMGE-5 (15). Unlike Gamide, Ggly does not act through the CCK-2 receptor but instead utilizes a novel receptor that is not sensitive to CCK-2 receptor antagonists (15, 16, 46). Little is known about the structure of this novel receptor, although binding studies have shown the existence of cell surface binding sites for Ggly (15, 16, 46). Recently, we have shown that the biological activity of Ggly is dependent on the presence of ferric ions (37) and that a truncated form of Ggly containing only nine amino acids [LE5AYG, Ggly-(5–13)] also stimulates IMGE-5 cell proliferation and migration in an iron-dependent manner (11). Ggly-induced signal transduction pathways have not been fully defined, although it has been reported that Ggly stimulates c-Jun NH2-terminal kinase activation independently of the MAPK pathway (53) and that PI3-K is involved in Ggly-induced dissociation and migration of IMGE-5 gastric epithelial cells (15). The observation that the signal transduction pathways for Ggly differ from Gamide even within the one cell type (15) further supports the notion of distinct receptors for Ggly and Gamide. Interaction of Ggly with the small GTP-binding proteins has not been reported previously.

In the present study, we determined the role of Rho-, ROCK-, and Rac/Cdc42-dependent pathways in Ggly-induced cell proliferation and migration. We tested the effects of Ggly on Rho/ROCK activities and the ability of C3, a specific inhibitor of Rho, and Y-27632, a specific inhibitor of ROCK, to inhibit cell proliferation and migration induced by Ggly and its truncated form LE5AYG. We have also examined the effects of these two inhibitors on Ggly-stimulated tyrosine phosphorylation of FAK. In parallel, we investigated the role of Rac/Cdc42-dependent pathways in the biological function of Ggly using dominant-negative mutants of Rac or Cdc42. Our data indicate that Ggly stimulates cell proliferation and migration through Rho- and ROCK-dependent pathway(s) by increasing Rho/ROCK activities and that Rac/Cdc42-dependent pathways are not required for these biological functions of Ggly.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cells and reagents. The IMGE-5 cell line was established from the gastric mucosa of mice transgenic for a temperature-sensitive mutant of the SV40 large T antigen as described previously (14). IMGE-5 cells were generally grown at 33°C in DMEM containing 1 U/ml {gamma}-interferon, 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin (permissive conditions). For all experiments, cells were transferred to the same medium without {gamma}-interferon at 39°C (nonpermissive conditions), at which temperature they display differentiated characteristics such as expression of functional adherens and tight junctions. All experiments were performed on cells between passages 20 and 30.

Ggly and LE5AYG were custom synthesized by Auspep (Melbourne, Australia). The ROCK inhibitor Y-27632 was purchased from Calbiochem (Croydon, Australia). The Rho activation assay kit was purchased from Cytoskeleton (Denver, CO). The cDNAs encoding Myc-tagged RacN17 and Cdc42N17 were generously given by Dr. Lewis (Univ. of Nebraska Medical Center, Omaha, NE). The glutathione S-transferase (GST)-PAK fusion protein and the cDNA encoding Clostridium botulinum C3 were gifts from Dr. Hiroshi Maruta (Ludwig Institute for Cancer Research, Melbourne, Australia). The C3 exoenzyme was obtained by cloning the C3 coding sequence in the vector pGEX-2TH. After purification of the GST-C3 exoenzyme fusion protein according to the method described previously (12), the fusion protein was digested with thrombin at 37°C and the free C3 exoenzyme was separated by centrifugation from GST, which remained bound to the glutathione-agarose beads. Anti-ROCK1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Rac1, Cdc42, and phosphorylated FAK (Tyr397) antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-FAK antibody was purchased from Transduction Laboratory (BD Biosciences, North Ryde, Australia).

Cell transfection. We transfected IMGE-5 cells with cDNAs encoding Myc-tagged RacN17 or Cdc42 N17 using Effectene transfection reagent (Qiagen, Melbourne, Australia) according to the manufacturer’s instructions. Stable clones were selected with G418 (800 µg/ml), and expression of the Myc-tagged proteins was confirmed by Western blot with anti-c-Myc antibody (Sigma, St. Louis, MO).

Proliferation assay. Cell proliferation was assayed by [3H]thymidine incorporation. IMGE-5 cells were seeded in a 96-well plate at a density of 2–5 x 103 cells/well in DMEM containing 10% FBS and 1 U/ml {gamma}-interferon and cultured at 33°C. On the following day, the cells were serum starved at 33°C for 24 h. The cells were then treated with full-length or truncated Ggly at the concentrations indicated in the text in the presence or absence of the Rho inhibitor C3 or the ROCK inhibitor Y-27632 in DMEM containing 1% FBS and 10 µCi/ml [methyl-3H]thymidine (Amersham Biosciences, Castle Hill, Australia). The cells were cultured at 39°C for 24 h and then harvested with the use of a Nunc cell harvester (Medos, Mt. Waverly, Australia). The amount of [3H]thymidine incorporated through DNA synthesis was detected with a {beta}-counter (Packard, Meriden, CT). The concentrations of full-length and truncated Ggly chosen were based on a previous study (11). Ggly, at 1 µM, did not compete with CCK-2 receptor binding at all (unpublished results). The concentration of C3 chosen was based on the dose-response study (Fig. 2) and reference to the literature (13, 18).



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Fig. 2. Inhibition of Rho by C3 suppresses both Ggly- and LE5AYG-induced cell proliferation and migration. In proliferation experiments (A and B), IMGE-5 cells were treated with or without 100 nM Ggly (A) or LE5AYG (B) in the presence or absence of different concentrations of C3 for 24 h. Ggly or LE5AYG alone significantly stimulated cell proliferation after 24-h treatment, and C3 suppressed Ggly- or LE5AYG-stimulated cell proliferation in a dose-dependent manner. In migration experiments (C and D), monolayers of IMGE-5 cells were wounded before treatment with or without 100 nM Ggly or LE5AYG in the presence or absence of C3 (5 µg/ml) for 24 h. Cells under different treatments were photographed at 0, 18, and 24 h (C). Wound sizes were expressed as a percentage of the wound size at 0 h (D). Ggly or LE5AYG alone significantly stimulated cell migration after 18 or 24 h of treatment, and C3 suppressed Ggly- or LE5AYG-stimulated cell migration. Statistical significance relative to unstimulated cells (*P < 0.05 and **P < 0.01) or relative to cells stimulated with Ggly (#P < 0.05 and ##P < 0.01) or LE5AYG ($P < 0.05 and $$P < 0.01) alone was determined by one-way ANOVA. Similar results were obtained in 3 separate experiments.

 
Migration experiments. To assess the effects of C3, Y-27632, and the expression of Myc-RacN17 and Myc-Cdc42N17 on Ggly- or LE5AYG-induced cell migration, wound healing experiments were performed as detailed elsewhere (14). Briefly, IMGE-5 cells were grown in 12-well plates in DMEM at 33°C until they reached 80% confluence; cells were then shifted to 39°C and serum starved for 24 h. After the confluent monolayer of cells had been wounded with a 20-µl pipette tip, cells were washed three times with PBS (2.7 mM KCl, 1.5 mM KH2PO4, 142 mM NaCl, and 10 mM Na2HPO4, pH 6.95) and treated with Ggly (100 nM) or LE5AYG (100 nM) in the presence or absence of C3 (5 µg/ml) or Y-27632 (10 µM) in DMEM containing 0.1% FBS. At this lower FBS concentration, Ggly-induced cell proliferation was greatly decreased; therefore, the observed wound closure would mainly result from migration. Morphology and migration of cells were observed and photographed immediately and after treatment for 18 and 24 h. Wound width was measured at six different positions on the photographs, and averages were calculated.

Rho, Rac, and Cdc42 activation assay. IMGE-5 cells were cultured in 15-mm-diameter dishes in DMEM containing 10% FBS and 1 U/ml {gamma}-interferon at 33°C until they reached 80% confluence; cells were then serum starved overnight and treated with Ggly (100 nM) in 1% FBS for the time indicated in the text. After the Ggly treatment, the cells were washed twice with PBS and lysed in cell lysis buffer (50 mM Tris, pH 7.5, 10 mM MgCl2, 0.5 M NaCl, and 1% Triton X-100). The cell lysates were clarified by centrifugation at 8,000 rpm for 5 min at 4°C. The resultant supernatants were incubated with Rhoteckin-RBD beads (GST-fusion protein beads from the Rho activation assay kit; Cytoskeleton) or GST-PAK fusion protein beads for 1 h at 4°C. The beads were washed once with cell lysis buffer, followed by one wash with wash buffer (25 mM Tris, pH 7.5, 30 mM MgCl2, and 0.5 M NaCl). The activated GTP forms of Rho, Rac, and Cdc42 bound to beads and the total Rho, Rac, and Cdc42 in cell lysates were detected by Western blotting using antibodies against Rho (Cytoskeleton), Rac, and Cdc42 (both from Upstate), respectively.

Kinase assays. ROCK kinase activity was determined on immunoprecipitates from cell extracts according to published methods (38). Serum-starved IMGE-5 cells were stimulated with 100 nM Ggly for the periods indicated in the text. The cells were washed twice with cold PBS and disrupted with lysis buffer. The cell lysates were cleared by centrifugation at 10,000 rpm for 10 min at 4°C. The protein concentrations in the supernatant were determined, and equal amounts of proteins were immunoprecipitated with anti-ROCK1 antibody (Santa Cruz) and protein A beads for 2 h at 4°C. The immunoprecipitates were washed twice with lysis buffer followed by two washes with kinase buffers (20 mM Tris·HCl, pH 7.5, 100 mM KCl, 0.1 mM DTT, 5 mM MgCl2, 1 mM EDTA, and 1 µM microcystin-LR). The immunoprecipitates were mixed with 10 µM myosin light chain 20 (MLC20, Sigma) and 1 mM ATP. The reactions were initiated by adding 100 µM [{gamma}-32P]ATP (Amersham Biosciences). After incubation at 30°C for 10 min, the reactions were stopped by the addition of 2x SDS sample buffer. The samples were heated at 95°C and electrophoresed on 15% SDS-polyacrylamide gels. After the gels were fixed and dried, the radioactive phosphorylated MLC20 bands were scanned with a ScanJet 5200C scanner (Hewlett Packard), and the density of each band was analyzed with the Tina 2.0 program (Berthold, Bundoora, Australia).

Western blot analysis. Cell lysates from the different treatments indicated in the text were boiled in SDS sample buffer and then electrophoresed on 10% SDS-polyacrylamide gels. After the proteins had been transferred onto nitrocellulose membranes, the membranes were blocked in 5% skim milk in PBST (0.1% Tween 20 in PBS) for 1 h at room temperature. Immunological blots were then performed overnight at 4°C in 1% BSA-PBST buffer containing antibodies specific for tyrosine-phosphorylated FAK, FAK, or ROCK1. After membranes were washed with PBST, they were incubated with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies (Bio-Rad, Regents Park, Australia). The bound antibodies were visualized using enhanced chemiluminescence reagents (Amersham Biosciences).

Statistical analysis. All values are expressed as means ± SE. Results were analyzed by one-way ANOVA. If there was a statistically significant difference in the data set, individual values were compared by Bonferroni’s t-test with the unstimulated control or with the values obtained in the presence of Ggly or LE5AYG, as appropriate. Differences between two means with P < 0.05 were considered significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previously, we reported that both Ggly and its truncated form LE5AYG stimulated cell proliferation and migration (11, 15). Because the Rho family G proteins (Rho, Rac, and Cdc42) regulate many cellular functions such as proliferation, migration, and growth through the activation of numerous signaling pathways (8, 19, 20, 23, 28, 29, 35, 41), the effects of Rho, Rac, and Cdc42 on Ggly- and LE5AYG-stimulated cell proliferation and migration were investigated.

Ggly increases Rho and ROCK activity in gastric epithelial cells. To determine whether Ggly could stimulate Rho or ROCK activity in gastric epithelial cells, the intracellular concentration of the active GTP-bound form of Rho was measured in the presence or absence of Ggly (100 nM). Ggly significantly increased Rho activation after stimulation of the cells for 5–15 min (Fig. 1A). Similarly, Ggly significantly increased ROCK kinase activity after stimulation of the cells for the same time (Fig. 1B). Ggly did not change the total concentrations of the Rho or ROCK proteins. These results demonstrated that Ggly significantly stimulates Rho activation and ROCK kinase activity.



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Fig. 1. Glycine-extended gastrin (Ggly) stimulates activation of Rho and ROCK kinase. After serum starvation, IMGE-5 cells were stimulated with or without 100 nM Ggly for 0, 5, 10, 15, 30, and 60 min. Activation of Rho (A) and ROCK kinase (B) was determined as described in MATERIALS AND METHODS by measurement of the GTP-bound form of Rho and phosphorylation of myosin light-chain kinase (MLC), respectively. The relative Rho or ROCK activation was calculated in arbitrary units, taking density of the unstimulated control as 1. Ggly significantly increased activation of both Rho and ROCK kinase after 5–15 min of stimulation. Data are means ± SE from 3 independent experiments. Cont, control. Statistical significance relative to the unstimulated control (*P < 0.05) was determined by one-way ANOVA.

 
Involvement of Rho in Ggly- and LE5AYG-induced cell proliferation and migration. The involvement of Rho in Ggly- or LE5AYG-induced cell proliferation was determined using a specific inhibitor of Rho, the C3 exoenzyme. C3 significantly inhibited Ggly-induced cell proliferation in a dose-dependent manner (Fig. 2A). C3 alone had no effect on cell proliferation (data not shown). Similar results were obtained when the effect of C3 on LE5AYG-induced cell proliferation was determined (Fig. 2B). The effect of Rho on Ggly- or LE5AYG- induced cell migration was also investigated using a wound healing assay, in which the closure of wounds in IMGE-5 cell monolayers maintained in serum-free medium was measured. In the presence of either Ggly (100 nM, Fig. 2C) or LE5AYG (100 nM, photographs not shown), wound closure was accelerated compared with control, leading to 80% closure by 24 h (Fig. 2D). The Rho-specific inhibitor C3, at 5 µg/ml, significantly slowed the wound-healing process induced by both Ggly (Fig. 2, C and D) or LE5AYG (Fig. 2D). The results indicate that activation of Rho is required for stimulation of cell proliferation and migration by either Ggly or its truncated analog LE5AYG.

Effects of the ROCK inhibitor Y-27632 on Ggly- and LE5AYG-induced cell proliferation and migration. ROCK, one of the downstream target proteins of Rho, mediates Rho-induced changes in the cytoskeleton and cell adhesion and migration (19, 20, 29). To elucidate the role of ROCK activation in Ggly- or LE5AYG-induced cell proliferation and migration, the effect of the ROCK inhibitor Y-27632 on the stimulation of cell proliferation and migration by Ggly or LE5AYG was investigated. Y-27632 significantly inhibited Ggly-induced cell proliferation in a dose-dependent manner (Fig. 3A). At 1 µM, Y-27632 completely blocked cell proliferation induced by Ggly. Similar results were observed when the effect of Y-27632 on LE5AYG-induced cell proliferation was tested, except that Y-27632 completely blocked LE5AYG-induced cell proliferation at a lower concentration (100 nM) (Fig. 3B). In the wound healing assay, both Ggly (Fig. 3C) and LE5AYG (photographs not shown) significantly reduced the wound sizes after 18 h of treatment (Fig. 3, D and E), and there were further decrements in the wound sizes after 24 h of treatment. Y-27632 decelerated the stimulation of wound closure by Ggly and LE5AYG, leaving significantly wider wounds at 18 and 24 h (Fig. 3, CE). These results suggest that a ROCK-dependent pathway is involved in Ggly-induced cell proliferation and migration.



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Fig. 3. Inhibition of ROCK by Y-27632 suppresses both Ggly- and LE5AYG-induced cell proliferation and migration. In proliferation experiments (A and B), IMGE-5 cells were treated with or without 100 nM Ggly (A) or LE5AYG (B) in the presence or absence of different concentrations of the ROCK inhibitor Y-27632 for 24 h. Both Ggly and LE5AYG stimulated IMGE-5 cell proliferation, and Y-27632 inhibited Ggly- and LE5AYG-induced cell proliferation in a dose-dependent manner. In migration experiments (CE), monolayers of IMGE-5 cells were wounded before treatment with or without Ggly (100 nM) or LE5AYG (100 nM) in the presence or absence of Y-27632 (10 µM) for 24 h. Cells under different treatments were photographed at 0, 18, and 24 h (C). Wound sizes were expressed as a percentage of the wound size at 0 h (D and E). Ggly or LE5AYG alone significantly stimulated cell migration after 18 or 24 h of treatment, and Y-27632 suppressed Ggly- or LE5AYG-stimulated cell migration. Statistical significance relative to the control value (*P < 0.05, **P < 0.01) or relative to cells stimulated with Ggly or LE5AYG alone (#P < 0.05 and ##P < 0.01) was determined by one-way ANOVA. Similar results were obtained in 3 separate experiments.

 
Role of Rac and Cdc42 in Ggly- and LE5AYG-stimulated cell proliferation and migration. To determine whether Ggly could stimulate Rac or Cdc42 activity in gastric epithelial cells, the intracellular concentrations of the active, GTP-bound forms of Rac and Cdc42 were measured in the presence or absence of Ggly (100 nM). Ggly did not significantly affect Rac or Cdc42 activation (Fig. 4A). To confirm that Rac and Cdc42 did not play a role in Ggly- or LE5AYG-stimulated cell proliferation and migration, dominant-negative mutants of RacN17 and Cdc42N17 were expressed in IMGE-5 cells. As shown in Fig. 4B, the expression of the dominant-negative mutants of RacN17 and Cdc42N17 blocked the activation of endogenous Rac and Cdc42. However, in the cells transfected with either RacN17 (Fig. 5A) or Cdc42N17 (Fig. 5B), Ggly (100 nM) stimulated cell proliferation to a similar degree to that shown in parental cells, when assayed by [3H]thymidine incorporation. Similar results were obtained when the cells were stimulated with LE5AYG. Likewise, stimulation of cell migration by either Ggly or its truncated form LE5AYG was not affected by the expression of dominant-negative mutants of Rac or Cdc42 (Fig. 6). The results indicate that activation of Rac and Cdc42 is not essential for stimulation of cell proliferation and migration by either Ggly or its truncated analog LE5AYG.



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Fig. 4. Activation of Rac and Cdc42 is unaffected by Ggly but is blocked by expression of Myc-RacN17 and Myc-Cdc42N17. A: after serum starvation, IMGE-5 cells were stimulated with or without Ggly (100 nM) for 0, 5, 10, 15, and 30 min. Activation of Rac and Cdc42 was determined as described in MATERIALS AND METHODS by measurement of the GTP-bound forms of Rac and Cdc42, respectively. Similar results were obtained from 3 individual experiments. B: activation of Rac and Cdc42 in parental and Myc-RacN17 and Myc-Cdc42N17 transfected cells was determined as described in MATERIALS AND METHODS. Lanes 1 and 4 represent samples from parental cells; lanes 2 and 3 represent samples from clones 2 and 11 of Myc-RacN17 transfected cells; and lanes 5 and 6 represent samples from clones 10 and 29 of Myc-Cdc42N17 transfected cells. Concentrations of the GTP-bound forms of Rac and Cdc42 were reduced in 2 independently isolated clones of either Myc-RacN17 or Myc-Cdc42N17 transfected cells, respectively. Similar results were obtained in at least 2 other experiments.

 


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Fig. 5. Neither Myc-RacN17 nor Myc-Cdc42N17 expression blocks the cell proliferation induced by Ggly or LE5AYG. Cell proliferation was measured by [3H]thymidine incorporation as described in MATERIALS AND METHODS. Both Ggly and LE5AYG stimulated cell proliferation in 2 independently isolated clones of either Myc-RacN17 transfected cells (A) or Myc-Cdc42N17 transfected cells (B) to a similar degree as in parental cells. Data are means from 3 separate sets of experiments, each in triplicate. Statistical significance relative to unstimulated cells was determined by one-way ANOVA (*P < 0.05 and **P < 0.01).

 


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Fig. 6. Neither Myc-RacN17 nor Myc-Cdc42N17 expression blocks the cell migration induced by Ggly and LE5AYG. Wound healing experiments were done in parental and in Myc-RacN17 and Myc-Cdc42N17 transfected cells as described in MATERIALS AND METHODS. Wound sizes were expressed as a percentage of the wound size at 0 h. Both Ggly and LE5AYG stimulated cell migration in either Myc-RacN17 or Myc-Cdc42N17 transfected cells to a similar degree as in parental cells. Statistical significance relative to the control value at 18 h (**P < 0.01) or 24 h (#P < 0.05 and ##P < 0.01) was determined by one-way ANOVA. Similar results were obtained in 3 separate experiments.

 
Ggly stimulated tyrosine phosphorylation of FAK in a Rho- and ROCK-dependent manner. Activation of Rho induces the formation of actomyosin stress fibers and focal adhesions, causing tyrosine phosphorylation of the focal adhesion proteins paxillin and FAK (1, 4, 41). In particular, Gamide and CCK induce Rho-dependent actin remodeling and coordinate tyrosine phosphorylation of FAK (7, 10, 27, 39, 51). To establish whether Ggly induced tyrosine phosphorylation of FAK, IMGE-5 cells were stimulated with Ggly or LE5AYG, and concentrations of phosphorylated and total FAK in cell extracts were determined by Western blotting. Both Ggly and LE5AYG (Fig. 7) stimulated the tyrosine phosphorylation of FAK. To ascertain the involvement of Rho and ROCK in the Ggly-induced tyrosine phosphorylation of FAK, IMGE-5 cells were stimulated with Ggly or LE5AYG for 15 min in the presence or absence of the Rho inhibitor C3 or the ROCK inhibitor Y-27632. C3 (Fig. 7A) or Y-27632 (Fig. 7B) reduced Ggly- or LE5AYG-induced tyrosine phosphorylation of FAK to control levels.



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Fig. 7. Stimulation of tyrosine phosphorylation of focal adhesion kinase (FAK) by Ggly and LE5AYG is Rho/ROCK-dependent. After serum starvation, IMGE-5 cells were treated with or without 100 nM Ggly or LE5AYG in the presence or absence of 5 µg/ml C3 (A) or 10 µM Y-27632 (B) for 15 min. The concentrations of tyrosine-phosphorylated FAK (pFAK) and total FAK (FAK) were determined by Western blotting, as described in MATERIALS AND METHODS. Relative amount of pFAK was calculated in arbitrary units, taking density of the unstimulated control as 1. Data are means ± SE from 3 independent experiments. Both Ggly and LE5AYG stimulated the tyrosine phosphorylation of FAK, and the stimulation was inhibited by either C3 or Y-27632. Statistical significance relative to the unstimulated control (*P < 0.05) or relative to cells stimulated with Ggly (#P < 0.05) or LE5AYG ($P < 0.05) alone was determined by one-way ANOVA.

 
Stimulation of FAK phosphorylation by Ggly requires ferric ions. We have previously demonstrated that the stimulation of IMGE-5 cell proliferation and migration by Ggly (37) and LE5AYG (11) is dependent on the presence of ferric ions. To confirm that ferric ions were also essential for Ggly-stimulated tyrosine phosphorylation of FAK, IMGE-5 cells were treated with or without Ggly (100 nM) in the presence or absence of the iron chelator desferrioxamine (DFO). The observation that Ggly stimulation of FAK tyrosine phosphorylation was blocked by DFO (Fig. 8A) indicated that ferric ions were required for this activity.



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Fig. 8. Stimulation of tyrosine phosphorylation of FAK by Ggly requires ferric ions. After serum starvation, IMGE-5 cells were treated with either 100 nM Ggly in the presence or absence of 1 µM desferrioxamine (DFO; A) or different truncated forms of Ggly (B), each at a concentration of 100 nM. Concentrations of pFAK and total FAK were detected by Western blotting as described in MATERIALS AND METHODS. Relative amounts of tyrosine-phosphorylated FAK were calculated in arbitrary units, taking density of the unstimulated control as 1. Data are means ± SE from 3 independent experiments. Observations that the chelating agent DFO blocked Ggly-stimulated FAK phosphorylation (A) and that Ggly fragments without the polyglutamate sequence were inactive (B) indicated that ferric ions were essential for Ggly-stimulated tyrosine phosphorylation of FAK. Statistical significance relative to the unstimulated control (**P < 0.01) or relative to cells stimulated with Ggly (#P < 0.05) was determined by one-way ANOVA.

 
The observation that the nonapeptide LE5AYG is as active as Ggly in stimulation of FAK phosphorylation (Fig. 7) suggested that the NH2 and COOH termini of Ggly were not required for this activity. The NH2- and COOH-terminal peptides of Ggly, Ggly-(1–4) and Ggly-(12–18), were therefore tested as stimulants of tyrosine phosphorylation of FAK (Fig. 8B). The fact that Ggly-(1–4) and Ggly-(12–18) were completely inactive is consistent with our previous conclusion based on cell proliferation and migration data: binding of ferric ions to the internal fragment LE5AYG [Ggly-(5–13)] is responsible for the biological activity of Ggly (11).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The intracellular signal transduction pathways activated by gastrin to induce cell proliferation and apoptosis have been the focus of numerous investigations (9, 15, 49, 50, 52, 54, 58). Most studies have been performed with Gamide, which executes its biological functions via the CCK-2 receptor (21, 25, 56). Gamide induces protein tyrosine kinase activity, activates the ERKs and MAPKs and PI3-K (6, 26, 49, 50, 52, 59), and promotes cell growth and survival through ERK- and PI3-K-dependent pathway(s) (5, 48, 50). Recent evidence has shown that Gamide stimulates cell growth and survival through the activation of multiple small GTP-binding proteins (49).

In contrast to Gamide, the signaling pathway involved in Ggly-induced cell proliferation and migration is poorly understood; so far, no receptor for Ggly has been characterized fully. Little is known about the role in Ggly signaling of small GTP-binding proteins, such as Rho family G proteins, although it is well established that Rho, Rac, and Cdc42 play important parts in cell growth, adhesion, and migration (8, 23, 28, 35, 41). In the present study, we demonstrated that Ggly induces cell proliferation and migration through a Rho- and ROCK-dependent pathway by activation of Rho and its downstream target ROCK kinase. Inhibition of Rho by C3, or of ROCK by Y-27632, completely blocked cell proliferation and migration induced by Ggly and its truncated form, LE5AYG. These results are similar to the report that Gamide activates Rho and that a dominant-negative mutant of Rho completely inhibited Gamide-stimulated cell proliferation and survival (49).

In many systems, activation of Rho induces the formation of actomyosin stress fibers and focal adhesions, causing tyrosine phosphorylation of the focal adhesion proteins paxillin and FAK (1, 4, 41). Our data clearly demonstrate that Ggly and LE5AYG are able to stimulate tyrosine phosphorylation of FAK and that stimulation is blocked by the Rho inhibitor C3 and the ROCK inhibitor Y-27632 (Fig. 7). The inhibition by C3 and Y-27632 of Ggly-induced tyrosine phosphorylation of FAK has further confirmed the involvement of Rho and ROCK in Ggly-induced cell migration and growth, as FAK forms a part of the focal adhesion complexes involved in cell adhesion, migration, and growth (17, 43, 47). Stimulation by Gamide of the CCK-2 receptor in NIH/3T3 cells into which the CCK-2 receptor had been introduced by transfection also induces Rho-dependent actin remodeling and tyrosine phosphorylation of FAK coordinately (51). Our results are consistent with the existence of a common Rho-dependent mechanism for stimulation of FAK phosphorylation by both Gamide and Ggly.

Many reports have shown that the Rho- and ROCK-dependent pathway is required for epithelial cell proliferation and migration (33, 36, 38, 42, 49). Activated Rho induces the kinase activity of ROCK, which in turn increases MLC phosphorylation, resulting in stimulation of stress fiber formation and cell migration during intestinal epithelial restitution (38). Interestingly, the Rho/ROCK pathway has also been involved in gastrin-releasing peptide-stimulated invasion and migration of Isrecol colon carcinoma cells (42). However, the involvement of the Rho/ROCK pathway in Ggly-stimulated proliferation and migration and the demonstration of activation of Rho and ROCK by Ggly have not been described previously. Ggly stimulates the growth of human gastric cancer cells through a receptor other than the CCK-1 or CCK-2 receptor (15, 16, 22, 37, 46). Ggly also stimulates colon cancer cell invasion (24) by increasing the expression of certain matrix metalloproteinases (2), but whether this is through a Rho/ROCK pathway remains to be determined. We cannot tell at this time which effector protein transmits the Ggly signal downstream of the Rho/ROCK pathway. Potential candidate Rho/ROCK effectors include Dia proteins, MLC kinase, lin-11 isl-1 mec-3 kinase, and proteins of the ezrin-radixin-moesin family or phospholipid metabolizing kinases (3, 44). Additional experiments are needed to understand the role of these proteins in Ggly-induced cell proliferation and migration. Similarly, the mechanism by which Ggly activates Rho and ROCK is still to be determined.

In contrast to the importance of Rho/ROCK in mediating the biological activity of Ggly, neither Rac nor Cdc42 is required for Ggly- or LE5AYG-induced cell proliferation and migration. The dominant-negative mutants of Rac and Cdc42 had no significant effect on cell proliferation (Fig. 5) and migration (Fig. 6) induced by Ggly or LE5AYG. Our findings with Ggly are different from a previous report that showed that Gamide can activate both Rac and Cdc42 in the rat pancreatic acinar cell line AR42J (49). Although a dominant-negative mutant of Rac had no effect on cell proliferation and survival induced by Gamide, expression of a dominant-negative mutant of Cdc42 completely blocked Gamide-stimulated cell proliferation and partially inhibited Gamide-induced cell survival (49). Although these studies were performed in different cell lines, the differences between our findings with Ggly and the previous report on Gamide are consistent with previous reports that Ggly and Gamide induce cell proliferation and migration through different cell surface receptors and through different postreceptor signal transduction pathways (5, 15, 16, 46, 49, 50).

The results in the present study are also consistent with our previous conclusion that binding of ferric ions to glutamates 7, 8, and 9 of Ggly is essential for biological activity (11, 37). First, stimulation of tyrosine phosphorylation of FAK by Ggly is abolished by the removal of ferric ions with the chelating agent DFO (Fig. 8A). Second, the observations (Fig. 8B) that Ggly-(5–18) retained full biological activity, that Ggly-(1–11) was partially active, and that Ggly-(1–4) and Ggly-(12–18) were inactive in the FAK phosphorylation assay confirm the importance of the midportion of Ggly containing the glutamates in biological activity (37). Third, the biological activity of LE5AYG in the proliferation and wound healing assays described in the present paper was similar to the activity of Ggly. All of the available data indicate that the complex between Ggly and ferric ions is responsible for the biological effects of the peptide.

In conclusion, Ggly significantly stimulates IMGE-5 cell proliferation and migration through a Rho- and ROCK-dependent pathway by increasing the activation of Rho and ROCK kinase activity. In contrast, the small GTP-binding proteins Rac and Cdc42 are not involved in the Ggly pathway. This knowledge of Ggly postreceptor signaling will facilitate identification and characterization of the Ggly receptor. The present study also provides new insights into the mechanisms responsible for the growth-promoting action of Ggly on both normal and neoplastic gastrointestinal tissues and may help in developing novel therapies to prevent or treat colon cancer.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by National Health and Medical Research Council of Australia Grants 114123 (to A. Shulkes), 208926 (to G. S. Baldwin), and 251581 (to G. S. Baldwin), National Institutes of Health Grant GM-65926 (to G. S. Baldwin and A. Shulkes), and by the Austin Hospital Medical Research Foundation (to G. S. Baldwin and A. Shulkes).


    ACKNOWLEDGMENTS
 
The authors thank D. Anthony for preparing the plasmid DNAs of Myc-RacN17 and Myc-Cdc42N17. We also thank H. Maruta and T. Nhue for the GST-PAK1 RBD fusion protein.


    FOOTNOTES
 

Address for reprint requests and other correspondence: H. He, Dept. of Surgery, Univ. of Melbourne, Austin Health, Studley Rd., Heidelberg, Victoria 3084, Australia (e-mail: hong.he{at}unimelb.edu.au)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
 GRANTS
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