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
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ABSTRACT |
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growth factor; wound healing; restitution; epithelium; stomach
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.
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MATERIALS AND METHODS |
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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 (1618 h). At specified times thereafter (024 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 (180%) 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.
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RESULTS |
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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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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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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
5899 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 5899 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 5899 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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To more rigorously define the mitogenic domain, the sequence of the 42-mer (peptide 5899) 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 5868 and 6785 were inactive. Growth was stimulated by peptide 8497, but required a higher molar concentration to reach a similar maximal value than did peptide 5899; this is reflected in the higher K1/2 (0.8 µM), which suggests that peptide 8497 (a 14-mer) has only 38% of the activity of the 42-mer.
Peptide 8497 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 7797 was synthesized by extending peptide 8497 by seven amino acids toward the NH2 terminus, whereas peptide 84101 was produced by extending peptide 8497 by four amino acids toward the COOH terminus. Adding seven amino acids to form peptide 7797 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 8497 by four residues produced peptide 84101, a less potent mitogen (K1/2: 1.0 µM). The similar K1/2 values (0.3 µM) for peptides 5899 and 7797 imply equivalent mitogenic potency, despite the twofold difference in their lengths. Amino acids 7797 appear, therefore, to represent the mitogenic domain contained within the 42-mer (peptide 5899). When peptide 7797 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 84101 (18-mer), 8497 (14-mer), 8997 (9-mer), and 8489 (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 7797, 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 7797 (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 7797, suggesting that the mitogenic 21-mer lacks the epitope(s). In AGS cells, growth stimulation by peptide 7797 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 7797 (Table 1) did not stimulate the growth of AGS cells (data not shown).
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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 7797 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 7797, 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 7797 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 7797, 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 7799 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 7797, 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 7797 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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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 6785 would block growth
stimulation by peptide 5899 if this mitogenic 42-mer exerts its effect
by a receptor-mediated mechanism. Peptide 5899 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 6785 was
raised progressively to 0.1 µg/ml, the growth-stimulatory effect of
peptide 5899 was nearly abolished (P < 0.001)
(Fig. 6), suggesting that the
two peptides compete for the same surface "receptor" site.
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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 7797 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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DISCUSSION |
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AMP-18 has now been found to exert pleiotropic effects that enhance mitogenesis and restitution, whether studied as AMP peptide 7797 (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 77101 (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: 0.31.0 µM) relative to the full-length protein for rhAMP-18 (K1/2: 5 nM). We would explain the specificity of antagonism by peptides 5868 and 6785 based on whether they overlap or not the agonist peptides 5899 and 8497; for example 5868 overlaps and inhibits 5899, but does not overlap or inhibit 8497. Finally, only peptide 5899 (the 42-mer) is recognized by the antisera; peptide 7797 (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 8497 (14-mer) (K1/2: 0.8 µM) than for peptide 5899 (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 5868 and 6785 can each block the mitogenic activity of peptide 5899 and the porcine antrum extract (not shown). Finally, peptide 6785, but not 5868, antagonizes the activity of peptide 8497; interestingly, peptide 6785 overlaps the adjacent 8497 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 -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
-defensin cryptidin 3 is known
to induce chloride secretion. Of particular interest is the relatively low
concentration (<1 µM) required for either AMP peptide 7797 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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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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