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Departments of 1 Medicine, 3 Pathology and Laboratory Medicine, and 6 Biochemistry, Emory University School of Medicine; 2 Nutrition and Health Sciences Program, Emory University, Atlanta, Georgia 30322; 5 Department of Pathology, Amgen Inc., Thousand Oaks, California 91320; and 4 Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The trefoil factor family
peptides TFF1, TFF2, and TFF3 are important for gut mucosal protection
and restitution. Keratinocyte growth factor (KGF) stimulates
proliferation and differentiation of epithelial cells with potent
effects on goblet cells. To investigate interactions between food
intake and KGF, rats were fed ad libitum (control), fasted for 72 h, or fasted for 72 h and then refed for 72 h with or without
KGF (3 mg · kg
1 · day1).
With fasting, goblet cell number in duodenum increased, TFF3 mRNA in
duodenum and jejunum decreased, and TFF3 protein did not change or
increased. KGF during fasting stimulated colonic growth, normalized
TFF3 mRNA in duodenum and jejunum, and broadly upregulated gut goblet
cell number and TFF3 protein expression. With fasting-refeeding, KGF
increased small bowel and colonic mucosal growth, goblet cell number,
and TFF3 protein but had variable effects on TFF3 mRNA. KGF induced
TFF2 mRNA and protein in duodenum and jejunum with both nutritional
regimens. We conclude that nutrient availability modifies rat
intestinal goblet cell number, TFF3 mRNA, and the gut-trophic effects
of KGF in a region-specific manner. KGF enhances TFF2 expression in
proximal small bowel and increases goblet cell number and TFF3 protein
content throughout the intestine independent of food intake.
colon; intestinal mucosa; keratinocyte growth factor
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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TOGETHER WITH MUCINS, trefoil factor family (TFF) peptides are major constituents of the mucus layer that protect the gastrointestinal mucosa from injurious agents (2, 5, 13, 15, 26, 29, 34, 38, 41). TFF1 (formerly pS2) is synthesized primarily in gastric mucosal pit cells (TFF1). TFF2 (formerly spasmolytic polypeptide or SP) is produced by gastric mucous neck cells and duodenal submucosal Brunner's glands (8, 22, 41). TFF3 (formerly intestinal trefoil factor) is synthesized by goblet cells throughout small intestine and colon (36, 40). Trefoil peptides are resistant to protease digestion, and their cellular localization is ideal for gut epithelial protection (7, 28, 41). In vitro and in vivo studies strongly link trefoil peptides with gastrointestinal mucosal restitution (7, 19, 28, 41). TFF1, TFF2, and TFF3 are upregulated at sites of gastric and intestinal mucosal injury, where these proteins stimulate gut epithelial cell migration and mucosal repair (16, 20, 25, 33, 45, 46). However, the role of TFF peptides in physiological and adaptive gut epithelial cell proliferation, apoptosis, and turnover remains uncertain (17, 28, 31, 38, 45). Recent studies in TFF2-deficient mice clearly show that TFF2 stimulates gastric mucosal cell proliferation (12); in contrast, epithelial cell proliferation and apoptosis in jejunum were unaltered in mice with targeted transgenic expression of TFF3 in jejunal mucosa (27).
Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family (FGF-7) synthesized by stromal cells (18). The presence of KGF and the KGF receptor in gastrointestinal mucosa suggests the involvement of the endogenous KGF action pathway in gut physiology (9, 18). KGF specifically stimulates the proliferation and differentiation of epithelial cells, including cells of the gastrointestinal mucosa (18, 24). KGF treatment augments small bowel and colonic mucosal growth, repair, and barrier function in models of malnutrition, parenteral nutrition, chemotherapy/irradiation, and intestinal inflammation (4, 10, 11, 23, 24, 32). KGF appears to have particularly trophic effects on intestinal goblet cell expression in vivo (10, 18). KGF also increased small intestinal TFF3 mRNA in a rat model of short bowel syndrome (23) and TFF3 protein in murine models of inflammatory bowel disease (4). In undifferentiated colonic HT-29 subclone H2 cells, KGF upregulated TFF3 mRNA and protein expression and stimulated differentiation into goblet cells through regulation of the goblet cell silencer inhibitor, a goblet cell-specific transcription factor (21).
Gut mucosal growth, function, and repair are highly dependent on nutrient availability (3, 14, 49). Nutritional status also regulates expression of various endogenous intestinal growth factors, including insulin-like growth factor-I and KGF and its receptor (9, 44, 47-49). However, there have been few studies on nutrient regulation of intestinal goblet cells (6, 30, 35, 37) or TFF peptide expression. Our hypotheses were as follows: 1) gut mucosal atrophy induced by fasting would be associated with a reduction in the number of goblet cells and decreased TFF expression; 2) KGF would increase gut mucosal growth, goblet cell number, and TFF expression; and 3) effects of KGF would be modified by food intake. Therefore, we used rat models of fasting and fasting-refeeding, with or without administration of KGF, to assess mucosal growth, goblet cell number, and expression of TFF peptide mRNA and protein in rat small bowel and colon.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animal Protocols
Male Sprague-Dawley rats weighing 170-210 g were housed in individual cages in the Emory University Animal Care Facility on a 12:12-h light-dark cycle. Animals were acclimatized to laboratory conditions for 3 days with ad libitum water and standard pelleted rat food (Laboratory Rodent Chow 5001, PMI Feeds, St. Louis, MO). Water was provided ad libitum to all rats, and animal food intake was measured daily. Initial body weight of each of the study groups was not different, and all control animals gained the expected amount of body weight over time (Table 1). The animal procedures were approved by the Institutional Animal Care and Use Committee of Emory University and followed the newest guiding principles of research (1).
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Study 1: food deprivation. Weight-matched rats (n = 8/group) were given food ad libitum (control) or were fasted for 3 consecutive days. Animals were not monitored for coprophagia. We previously showed in rats that a 3-day period of fasting induces gut mucosal atrophy (47). Rats were also given daily intraperitoneal injections of recombinant human KGF (Amgen, Thousand Oaks, CA; 3 mg/kg, fasted-KGF) or saline (fasted-Sal). The fed control group was given daily intraperitoneal saline injections. Animals were killed on the morning of day 4. As expected, at death, fasted animals had lost ~20% of their initial body weight, whereas control animals had continued to gain weight (Table 1).
Study 2: food deprivation and refeeding. Weight-matched rats (n = 8/group) were fed ad libitum for 6 consecutive days (control) or fasted for 3 days and then refed ad libitum for 3 days. We previously showed in rats that gut mucosal atrophy induced by 3 days of fasting can be reversed by 3 days of oral refeeding (10, 47). During the entire 6-day protocol, fasted-refed rats were given daily intraperitoneal injections of KGF (3 mg/kg, refed-KGF) or saline (refed-Sal); control rats were given intraperitoneal saline. To ensure identical food intake between groups during refeeding, the refed-KGF rats were pair fed the average daily food intake consumed by the refed-Sal rats. Animals were killed on the morning of day 7.
Tissue Preparation
At the end of the individual study periods, animals were anesthetized with a mixture of ketamine (100 mg/ml) and xylazine (20 mg/ml) administered at 0.1-0.15 ml/100 g body wt ip. The abdomen was opened by a midline incision, and the stomach antrum, ligament of Treitz, and ileal-cecal junction were identified and marked. The small intestine and colon were removed sequentially from the peritoneal cavity, and the lumen was flushed with ice-cold saline to clear intestinal contents. The small bowel and colon were individually suspended from a ring stand with a constant distal weight, and defined intestinal segments were excised. Full-thickness sections (2 and 4 cm) were obtained from defined segments of proximal duodenum, proximal jejunum, distal ileum, and proximal colon for determination of TFF protein and mRNA content, respectively. Full-thickness samples of stomach antrum were obtained from ad libitum-fed rats for use as positive controls in TFF1 and TFF2 mRNA and protein expression studies. All tissue samples were weighed, snap-frozen in liquid nitrogen, and stored at
80°C for later analysis. Additional 0.5-cm sections from
each intestinal segment were fixed in 4% paraformaldehyde and embedded
in paraffin for later morphological analysis and immunohistochemistry.
Gut Mucosal Growth Indexes
Paraffin-embedded sections of duodenum, jejunum, ileum, and colon were stained with hematoxylin and eosin. Crypt depth and villus height were measured manually using a viewing Olympus BH-2 light microscope and calibrated ocular micrometer by two pathologist coinvestigators who were blinded to study group. A total of 15-25 well-oriented crypts and villi from each small bowel segment and 15-25 colonic crypts from each colon segment per animal were measured and averaged.Gut Mucosal Goblet Cell Number
Goblet cells were identified by classical morphology in each small bowel and colonic section in all animals. Total goblet cell number was determined along the combined crypt-villus axis of 10 well-oriented crypt-villus units in each small bowel section and 10 well-oriented colonic crypt units, from the crypt base to the luminal surface. To confirm goblet cell numbers identified by morphology, goblet cells in duodenal and colonic sections from the same animals (n = 5-6/group) were localized using Alcian blue staining (19). Positive staining with Alcian blue indicates the presence of acid mucins within goblet cells and was quantitated as outlined for morphological assessment.RNA Preparation and Northern Blot Analysis
Northern blotting was performed as previously described (49). Total cellular RNA was isolated using TRI-reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions. RNA integrity was confirmed by ethidium bromide staining.The rat TFF1 cDNA was a 135-bp KpnI/SacI fragment
based on the rat TFF1 cDNA sequence made by PCR and subcloned into
pGEMT (Promega, Madison, WI) (20). The TFF2 cDNA was a
267-bp XbaI fragment of the rat TFF2 cDNA sequence
(22) made by RT-PCR and subcloned into pGEMT. The TFF3
cDNA probe used for hybridization was a 500-bp
ApaI/BstXI fragment of the rat TFF3 cDNA sequence (36) subcloned into pGEMT. The cDNAs were labeled with
[32P]dCTP by random priming (Prime-It II Random Primer
Labeling Kit, Stratagene, La Jolla, CA) followed by spin column removal
of unincorporated nucleotides. Total RNA (25 µg/lane) was loaded onto
1% agarose gels, electrophoresed, and transferred to nylon membranes.
The membranes were hybridized with the respective TFF probes for
16-18 h at 42°C in hybridization solution, as described
elsewhere (49), washed under high-stringency conditions,
and exposed for various times at 80°C using Kodak X-OMAT film with
intensifying screens. To control for RNA loading, all membranes were
stripped and rehybridized with a mouse 18S cDNA probe (American Type
Culture Collection, Rockville, MD). Bands were quantitated by laser densitometry.
Western Immunoblotting
A rabbit monoclonal antibody against human TFF2 (provided by Dr. Lars Thim, Novo Nordisk, Malov, Denmark) and a rabbit anti-rat polyclonal TFF3 antibody raised against a 21-residue synthetic peptide from the predicted COOH-terminal sequence of rat TFF3 were used (12, 36). The anti-human TFF2 antibody has been shown to be specific for TFF2 in rat stomach and intestine (12). Tissues were suspended in ice-cold lysis buffer containing PBS, NP-40, sodium deoxycholate, SDS, EDTA, chymotrypsin, thermolysin, pronase, papain, and pancreas extract. Samples were homogenized and centrifuged, and supernatants were stored at70°C. Protein concentration of each
preparation was determined by the Bradford method, performed in
duplicate, with BSA as a standard. Solubilized protein (30 µg/sample)
was resolved by SDS-PAGE in 16.5% Tris-tricine gels (Bio-Rad,
Hercules, CA). Identical gels were checked for equality of protein
loading using Coomassie blue staining. Proteins were transferred to
Hybond ECL nitrocellulose membranes (Amersham, Arlington Heights, IL).
Membranes were blocked for 60 min at 22°C with 5% dry milk, 0.1%
BSA, 1× PBS, and 0.05% Tween 20 and incubated with TFF2 or TFF3
antibody (1:500) in blocking solution. Bound antibody was detected with
anti-rabbit horseradish peroxidase-conjugated secondary antibody
(anti-rabbit IgG; Santa Cruz Biotechnology, Santa Cruz, CA) and ECL
detection system reagents (Amersham). Protein bands were quantified by densitometry.
Immunohistochemistry
Deparaffinized intestinal sections were rehydrated, proteolyzed with proteinase K, washed, blocked with 1% gelatin-p-aminobenzoic acid, and incubated with primary anti-human TFF2 and anti-rat TFF3 antibody (1:500 dilution), respectively. Binding of primary antibody was visualized by incubation with goat anti-rabbit immunoglobulin G (1:500; Vector Laboratories, Burlingame, CA) and use of avidin, biotinylated horseradish peroxidase, and diaminobenzidine tetrahydrochloride reagents according to the manufacturer's instructions (ABC-Peroxidase Elite, Vector Laboratories). The sections were counterstained with hematoxylin. Tissue sections processed without primary antibody or with overnight incubation of primary antibody with TFF2 or TFF3 protein (10 µM at 4°C) were used as negative controls.Statistical Analysis
The abundance of TFF mRNA and protein was expressed in densitometry units, normalized against values obtained for control animals within each experiment. mRNA data from Northern blotting experiments were normalized to 18S mRNA expression. One-factor ANOVA was used to detect significant intergroup differences, in which case individual study groups were compared using Fisher's protected least significant difference test. P < 0.05 was considered statistically significant in all analyses. Values are means ± SE.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Gut Mucosal Growth Indexes
Fasting for 3 days induced site-specific effects on gut mucosal growth indexes (Table 2). The significant changes were a modest decrease in jejunal and increase in ileal villus height (to 83 and 112% of control values, respectively) and a decrease in jejunal, ileal, and colonic crypt depth (to 85, 85, and 88% of control values, respectively). KGF administration completely prevented the decrease in colonic crypt depth during food deprivation but did not alter the small bowel mucosal growth indexes. Rats in the fasting-refeeding protocol exhibited site-specific effects on gut mucosal growth parameters, and KGF induced small bowel and colonic mucosal growth responses (Table 3).
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Goblet Cell Number
Goblet cell number increased in a proximal-to-distal intestinal gradient in the control, nutrient-restricted, and refed rats (Table 4). Fasting alone increased duodenal goblet cell number by 25% compared with control fed values but did not modify goblet cell number in the other gut segments. KGF markedly increased goblet cell proliferation in all gut segments during food deprivation (Table 4). In fasted-refed rats, goblet cell number in duodenum and colon increased (to 118 and 125% of control, respectively). In this model, KGF markedly increased goblet cell number in duodenum, jejunum, ileum, and colon (Table 4). The magnitude of the goblet cell response to KGF in refed rats was quantitatively similar to the response to KGF when given during food deprivation alone.
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Alcian blue staining detects acid mucin-positive goblet cells and was performed in duodenal and colonic mucosal sections to confirm the morphological assessment. Results using Alcian blue were statistically similar to data derived from morphological determination of goblet cell number. The number of Alcian blue-positive cells per 10 crypt units in duodenal mucosa was 169 ± 18, 209 ± 3, and 343 ± 13 in control, fasted-Sal, and fasted-KGF groups, respectively (P < 0.05, fasted-Sal vs. control; P < 0.01, fasted-KGF vs. control and fasted-Sal). In colonic mucosa, the number of Alcian blue-positive cells was 294 ± 15, 295 ± 8, and 539 ± 9 in control, fasted-Sal, and fasted-KGF groups, respectively (P < 0.01, fasted-KGF vs. control and fasted-Sal). In the fasting-refeeding model, the number of Alcian blue-positive cells in duodenal mucosa was 156 ± 4, 184 ± 3, and 397 ± 13 in control, refed-Sal, and refed-KGF groups, respectively (P < 0.08, refed-Sal vs. control; P < 0.01, refed-KGF vs. control and refed-Sal). In colon, the number of Alcian blue-positive cells was 303 ± 7, 358 ± 16, and 578 ± 3 in control, refed-Sal, and refed-KGF groups, respectively (P < 0.05, refed-Sal vs. control; P < 0.01, refed-KGF vs. control and refed-Sal).
TFF1 and TFF2 Expression
The expected 0.5-kb TFF1 mRNA transcript was demonstrated in stomach (not shown), but TFF1 mRNA was not detected in duodenum, jejunum, ileum, or colon in any of the study groups, even after 8 days of autoradiographic exposure. The 0.6-kb rat TFF2 mRNA transcript was evident in stomach after 3 h of exposure (Fig. 1A, lane 1). TFF2 mRNA was not detected after 48 h of exposure in duodenum (Fig. 1, A and C) or jejunum (Fig. 1, C and D) in control, fasted, or fasted-refed animals given saline. However, KGF treatment induced expression of TFF2 mRNA in duodenum and jejunum in both nutritional models (Fig. 1). Upregulated TFF2 gene expression with KGF was particularly marked in the duodenum of food-deprived rats (Fig. 1A, lanes 4 and 7). TFF2 mRNA was not detected in ileum or colon after 8 days of exposure (not shown).
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TFF2 immunoreactivity was consistently detected in duodenal submucosal
Brunner's glands in all study groups (Fig. 2, A and B). In jejunum, faint
expression of TFF2 protein was present, but cellular localization was
variable between animals; low-level staining was observed in goblet
cells, along the apical border of villus epithelial cells, and in
secreted mucus (not shown). TFF2 was not observed in the duodenal
mucosa of saline-treated rats; however, KGF administration in both
nutritional models ectopically induced TFF2 protein expression in
goblet cells of duodenum and, to a lesser extent, jejunum (Fig. 2,
C and D). TFF2 staining after KGF treatment
appeared heaviest in the crypt and lower villus region, with decreased
staining in the mid- to upper villus goblet cells.
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Using Western blotting, we detected a ~16-kDa band in duodenal and
jejunal tissue that was identical in size to purified TFF2 protein and
the band observed in rat stomach (Fig.
3). Food deprivation alone did not alter
TFF2 protein abundance in duodenum and jejunum; however, KGF given in
this setting increased TFF2 abundance in duodenum and, to a lesser
extent, jejunum compared with controls (Fig. 3). In the
fasting-refeeding protocol, KGF did not significantly alter TFF2
protein abundance in duodenum (100 ± 5, 121 ± 4, and 137 ± 24 in control, fasted-Sal, and fasted-KGF, respectively; not significant) or jejunum (100 ± 14, 153 ± 19, and
140 ± 29 in control, fasted-Sal, and fasted-KGF, respectively;
not significant).
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TFF3 Expression
Fasting decreased steady-state expression of TFF3 mRNA in duodenum and jejunum (to 55 and 30% of control values, respectively) but did not alter TFF3 mRNA values in ileum or colon (Fig. 4A). KGF treatment during food deprivation prevented the decrease in duodenal and jejunal TFF3 mRNA but did not alter TFF3 mRNA expression in ileum or colon. In contrast to changes in duodenal and jejunal TFF3 mRNA, fasting increased TFF3 protein levels in jejunum and did not alter levels in the other intestinal segments (Fig. 4B). KGF treatment in this nutritional model also increased TFF3 protein abundance in duodenum, ileum, and especially colon (to 135, 263, and 1,578% of control values, respectively) and tended to increase values in jejunum (not significant).
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Fasting-refeeding did not modify intestinal TFF3 mRNA levels compared
with continuously fed control animals (Fig.
5A). KGF treatment in this
nutritional setting increased TFF3 mRNA abundance in duodenum, ileum,
and colon (to 238, 686, and 244% of control values) but did not alter
values in jejunum. TFF3 protein levels in fasted-refed rats were
similar to those in fed controls (Fig. 5B). KGF
administration in this model increased TFF3 protein expression by two-
to fourfold in duodenum, jejunum, ileum, and colon compared with
control values (Fig. 5B).
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Immunoreactive TFF3 protein was readily detected within goblet cells in
all bowel segments. In ileum, TFF3 protein was localized within goblet
cells and along the overlying mucosal surface, reflecting secretion
into the lumen (Fig. 6). Consistent with
the Western blot data shown in Fig. 5, ileal mucosa TFF3 protein
abundance and cellular expression were similar in fasted-refed and
control rats (Fig. 6, A and B). However, KGF
treatment in this model increased ileal goblet cell number and TFF3
immunoreactivity approximately twofold (Fig. 6C). TFF3
staining was eliminated by overnight adsorption of primary antibody
with TFF3 peptide or omission of primary antibody (not shown). TFF3
protein was localized to colonic goblet cells and was unaltered by
fasting-refeeding (Fig. 7, A and B). KGF treatment increased goblet cell number and TFF3 immunoreactivity in
colon (Fig. 7C).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Nutritional status markedly influences gut mucosal growth and function, but surprisingly little is known about the effects of nutritional factors on intestinal goblet cells or their secreted proteins (6, 33, 42). Little is known regarding the influence of nutrient intake on gut mucosal responses to recombinant growth factors, including KGF (44, 48, 49). To our knowledge, this is the first study to evaluate effects of diet and KGF on concomitant gut goblet cell and TFF peptide expression and to determine whether KGF regulates TFF1 and TFF2 expression.
Gut Mucosal Growth Indexes
Enteral nutrient availability induced segment-specific effects on small intestinal and colonic mucosal growth, consistent with previous studies (10, 44, 47). In the absence of exogenous nutrients (fasted model), KGF did not increase small bowel crypt depth or villus height but prevented the decrease in colonic crypt depth. When given during fasting followed by refeeding, KGF increased duodenal, jejunal, and ileal villus height and duodenal and colonic crypt depth. We did not investigate the time course of response to KGF or administer KGF to control fed rats. However, KGF has been shown to increase small bowel mucosal growth as early as 1 day after treatment in fed rats (18). Also, we previously showed that KGF administration during refeeding induced greater duodenal and ileal mucosal growth when animals were refed at 25% of ad libitum intake (10). Thus the level of nutrient intake appears to influence KGF gut-trophic responses in small bowel, with the presence of some luminal food being important. In contrast, this study and our previous work in different nutritional models demonstrate that KGF potently stimulates colonic growth independent of the level of nutrition (10). Additional studies to determine responses to KGF in parenterally fed animals, in which luminal food is absent, would be of interest (32).Goblet Cell Number
Intestinal goblet cell number and crypt depth/villus height during altered nutrient availability alone were generally dissociated (Tables 2-4). Goblet cell number increased in duodenum after fasting alone or fasting-refeeding. To our knowledge, this is the first demonstration of increased intestinal goblet cell number during altered nutrient availability. These data are consistent with another study showing increased mucus production in proximal small bowel after a 48-h fast in rats (37). Earlier investigations indicated that chronic protein depletion or protein-energy undernutrition decreased goblet cell number or mucin synthesis in rodent and piglet proximal small intestine, respectively (30, 35). Differences between our results and these latter studies may relate to the type or duration of malnutrition or the animal species utilized. Colonic goblet cell number increased in the fasting-refeeding model but was unaltered with fasting alone. Different local or systemic nutrition-related factors thus govern expression of the goblet cell lineage in duodenum compared with colon. Increased goblet cell expression in these gut segments may represent a protective adaptation to diminished nutrient availability. KGF increased goblet cell number uniformly throughout the small and large intestine, and this effect was unrelated to nutrient availability. In vivo studies to determine whether malnutrition, enteral vs. parenteral feeding, or KGF alters goblet cell differentiation, proliferation, apoptosis, and production of specific mucins (6, 43) would be of interest.TFF1 and TFF2 Responses
As expected, TFF1 and TFF2 mRNA were abundant in rat stomach tissue by Northern blotting but were not detected in intestine, with or without oral intake. KGF treatment did not induce TFF1 mRNA in any bowel segment or TFF2 mRNA in ileum and colon. Thus KGF stimulation of trefoil peptides is region and TFF specific. It is possible that a more sensitive assay, such as PCR or in situ hybridization, may have enabled detection of these mRNAs in the intestine.We confirmed cellular localization of TFF2 protein in duodenal submucosal Brunner's glands, as previously reported by others (22, 33, 34, 40, 45). However, we show for the first time that KGF upregulates TFF2 expression in rat proximal small bowel, with ectopic expression in goblet cells and secretion of the peptide into the lumen. Ectopic local upregulation of TFF2 was previously shown to occur in ulcer-associated cell lineages and in goblet cells at sites of gut mucosal injury (20, 25, 33, 46), suggesting a role for TFF2 in bowel mucosal restitution. Local KGF mRNA in gut mucosa is upregulated at sites of injury in inflammatory bowel disease and with mucosal ulceration (4, 48). Our results showing that exogenous KGF increased goblet cell TFF2 suggest that local upregulation of TFF2 in areas of mucosal inflammation may be due, in part, to increased local levels of KGF.
TFF3 Responses
The broad pattern of upregulated TFF3 protein after KGF injection occurred concomitantly with increased TFF mRNA in most gut regions. However, in ileum and colon after food deprivation (Fig. 4) and in jejunum after fasting-refeeding (Fig. 5), TFF3 mRNA levels were unchanged, despite an increase in TFF3 protein abundance. In addition, with fasting alone, TFF3 mRNA decreased in duodenum and ileum, whereas protein levels at these sites were maintained. Taken together, these data suggest that TFF3 expression is controlled at a posttranscriptional level in a region-specific manner and differently depending on nutrient supply or KGF administration.KGF-stimulated TFF3 production was localized to goblet cells, and abundant secreted TFF3 was present in the gut lumen. TFF3 mRNA and/or protein levels in some gut segments varied independently from changes in goblet cell number during altered nutrition (e.g., duodenum in both models and in colon with fasting-refeeding). Discordance between goblet cell number and TFF3 production was previously observed in mice with partial ablation of intestinal goblet cells, in which chemical mucosal injury increased goblet cell TFF3 expression (19). Increased goblet cell number with KGF treatment was invariably associated with increased TFF3 protein abundance in all gut segments. Previous studies show that KGF increases muc2 mRNA levels in HT-29 subclone H2 cells (21). Given the emerging roles for mucins such as muc2 in intestinal mucosal growth, protection, and barrier function (43, 45), in vivo studies to determine whether KGF or specific dietary factors differentially regulate goblet cell production of specific mucins would be of interest (43).
Changes in local TFF2 or TFF3 expression and mucosal crypt depth and villus height did not generally correlate in the present study. However, our study was not designed to test whether intestinal growth responses to diet or KGF are mediated by changes in goblet cell number or local TFF production. Studies of gut growth responses to nutritional alterations and KGF in TFF2 and TFF3 knock-out and transgenic mice are needed to address these questions.
In conclusion, in rat intestine, nutrient availability modulated goblet cell number, small intestinal TFF3 mRNA abundance, and trophic effects of KGF, but these effects were region specific. KGF markedly increased goblet cell number and TFF3 expression throughout the intestine, despite short-term lack of food intake. In addition, KGF induced ectopic TFF2 expression in proximal small bowel goblet cells. Further studies are necessary to determine the mechanisms involved in the nutritional and KGF modulation of goblet cells and their trefoil peptide products. In addition, more information on the specific roles of goblet cells and TFF peptides in mediating actions of nutrients and KGF in the gut is needed. Increased goblet cell number and TFF2 and TFF3 expression may mediate some of the beneficial effects of KGF observed previously (4, 10, 11, 24). Our results extend these studies and suggest that KGF may have potential therapeutic roles in disorders associated with malnutrition, including intestinal inflammation and short gut syndrome.
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ACKNOWLEDGEMENTS |
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C. Fernández-Estívariz gratefully acknowledges the support of Dr. J. Moreiro (Son Dureta Hospital, Mallorca, Spain, and Nutricia).
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FOOTNOTES |
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This work was supported, in part, by National Institutes of Health Grants DK-55850 and RR-00039 (to T. R. Ziegler), ES-009047 (to D. P. Jones), and DK-43351, DK-46906, and DK-43351 (to D. K. Podolsky) and Spanish Instituto de Salud Carlos III del Ministerio de Sanidad y Consumo Grants BAE 97/5082 and 98/5057 (to C. Fernández-Estívariz).
Address for reprint requests and other correspondence: T. R. Ziegler, Suite GG-23, General Clinical Research Center, Emory University Hospital, 1364 Clifton Rd., Atlanta, GA 30322 (E-mail: tzieg01{at}emory.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.
First published October 10, 2002;10.1152/ajpregu.00428.2002
Received 17 July 2002; accepted in final form 3 October 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
American Physiological Society.
Guiding principles for research involving animals and human beings.
Am J Physiol Regul Integr Comp Physiol
283:
R281-R283,
2002
2.
Babyatsky, MW,
deBeaumont M,
Thim L,
and
Podolsky DK.
Oral trefoil peptides protect against ethanol- and indomethacin-induced gastric injury in rats.
Gastroenterology
110:
489-497,
1996[Web of Science][Medline].
3.
Butler, RN,
Lawson MJ,
Goland GJ,
Jarrett IG,
and
Roberts-Thomson IC.
Proliferative activity in the proximal and distal colon of the rat after fasting and refeeding.
Immunol Cell Biol
66:
193-198,
1988.
4.
Byrne, FR,
Farrell CL,
Aranda R,
Rex KL,
Scully S,
Brown HL,
Flores SA,
Gu LH,
Danilenko DM,
Lacey DL,
Ziegler TR,
and
Senaldi G.
rHuKGF ameliorates symptoms in DSS and DCD4+CD45RHHiT cell transfer mouse modes of inflammatory bowel disease.
Am J Physiol Gastrointest Liver Physiol
282:
G690-G701,
2002
5.
Chinery, R,
and
Playford RJ.
Combined intestinal trefoil factor and epidermal growth factor is prophylactic against indomethacin-induced gastric damage in the rat.
Clin Sci (Lond)
88:
401-403,
1995[Medline].
6.
Claustre, J,
Toumi F,
Trompette A,
Jourdan G,
Guignard H,
Chayvialle JA,
and
Plaisancie P.
Effects of peptides derived from dietary proteins on mucus secretion in rat jejunum.
Am J Physiol Gastrointest Liver Physiol
283:
G521-G528,
2002
7.
Dignass, A,
Lynch-Devaney K,
Kindon H,
Thim L,
and
Podolsky DK.
Trefoil peptides promote epithelial migration through a transforming growth factor-
-independent pathway.
J Clin Invest
94:
376-383,
1994[Web of Science][Medline].
8.
Elia, G,
Chinery R,
Hanby AM,
Poulsom R,
and
Wright NA.
The production and characterization of a new monoclonal antibody to the trefoil peptide human spasmolytic polypeptide.
Histochem J
26:
644-647,
1994[Web of Science][Medline].
9.
Estívariz, CF,
Gu LH,
Scully S,
Eli E,
Jonas CR,
Farell CL,
and
Ziegler TR.
Regulation of keratinocyte growth factor (KGF) and KGF receptor mRNAs by nutrient intake and KGF administration in rat intestine.
Dig Dis Sci
45:
736-743,
2000[Web of Science][Medline].
10.
Estivariz, CF,
Jonas CR,
Gu LH,
Diaz EE,
Wallace TM,
Pascal RR,
Farrell CL,
and
Ziegler TR.
Gut-trophic effects of keratinocyte growth factor in rat small intestine and colon during enteral refeeding.
JPEN J Parenter Enteral Nutr
22:
259-267,
1998
11.
Farrell, CL,
Bready JV,
Rex KL,
Chen JN,
DiPalma CR,
Whitcomb KL,
Yin S,
Hill DC,
Wiemann B,
Starnes CO,
Havill AM,
Lu ZN,
Aukerman SL,
Pierce GF,
Thomason A,
Potten CS,
Ulich TR,
and
Lacey DL.
Keratinocyte growth factor protects mice from chemotherapy and radiation-induced gastrointestinal injury and mortality.
Cancer Res
58:
933-939,
1998
12.
Farrell, JJ,
Taupin D,
Koh TJ,
Chen D,
Zhao CM,
Podolsky DK,
and
Wang TC.
TFF2/SP-deficient mice show decreased gastric proliferation, increased acid secretion, and increased susceptibility to NSAID injury.
J Clin Invest
109:
193-204,
2002[Web of Science][Medline].
13.
Furuta, GT,
Turner JR,
Taylor CT,
Hershberg RM,
Comerford K,
Narravula S,
Podolsky DK,
and
Colgan SP.
Hypoxia-inducible factor 1-dependent induction of intestinal trefoil factor protects barrier function during hypoxia.
J Exp Med
193:
1027-1034,
2001
14.
Goodlad, RA,
and
Wright NA.
The effects of starvation and refeeding on intestinal cell proliferation in the mouse.
Virchows Arch
45:
63-73,
1984[Web of Science].
15.
Grisham, MB,
Von Ritter C,
Smith BF,
Lamont JT,
and
Granger DN.
Interaction between oxygen radicals and gastric mucin.
Am J Physiol Gastrointest Liver Physiol
253:
G93-G96,
1987
16.
Hanby, AM,
Jankowski JA,
Elia G,
Poulsom R,
and
Wright NA.
Expression of the trefoil peptides pS2 and human spasmolytic polypeptide (hSP) in Barrett's metaplasia and the native oesophageal epithelium: delineation of epithelial phenotype.
J Pathol
173:
213-219,
1994[Web of Science][Medline].
17.
Hoosein, NM,
Thim L,
Jorgensen KH,
and
Brattain MG.
Growth stimulatory effect of pancreatic spasmolytic polypeptide on cultured colon and breast tumor cells.
FEBS Lett
247:
303-306,
1989[Web of Science][Medline].
18.
Housley, RM,
Morris CF,
Boyle W,
Ring B,
Biltz R,
Tarpley JE,
Aukerman SL,
Devine PL,
Whitehead RH,
and
Pierce GF.
Keratinocyte growth factor induces proliferation of hepatocytes and epithelial cells throughout the rat gastrointestinal tract.
J Clin Invest
94:
1764-1777,
1994[Web of Science][Medline].
19.
Itoh, H,
Beck PL,
Inoue N,
Xavier R,
and
Podolsky DK.
A paradoxical reduction in susceptibility to colonic injury upon targeted transgenic ablation of goblet cells.
J Clin Invest
104:
1539-1547,
1999[Web of Science][Medline].
20.
Itoh, H,
Tomita M,
Uchino H,
Kobayashi T,
Kataoka H,
Sekiya Y,
and
Nawa Y.
cDNA cloning of rat pS2 peptide and expression of trefoil peptides in acetic acid-induced colitis.
Biochem J
318:
939-944,
1996.
21.
Iwakiri, D,
and
Podolsky DK.
Keratinocyte growth factor promotes goblet cell differentiation through regulation of goblet cell silencer inhibitor.
Gastroenterology
120:
1372-1380,
2001[Web of Science][Medline].
22.
Jeffrey, GP,
Oates PS,
Wang TC,
Babyatsky MW,
and
Brand SJ.
Spasmolytic polypeptide: a trefoil peptide secreted by rat gastric mucous cells.
Gastroenterology
106:
336-345,
1994[Web of Science][Medline].
23.
Johnson, WF,
DiPalma CR,
Ziegler TR,
Scully S,
and
Farrell CL.
Keratinocyte growth factor enhances early gut adaptation in a rat model of short bowel syndrome.
Vet Surg
29:
17-27,
2000[Web of Science][Medline].
24.
Jonas, CR,
Estivariz CF,
Jones DP,
Gu LH,
Wallace TM,
Diaz EE,
Pascal RR,
Cotsonis GA,
and
Ziegler TR.
Keratinocyte growth factor enhances glutathione redox state in rat intestinal mucosa during nutritional repletion.
J Nutr
129:
1278-1284,
1999
25.
Khulusi, S,
Hanby AM,
Marrero JM,
Patel P,
Mendall MA,
Badve S,
Poulsom R,
Elia G,
Wright NA,
and
Northfield TC.
Expression of trefoil peptides pS2 and human spasmolytic polypeptide in gastric metaplasia at the margin of duodenal ulcers.
Gut
37:
205-209,
1995
26.
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[Web of Science][Medline].
27.
Marchbank, T,
Cox HM,
Goodlad RA,
Giraud AS,
Moss SF,
Poulsom R,
Wright NA,
Jankowski J,
and
Playford RJ.
Effect of ectopic expression of rat trefoil factor family 3 (intestinal trefoil factor) in the jejunum of transgenic mice.
J Biol Chem
276:
24088-24096,
2001
28.
Mashimo, H,
Wu DC,
Podolsky DK,
and
Fishman MC.
Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor.
Science
274:
262-265,
1996
29.
McKenzie, C,
Marchbank T,
Playford RJ,
Otto W,
Thim L,
and
Parsons ME.
Pancreatic spasmolytic polypeptide protects the gastric mucosa but does not inhibit acid secretion or motility.
Am J Physiol Gastrointest Liver Physiol
273:
G112-G117,
1997
30.
Neutra, MR,
Maner JH,
and
Mayoral LG.
Effects of protein-calorie malnutrition on the jejunal mucosa of tetracycline-treated pigs.
Am J Clin Nutr
27:
287-295,
1974[Web of Science][Medline].
31.
Otto, WR,
Rao J,
Cox HM,
Kotzian E,
Lee CY,
Goodlad RA,
Lane A,
Gorman M,
Freemont PA,
Hansen HF,
Pappin D,
and
Wright NA.
Effects of spasmolytic polypeptide on epithelial cell function.
Eur J Biochem
235:
64-72,
1996[Web of Science][Medline].
32.
Playford, RJ,
Marchbank T,
Mandir N,
Higham A,
Meeran K,
Ghatei MA,
Bloom SR,
and
Goodlad RA.
Effects of keratinocyte growth factor (KGF) on gut growth and repair.
J Pathol
184:
316-322,
1998[Web of Science][Medline].
33.
Poulsom, R,
Chinery R,
Sarraf C,
Van Noorden S,
Stamp GW,
Lalani EN,
Elia G,
and
Wright NA.
Trefoil peptide gene expression in small intestinal Crohn's disease and dietary adaptation.
J Clin Gastroenterol
17:
78-91,
1993.
34.
Sands, BE,
and
Podolsky DK.
The trefoil peptide family.
Annu Rev Physiol
58:
253-273,
1996[Web of Science][Medline].
35.
Sherman, P,
Forstner J,
Roomi N,
Khatri I,
and
Forstner G.
Mucin depletion in the intestine of malnourished rats.
Am J Physiol Gastrointest Liver Physiol
248:
G418-G423,
1985
36.
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
37.
Szentkuti, L,
and
Lorenz K.
The thickness of the mucus layer in different segments of the rat intestine.
Histochem J
27:
466-472,
1995[Web of Science][Medline].
38.
Tan, XD,
Chen YH,
Liu QP,
Gonzalez-Crussi F,
and
Liu XL.
Prostanoids mediate the protective effect of trefoil factor 3 in oxidant-induced intestinal epithelial cell injury: role of cyclooxygenase-2.
J Cell Sci
113:
2149-2155,
2000[Abstract].
39.
Taupin, DR,
Kinoshita K,
and
Podolsky DK.
Intestinal trefoil factor confers colonic epithelial resistance to apoptosis.
Proc Natl Acad Sci USA
97:
799-804,
2000
40.
Taupin, DR,
Pang KC,
Green SP,
and
Giraud AS.
The trefoil peptides spasmolytic polypeptide and intestinal trefoil factor are major secretory products of the rat gut.
Peptides
16:
1001-1005,
1995[Web of Science][Medline].
41.
Tran, CP,
Cook GA,
Yeomans ND,
Thim L,
and
Giraud AS.
Trefoil peptide TFF2 (spasmolytic polypeptide) potently accelerates healing and reduces inflammation in a rat model of colitis.
Gut
44:
636-642,
1999
42.
Tran, CP,
Familari M,
Parker LM,
Whitehead RH,
and
Giraud AS.
Short-chain fatty acids inhibit intestinal trefoil factor gene expression in colon cancer cells.
Am J Physiol Gastrointest Liver Physiol
275:
G85-G94,
1998
43.
Velcich, A,
Yang W,
Heyer J,
Fragale A,
Nicholas C,
Viani S,
Kucherlapati R,
Lipkin M,
Yang K,
and
Augenlicht L.
Colorectal cancer in mice genetically deficient in the mucin Muc2.
Science
295:
1726-1729,
2002
44.
Winesett, DE,
Ulshen MH,
Hoyt EC,
Mohapatra NK,
Fuller CR,
and
Lund PK.
Regulation and localization of the insulin-like growth factor system in small bowel during altered nutrient status.
Am J Physiol Gastrointest Liver Physiol
268:
G631-G640,
1995
45.
Wong, WM,
Poulsom R,
and
Wright NA.
Trefoil peptides.
Gut
44:
890-895,
1999
46.
Wright, NA,
Poulsom R,
Stamp G,
Van Noorden S,
Sarraf C,
Elia G,
Ahnen D,
Jeffery R,
Longcroft J,
Pike C,
Rio MC,
and
Chambon P.
Trefoil peptide gene expression in gastrointestinal epithelial cells in inflammatory bowel disease.
Gastroenterology
104:
12-20,
1993[Web of Science][Medline].
47.
Ziegler, TR,
Almahfouz A,
Pedrini MT,
and
Smith RJ.
A comparison of rat small intestinal insulin and insulin-like growth factor I receptors during fasting and refeeding.
Endocrinology
136:
5148-5154,
1995[Abstract].
48.
Ziegler, TR,
Jonas CR,
Estívariz CF,
Gu LH,
Jones DP,
and
Leader LM.
Interactions between nutrients and growth factors in intestinal growth, repair and function.
JPEN J Parenter Enteral Nutr
23:
S174-S183,
1999.
49.
Ziegler, TR,
Mantell MP,
Chow JC,
Rombeau JL,
and
Smith RJ.
Gut adaptation and the insulin-like growth factor system: regulation by glutamine and insulin-like growth factor-I administration.
Am J Physiol Gastrointest Liver Physiol
271:
G866-G875,
1996
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P < 0.05 vs. fasted-Sal.
