Peptide fragments of AMP-18, a novel secreted gastric antrum mucosal protein, are mitogenic and motogenic

F. Gary Toback,1 Margaret M. Walsh-Reitz,1 Mark W. Musch,1 Eugene B. Chang,1 John Del Valle,2 Hongyu Ren,1 Erick Huang,1 and Terence E. Martin3

Departments of 1Medicine and 3Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637; and 2Department of Medicine, University of Michigan, Ann Arbor, Michigan 48109

Submitted 23 October 2002 ; accepted in final form 18 March 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antrum mucosal protein (AMP)-18 is a novel 18-kDa protein synthesized by cells of the gastric antrum mucosa. The protein is present in secretion granules of murine gastric antrum epithelial cells and is a component of canine antrum mucus, suggesting that it is secreted into the viscoelastic gel layer on the mucosal surface. Release of the protein appears to be regulated because forskolin decreased the amount of immunoreactive AMP-18 in primary cultures of canine antrum mucosal epithelial cells, and indomethacin gavaged into the stomach of mice reduced AMP-18 content in antrum mucosal tissue before inducing histological injury. A functional domain of the protein was identified by preparing peptides derived from the center of human AMP-18. A 21-mer peptide stimulated growth of gastric and intestinal epithelial cells, but not fibroblasts, and increased restitution of scrape-wounded gastric epithelial monolayers. These functions of AMP-18 suggest that its release onto the apical cell surface is regulated and that the protein and/or peptide fragments may protect the antral mucosa and promote healing by facilitating restitution and proliferation after injury.

growth factor; wound healing; restitution; epithelium; stomach


CELLS OF THE GASTRIC MUCOSA are constantly subjected to the stresses of an acidic pH, the proteolytic enzyme pepsin, and high pressures that develop in the stomach lumen during digestion. Identification of endogenous molecules that protect the mucosa in this hostile environment and facilitate repair of surface epithelial cells when they are injured would provide fresh insight into stomach physiology. A cDNA clone encoding a protein with these characteristics was identified, but not recognized as such, during the cloning of the peptide hormone gastrin, more than a quarter century ago. During the original isolation of cDNA clones that encode gastrin, which is expressed specifically in cells of the gastric antrum (31), another antral-specific mRNA was identified (21). Its open-reading frame, which is highly conserved between human and pig, predicted a novel conserved protein of no readily predicted function (11). The cDNA was expressed in E. coli, and the protein product was used to prepare two specific polyclonal antisera in rabbits. The antisera were subsequently used to localize the protein in the antral mucosa of all seven mammals tested to date (11). Given the tissue specificity of expression of the cDNA sequence and the apparent ubiquitous presence of the protein in the antrum mucosa of mammalian species, the molecule was named AMP-18 for 18-kDa antrum mucosal protein. A cDNA clone called CA11, which predicts an amino acid sequence that differs from human AMP-18 in only a single residue (11), was reported in normal human gastric mucosa, but not in most gastric cancers, by using a differential display technique (25, 32). Although a function for CA11 protein was not identified by the investigators who discovered it, they did suggest that loss of CA11 expression may play a role in gastric carcinogenesis.

Expression of AMP-18 was localized to mouse gastric antrum by using immunohistochemistry and immunoblotting and by Northern blot hybridization of RNAs from porcine gut mucosal tissues (11). Immunoelectron microscopy indicated that the protein is localized within granules just under the apical plasma membrane, suggesting that it is a secreted rather than an integral membrane protein. Initial studies to identify a function of the protein showed that porcine and murine antrum extracts were mitogenic for epithelial cells in culture, and that this growth-promoting effect was blocked by each of two specific antisera (11). A recombinant human (rh) protein was also found to be mitogenic. These observations stimulated us to better characterize release of AMP-18, seek a mitogenic domain within its primary structure, and determine whether it was capable of restitution to learn more about how it could maintain and/or repair the gastric epithelium.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SDS-PAGE and immunoblotting. Proteins in canine gastric antral mucus, the medium of primary cultures of canine antral cells, and mouse antral tissue (obtained as described below) were homogenized in lysis buffer (10 mM Tris, pH 7.4, 5 mM MgCl2, 50 U/ml DNase and RNase, 1 mM phenylmethyl sulfonylfluoride, and 10 µg/ml each of leupeptin, aprotinin, and pepstatin), solubilized in a half-volume of 3x Laemmli sample buffer containing {beta}-mercaptoethanol, separated by SDS-PAGE (12.5%), and blotted onto a polyvinylidene difluoride (Immobilon PSQ; Millipore, New Bedford, MA) membrane by using Towbin buffer (25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3). Nonspecific binding sites were blocked by using 5% (wt/vol) nonfat dry milk in Tris-buffered saline (TBS; 100 mM Tris, 137 mM NaCl, 10 mM KCl, pH 7.3) with 0.05% (vol/vol) Tween 20 (T-TBS) for 1 h at room temperature, as described previously (1). The membrane was then incubated with primary antibody [rabbit antiserum to rh AMP-18 precursor (rh-pre-AMP-18)], described previously (11), or mouse monoclonal anti-cytokeratin peptide 18 clone CY-90 diluted in T-TBS. It was rocked gently overnight at 4°C and then washed four times in T-TBS at room temperature for a total of 30 min. The immunoblot was incubated with a secondary antibody conjugated with horseradish peroxidase for 1 h and washed three times with T-TBS and once with TBS; the labeled proteins were visualized with an enhanced chemiluminescent reagent (SuperSignal West Pico, Pierce, West Rockford, IL).

Release of AMP-18. Lysates of confluent cultures of different gastrointestinal (GI) epithelial cell lines were prepared to study the release of AMP-18. Ten micrograms of total cell protein were assayed for the presence of AMP-18 by immunoblotting lysates prepared from several established gastric lines [AGS, human antral epithelial (HAE), KATO III, RGM-1, NCI-N87, SK-GT5], a gastric line derived from the Immortomouse (30) (gift of R. H. Whitehead, Vanderbilt University) at the permissive or nonpermissive temperature, human colonic adenocarcinoma lines (HT29A1 and CaCo2/bbe, subclone C2), and nontransformed monkey and canine renal (BSC-1, Madin-Darby canine kidney) epithelial cells, none of which expressed the protein. As the protein was not detected in tissue samples from primary or metastatic human gastric carcinomas by immunohistochemical staining, and a nontransformed human gastric epithelial cell line does not exist, preliminary studies were performed by using mouse gastric antrum explants and, subsequently, in primary canine antrum cell cultures. AMP-18 in the medium bathing antrum explants was concentrated with a YM-10 membrane (Amicon), the protein concentration of the medium was measured using the bicinchoninic acid procedure (Pierce), and the proteins were analyzed by immunoblotting. To determine whether AMP-18 is released from cells in vivo, mucus overlying the gastric antrum mucosal surface was aspirated under direct vision from each of three fasted, anesthetized dogs; care was taken not to perturb the mucosal surface during aspiration of the mucus. Proteins in the mucus were separated by SDS-PAGE, and immunoblots were then probed with antisera to AMP-18 and to cytokeratin-18, an intermediate filament protein that served as a marker for desquamated gastric epithelial mucosal cells.

Primary cultures of canine antral mucosal cells. Under general anesthesia, the dog stomach was removed between clamps. The antrum mucosal layer was bluntly separated from the submucosa and rinsed in cold Hanks' balanced salt solution containing 0.1% BSA, 100 mg/l penicillin, and 100 mg/l streptomycin (2). Cells were then dispersed by sequential exposure to collagenase (0.35 mg/ml) and EDTA (1 mM). Mucosal epithelial cells were enriched by centrifugal elutriation, plated at a concentration of 2 x 106 cells/well in 12-well tissue culture plates that had been coated with Matrigel (Becton Dickinson, Bedford, MA) (diluted 1:5 with water) in Ham's F-12/DMEM (50:50 vol/vol) medium containing 10% heat-inactivated dog serum, insulin (1 mg/ml), hydrocortisone (1 mg/ml), and gentamicin (100 mg/l); and allowed to attach in an incubator (5% CO2). After 24 h of stabilization, the cells were washed three times with Earle's balanced salt solution containing 10 mM HEPES (pH 7.4) and 0.1% BSA to remove dead and nonadherent cells and then incubated in medium containing the adenylate cyclase activator forskolin to raise the intracellular level of cAMP (n = 6 cultures) or vehicle (dimethylsulfoxide). One hour later, cells were scraped into a tube, lysed, and assayed for AMP-18 by immunoblotting. Equal protein loading in each lane was confirmed by reprobing the blots with an antibody to rat heat shock cognate 73 (SPA815; Stressgen, Victoria, British Columbia), which is constitutively expressed by these cells. Relative values for AMP-18 immunoreactivity were analyzed with a Macintosh computer by using the public domain National Institutes of Health Image 1.54 program (http://rsb.info.nih.gov/nih-image/). Proteins in culture medium (including released AMP-18) were separated by SDS-PAGE; blots were prepared and then probed with antisera to AMP-18 and heat shock cognate 73.

Effect of intragastric administration of indomethacin on content of AMP-18 in mouse gastric antrum tissue in vivo. To determine whether the amount of AMP-18 in cells of the gastric antrum of mice could be altered by a nonsteroidal anti-inflammatory drug, 20 mg/kg of either indomethacin, a nonselective cyclooxygenase (COX) inhibitor, or rofecoxib (Vioxx; Merck), a selective COX-2 inhibitor, were administered in a solution of 5% sodium bicarbonate by gavage to 20-g, male C57BL/6 mice (Taconic Farms, Germantown, NY). Each drug or the vehicle alone (250 µl) was gavaged into mice deprived of food, but not water, overnight (16–18 h). At specified times thereafter (0–24 h), animals were killed, the stomach was removed and rinsed, and the antrum mucosa was scraped with a glass slide into an homogenizer tube containing ice-cold PBS and then centrifuged (14,000 g for 30 s). Proteins in the pellet were separated by SDS-PAGE and immunoblotted with antibody to rh-pre-AMP-18 (11), as described above. All procedures involving the use of animals were approved by, and in accordance with, the guidelines of the University of Chicago and University of Michigan Animal Care and Use Resources Committees.

Mitogenesis. To measure mitogenic activity, AGS human gastric adenocarcinoma cells, HAE (human gastric antrum epithelial primary cultures transformed with SV40 large T antigen; kindly provided by Dr. Duane Smoot, Howard University College of Medicine), rat diploid small intestinal epithelial cells (IEC) of the IEC-6 and IEC-18 lines, NCI N-87 human gastric carcinoma cells, SK-GT5 human gastroesophageal adenocarcinoma cells, and monkey kidney epithelial BSC-1 cells (11) were studied. Human WI-38 fibroblasts and HeLa cells served as non-GI control cell lines. Mitogenesis was assayed by performing cell counts 4 days after exposing a confluent culture to the agent of interest, adding trypsin to prepare a suspension of single cells, and confirming cell separation while counting them in a hemocytometer, as reported previously (28). To measure DNA synthesis, IEC-6 cells were plated at a density of 3 x 106 cells per 60-mm dish and grown to high density in DMEM containing 1% calf serum (CS) and insulin (100 U/l). The medium was replaced with fresh medium containing 0.01% CS and insulin; cells became quiescent and were used for study 2 days later. The agent of interest (in water) or vehicle (water) was added to the culture medium, and, 20 h afterwards, 12.5 µCi of [methyl-3H]thymidine were added. Five hours later, radioactivity in the trichloroacetic acid-insoluble fraction was measured, as described previously (28).

Mitogenic activity was assessed in each of the following preparations: native AMP-18 in pig antral extracts (11), rhAMP-18 produced by transformed E. coli obtained as described previously (11), and synthetic peptides derived from a central domain of the predicted sequence of mature human AMP-18 prepared as outlined below.

To identify a mitogenic domain within processed, mature human AMP-18, five ~40-mer peptides were synthesized in the University of Chicago Peptide Core Facility. Synthetic AMP peptides were each purified by reversed-phase HPLC by using a gradient of acetonitrile (1–80%) in 0.09% trifluoroacetic acid. The sequence of each peptide was confirmed by microsequencing, and its predicted size was confirmed by mass spectrometry. The purified peptide was then dissolved in water, and its capacity to stimulate growth of cells in culture was assessed, as described above.

Restitution in scrape-wounded monolayer cultures. To measure migration after scrape wounding (5, 6, 18), HAE or IEC-18 cells were grown to high density in A-50 culture medium (Biosource International, Rockville, MD) or DMEM, respectively, containing 1% CS, in 60-mm dishes. Each medium was aspirated and then replaced with fresh medium containing 0.01% CS. The monolayer was mechanically wounded by scraping off a section of it with a razor blade. Detached cells were removed by aspirating the medium and rinsing the remaining cells twice with fresh medium containing 0.01% CS. Fresh medium (5 ml) containing CS (0.01%) and insulin (100 U/l) was added to scrape-wounded cultures. Either a synthetic AMP peptide, EGF, or both were added to duplicate cultures. Migration was assessed at 24, 48, and 72 h after wounding by measuring the distance (in mm) that cells had migrated from the wound edge by using a microscope eyepiece reticle (10 mm long; 0.1-mm markings). The distance traveled by migrating cells at 12 randomly chosen sites along a 0.25-mm segment of the wound edge was measured at 40-fold magnification. Migration was assessed at different sites in two separate wounds made in each culture.

Statistics. Data were compared by Student's t-test; P < 0.05 was accepted as significant. Values are means ± SE.

Reagents were purchased from Sigma (St. Louis, MO), unless otherwise specified.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Secretion of AMP-18 by gastric mucosal epithelial cells. Evidence for secretion of AMP-18 in vivo was sought by aspirating aliquots of mucus overlying the gastric antrum of three fasted, anesthetized dogs without perturbing the mucosal surface. As shown in the right lane of Fig. 1, a representative immunoblot of this mucus revealed abundant AMP-18 with only a very weak signal for cytokeratin-18, a marker protein used to detect desquamated gastric epithelial cells. These results, taken together with the previous immunoelectron microscopic localization of AMP-18 in secretion granules (11), suggest that AMP-18 is released from the cells into the mucus that overlies the antral mucosa, rather than being deposited there when detached surface cells degenerate.



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Fig. 1. Detection of immunoreactive 18-kDa antrum mucosal protein (AMP-18) in mucus overlying the canine gastric antrum. Right lane: mucus was aspirated from the gastric antrum of an anesthetized dog, and its protein content was measured. Left lane: canine antrum mucosal epithelial cells were isolated, and primary cultures were prepared as described in MATERIALS AND METHODS. After 1 day in culture, the cells were collected and lysed, and the protein content of the lysate was measured. Mucous and cell lysate proteins were separated by SDS-PAGE. Each lane was loaded with 10 µg protein. Blots were prepared and probed with the rabbit anti-human pre-AMP-18 serum and with an antiserum to cytokeratin-18. As expected, the abundance of cytokeratin-18 was far greater in cell lysate than in mucus. Images shown are representative of those obtained on 3 occasions.

 

Next we asked if secretion of intracellular AMP-18 is subject to regulation. Although we were unable to identify an established gastric, colonic, or renal epithelial cell line that contained the protein, a pilot study using mouse antrum explants was employed to test the hypothesis that AMP-18 is released by the cells. Immunoblot analysis of proteins in the explant bathing medium revealed that AMP-18 was present in the serum-free, buffered salt solution at pH 7, as well as at pH 3, and, to a greater extent, at 37°C than at room temperature (data not shown). Next, primary cultures of canine antral epithelial cells were prepared (2) and shown to contain AMP-18 when immunoblotting was performed on extracts of cell monolayers. Forskolin, a compound known to raise intracellular cAMP, was added to the monolayer to determine whether this second messenger acts as a secretogogue for AMP-18 as it does for parathyroid hormone (3). Measurements based on immunoblots of cell lysates indicated that AMP-18 immunoreactivity declined by 38% (P < 0.05) 1 h after exposure to forskolin (Fig. 2).



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Fig. 2. Content of AMP-18 in primary cultures of canine antral epithelial cells is reduced after exposure of cells to forskolin. Freshly isolated cells were plated onto matrigel-coated wells. After 24 h, forskolin (10 µM) was added to the culture medium, and, 1 h later, cells were harvested, lysed, and assayed for AMP-18 by immunoblotting. AMP-18 immunoreactivity in cells after exposure to forskolin in 2 experiments was lower than in control cultures (P < 0.05). Equal protein loading in each lane was documented by probing the blots with an antibody to heat shock cognate 73 (hsc73), which is constitutively expressed by these cells. Values are means ± SE.

 

To determine whether AMP-18 release is triggered in vivo by an agent known to act on the gastric antrum of humans and rodents (13, 24), indomethacin, a nonselective COX inhibitor, was gavaged into C57BL/6 mice. Immunoreactive AMP-18 in antral mucosal scrapings was reduced by 70% in animals given indomethacin compared with control animals at 4 h (P < 0.02) (Fig. 3). However, no histological evidence of gastric mucosal injury in the treated mice was detected before 18 h, as reported previously (13). In addition to the negative histological findings, further evidence that indomethacin did not induce cell detachment was obtained when immunoblots revealed no differences between control and indomethacin-treated tissue when probed with an antiserum to cytokeratin-18. As in the immunoblots (Fig. 3), immunohistochemical analysis of tissue from mice exposed to indomethacin for 8 h revealed less AMP-18 within cells of the antral surface and upper crypts than in control animals gavaged with the vehicle (not shown). The apparent absence of mucosal cell detachment by histological and immunohistochemical techniques suggests that exposure to a nonselective COX inhibitor decreases mucosa cell content of AMP-18, possibly by stimulating its secretion. Rofecoxib, a COX-2-selective inhibitor, was not associated with a fall in the level of AMP-18 in antral tissue for up to 18 h after it was gavaged (n = 3 mice) (data not shown). In summary, these and previous observations (11) suggest that AMP-18 is located in secretion granules in gastric antrum epithelial cells and that its release may be triggered by physiological and pharmacological agents.



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Fig. 3. Intragastric administration of indomethacin to mice reduces antrum mucosal content of AMP-18. C57BL/6 mice were gavaged with indomethacin (20 mg/kg) in sodium bicarbonate (5%). At specific times thereafter, antrum mucosal tissue was assayed for immunoreactive AMP-18. Indomethacin administration reduced AMP-18 content in antrum epithelium at 4 and 8 h (*P < 0.02). At 24 h, AMP-18 immunoreactivity returned to a value similar to that observed in control tissue. Equal protein loading in each lane was confirmed by probing blots with an antibody to hsc73. AMP-18 immunoreactivity in different mice was compared after standardizing each sample for hsc73 immunoreactivity. Values for AMP-18 immunoreactivity are means ± SE for at least 3 mice, and each band at top depicts a blot for a typical mouse at the designated time.

 

A growth-stimulatory domain of human AMP-18. The renewal time of pit cells in the mouse gastric antrum is only 2.8 days (9), possibly in response to the low pH, the action of pepsin, and high intraluminal pressures developed during digestion. Previous studies showing the mitogenic effect of porcine and murine antral tissue extracts and rhAMP-18 (11) support the hypothesis that localization of AMP-18 in surface epithelial cells and its apparent release (Figs. 1, 2, 3) allow it to function as an autocrine growth factor that could maintain and repair the gastric mucosa under these adverse conditions. To look for a mitogenic domain within AMP-18, five relatively large oligopeptides (of ~40 amino acids each), spanning the 165 amino acids of the mature protein (not including the signal peptide), were demarcated within the open-reading frame of the human cDNA clone (11). Growth stimulation was assessed by counting the number of BSC-1 cells 4 days after exposure to different concentrations of the peptide under study. Mitogenic peptides each showed growth stimulation of ~230% compared with 165% in control cultures (P < 0.001) and were compared with each other by calculating the peptide concentration at which proliferation was half-maximal (K1/2). One peptide, a 42-mer spanning amino acid 58–99 of the mature form of the protein shown in Ref. 11 (equivalent to lysine-78 to leucine-119 of the pre-AMP-18 sequence), exhibited mitogenic activity (K1/2 = 0.3 µM) (Table 1). Its growth-promoting activity was totally blocked by the specific antisera, but not the preimmune sera, and immunoblots showed that the antisera recognized epitope(s) on the synthetic peptide (not shown). The reaction of AMP peptide 58–99 with the antibodies was not unexpected, because this region of the sequence is predicted to be exposed on the surface of the protein and to be antigenic. Synthetic peptide 58–99 appears to exert its growth-promoting effect via the same pathway as native AMP-18, because their maximal mitogenic effects are not additive (not shown).


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Table 1. Analysis of the mitogenic domain of AMP-18

 

To more rigorously define the mitogenic domain, the sequence of the 42-mer (peptide 58–99) was divided so that a lysine-lysine (K-K) doublet was at the NH2 terminus, and a single K was at the carboxy (C)-terminus of each of three new peptides that were synthesized, HPLC purified, and assayed for mitogenic activity (Table 1). Peptides 58–68 and 67–85 were inactive. Growth was stimulated by peptide 84–97, but required a higher molar concentration to reach a similar maximal value than did peptide 58–99; this is reflected in the higher K1/2 (0.8 µM), which suggests that peptide 84–97 (a 14-mer) has only 38% of the activity of the 42-mer.

Peptide 84–97 was then extended in the NH2-terminal or COOH-terminal direction to determine whether a slightly longer peptide would replicate the greater mitogenic potency of the 42-mer. Peptide 77–97 was synthesized by extending peptide 84–97 by seven amino acids toward the NH2 terminus, whereas peptide 84–101 was produced by extending peptide 84–97 by four amino acids toward the COOH terminus. Adding seven amino acids to form peptide 77–97 resulted in a K1/2 of 0.3 µM, suggesting that the affinity of this 21-mer for a putative receptor was similar to that of the 42-mer (K1/2: 0.3 µM). Extending peptide 84–97 by four residues produced peptide 84–101, a less potent mitogen (K1/2: 1.0 µM). The similar K1/2 values (0.3 µM) for peptides 58–99 and 77–97 imply equivalent mitogenic potency, despite the twofold difference in their lengths. Amino acids 77–97 appear, therefore, to represent the mitogenic domain contained within the 42-mer (peptide 58–99). When peptide 77–97 was divided into smaller fragments, peptides that were 6, 9, 14, and 18 amino acids in length were each mitogenic, but their K1/2 values were higher than for the 21-mer, indicating that they were not as potent (Table 1). A 4-mer was not mitogenic at concentrations up to 120 µM. The small size of peptides 84–101 (18-mer), 84–97 (14-mer), 89–97 (9-mer), and 84–89 (6-mer) suggest that they exert their mitogenic effects via a receptor-mediated mechanism, because none of them is long enough to extend through the plasma membrane, which usually requires a minimum of 20 amino acids. Peptide 77–97, a 21-mer, seems unlikely to insert into the plasma membrane, because its amino acid composition predicts a strongly hydrophilic character; its relative hydrophobicity value (14, 28) is -44.4 kcal/mol.

AMP-18 and its derived peptides stimulate growth of stomach and IECs. To assess the role of AMP-18 as a gastric cell growth factor, its effect on proliferation of four human stomach lines (AGS, HAE, NCI N-87, SKGT5) was studied. Mitogenic stimulation of AGS cells was observed with porcine antrum mucosal tissue extract and synthetic human AMP peptide 77–97 (Fig. 4, top). As expected, rabbit antiserum to AMP-18 precursor protein inhibited growth-promoting activity of the antrum extract, but not of the much smaller peptide 77–97, suggesting that the mitogenic 21-mer lacks the epitope(s). In AGS cells, growth stimulation by peptide 77–97 was additive with the maximal mitogenic concentration of EGF (P < 0.001), suggesting that the two mitogens do not use the same receptor and/or utilize different signaling pathways (Fig. 4, top right). The scrambled isoform of peptide 77–97 (Table 1) did not stimulate the growth of AGS cells (data not shown).



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Fig. 4. Effect of porcine antrum mucosal extract, human AMP peptide 77–97, and EGF on growth of gastric (AGS) and intestinal (IEC-6) cells. Different amounts of antrum extract, peptide 77–97, and/or EGF were added to the culture medium, and 4 days later the no. of cells was counted. Antrum extract and AMP peptide each stimulated growth of both AGS (top left and middle) and IEC-6 (bottom left and middle) cells (P < 0.001 at 2 µg/ml) in a concentration-dependent manner. Values are means ± SE for at least 4 cultures. When antrum extract (2 µg/ml) was preincubated for 30 min with rabbit antiserum to human AMP-18 (1:100 dilution; +Ab) before addition to the culture medium, growth stimulation was reduced by 90% (top left, {triangleup}) (P < 0.001); preimmune serum had no effect (data not shown). The antiserum did not alter the mitogenic effect of peptide 77–97 (2 µg/ml) (top middle, {triangleup}). The mitogenic potency of peptide 77–97 (8 µg/ml) (P < 0.001) was similar to EGF (50 ng/ml) (P < 0.001) in AGS cells (top right), but the peptide (1 µg/ml) appeared more potent than EGF in IEC-6 cells (bottom right) (P < 0.001). Cont, control.

 

HAE cells were studied to test whether AMP-18 could exert its mitogenic effect on epithelial cells that exist in the local environment of its synthesis in vivo. Fig. 5, left, shows that AMP peptide 77–97 stimulated growth of these cells, as did EGF (P < 0.001). Growth of NCI N-87 cells and SK-GT5 cells was also stimulated by porcine or murine antrum extract, peptide 77–97, or EGF in a concentration-dependent manner (data not shown). Antiserum to AMP-18 blocked the mitogenic effect of antrum extract on these two gastric epithelial cell lines, but not the proliferative effects of peptide 77–97 or EGF. As each of the four human stomach epithelial cell lines studied is transformed, and a nontransformed gastric epithelial cell line is not available, we also studied nontransformed, epithelial lines from two other species: rat intestinal IEC-6 and IEC-18 cells, and monkey kidney BSC-1 cells. As with the gastric epithelial cells, growth of rat diploid IEC-6 cells was also stimulated by the antrum extract, peptide 77–97, and EGF, although the peptide appeared to be a more potent mitogen than EGF (Fig. 4, bottom right) (P < 0.001). The mitogenic effect of peptide 77–99 was corroborated by measuring [3H]thymidine incorporation into DNA in confluent cultures of IEC-6 cells, which was stimulated by 68% (P < 0.001), from 16,668 ± 616 counts per minute/3 x 106 cells in control cultures to 28,036 ± 882 counts per minute/3 x 106 in cells exposed to AMP peptide (8 µg/ml) for 25 h. Preimmune sera had no effect on growth. Purified rhAMP-18 stimulated growth of IEC-18 cells to the same extent as did AMP peptide 77–97, but the K1/2 required by the peptide (300 nM) (Table 1) was far greater than that for the recombinant protein (5 nM) (11). Scrambled AMP peptide (Table 1) did not increase cell number in cultures of either IEC-18 or BSC-1 cells at concentrations up to 8 µg/ml (3.7 µM), and AMP peptide 77–97 did not stimulate growth of human fibroblastic (WI-38) or epidermoid (HeLa) cells at concentrations up to 8 µg/ml. These observations imply that AMP peptide could exert its mitogenic effect on certain epithelial cells via a common surface receptor, although gastric, possibly intestinal, but not renal cells, would be exposed to AMP peptides in vivo.



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Fig. 5. Effect of AMP peptide 77–99 and EGF on growth and wound restitution of human antrum epithelial cells. To measure growth (left), human antrum epithelial (HAE) cells were plated in 60-mm dishes. Peptide 77–97 (8 µg/ml) or EGF (50 ng/ml) or both were added to the medium, and the no. of cells was counted 4 days later. AMP peptide and EGF each stimulated proliferation (P < 0.001). Values are means ± SE for at least 5 cultures. To measure migration (right), cells were grown in 60-mm dishes to obtain a confluent monolayer. The medium was aspirated and replaced with fresh medium containing 0.01% calf serum (CS), and the monolayer was mechanically wounded by scraping with a razor blade. Detached cells were removed by aspirating the medium and rinsing the wounded monolayer twice with fresh medium containing 0.01% CS. Fresh medium (5 ml) containing 0.01% CS alone or with either peptide 77–97 (8 µg/ml), EGF (50 ng/ml), or both was added to each of 2 wounded cultures. Migration was assessed at 72 h after wounding by measuring the distance (in mm) that cells had migrated from the wound edge, as described in MATERIALS AND METHODS. Values are the mean distance that cells migrated into the denuded area from the edge of 2 different wounds in each of 2 cultures ± SE. Cells exposed to peptide 77–97 migrated further from the wound edge than those exposed to vehicle (P < 0.001). EGF also stimulated wound resurfacing, and the 2 agents appeared to act in an additive manner to enhance migration (P < 0.001). C, control.

 

Competitive mitogenic inhibition by AMP-18-derived peptides. To better characterize the apparent interaction between AMP peptides and their binding site(s) on the cell surface, nontransformed rat IEC-18 cells were studied. We tested the hypothesis that progressively increasing the concentration of nonmitogenic peptide 67–85 would block growth stimulation by peptide 58–99 if this mitogenic 42-mer exerts its effect by a receptor-mediated mechanism. Peptide 58–99 stimulated an increase in cell number of 407% compared with 290% by the vehicle in a 3-day assay (P < 0.001). As the concentration of peptide 67–85 was raised progressively to ~0.1 µg/ml, the growth-stimulatory effect of peptide 58–99 was nearly abolished (P < 0.001) (Fig. 6), suggesting that the two peptides compete for the same surface "receptor" site.



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Fig. 6. Effect of AMP peptide 67–85 on growth of intestinal epithelial cells stimulated by peptide 58–99. Confluent cultures of IEC-18 cells were prepared. One day later, medium was aspirated and replaced with 5 ml of DMEM containing CS (0.5%) and insulin, without (control) or with mitogenic peptide 58–99 (8 µg/ml). Sister plates receiving 1 ml medium and different amounts of peptide 67–85 were incubated for 1 h at 38°C in a CO2 incubator, and then an additional 4 ml of medium were added to each dish. Peptide 58–99 was added to 2 of the 4 sister plates at each concentration of peptide 67–85, and the no. of cells was counted. In the absence of peptide 67–85, cell no. increased by 290%, whereas cells exposed to peptide 58–99 (P < 0.001) increased in number by 407%, and EGF-treated (50 ng/ml) cells increased by 402% (not shown) during the next 3 days. The 40% relative stimulation of cell growth by mitogenic peptide 58–99 was completely abolished by preincubation of cells with peptide 67–85 (0.25 µg/ml). When added alone, peptide 67–85 (0.25–8 µg/ml) had no effect on cell proliferation. Values for the no. of cells per culture (n = 4) are shown relative to multiplication of cells exposed to the vehicle during the same period.

 

Restitution after scrape wounding. As restitution is an important component of wound repair, and mitogenic proteins such as EGF are also motogenic (5), we added AMP peptide to scrape-wounded monolayer cultures of HAE cells and found that it stimulated migration of cells at the wound edge at 72 h (Fig. 5, right). This enhancement of wound restitution was also detected after 24 or 48 h of exposure to AMP peptide (Fig. 7), before any mitogenic effect can be detected by an increase in cell number. AMP peptide (Fig. 7B) and rhAMP-18 (not shown) also enhanced migration in nontransformed rat intestinal cells of the IEC-18 line after scrape wounding. This motogenic effect of peptide 77–97 was additive with EGF (Fig. 5, right). Whether there is synergism or not in vivo, the observed additivity suggests that AMP-18 may play an important role in maintaining an intact stomach mucosal epithelium and in facilitating its repair after injury.



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Fig. 7. Time course of the effect of AMP peptide 77–97 on wound restitution in HAE (A) and rat IEC-18 (B) cells. Confluent monolayer cultures were mechanically wounded by scraping with a razor blade, and the distance that cells migrated from the wound edge was measured, as described in Fig. 5 legend. Cells migrated further in the presence of AMP peptide at each time point studied (P < 0.005). No difference in cell number was detected in nonwounded cultures in the presence or absence of AMP peptide at 48 h. Values are means ± SE.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies using immunohistochemistry and immunoelectron microscopy suggested that AMP-18 is packaged within secretory granules, which are abundant just under the apical surface of gastric epithelial cells that comprise the antral mucosa (11). The protein appears to be secreted because it was detected (by immunoblotting) in the bathing medium of mouse antral explants and in gastric mucus aspirated from the antrum surface of anesthetized dogs (Fig. 1). Furthermore, decreased AMP-18 content in primary cultures of canine antrum cells exposed to forskolin (Fig. 2) and structurally intact mouse gastric antrum tissue after indomethacin gavage (Fig. 3) suggest that secretion of the protein is subject to regulation. These observations imply that AMP-18 cosecretion with mucins into the viscoelastic gel that overlies the antrum epithelium may be regulated by at least two different signals: an increase in intracellular cyclic AMP and exposure to indomethacin, a nonselective COX inhibitor, but not an agent that is COX-2 selective (rofecoxib). However, the results do not exclude the possibility that exposure to forskolin and/or indomethacin reduces AMP-18 immunoreactivity by stimulating degradation of the protein. Defining the mechanisms by which each of these agents modulates AMP-18 release, degradation, and/or production will require further study.

AMP-18 has now been found to exert pleiotropic effects that enhance mitogenesis and restitution, whether studied as AMP peptide 77–97 (Table 1, Figs. 4 and 5), rhAMP-18 (11), or the native protein in antrum tissue extracts (Fig. 4). As a component of the viscoelastic gel, AMP-18, or possibly a peptide fragment of it in vivo, could protect the antrum epithelium against stresses, such as the action of pepsin, acidic pH, mechanical forces, and high pressures that develop in the gastric lumen during digestion. As a gastric epithelial cell growth factor, AMP-18 could facilitate replenishment of the surface luminal epithelial cell layer to maintain mucosal integrity, if it gains access to the proliferative zone in gastric crypts by back diffusion after injury following damage by nonsteroidal anti-inflammatory drugs, ethanol, or pathogens. After injury of the gastric mucosal surface, restitution occurs very rapidly (8), followed by proliferation and differentiation to reestablish epithelial integrity (15, 20), processes in which AMP-18 and other endogenous molecules (4, 22, 27) could play a role.

Comparison of the predicted secondary structures for AMP-18 proteins of human, pig, and mouse presented in the companion report (11) suggests a conserved helix-loop-sheet domain in a central region now shown to encompass a bioactive peptide, i.e., amino acids 77–101 (Table 1). Studies of peptides within this domain suggest a relatively simple linear model for the growth-stimulatory region: there is an N-terminal extended binding domain (predicted to be largely helical in character, the relative rigidity of which may explain the linear organization of the relevant sequences as determined in the cell growth studies), followed by a region rich in glycine and proline predicted to be a loop structure (Table 1). Although it is unlikely that bioactive peptides assume a stable structure in aqueous solution, we take the conserved predictions to indicate that the structural potentials of this region of AMP-18 may be important for its biological function. It seems reasonable to predict that the interaction of mitogenic peptides with a cell surface receptor could stabilize the active conformation and that the requirement to transiently form the appropriate conformation in solution would explain the lower activity of peptides (K1/2: 0.3–1.0 µM) relative to the full-length protein for rhAMP-18 (K1/2: 5 nM). We would explain the specificity of antagonism by peptides 58–68 and 67–85 based on whether they overlap or not the agonist peptides 58–99 and 84–97; for example 58–68 overlaps and inhibits 58–99, but does not overlap or inhibit 84–97. Finally, only peptide 58–99 (the 42-mer) is recognized by the antisera; peptide 77–97 (a 21-mer) apparently does not contain or cannot form the epitope.

Although a receptor for AMP peptide/AMP-18 has not been identified, data presented in Table 1 are consistent with the hypothesis that peptide-mediated mitogenesis is mediated via a cell surface binding site. The higher K1/2 value for peptide 84–97 (14-mer) (K1/2: 0.8 µM) than for peptide 58–99 (42-mer) (K1/2: 0.3 µM) (Table 1) suggests that the size and/or sequence of the smaller peptide limits its capacity to bind to a surface site, perhaps due to a reduced ability to form the correct conformation, or possibly because of the loss of ancillary binding regions. The latter notion also is supported by our observations that the nonmitogenic peptides 58–68 and 67–85 can each block the mitogenic activity of peptide 58–99 and the porcine antrum extract (not shown). Finally, peptide 67–85, but not 58–68, antagonizes the activity of peptide 84–97; interestingly, peptide 67–85 overlaps the adjacent 84–97 sequence by two residues.

In summary, AMP-18 may play an important role as a gastrokine in maintaining gastric mucosal integrity and mediating repair after injury, as described for other endogenous proteins synthesized by cells of the GI epithelium, such as trefoil peptides (7, 12, 19, 23, 26) and {alpha}-defensins (10, 16, 17). Some structural and functional characteristics of these molecules are compared in Table 2. Each of them is secreted by a specific type of GI epithelial cell and differs with regard to the size of its propeptide, mature processed protein, and cDNA. In terms of biological function, only AMP-18 is mitogenic, although it apparently shares with trefoil peptides (i.e., intestinal trefoil factor) the capacity to stimulate restitution, whereas only the {alpha}-defensin cryptidin 3 is known to induce chloride secretion. Of particular interest is the relatively low concentration (<1 µM) required for either AMP peptide 77–97 or rhAMP-18 protein to exert its biological effects, compared with trefoil peptides and cryptidin 3 (>100 µM), a characteristic more typical of a growth factor or cytokine than a general environmental factor. Additional studies will be required to define these and other roles of AMP-18 in physiological and pathological states.


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Table 2. Comparison of structural and functional characteristics of trefoil peptides, an {alpha}-defensin, and AMP-18

 


    ACKNOWLEDGMENTS
 
This work was supported by a grant from the Crohn's and Colitis Foundation of America (to F. G. Toback) and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-42086 and DK-47722 (to E. B. Chang).


    FOOTNOTES
 

Address for reprint requests and other correspondence: F. G. Toback, The Univ. of Chicago, Dept. of Medicine, MC5100, 5841 South Maryland Ave., Chicago, IL 60637 (E-mail: gtoback{at}medicine.bsd.uchicago.edu).

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Aithal HN, Walsh-Reitz MM, Kartha S, Gluck SL, Franklin WA, Knigge KM, and Toback FG. Kinetics of a novel cytosolic protein during the onset of renal epithelial cell growth. Am J Physiol Renal Fluid Electrolyte Physiol 255: F868–F873, 1988.[Abstract/Free Full Text]
  2. Beales I, Blaser MJ, Srinivasan S, Calam J, Perez-Perez GI, Yamada T, Scheiman J, Post L, and Del Valle J. Effect of Helicobacter pylori products and recombinant cytokines on gastrin release from cultured canine G cells. Gastroenterology 113: 465–471, 1997.[ISI][Medline]
  3. Chen CJ, Anast CS, and Brown EM. High osmolality: a potent parathyroid hormone secretogogue in dispersed parathyroid cells. Endocrinology 121: 958–964, 1987.[Abstract]
  4. Dignass AU, Becker A, Spiegler S, and Goebell H. Adenine nucleotides modulate epithelial wound healing in vitro. Eur J Clin Invest 28: 554–561, 1998.[ISI][Medline]
  5. Kartha S and Toback FG. Adenine nucleotides stimulate migration in wounded cultures of kidney epithelial cells. J Clin Invest 90: 288–292, 1992.[ISI][Medline]
  6. Kartha S and Toback FG. Migration of kidney epithelial cells during recovery from acute renal failure. In: Acute Renal Failure. New Concepts and Therapeutic Strategies, edited by Goligorsky MS. New York: Churchill Livingstone, 1995, p. 287–319.
  7. Kindon H, Pothoulakis C, Thim L, Lynch-Devaney K, and Podolsky DK. Trefoil peptide protection of intestinal epithelial barrier function: cooperative interaction with mucin glycoprotein. Gastroenterology 109: 516–523, 1995.[ISI][Medline]
  8. Lacy ER, Morris GP, and Cohen MM. Rapid repair of the surface epithelium in human gastric mucosa after acute superficial injury. J Clin Gastroenterol 17: S125–S135, 1993.[ISI][Medline]
  9. Lee ER. Dynamic histology of the antral epithelium in the mouse stomach. III. Ultrastructure and renewal of pit cells. Am J Anat 172: 225–240, 1985.[ISI][Medline]
  10. Lencer WI, Cheung G, Strohmeier GR, Currie MG, Ouellette AJ, Selsted ME, and Madara JL. Induction of epithelial chloride secretion by channel-forming cryptdins 2 and 3. Proc Natl Acad Sci USA 94: 8585–8589, 1997.[Abstract/Free Full Text]
  11. Martin TE, Powell CT, Wang Z, Bhattaacharyya S, Walsh-Reitz MM, Agarwal K, and Toback FG. A novel mitogenic protein that is highly expressed in cells of the gastric antrum mucosa. Am J Physiol Gastrointest Liver Physiol 285: G332–G343, 2003.[Abstract/Free Full Text]
  12. May FE and Westley BR. Trefoil proteins: their role in normal and malignant cells. J Pathol 183: 4–7, 1997.[ISI][Medline]
  13. Morise Z, Granger DN, Fuseler JW, Anderson DC, and Grisham MB. Indomethacin induced gastropathy in CD18, intercellular adhesion molecule 1, or P-selectin deficient mice. Gut 45: 523–528, 1999.[Abstract/Free Full Text]
  14. Nozaki Y and Tanford C. The solubility of amino acids and two glycine peptides in aqueous ethanol and dioxane solutions. Establishment of a hydrophobicity scale. J Biol Chem 246: 2211–2217, 1971.[Abstract/Free Full Text]
  15. Nusrat A, Delp C, and Madara JL. Intestinal epithelial restitution. Characterization of a cell culture model and mapping of cytoskeletal elements in migrating cells. J Clin Invest 89: 1501–1511, 1992.[ISI][Medline]
  16. Ouellette AJ IV. Paneth cell antimicrobial peptides and the biology of the mucosal barrier. Am J Physiol Gastrointest Liver Physiol 277: G257–G261, 1999.[Abstract/Free Full Text]
  17. Ouellette AJ and Selsted ME. Paneth cell defensins: endogenous peptide components of intestinal host defense. FASEB J 10: 1280–1289, 1996.[Abstract/Free Full Text]
  18. Pawar S, Kartha S, and Toback FG. Differential gene expression in migrating renal epithelial cells after wounding. J Cell Physiol 165: 556–565, 1995.[ISI][Medline]
  19. Podolsky DK. Healing the epithelium: solving the problem from two sides. J Gastroenterol 32: 122–126, 1997.[ISI][Medline]
  20. Podolsky DK. Mucosal immunity and inflammation. V. Innate mechanisms of mucosal defense and repair: the best offense is a good defense. Am J Physiol Gastrointest Liver Physiol 277: G495–G499, 1999.[Abstract/Free Full Text]
  21. Powell CT. Characterization of a Novel Messenger RNA and Immunochemical Detection of its Protein From Porcine Gastric Mucosa (PhD Dissertation). Chicago, IL: University of Chicago Press, 1987.
  22. Rutten MJ, Dempsey PJ, Solomon TE, and Coffey RJ Jr. TGF-{alpha} is a potent mitogen for primary cultures of guinea pig gastric mucous epithelial cells. Am J Physiol Gastrointest Liver Physiol 265: G361–G369, 1993.[Abstract/Free Full Text]
  23. Sands BE and Podolsky DK. The trefoil peptide family. Annu Rev Physiol 58: 253–273, 1996.[ISI][Medline]
  24. Satoh H, Inada I, Hirata T, and Maki Y. Indomethacin produces gastric antral ulcers in the refed rat. Gastroenterology 81: 719–725, 1981.[ISI][Medline]
  25. Shiozaki K, Nakamori S, Tsujie M, Okami J, Yamamoto H, Nagano H, Dono K, Umeshita K, Sakon M, Furukawa H, Hiratsuka M, Kasugai T, Ishiguro S, and Monden M. Human stomach-specific gene, CA11, is down-regulated in gastric cancer. Int J Oncol 19: 701–707, 2001.[ISI][Medline]
  26. Suemori S, Lynch-Devaney K, and Podolsky DK. Identification and characterization of rat intestinal trefoil factor: tissue- and cell-specific member of the trefoil protein family. Proc Natl Acad Sci USA 88: 11017–11021, 1991.[Abstract]
  27. Takahashi M, Ota S, Shimada T, Hamada E, Kawabe T, Okudaira T, Matsumura M, Kaneko N, Terano A, Nakamura T, and Omata M. Hepatocyte growth factor is the most potent endogenous stimulant of rabbit gastric epithelial cell proliferation and migration in primary culture. J Clin Invest 95: 1994–2003, 1995.[ISI][Medline]
  28. Toback FG. Induction of growth in kidney epithelial cells in culture by Na+. Proc Natl Acad Sci USA 77: 6654–6656, 1980.[Abstract]
  29. Von Heijne G and Blomberg C. Trans-membrane translocation of proteins. The direct transfer model. Eur J Biochem 97: 175–181, 1979.[Abstract]
  30. Whitehead RH, VanEeden PE, Noble MD, Ataliotis P, and Jat PS. Establishment of conditionally immortalized epithelial cell lines from both colon and small intestine of adult H-2KbtsA58 transgenic mice. Proc Natl Acad Sci USA 90: 587–591, 1993.[Abstract]
  31. Yoo OJ, Powell CT, and Agarwal KL. Molecular cloning and nucleotide sequence of full-length of cDNA coding for porcine gastrin. Proc Natl Acad Sci USA 79: 1049–1053, 1982.[Abstract]
  32. Yoshikawa Y, Mukai H, Hino F, Asada K, and Kato I. Isolation of two novel genes, down-regulated in gastric cancer. Jpn J Cancer Res 91: 459–463, 2000.[ISI][Medline]