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The Journal of Biological Chemistry logoLink to The Journal of Biological Chemistry
. 2012 Oct 10;287(49):41458–41468. doi: 10.1074/jbc.M112.382465

Identification of the F1-ATPase at the Cell Surface of Colonic Epithelial Cells

ROLE IN MEDIATING CELL PROLIFERATION*

Aline Kowalski-Chauvel ‡, Souad Najib ‡, Irina G Tikhonova §, Laurence Huc , Fredéric Lopez ‡, Laurent O Martinez ‖, Elizabeth Cohen-Jonathan-Moyal ‡,**, Audrey Ferrand ‡, Catherine Seva ‡,1
PMCID: PMC3510843  PMID: 23055519

Background: The F1 domain of F1Fo-ATPase was initially believed to be strictly expressed in the mitochondrial membrane.

Results: F1-ATPase is present at the cell surface of colonic epithelial cells and serves as a receptor for a gastrointestinal peptide mediating cell growth.

Conclusion: We identified a new localization and a new function for the F1-ATPase in colonic cells.

Significance: Cell surface F1-ATPase represents a new target in anti-proliferative strategies.

Keywords: Cell Proliferation, Colorectal Cancer, Epithelial Cell, F1Fo ATPase, Peptides, Colonic Epithelial Cells

Abstract

F1 domain of F1Fo-ATPase was initially believed to be strictly expressed in the mitochondrial membrane. Interestingly, recent reports have shown that the F1 complex can serve as a cell surface receptor for apparently unrelated ligands. Here we show for the first time the presence of the F1-ATPase at the cell surface of normal or cancerous colonic epithelial cells. Using surface plasmon resonance technology and mass spectrometry, we identified a peptide hormone product of the gastrin gene (glycine-extended gastrin (G-gly)) as a new ligand for the F1-ATPase. By molecular modeling, we identified the motif in the peptide sequence (E(E/D)XY), that directly interacts with the F1-ATPase and the amino acids in the F1-ATPase that bind this motif. Replacement of the Glu-9 residue by an alanine in the E(E/D)XY motif resulted in a strong decrease of G-gly binding to the F1-ATPase and the loss of its biological activity. In addition we demonstrated that F1-ATPase mediates the growth effects of the peptide. Indeed, blocking F1-ATPase activity decreases G-gly-induced cell growth. The mechanism likely involves ADP production by the membrane F1-ATPase, which is induced by G-gly. These results suggest an important contribution of cell surface F1-ATPase in the pro-proliferative action of this gastrointestinal peptide.

Introduction

F1Fo ATPase is a multisubunit transmembrane enzyme that was initially localized in the inner side of the mitochondrial membrane. Interestingly, it is now recognized that the F1 complex, composed by α,β,γ,δ,ϵ subunits, that contains the catalytic site for ATP synthesis or hydrolysis is expressed at the plasma membrane of different cell types and represents a cell surface receptor for apparently unrelated ligands (1). In particular, F1-ATPase at the cell surface of human umbilical vascular endothelial cells (HUVEC)2 or hepatocarcinoma and lung cancer cells has been described as a receptor for angiostatin, a proteolytic fragment of plasminogen. In these cells, angiostatin binds to the cell surface F1-ATPase and inhibits ATP production, leading to cell death, anti-tumorigenic, or anti-angiogenic effects (2–5). Endothelial cell surface F1-ATPase also serves as a receptor for apolipoprotein AI (apoAI). This lipoprotein stimulates cell surface F1-ATPase activity, leading to ADP production and an increase in proliferation of HUVEC (6). In addition, apoA-I interacts with the F1-ATPase at the cell surface of hepatocytes playing a role in HDL endocytosis (7). In the present study using immunofluorescence approaches and confocal microscopy, we demonstrate for the first time the presence of the F1-ATPase at the cell surface of colonic epithelial cells. More interestingly, using surface plasmon resonance (SPR) technology coupled to mass spectrometry we have identified a new ligand of the F1-ATPase, a peptide hormone product of the gastrin gene, the glycine-extended form of gastrin (G-gly). G-gly is the first gastrin precursor for which growth factor properties were demonstrated (8). Subsequently, numerous publications have reported the proliferative effects of G-gly on normal and cancerous gastrointestinal cells in vitro and in vivo (9–18). In addition, G-gly induces tubule formation by human vascular endothelial cells in vitro in a similar manner to VEGF, suggesting a potential proangiogenic role for this peptide (19). So far, the membrane receptor for G-gly has not been identified. This gastrin precursor does not bind the receptor specific for the mature form of the hormone, and several publications show that G-gly proliferative effects are independent from the gastrin receptor (8, 11, 20). However, we and others have previously demonstrated the presence of high affinity G-gly binding sites on gastrointestinal cells, suggesting the existence of G-gly receptors at the cell surface (8, 11). Here by molecular modeling and validation of this model with mutagenesis and biological analysis, we have characterized the molecular interaction between G-gly and the F1-ATPase. We have also identified the function of the F1-ATPase activated by G-gly at the cell surface of colonic epithelial cells.

EXPERIMENTAL PROCEDURES

Cell Culture

HCT116, HT29, and CACO-2 cells were obtained from the American Type Culture Collection (LGC Standards), HUVEC were from Millipore (Tebu Bio), and YAMC cells were kindly provided by Robert H. Whitehead (Vanderbilt University Medical Center, Nashville, TN) and grown as previously described (4, 13, 45).

Purified Membranes

Cells were scraped and lysed in a phosphate buffer, pH 7.4, through freeze/thaw cycles. After centrifugation for 15 min at 1000 rpm, the pellet was homogenized in 0.3 m sucrose then centrifuged for 2 h 15 min at 27,000 rpm in 10 ml of 1.63 m sucrose (final sucrose molarity, 1.56). The purified plasma membrane were collected at the interface of the sucrose gradient and diluted in 1 ml of a Tris buffer (20 mm, pH 7.4, supplemented by soybean trypsin inhibitor (0.1 mg/ml). The proteins concentration was determined by BCA assay kit (Pierce).

F1-ATPase and IF1 Preparation

F1-ATPase was purified from bovine heart mitochondria as previously described (21) and kindly provided by J. Walker (MRC Mitochondrial Biology Unit, Cambridge, UK). The plasmid encoding the bovine mutated form IF1 (IF1-H49K, histidine 49 switched to lysine) was kindly provided by J. Walker, and protein expression and purification were carried out as previously described (22).

Surface Plasmon Resonance Assays

Studies based on SPR technology were performed on a BIAcore 3000 optical biosensor instrument (BIAcore AB, Uppsala, Sweden). Immobilization of biotinylated peptides was performed on a streptavidin-coated sensorchip in HBS-EP buffer (10 mm Hepes, pH 7.4, 150 mm NaCl, 3 mm EDTA, 0.005% surfactant P20). All immobilization steps were performed at a final peptide concentration of 50 ng/ml (flow rate 10 μl/min). Injections were stopped when a level of 350 RU was obtained. A channel was left empty and used as a reference surface for nonspecific binding measurements. The analyte was injected over the immobilized peptides for 4 min (flow rate 30 μl/min) in a K-Inject mode. Kinetics constants (Ka and Kd) were calculated using BIAevaluation 4.0.1 software. Kd was calculated as the ratio of Kd/Ka. For recovery strategy, biotinylated G-Gly peptide was immobilized on 3 channels of a SA sensorchip at a level of 900 RU per channel with an empty channel as reference surface. Solubilized membrane proteins were injected for 6 min (20 μl/min flow rate). Four cycles of recovery with a 50 mm triethylammonium, 0.5 m urea solution were performed to obtain 27,000 RU of recovered proteins corresponding to 27 ng of proteins. Recovered proteins were subjected to mass spectrometry analysis for identification (NanoLC/ESI QStar MS/MS, IPBS Toulouse Genopole).

Molecular Modeling

The sequence of G-gly was aligned to several species of IF1 using the ClustalX program (40) and adapted for the figure using the GeneDoc program. The amino acid sequence files with the code P01096, Q9UII2, Q03344, O35143, P01097, and P09940 were downloaded from the Expasy server. The secondary structure and three-dimensional model of G-gly are predicted using the PEP-FOLD server (26). The model of G-gly was rigidly docked to the crystal structure of F1-ATPase (PDB code 2V7Q) using the Glide module of Schrodinger software (Glide, Version 5.5, Schrödinger, LLC, New York, 2009). The box was centered based on the position of IF1 in the crystal structure of 2V7Q, and the size of the docking box, 30 × 30 × 30, was used. The SP scoring function of Glide was chosen, and 40 poses were generated. The selected docking pose was chosen for the optimization of the peptide-enzyme interactions using 500-ps molecular dynamics simulations in implicit solvent using the Schrodinger software. The picture was prepared in the Schrodinger software (Maestro, Version 9.1, Schrödinger, LLC, New York, 2010).

Immunofluorescence and Confocal Microscopy

CACO-2 cells were plated on Transwell-ClearTM filters (Corning, Costar) at high density to obtain confluence in 5–8 days. 8–10 days post-confluent CACO-2 cells (to obtain well polarized cells) were fixed and permeabilized using standard methods. For all other lines, cells were grown in Lab-Tek chambered cover-glass, and immuno-stainings were performed on intact cells, non-fixed, and non-permeabilized using standard methods. Antibodies used were: anti-β-ATPase (Molecular Probes), anti-EGF receptor and anti-E-cadherin (Cell Signaling), and DPP-IV (R&D Systems). When mentioned, cells were incubated with 10 nm MitoTracker Red (Molecular Probes) for 15 min at 37 °C. After washes in PBS, the cells were visualized by confocal microscopy. For immunostaining on the formaldehyde-fixed, paraffin-embedded tissues (colonic tissue of mouse), heat-induced epitope retrieval was performed in 10 mm citrate buffer, and primary antibodies were applied overnight (anti-β-ATPase from Molecular Probes and anti E-cadherin from Cell Signaling). The detection was done using Alexa Fluor 488 goat anti-mouse antibody and Alexa Fluor 647 donkey anti-rabbit (Molecular Probes). Control slides, where the primary antibody was replaced by non-immune IgG, were checked for nonspecific reactivity. Slides were analyzed by confocal microscopy (magnificence ×40).

ADP Generation Assay

HUVEC in 6-well plates were washed and incubated for 1 h at 37 °C in serum-free medium, then [α-32P]ATP (0.5 μCi/well) (PerkinElmer Life Sciences) was added for 10 min at 37 °C with or without treatments. Nucleotides were then separated by HPLC and quantified as previously described (12).

Measurements of pHi by Flow Cytometry

The pHi was monitored using the pH-sensitive fluorescent probe carboxy-SNARF-1 (Invitrogen) and flow cytometry analysis. Cells were loaded with SNARF by incubating them in a 5 μm solution for 20 min at 37 °C in cell suspension buffer (CSB; 124.8 mm NaCl, 4.7 mm KCl, 1.2 mm KH2PO4, 10 mm HEPES, pH 7.4, 1 mm MgCl2, 1 mm CaCl2, 10 mm glucose). After trypsinization and resuspension in CSB, cells were stimulated with G-gly. The mean fluorescence intensity of 10,000 cells was determined after 10 min of treatment on MACSQuant analyzer (Miltenyi Biotec, Bergish Gladbach, Germany) (excitation 488 nm; emissions B2 channel, 585 nm, and B3 channel, 655 nm). The emission ratio 585/655 was then converted into pH value by using the calibration curve obtained in situ on control cells exposed to calibration buffers containing 10 μm nigericin (135 mm KCl, 0.83 mm MgSO4, 1.26 mm CaCl2, 1.05 mm MgCl2, 10 mm glucose, and 10 mm MES, pH 5.5, 10 mm HEPES, pH 7.4, or 10 mm CAPSO, pH 8.7) (23). The dimethyl amiloride (Sigma), an inhibitor of the Na+/H+ exchanger, was used as an acidifying positive control (24).

Proliferation Assays

Proliferation analyses were carried out by using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay (Sigma) as previously described (13).

Statistical Analysis

Means ± S.E. and Student's t tests were performed using Excel. ***, p < 0.001; **, 0.001<p < 0.01; *, 0.01<p < 0.05; not significant (ns), p > 0.05.

RESULTS

Identification of the Plasma Membrane F1Fo-ATPase as a Potential Binding Protein for G-gly

We previously reported G-gly proliferative effects on the human colon cancer cells HCT116 (9). Here we have purified plasma membranes from the HCTT16 cells. Solubilized membrane proteins were then tested for their interaction with G-gly by SPR (BIAcore 3000) using biotinylated G-gly immobilized on the BIAcore sensor chip. Biotin alone was coated as a control. As shown by the sensogram (Fig. 1), we observed a remarkable increase in the interaction with biotinylated G-gly compare with biotin alone indicating the presence of potential G-gly-binding proteins in the purified plasma membranes of HCT116 cells. After a recovery procedure, membrane proteins bound to G-gly were analyzed by mass spectrometry (nanoLC/ESI QStar MS/MS), and we identified, using the Mascot search engine, different subunits that compose the F1 complex of the F1Fo-ATPase (Table 1).

FIGURE 1.

FIGURE 1.

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Surface plasmon resonance analysis of the interaction of biotinylated G-gly (Biot-G-gly) or biotin alone (Biot) immobilized on a sensor chip with solubilized plasma membrane proteins from colon cancer cells HCT116. Sensorgrams represent the RU as a function of time. Results are representative of three independent experiments.

TABLE 1.

Identification by mass spectrometry (nanoLC/ESI QStar MS/MS) after micro-recovery of the solubilized plasma membrane proteins bound to G-gly immobilized on a Biacore sensor chip

MW, molecular weight; pI, isoelectric point.

Protein ID Score MW pI % Covered Peptides
ATP-synthase -α-chain 235 58,790 9.22 27 12
ATP-synthase -δ-chain 167 23,383 10.03 22 6
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Because several publications have demonstrated that binding of two ferric ions to G-gly is essential for the biological activity of the peptide (25, 26), we analyzed the interaction between the solubilized membrane proteins and G-gly employing SPR in the presence or absence of desferrioxamine (DFO), a ferric ions chelator, which is known to inhibit the proliferative effect of G-gly in the HCT116 cells (9). As expected, the sensogram in Fig. 2A shows a decrease (42%) of the G-gly binding to the solubilized proteins in the presence of DFO, suggesting a role of ferric ions in the interaction of G-gly to its potential binding proteins. Eluted proteins were resolved by SDS-PAGE and silver-stained (Fig. 2B). Interestingly, staining of the bands around 50 kDa was decreased by the pretreatment with DFO, indicating a decrease in the interaction of G-gly with these proteins. We specifically cut the corresponding bands from the gel and identified the proteins by mass spectrometry. The α- and β-subunits of the F1-ATPase were clearly identified with a good score (Table 2).

FIGURE 2.

FIGURE 2.

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Interaction between the solubilized membrane proteins and G-gly in the presence or absence of DFO. A, shown is surface plasmon resonance analysis of the interaction of biotinylated G-gly immobilized on a sensor chip with solubilized plasma membrane proteins from colon cancer cells (HCT116) incubated in the presence of 1 μm DFO (+DFO) or in the absence of DFO (−DFO). All sensorgrams represent the RU as a function of time. B, HCT116 membrane proteins incubated in the presence of 1 μm DFO (+DFO) or in absence of DFO (−DFO) bound to G-gly on the sensor chip were eluted from the Biacore, resolved by SDS-PAGE, and silver-stained. The arrows indicate the bands that have been cut for mass spectrometry analysis. Results are representative of two independent experiments.

TABLE 2.

Proteins identification by mass spectrometry (nanoLC/ESI QStar MS/MS)

Solubilized plasma membrane proteins bound to G-gly immobilized on a sensor chip in the presence or absence of DFO were eluted, resolved by SDS-PAGE, and silver-stained. The bands around 50 kDa were cut and analyzed by mass spectrometry. MW, molecular weight; pI, isoelectric point.

Protein ID Score MW pI % Covered Peptides
−DFO
    ATP-synthase -α-chain 297 58904 9.22 13 8
    ATP-synthase -β-chain 265 56318 5.19 12 6
+DFO
    ATP-synthase-α-chain 275 58904 9.22 11 7
    ATP-synthase-β-chain 97 56318 5.19 6 3
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Direct Interaction of G-gly with F1-ATPase

We used the SPR technology to verify the direct interaction between purified F1-ATPase (Fig. 3A) and G-gly. The sensograms in Fig. 3B show a direct interaction between G-gly coated on the BIAcore sensor chip and the purified F1-ATPase used as an analyte. Binding of F1-ATPase to the immobilized G-gly was dose-dependent (3–50 nm) allowing us to determine the affinity between this multisubunit complex and the gastrin precursor, G-gly (KD ∼ 1.7 nm).

FIGURE 3.

FIGURE 3.

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A, F1-ATPase was purified from bovine heart mitochondria as described under “Experimental Procedures” and detected by Coomassie Blue on SDS-polyacrylamide gel electrophoresis. B, surface plasmon resonance analysis of the interaction of G-gly with purified F1-ATPase is shown. Dose-dependent binding of the purified F1-ATPase, used as an analyte, to the biotinylated G-gly immobilized on the sensor chip is shown. All sensorgrams represent the RU as a function of time. Results are representative of three independent experiments.

Computational Study of the G-gly Interactions with the F1-ATPase

We examined the molecular basis of interactions between G-gly and F1-ATPase by molecular modeling. The prediction of the secondary and three-dimensional structures of G-gly using the peptide structure prediction PEP-FOLD server shows that G-gly is a likely α-helix. We noted that G-gly has an E(E/D)XY motif also found in IF1, an α-helical natural inhibitor of F1-ATPase. Fig. 4A shows the sequence alignment of IF1 from different species and human G-gly in which the E(E/D)XY motif can be identified. Because the sequence fragment of IF1 corresponding to G-gly is a helix, it additionally supports the helical nature of G-gly predicted by the web server. The crystal structure of IF1 in the complex with F1-ATPase in which IF1 is bound to α and β subunits and makes few contacts with the δ-subunit is available (PDB code 2V7Q). Given that our experimental studies predicted the direct interactions of G-gly with mainly α and β subunits and possibly the δ-subunit, we docked the α-helical structure of G-gly to F1-ATPase, specifying the docking box around the position of IF1 in the crystal structure. Among generated docking poses of G-gly, we selected for detailed examinations, the pose in which the residues of the E(E/D)XY motif form contacts similar to IF1, thus preserving the molecular recognition pattern, which has been recently shown to be an essential component to the binding energy that holds IF1 and F1-ATPase together (27). The position of G-gly and its interactions with F1-ATPase are shown in Fig. 4, B–D). We propose that G-gly is bounded to the β-subunit through several interactions: (i) the salt bridges involving Glu-9 and -10 of G-gly and Arg-408 of the β-subunit, (ii) a hydrogen bond between Glu-9 and Tyr-381, (iii) hydrophobic and van der Waals interactions of Leu-5, Tyr-12, and Phe-17 with Met-393, Lys-401, Ile-388, Pro-453, and Gln-455 of the β-subunit, and (iv) hydrogen bonds between Gly-18 and His-477 and Gly-7 and Ala-401. G-gly also interacts with the α-subunit through hydrophobic and van der Waals interactions of Trp-4, Ala-11, and Trp-14 with Phe-403, Val-400, Leu-417, and Gln-396 of the α-subunit. There is no direct contact of G-gly with the δ-subunit in this binding mode, although some docking solutions provided few contacts with the δ-subunit.

FIGURE 4.

FIGURE 4.

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Probing G-gly-F1-ATPase interactions using molecular modeling. A, shown is sequence alignment of IF1 from Bos taurus, Homo sapiens, Mus musculus, Rattus norvegicus, Saccharomyces cerevisiae, Pichia jadinii, and the human G-gly. B, shown is the hypothetical location of G-gly in the x-ray structure of F1-ATPase bound to IF1. The x-ray structure is shown in ribbon-like representation, where α-subunits binding ATP or ADP or an α-nucleotide low affinity subunit are in red, green, and gray, respectively; β-subunits binding ATP or ADP or a β-nucleotide low affinity subunit are in brown, blue, and yellow, respectively; δ-subunit is in cyan, and G-gly is in purple. The G-gly carbon atoms of backbone and side chains are shown in gray stick. C and D, shown is a close view of G-gly interactions with the α and β subunits. The hydrogen bond interactions are highlighted with the dotted purple line in C, and all favorable contacts are in the dotted green line in D.

The salt bridge between Glu-9 and Arg-408 corresponds to similar interactions between Glu-30 of IF1 and Arg-408. These interactions have been recently shown by mutagenesis to play an important role in bovine and yeast IF1 binding to F1-ATPase (27). In particular, mutation of bovine Glu-30 (yeast E25) to alanine strongly decreases the binding (27).

Loss of Interaction between the F1-ATPase and G-gly with a Mutation in the E(E/D)XY Domain

On the basis of our SPR studies and molecular modeling, we propose that binding of G-gly into F1-ATPase may occur in a similar fashion to IF1. To confirm this hypothesis, we mutated the Glu-9 residue of G-gly to alanine and analyzed by SPR the interaction between this mutated form of the peptide and the F1-ATPase. As shown by the sensograms in Fig. 5, the binding of G-gly coated on the BIAcore sensor chip to the purified F1-ATPase used as an analyte is abolished when Glu-9 of G-gly is mutated to alanine. The natural receptor of the mature amidated form of gastrin is the CCK2 receptor, a G protein-coupled receptor mainly localized in the stomach. However, we verified the potential interaction between gastrin-amide (G-NH2) and the F1-ATPase. As expected, we did not observe any interaction by SPR between the F1-ATPase and G-NH2 (Fig. 5).

FIGURE 5.

FIGURE 5.

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Loss of interaction between the F1-ATPase and G-gly with a mutation in the E(E/D)XY domain. The graph represents surface plasmon resonance analysis of the interaction of purified F1-ATPase used as an analyte with G-gly, G-gly mutated on Glu-9 to alanine in the E(E/D)XY motif (G-gly-E9A), or G-NH2, the mature amidated form of gastrin, immobilized on the sensor chip. All sensorgrams represent the RU as a function of time. Results are representative of three independent experiments.

Localization of F1-ATPase at the Cell Surface of Normal and Tumor Colonic Cells

The expression of F1-ATPase at the plasma membrane of normal and tumor colonic epithelial cells has never been reported. We performed immuno-staining with an antibody against the β-subunit of F1-ATPase on non-fixed non-permeabilized living cells. In these conditions, the βF1 antibody interacts only with proteins localized at the cell surface. Analysis by confocal microscopy shows a βF1 staining on the cell surface of two colon cancer cell lines, HCT116 and HT29, in which we previously reported a biological effect of G-gly (Fig. 6, a and d) (9, 28). These results suggest that the orientation of the F1-ATPase in the membrane is such that the F1 component is extracellular and available to the antibody. These patch-like structures did not colocalize with MitoTracker Red, a specific marker of mitochondria (Fig. 6, g and h). Using an antibody directed against a membrane receptor (EGF receptor) as a control, we observed a staining at the cell surface similar to that of plasma membrane βF1 (Fig. 6, b and e). In contrast, when cells were permeabilized (Fig. 6i), the F1-ATPase staining was intracellular, localized to mitochondria as expected.

FIGURE 6.

FIGURE 6.

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Localization of the β-subunit of F1-ATPase on colonic epithelial cells by confocal microscopy. Cells were stained for β-F1-ATPase (green, a, d, g, h, i, and j), mitochondria (MitoTracker: red, g, h, and j), or a plasma membrane protein (EGF receptor ((EGFR)) (red, b and e) as described under “Experimental Procedures.” Experiments (a–h and j) were performed on intact, non-fixed, non-permeabilized colonic epithelial cells normal (YAMC) or cancerous (HCT116, HT29) cells. Experiment i was performed on fixed and permeabilized HCT116 cells. Magnification, ×40). Results are representative of three-six independent experiments.

Because the proliferative effects of G-gly have also been observed on normal colonic epithelial cells (11, 29–31), we analyzed the expression of F1-ATPase at the cell surface of a nontransformed colon cell line, YAMC, on which a previous report has demonstrated the presence of specific binding sites for G-gly and a proliferative effect of the peptide (11). Similarly to what we observed for colon cancer cell lines, confocal microscopy analysis on non-fixed, non-permeabilized living cells show βF1 staining on the cell surface of YAMC that does not colocalize with MitoTracker Red (Fig. 6j). In addition, G-gly proliferative effects have been previously reported on normal epithelial cells from the colonic mucosa of mice (22). Here we confirmed on mouse colonic tissue sections the plasma membrane localization of the F1-ATPase on normal colonic epithelial cells. Indeed, by confocal microscopy using antibodies that detect specifically, βF1 or E-cadherin, known to be localized on the basolateral membrane, we observed a co-staining of the two proteins at the membrane of colonic epithelial cells (Fig. 7A). To confirm the location of the F1-ATPase on the basolateral membranes of polarized colonic epithelial cells, we used specific markers of, respectively, the apical or basolateral membranes on a monolayer of polarized CACO-2 cells. Confocal X-Y (top and bottom of the cells) (Fig. 7B) and Z sections (Fig. 7C) show the absence of apical labeling with the βF1 antibody, whereas the staining for DPP-IV, a marker of apical membranes, is positive. On the contrary, we observed the presence of basolateral labeling for F1-ATPase as well as for E-cadherin.

FIGURE 7.

FIGURE 7.

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Localization of the β-subunit of F1-ATPase on colonic epithelial cells by confocal microscopy. Cells or tissue sections were labeled with the indicated antibodies (β-F1-ATPase, E-cadherin, DPP-IV) as described under “Experimental Procedures.” A, experiments were performed on fixed colonic tissue sections of mouse. Confocal X-Y sections (top and bottom) (B) or Z sections (C) of fixed confluent polarized CACO-2 cells grown on Transwell-ClearTM filters (Corning, Costar). Arrows in A and B show the membrane colocalization of F1-ATPase and a plasma membrane protein (E-cadherin). Magnification, ×60. Results are representative of three independent experiments.

Cell Surface F1-ATPase Activity Is Stimulated by G-gly

Several works have reported the presence F1-ATPase at the cell surface of HUVEC and demonstrated its role in angiogenesis. In particular, Radojkovic et al. (6) reported that F1-ATPase on the surface of endothelial cells serves as a receptor for apoAI. This lipoprotein stimulates cell surface F1-ATPase activity, leading to ADP production and an increase in proliferation of HUVEC. Recent work showed that G-gly is capable to increase tubule and node formation by HUVEC (19). We, therefore, hypothesize that G-gly might stimulate the cell surface F1-ATPase activity in a manner similar to what is observed with apoAI.

Extracellular ADP production by HUVEC or colon cancer cells were analyzed by measuring the generation of [α-32P]ADP from [α-32P]ATP in the cell media 10 min after the addition of the peptide, as previously described (6). In the presence of G-gly we observed an increased hydrolytic activity on HUVEC (+46% ± 6) (Fig. 8A) similar to that previously described for apoAI on the same cells (6). The ability of IF1 protein to specifically inhibit the hydrolytic activity of F1-ATPase is well documented. The efficiency of this natural inhibitor is pH-dependent. We, therefore, used a mutated form of IF1 (IF1-H49K) known to be active at all pH values. As previously reported in the HUVEC cells, IF1-H49K decreases the basal extracellular ADP production (6) but more interestingly reverses ATP hydrolysis and ADP production in response to G-gly (Fig. 8A). Similar results were obtained on colonic epithelial cancer cells (+21.5 ± 2.3) (Fig. 8B), indicating that the membrane F1-ATPase is also functional on this cellular model and leads to ATP hydrolysis.

FIGURE 8.

FIGURE 8.

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Functionality of Cell surface F1-ATPase. Extracellular ADP production by HUVEC (A) or colon cancer cells (B) were analyzed for 10 min at 37 °C as described under “Experimental Procedures” in the presence of G-gly (10 nm for HUVEC or 1 nm for HCT116) with or without IF1 (1 μm). Control experiment was done with PBS. Data are expressed as the percentage of change in extracellular ADP level in treated cells as compared with the control (set at 0). The results are representative of two independent experiments performed on two different set of cells. C, pHi was measured on HCT116 cells in the absence (control) or presence of 1 nm G-gly or dimethyl amiloride (DMA) using the pH-sensitive fluorescent probe carboxy-SNARF-1 and flow cytometry analysis as described under “Experimental Procedures.” Experiments were performed in neutral extracellular pH. Mean ± S.E. (n = 3).

Cell surface ATPase on endothelial cells has been previously shown to regulate the intracellular pH (32). Here we have analyzed whether G-gly induces intracellular acidification on colonic epithelial cell. pHi was measured using the pH-sensitive fluorescent probe carboxy-SNARF-1 and flow cytometry analysis as described under “Experimental Procedures.” Experiments were performed in neutral extracellular pH conditions for which we observed ADP production and cell proliferation in response to G-gly. As shown in Fig. 8C, pHi is not significantly modified after 10 min of G-gly treatment as compared with control (respectively, 7.23 ± 0.03 and 7.25 ± 0.05). Similar results were obtained for longer times of stimulation (1–48 h; data not shown). The dimethyl amiloride, an inhibitor of the Na+/H+ exchanger, was used as an acidifying positive control. As expected we observed at 10 min an acidification of the pHi with dimethyl amiloride compared with the control condition (6.84 versus 7.29, p value = 0.0001).

Cell Surface F1-ATPase Mediates G-gly Proliferative Effects on Endothelial Cells and Colon Cancer Cells

To demonstrate the role of F1-ATPase in mediating the growth factor effect of G-gly, we performed proliferative assays on HUVEC and colon cancer cells in the presence of the F1-ATPase inhibitor, IF1-H49K. Stimulation of HUVEC (Fig. 9A) or HCT116 cells (Fig. 9B) by G-gly induced a significant increase of cell proliferation. These effects were completely abolished when the cells were pretreated with the F1-ATPase inhibitor, indicating that the effects of G-gly on cell survival are lost when the F1-ATPase is inhibited. To confirm the involvement of cell surface F1-ATPase in the growth factor effect of G-gly, proliferative assays were also performed in the presence of an anti-β-F1-ATPase blocking antibody that was shown to bind to the cell surface F1-ATPase. As shown in Fig. 9, the blocking antibody could abolish G-gly-induced cell proliferation on endothelial cells (Fig. 9A) and colon cancer cells (Fig. 9B) similarly to what we observed with IF1 on these cellular models. These results confirm that the cell surface F1-ATPase plays an important role in mediating growth factor effects of G-gly in both endothelial cells and colon cancer cells. We also used angiostatin, another ligand of membrane F1-ATPase known to inhibit endothelial cell proliferation. By treating colon cancer cells with a concentration of angiostatin previously used in other studies (6), we confirmed its ability to inhibit cell proliferation. In addition, G-gly proliferative effects were abolished when the colon cancer cells were pretreated with angiostatin (Fig. 9B).

FIGURE 9.

FIGURE 9.

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Cell surface F1-ATPase mediates G-gly proliferative effects on HUVEC and colon cancer cells. Proliferation of HUVEC (A) or HCT116 cells (B and C) was measured as described under “Experimental Procedures” by using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test. Cells were treated with the native form of G-gly at 10 nm for HUVEC (A) or 1 nm for HCT116 (B and C). Cells were also treated with G-gly mutated on Glu-9 at 1 nm (C) or the amidated form of gastrin (G-NH2) at 1 nm. When indicated, cells were pretreated with IF1-H49K, a natural inhibitor of the hydrolytic activity of F1-ATPase, an antibody against the β-subunit of F1-ATPase (50 μg/ml) or angiostatin (1 μm). Results are expressed as the percentage of change in cell proliferation as compared with untreated cells (set at 0). Data are the mean ± S.E.; n = 4–6.

On the basis of SPR studies and molecular modeling, we proposed that binding of G-gly into F1-ATPase involves the E(E/D)XY motif conserved in the G-gly and IF1 sequences. Because mutation of the Glu-9 residue of G-gly into alanine strongly decreases the binding of the peptide to the F1-ATPase, we also tested the ability of the mutated G-gly to stimulate cell proliferation. As shown in Fig. 9C, the proliferative effect observed when colon cancer cells are stimulated with 1 nm G-gly is totally absent when a similar concentration of the mutated peptide is used (G-gly-E9A). Higher concentrations of G-gly-E9A were also tested in proliferation assays (10–1000 nm), but none was efficient to stimulate cell proliferation (data not shown). As expected, the proliferation of HCT116 cells that do not express the CCK2 receptor was not stimulated by gastrin-amide (Fig. 9C).

DISCUSSION

Although F1Fo ATPase was initially believed to be strictly expressed in the inner membrane of mitochondria, recent studies have shown that the F1 complex, containing the catalytic site for ATP synthesis or hydrolysis, is expressed at the cell surface of endothelial cells and hepatocytes as well as hepatoma and lung cancer cells (1). This complex has been characterized as a cell surface receptor for different ligands. In the present study we have identified by mass spectrometry in the purified plasma membranes from colorectal cancer cells different subunits of the F1 complex (α, β, δ). In addition, using immunofluorescent approaches and confocal microscopy, we confirmed the presence of the F1-ATPase at the cell surface of different colon cancer cell lines and in the plasma membrane of normal colonic epithelial cells.

F1-ATPase, at the cell surface of endothelial cells as well as hepatoma and lung cancer cells, has been described as a receptor for angiostatin that mediates cell death through the inhibition of ATP production (2–5). In contrast, apoAI, another ligand of endothelial cell surface F1-ATPase, induces cell proliferation and survival by stimulating ADP production (6). Here, using SPR technology coupled to mass spectrometry, we have identified a gastrointestinal peptide, the glycine-extended form of gastrin, as a new ligand for the F1-ATPase that is localized at the cell surface of colonic epithelial cells. Because the gastrin precursor, G-gly, does not stimulate gastric acid secretion as does the mature form of the hormone, it was initially considered without biological activity. However, we were the first to demonstrate a proliferative effect of this hormonal precursor on tumor cells (8). G-gly is now clearly established as a growth factor for normal and cancerous gastrointestinal cells particularly from colon origin. However, the receptor that mediates the biological activity of this precursor peptide and might represent a new potential target in gastrointestinal cancers was unknown. In the present study we clearly demonstrate using purified F1-ATPase and synthetic G-gly in a SPR approach the direct interaction between G-gly and the F1-ATPase. Interestingly the affinity calculated in these experiments was similar to that previously reported in pharmacological studies for the G-gly high affinity binding sites (8, 11). In addition, by molecular modeling we identified the motif in the G-gly sequence (E(E/D)XY) that directly interacts with F1-ATPase as well as the amino acid residues in the β-and α-subunits of the F1-ATPase that bind this motif. We confirmed this molecular model by mutation of the E(E/D)XY motif, in particular, replacement of the Glu-9 residue by an alanine resulted in a strong decrease of G-gly binding to the F1-ATPase.

We have also demonstrated the role of the F1-ATPase in mediating the growth factor effect of G-gly on both colon cancer cells and HUVEC. Indeed, blocking the F1-ATPase with a specific antibody or IF1, a natural inhibitor of the enzyme, abolished G-gly proliferative effects in these cells. The mechanism likely involves ADP production by the membrane F1-ATPase. Indeed, in the presence of G-gly the membrane F1-ATPase functions as an ATP hydrolase and produces extracellular ADP. These effects are reversed in the presence of IF1, known to inhibit the hydrolytic activity of F1-ATPase. In addition, G-gly activity is also abolished by mutation of the E(E/D)XY motif in the G-gly sequence, which directly interacts with the F1-ATPase. In contrast, G-gly does not regulate pHi in the experimental conditions (neutral pHi) for which we observed ADP production and cell proliferation. Some membrane F1-ATPase ligands such as angiostatin have been previously reported to induce cell death by decreasing pHi but only in a low extracellular pH environment (33).

In vivo, transgenic mice overexpressing circulating G-gly exhibited increased colonic proliferation compared with wild-type controls (31). Similarly, continuous infusion of G-gly into rats or gastrin-deficient mice also resulted in an increase of colonic proliferation (29). These results suggest the existence on colonic epithelium of a putative receptor or binding protein for G-gly that mediates the proliferative effects of the peptide. Here we have shown that the F1-ATPase, which directly binds G-gly, is present at the plasma membrane of colonic epithelial cells in a mouse model. It is also interesting to notice that in both in vivo models (G-gly transgenic mice or G-gly infusion), treatment of the animals with DFO, a ferric ions chelating agent, reverses the proliferative effects induced by G-gly, suggesting that the biological activity of the peptide is dependent on the presence of ferric ions (34). In vitro studies confirm that G-gly binds two ferric ions necessary to its biological activity (9, 25, 26). In accordance with these previous data, our results show that the binding of G-gly to its potential receptor is decreased in the presence of ferric ions chelator.

Several studies have suggested a link between F1-ATPase and colorectal cancer. Indeed, F1-ATPase has been shown to be up-regulated in primary colorectal tumors compared with the adjacent normal mucosa (35). In addition, an association between F1-ATPase overexpression with poor survival of the patients has been reported in stage III colorectal cancer (36). F1-ATPase expression is also up-regulated in liver metastasis of colorectal cancer as compared with primary tumor as well as in tumor spheroids of colon carcinoma cells compared with cells grown in monolayer (36, 37). It is, therefore, interesting to notice that the new ligand of F1-ATPase that we have identified in the present study is also overexpressed in these tumor cells. Indeed, the gene coding for gastrin is a target of oncogenic pathways frequently activated in colon cancer such as APC/β-catenin or Ras pathways (38–41). Therefore, high concentrations of gastrin precursors, including G-gly have been observed in colon tumors and in blood of patients with colorectal cancer (42–45). These data suggest that the cell surface F1-ATPase may contribute to a pro-tumoral autocrine loop in colorectal cancer cells. Interestingly, an anti-tumor strategy using monoclonal antibodies against the β-subunit of F1-ATPase has been previously proposed for hepatoma because these antibodies have been shown to reduce growth of these tumors (46). In view of our results, targeting cell surface F1-ATPase might be also a new therapeutic approach for colon cancer. In colon cancer, both F1-ATPase and its ligand G-gly seem to be overexpressed; an alternative or complementary strategy to F1-ATPase blocking could be the use of antibodies against the gastrin precursors.

Acknowledgments

We thank M. Montgomery and J. Walker (MRC Mitochondrial Biology Unit, Cambridge, UK) for providing the purified F1-ATPase We thank Danielle Daviaud and the Cellular Imaging facility of I2MC (Toulouse, France) for technical assistance in confocal microscopy.

*

This work was supported by INSERM, the French Association for Cancer Research (ARC-SFI-201112-03828).

2
The abbreviations used are:
HUVEC
human umbilical vascular endothelial cells
apoAI
apolipoprotein AI
G-gly
glycine-extended form of gastrin
SPR
surface plasmon resonance
RU
resonance units
DFO
desferrioxamine.

REFERENCES

  • 1. Mowery Y. M., Pizzo S. V. (2008) Targeting cell surface F1F0 ATP synthase in cancer therapy. Cancer Biol. Ther. 7, 1836–1838 [DOI] [PubMed] [Google Scholar]
  • 2. Arakaki N., Nagao T., Niki R., Toyofuku A., Tanaka H., Kuramoto Y., Emoto Y., Shibata H., Magota K., Higuti T. (2003) Possible role of cell surface H+ -ATP synthase in the extracellular ATP synthesis and proliferation of human umbilical vein endothelial cells. Mol. Cancer Res. 1, 931–939 [PubMed] [Google Scholar]
  • 3. Chi S. L., Pizzo S. V. (2006) Angiostatin is directly cytotoxic to tumor cells at low extracellular pH. A mechanism dependent on cell surface-associated ATP synthase. Cancer Res. 66, 875–882 [DOI] [PubMed] [Google Scholar]
  • 4. Moser T. L., Kenan D. J., Ashley T. A., Roy J. A., Goodman M. D., Misra U. K., Cheek D. J., Pizzo S. V. (2001) Endothelial cell surface F1-F0 ATP synthase is active in ATP synthesis and is inhibited by angiostatin. Proc. Natl. Acad. Sci. U.S.A. 98, 6656–6661 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Moser T. L., Stack M. S., Asplin I., Enghild J. J., Højrup P., Everitt L., Hubchak S., Schnaper H. W., Pizzo S. V. (1999) Angiostatin binds ATP synthase on the surface of human endothelial cells. Proc. Natl. Acad. Sci. U.S.A. 96, 2811–2816 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Radojkovic C., Genoux A., Pons V., Combes G., de Jonge H., Champagne E., Rolland C., Perret B., Collet X., Tercé F., Martinez L. O. (2009) Stimulation of cell surface F1-ATPase activity by apolipoprotein A-I inhibits endothelial cell apoptosis and promotes proliferation. Arterioscler. Thromb. Vasc. Biol. 29, 1125–1130 [DOI] [PubMed] [Google Scholar]
  • 7. Martinez L. O., Jacquet S., Esteve J. P., Rolland C., Cabezón E., Champagne E., Pineau T., Georgeaud V., Walker J. E., Tercé F., Collet X., Perret B., Barbaras R. (2003) Ectopic β-chain of ATP synthase is an apolipoprotein A-I receptor in hepatic HDL endocytosis. Nature 421, 75–79 [DOI] [PubMed] [Google Scholar]
  • 8. Seva C., Dickinson C. J., Yamada T. (1994) Growth-promoting effects of glycine-extended progastrin. Science 265, 410–412 [DOI] [PubMed] [Google Scholar]
  • 9. Ferrand A., Kowalski-Chauvel A., Pannequin J., Bertrand C., Fourmy D., Dufresne M., Seva C. (2006) Glycine-extended gastrin activates two independent tyrosine kinases in upstream of p85/p110 phosphatidylinositol 3-kinase in human colonic tumor cells. World J. Gastroenterol. 12, 1859–1864 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. He H., Pannequin J., Tantiongco J. P., Shulkes A., Baldwin G. S. (2005) Glycine-extended gastrin stimulates cell proliferation and migration through a Rho- and ROCK-dependent pathway, not a Rac/Cdc42-dependent pathway. Am. J. Physiol. Gastrointest. Liver Physiol. 289, G478–G488 [DOI] [PubMed] [Google Scholar]
  • 11. Hollande F., Imdahl A., Mantamadiotis T., Ciccotosto G. D., Shulkes A., Baldwin G. S. (1997) Glycine-extended gastrin acts as an autocrine growth factor in a nontransformed colon cell line. Gastroenterology 113, 1576–1588 [DOI] [PubMed] [Google Scholar]
  • 12. Iwase K., Evers B. M., Hellmich M. R., Guo Y. S., Higashide S., Kim H. J., Townsend C. M., Jr. (1997) Regulation of growth of human gastric cancer by gastrin and glycine-extended progastrin. Gastroenterology 113, 782–790 [DOI] [PubMed] [Google Scholar]
  • 13. Koh T. J., Field J. K., Varro A., Liloglou T., Fielding P., Cui G., Houghton J., Dockray G. J., Wang T. C. (2004) Glycine-extended gastrin promotes the growth of lung cancer. Cancer Res. 64, 196–201 [DOI] [PubMed] [Google Scholar]
  • 14. Ogunwobi O. O., Beales I. L. (2006) Glycine-extended gastrin stimulates proliferation and inhibits apoptosis in colon cancer cells via cyclo-oxygenase-independent pathways. Regul. Pept. 134, 1–8 [DOI] [PubMed] [Google Scholar]
  • 15. Ogunwobi O. O., Beales I. L. (2008) Glycine-extended gastrin stimulates proliferation via JAK2- and Akt-dependent NF-κB activation in Barrett's oesophageal adenocarcinoma cells. Mol. Cell Endocrinol. 296, 94–102 [DOI] [PubMed] [Google Scholar]
  • 16. Pannequin J., Kovac S., Tantiongco J. P., Norton R. S., Shulkes A., Barnham K. J., Baldwin G. S. (2004) A novel effect of bismuth ions. Selective inhibition of the biological activity of glycine-extended gastrin. J. Biol. Chem. 279, 2453–2460 [DOI] [PubMed] [Google Scholar]
  • 17. Stepan V. M., Krametter D. F., Matsushima M., Todisco A., Delvalle J., Dickinson C. J. (1999) Glycine-extended gastrin regulates HEK cell growth. Am. J. Physiol. 277, R572–R581 [DOI] [PubMed] [Google Scholar]
  • 18. Watson S. A., Michaeli D., Grimes S., Morris T. M., Robinson G., Varro A., Justin T. A., Hardcastle J. D. (1996) Gastrimmune raises antibodies that neutralize amidated and glycine-extended gastrin-17 and inhibit the growth of colon cancer. Cancer Res. 56, 880–885 [PubMed] [Google Scholar]
  • 19. Clarke P. A., Dickson J. H., Harris J. C., Grabowska A., Watson S. A. (2006) Gastrin enhances the angiogenic potential of endothelial cells via modulation of heparin-binding epidermal-like growth factor. Cancer Res. 66, 3504–3512 [DOI] [PubMed] [Google Scholar]
  • 20. Dufresne M., Seva C., Fourmy D. (2006) Cholecystokinin and gastrin receptors. Physiol. Rev. 86, 805–847 [DOI] [PubMed] [Google Scholar]
  • 21. Lutter R., Abrahams J. P., van Raaij M. J., Todd R. J., Lundqvist T., Buchanan S. K., Leslie A. G., Walker J. E. (1993) Crystallization of F1-ATPase from bovine heart mitochondria. J. Mol. Biol. 229, 787–790 [DOI] [PubMed] [Google Scholar]
  • 22. Cabezon E., Butler P. J., Runswick M. J., Walker J. E. (2000) Modulation of the oligomerization state of the bovine F1-ATPase inhibitor protein, IF1, by pH. J. Biol. Chem. 275, 25460–25464 [DOI] [PubMed] [Google Scholar]
  • 23. Lemarie A., Huc L., Pazarentzos E., Mahul-Mellier A. L., Grimm S. (2011) Specific disintegration of complex II succinate:ubiquinone oxidoreductase links pH changes to oxidative stress for apoptosis induction. Cell Death Differ. 18, 338–349 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Soleimani M., Singh G., Bizal G. L., Gullans S. R., McAteer J. A. (1994) Na+/H+ exchanger isoforms NHE-2 and NHE-1 in inner medullary collecting duct cells. Expression, functional localization, and differential regulation. J. Biol. Chem. 269, 27973–27978 [PubMed] [Google Scholar]
  • 25. Pannequin J., Barnham K. J., Hollande F., Shulkes A., Norton R. S., Baldwin G. S. (2002) Ferric ions are essential for the biological activity of the hormone glycine-extended gastrin. J. Biol. Chem. 277, 48602–48609 [DOI] [PubMed] [Google Scholar]
  • 26. Pannequin J., Tantiongco J. P., Kovac S., Shulkes A., Baldwin G. S. (2004) Divergent roles for ferric ions in the biological activity of amidated and non-amidated gastrins. J. Endocrinol. 181, 315–325 [DOI] [PubMed] [Google Scholar]
  • 27. Bason J. V., Runswick M. J., Fearnley I. M., Walker J. E. (2011) Binding of the inhibitor protein IF(1) to bovine F(1)-ATPase. J. Mol. Biol. 406, 443–453 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Bertrand C., Kowalski-Chauvel A., Do C., Résa C., Najib S., Daulhac L., Wang T. C., Ferrand A., Seva C. (2010) A gastrin precursor, gastrin-gly, up-regulates VEGF expression in colonic epithelial cells through an HIF-1-independent mechanism. Int. J. Cancer 126, 2847–2857 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Aly A., Shulkes A., Baldwin G. S. (2001) Short term infusion of glycine-extended gastrin(17) stimulates both proliferation and formation of aberrant crypt foci in rat colonic mucosa. Int. J. Cancer 94, 307–313 [DOI] [PubMed] [Google Scholar]
  • 30. Houli N., Loh S. W., Giraud A. S., Baldwin G. S., Shulkes A. (2006) Mitogenic effects of both amidated and glycine-extended gastrin-releasing peptide in defunctioned and azoxymethane-treated rat colon in vivo. Regul. Pept. 134, 9–16 [DOI] [PubMed] [Google Scholar]
  • 31. Koh T. J., Dockray G. J., Varro A., Cahill R. J., Dangler C. A., Fox J. G., Wang T. C. (1999) Overexpression of glycine-extended gastrin in transgenic mice results in increased colonic proliferation. J. Clin. Invest. 103, 1119–1126 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Alard J. E., Hillion S., Guillevin L., Saraux A., Pers J. O., Youinou P., Jamin C. (2011) Autoantibodies to endothelial cell surface ATP synthase, the endogenous receptor for hsp60, might play a pathogenic role in vasculatides. PLoS One 6, e14654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Champagne E., Martinez L. O., Collet X., Barbaras R. (2006) Ecto-F1Fo ATP synthase/F1 ATPase. Metabolic and immunological functions. Curr. Opin. Lipidol 17, 279–284 [DOI] [PubMed] [Google Scholar]
  • 34. Ferrand A., Lachal S., Bramante G., Kovac S., Shulkes A., Baldwin G. S. (2010) Stimulation of proliferation in the colorectal mucosa by gastrin precursors is blocked by desferrioxamine. Am. J. Physiol. Gastrointest. Liver Physiol. 299, G220–G227 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Tomonaga T., Matsushita K., Yamaguchi S., Oh-Ishi M., Kodera Y., Maeda T., Shimada H., Ochiai T., Nomura F. (2004) Identification of altered protein expression and post-translational modifications in primary colorectal cancer by using agarose two-dimensional gel electrophoresis. Clin. Cancer Res. 10, 2007–2014 [DOI] [PubMed] [Google Scholar]
  • 36. Chang H. J., Lee M. R., Hong S. H., Yoo B. C., Shin Y. K., Jeong J. Y., Lim S. B., Choi H. S., Jeong S. Y., Park J. G. (2007) Identification of mitochondrial FoF1-ATP synthase involved in liver metastasis of colorectal cancer. Cancer Sci. 98, 1184–1191 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Dardousis K., Voolstra C., Roengvoraphoj M., Sekandarzad A., Mesghenna S., Winkler J., Ko Y., Hescheler J., Sachinidis A. (2007) Identification of differentially expressed genes involved in the formation of multicellular tumor spheroids by HT-29 colon carcinoma cells. Mol. Ther. 15, 94–102 [DOI] [PubMed] [Google Scholar]
  • 38. Chakladar A., Dubeykovskiy A., Wojtukiewicz L. J., Pratap J., Lei S., Wang T. C. (2005) Synergistic activation of the murine gastrin promoter by oncogenic Ras and β-catenin involves SMAD recruitment. Biochem. Biophys. Res. Commun. 336, 190–196 [DOI] [PubMed] [Google Scholar]
  • 39. Finley G. G., Koski R. A., Melhem M. F., Pipas J. M., Meisler A. I. (1993) Expression of the gastrin gene in the normal human colon and colorectal adenocarcinoma. Cancer Res. 53, 2919–2926 [PubMed] [Google Scholar]
  • 40. Koh T. J., Bulitta C. J., Fleming J. V., Dockray G. J., Varro A., Wang T. C. (2000) Gastrin is a target of the β-catenin/TCF-4 growth-signaling pathway in a model of intestinal polyposis. J. Clin. Invest. 106, 533–539 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Nakata H., Wang S. L., Chung D. C., Westwick J. K., Tillotson L. G. (1998) Oncogenic ras induces gastrin gene expression in colon cancer. Gastroenterology 115, 1144–1153 [DOI] [PubMed] [Google Scholar]
  • 42. Ciccotosto G. D., McLeish A., Hardy K. J., Shulkes A. (1995) Expression, processing, and secretion of gastrin in patients with colorectal carcinoma. Gastroenterology 109, 1142–1153 [DOI] [PubMed] [Google Scholar]
  • 43. Nemeth J., Taylor B., Pauwels S., Varro A., Dockray G. J. (1993) Identification of progastrin derived peptides in colorectal carcinoma extracts. Gut 34, 90–95 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Van Solinge W. W., Nielsen F. C., Friis-Hansen L., Falkmer U. G., Rehfeld J. F. (1993) Expression but incomplete maturation of progastrin in colorectal carcinomas. Gastroenterology 104, 1099–1107 [DOI] [PubMed] [Google Scholar]
  • 45. Ferrand A., Wang T. C. (2006) Gastrin and cancer. A review. Cancer Lett 238, 15–29 [DOI] [PubMed] [Google Scholar]
  • 46. Wang J., Han Y., Liang J., Cheng X., Yan L., Wang Y., Liu J., Luo G., Chen X., Zhao L., Zhou X., Wu K., Fan D. (2008) Effect of a novel inhibitory mAb against β-subunit of F1F0 ATPase on HCC. Cancer Biol. Ther. 7, 1829–1835 [DOI] [PubMed] [Google Scholar]

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