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Temporal changes in TFF3 expression andjejunal morphology during methotrexate-induceddamage and repair

C. J. XIAN,1 G. S. HOWARTH,1 C. E. MARDELL,1 J. C. COOL,1M. FAMILARI,2 L. C. READ,1 AND A. S. GIRAUD2

1Child Health Research Institute and Cooperative Research Centre for Tissue Growthand Repair, North Adelaide, South Australia 5006; and 2University of MelbourneDepartment of Medicine, Western Hospital, Footscray, Victoria 3011, Australia

Xian, C. J., G. S. Howarth, C. E. Mardell, J. C. Cool, M.Familari, L. C. Read, and A. S. Giraud. Temporal changesin TFF3 expression and jejunal morphology during methotrex-ate-induced damage and repair. Am. J. Physiol. 277 (Gastro-intest. Liver Physiol. 40): G785–G795, 1999.—Trefoil factorTFF3 has been implicated in intestinal protection and repair.This study investigated the spatiotemporal relationship be-tween TFF3 expression and morphological changes duringintestinal damage and repair in a rat model of methotrexate-induced small intestinal mucositis. Intestinal tissues fromrats with mucositis were collected daily for 10 days. Mucosaldamage was characterized by an initial decrease in cellproliferation resulting in crypt loss, villus atrophy, anddepletion of goblet cells, followed by hyperproliferation thatlead to crypt and villus regeneration and mucous cell repopu-lation. TFF3 mRNA levels increased marginally during histo-logical damage, and the cell population expressing TFF3mRNA expanded from the usual goblet cells to include somenongoblet epithelial cells before goblet cell repopulation.TFF3 peptide, however, was depleted during histologicaldamage and normalized during repair, mirroring the disap-pearance and repopulation of goblet cells. Although there isno temporal relationship between TFF3 levels and crypthyperproliferation, confirming the nonmitogenic nature ofTFF3, the coincidental normalization of TFF3 peptide withrepopulation of goblet cells and mucin production after prolif-erative overshoot suggests that TFF3 may play a role in theremodeling phase of repair.

mucositis; intestinal trefoil peptide; intestinal mucosal in-jury; regeneration

INTESTINAL TREFOIL FACTOR (ITF, also known as TFF3)and the other two known mammalian members of thetrefoil peptide family, pS2 (also known as TFF1) andspasmolytic polypeptide (SP, also known as TFF2),share a three-loop (or ‘‘trefoil’’) structural motif (forreviews, see Refs. 23, 27–29, 33, and 34). Formation ofthe three intrachain loops in trefoil peptides, securedby three disulfide bonds from six cysteine residues,results in rigidity of the structural motif and stabilityagainst acid hydrolysis and proteolysis in the gut (12,35). In mammals, trefoil peptides are produced predomi-nantly in the gastrointestinal tract, generally with aregional specificity. TFF1 (pS2) and TFF2 (SP) are

mainly produced by mucous cells of the gastric mucosaand biliary-pancreatic ducts; however, TFF3 (ITF) ispredominantly synthesized by the goblet cells in thesmall and large intestine (22, 30, 32). Trefoil peptidesare secreted in association with mucins into gut lumenand are concentrated within the mucus layer (15).

Because of their stability against gastric acid andluminal proteolysis as well as their abundance in thegut lumen, trefoil peptides are well suited to exertfunctions at the luminal-mucosal interface. However,the functional effects and mechanisms of action oftrefoil peptides in the gut are not completely clear.Evidence from in vitro and in vivo experiments sug-gests that trefoil peptides may play a key role inprotecting the gastrointestinal mucosa from variousinsults (4, 6, 8), probably by preserving the integrity ofthe epithelial barrier by promoting the formation of acontinuous gel with mucin glycoproteins on the muco-sal surface (16). Previous studies have also shown thattrefoil peptides enhance mucosal repair by stimulatingcell restitution immediately after damage (10, 20). Insupport of these maintenance and reparative functionsof trefoil peptides in the gut are studies demonstratingoverexpression of trefoil peptides at sites of gastrointes-tinal ulceration and inflammation (26, 39), an in-creased resistance to intestinal damage in transgenicmice overexpressing TFF1 (21), and a higher suscepti-bility to gut-related injury in mice that lack eitherTFF1 (18) or TFF3 (19) genes.

We attempted to further our understanding of therole of trefoil peptides in intestinal mucosal injury andrepair by examining the spatiotemporal relationshipbetween expression of TFF3, the major trefoil peptideexpressed in the intestine, and the small intestinalmucosal damage and repair processes in a rat model ofmethotrexate-induced mucositis. Methotrexate, as achemotherapeutic agent, exerts its toxicity to intestinalepithelial cells by directly inhibiting DNA synthesis,leading to a reduction of mitosis in the crypts andshortening of the villi (3, 38). This model of intestinalmucositis is characterized histologically by crypt loss,villus fusion and atrophy, gross capillary dilatation, amixed cellular infiltrate, and a rapid recovery (13, 31).These features resemble the gut mucositis experiencedas a common side effect by patients undergoing chemo-therapy or radiotherapy (3). In the present study, acomplete time course of methotrexate-induced intesti-nal damage and repair in rats was used to investigatethe timing and location of TFF3 protein and mRNA

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expression in the intestinal tissues, as related to histo-pathological and cellular changes.

MATERIALS AND METHODS

Time course of methotrexate-induced intestinal mucositis.Intestinal mucositis was induced in 120-g body wt maleSprague-Dawley rats using methotrexate as described previ-ously (13). Briefly, methotrexate at 2.5 mg/kg was subcutane-ously administered to rats in the suprascapular region oncedaily for 3 consecutive days. One group of ad libitum-fednormal rats without methotrexate injection (n 5 8) were usedas control animals (day 0). Daily for 10 days, groups of rats(n 5 8) were injected intraperitoneally with bromodeoxyuri-dine (BrdU) (an analog of thymidine that is incorporated intoDNA during the S phase of the cell cycle) at 50 mg/kg andwere killed 1 h later. Two groups of pair-fed rats (n 5 4)without methotrexate injection but consuming daily amountsof food similar to the methotrexate-injected animals werekilled on day 5 and day 8 for comparison with their methotrex-ate-injected counterparts. Two 3-cm-long tissue specimensfrom the proximal jejunum, the region of the intestine withmaximal damage, were freshly collected, with the proximalsegment immediately snap-frozen in liquid nitrogen (for RNAand protein extraction) and stored at 280°C and with thedistal segment fixed in methacarn fixative for 2 h. Each fixedintestinal specimen was dehydrated by passage throughgraded alcohol, cut into four equal lengths, and embedded inparaffin wax. Paraffin-embedded, transverse sections (4 µmthick, each with 4 intestinal segments) from these processedtissues were used for histological analysis, cell proliferationstudy, TFF3 immunohistochemistry, and in situ hybridiza-tion.

Semiquantitative histopathological analysis of intestinaldamage and repair. Hematoxylin- and eosin-stained tissuesections from the proximal jejunum were examined with alight microscope. An overall score of intestinal damage sever-ity was semiquantitatively assessed as described (13), usingcriteria that included villus fusion and stunting (atrophy),disruption of brush-border and surface enterocytes, reductionin numbers of mitotic figures, crypt loss/architectural disrup-tion, disruption or distortion of crypt cells, crypt abscessformation, infiltration of polymorphonuclear cells and lympho-cytes, and dilatation of lymphatics and capillaries.

Periodic acid-Schiff staining and goblet cell counting. As anadditional means to assess intestinal damage, goblet cellnumbers were derived from the 4-µm paraffin sections of theproximal jejunum specimens. To highlight the goblet cells,sections were stained for mucins by the periodic acid-Schiff(PAS) technique (5). Counting PAS-stained goblet cells inboth villi and crypts was performed using computer-aidedvideo image analysis. For each specimen, a total of 20microscopic fields of crypts and 20 fields of villi under a 320objective were measured per animal. For each field, the totalarea of crypts (including areas of epithelium, crypt lumen,and stroma) or the total area of villus epithelium only(excluding lumen and stroma) was traced to provide an areaof measurement, and the number of goblet cells within thatfield was counted. Goblet cell counts per unit area were thenderived from the two values.

TFF3 Western blotting. To demonstrate potential changesin TFF3 protein expression, Western blotting was performedon total protein samples isolated from proximal jejunaltissues using RNA-DNA-protein separation reagent (ProgenIndustries, Brisbane, Australia). First, the protein concentra-tion was quantitated using Bradford reagent (Sigma, St.Louis, MO) with BSA (Sigma) as a standard. Equal amountsof protein (200 µg) from each sample, 200 ng of rat recombi-

nant TFF3 (used as a standard, a generous gift from Dr. LarsThim, Novo Nordisk), and 2 µg of a biotinylated broad rangeof protein molecular weight markers (Bio-Rad, Hercules, CA)were treated with a reducing sample buffer, separated on a12.5% SDS-PAGE minigel, and electroblotted onto a 0.2-µmnitrocellulose filter. The filters were probed with a rabbitanti-rat TFF3 antiserum (32) at 1:12,500 dilution, detectedwith a swine anti-rabbit biotinylated IgG and avidin/biotinylatedhorseradish peroxidase reagents (Dako, Carpinteria, CA).Immunoreactivity was developed as enzyme-chemilumines-cence signal using the enhanced chemiluminescence Westernblotting system (Amersham, Buckinghamshire, UK). Thespecificity of this anti-TFF3 antiserum has been demon-strated previously by RIAs and Western blotting, revealing nocross-reaction with other trefoil peptides (9).

TFF3 immunostaining. To examine any potential site-specific changes of TFF3 protein in the intestine during thedamage/repair time course, immunohistochemical detectionof TFF3 was performed on paraffin sections of the proximaljejunum. After nonspecific binding sites were blocked with 5%pig serum, sections were incubated overnight at 4°C with theabove rabbit anti-TFF3 serum at 1:600 dilution in Tris-buffered saline (TBS, pH 7.4) containing 1% BSA. Aftersections were washed, staining was visualized with a swineanti-rabbit biotinylated IgG and avidin/biotinylated horserad-ish peroxidase reagents (Dako) and 3,38-diaminobenzidine(DAB) substrate (Sigma). A normal rabbit serum was used asa negative control.

TFF3 RNase protection assay. An RNase protection assaywas used to quantitate the changes in TFF3 gene expressionduring the mucositis time course, according to a previouslydescribed method (9, 11, 17). Total RNA was extracted usingthe RNA-DNA-protein separation reagent from the speci-mens of proximal jejunal tissues used for protein extraction.Antisense [32P]UTP-labeled riboprobes for TFF3 and glyceral-dehyde-3-phosphate dehydrogenase (GAPDH) (used as aninternal loading control) were made, respectively, from a ratTFF3 cDNA (423 bp/Bluescript, a generous gift from Profes-sor D. K. Podolsky, Massachusetts General Hospital, Boston,MA) and a rat GAPDH cDNA (300 bp/Bluescript, a gift fromDr. P. Fuller, Prince Henry’s Institute for Medical Research,Melbourne, Australia). Probes were incubated with 2 µg oftotal RNA and hybridized overnight at 45°C, and the hybridswere digested with 20 µg/ml RNase A and 1 µg/ml RNase T1(Sigma) at 37°C for 30 min. Protected fragments were re-solved on 5% acrylamide-8 M urea gels. Densitometry ofautoradiographs was carried out on a Molecular Dynamicsscanning laser densitometer using ImageQuant software.Control digests without RNA (water control) or with 20 µg ofyeast tRNA resulted in no protected bands, as we havepreviously demonstrated (data not shown here) (11). Thespecificity of this assay was also built into the probe we used,which was designed from the 38 end of the gene (11). Thisregion of the gene is known to be the most variable partamong the several members of the trefoil peptide family.

TFF3 in situ hybridization. A nonradioactive in situ hybrid-ization technique was used to examine the cellular distribu-tion of TFF3 mRNA expression in the intestine over thedamage and recovery time course. Digoxigenin (DIG)-labeledriboprobes were generated from a pKS1 ribovector contain-ing 348-bp rat TFF3 cDNA (11). The plasmid DNA waslinearized with Not I and HinD III restriction enzymes andwas used to make sense and antisense probes by in vitrotranscription with T3 and T7 RNA polymerase, respectively,using a DIG RNA labeling kit (Boehringer Mannheim, Mann-heim, Germany). The efficiency of DIG labeling was quanti-tated by dot blotting using a DIG detection kit (Boehringer

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Mannheim). In situ hybridization was performed on 4-µmparaffin sections of the proximal jejunum mounted on 3-ami-nopropyltriethoxysilane-coated glass slides. Sections weredewaxed, hydrated, and treated with 0.2 N HCl for 20 min atroom temperature. Sections were then permeabilized with10 µg/ml proteinase K (Sigma) for 15 min at 37°C. Afterpostfixation for 5 min with 2% paraformaldehyde, sectionswere neutralized for 10 min with 0.2% glycine in PBS (pH7.4). After washing and dehydration, sections were coveredwith 25 µl of hybridization mix (preheated for 5 min at 85°C)containing 0.5 µg/ml sense (used as a negative control) orantisense probe, 50% formamide, 10% dextran sulfate, 0.05%Triton X-100, 500 µg/ml herring sperm DNA (BoehringerMannheim), 0.05% polyvinylpyrrolidone, and 53 SSC (750mM NaCl and 75 mM sodium citrate, pH 7.0). Sections werecovered with glass coverslips and incubated in a humidifiedchamber for 18 h at 58°C. Unhybridized probes were washedoff with 23 SSC for 30 min at room temperature, 23 SSC for1 h at 65°C, and 0.13 SSC for 1 h at 65°C. After nonspecificbinding sites were blocked with 10% normal sheep serum for30 min, the sections were incubated for 30 min at 37°C withan alkaline phosphatase-coupled sheep anti-DIG IgG (Boeh-ringer Mannheim) and developed by incubating in nitro bluetetrazolium-X-phosphate substrate for 18 h in the dark(Boehringer Mannheim). For negative controls, apart fromthe use of the sense probe in place of the antisense probe inthe hybridization described above, sections that had beenpretreated with RNase A (Boehringer Mannheim) (100 µg/mlin 23 SSC) for 1 h at 37°C were also used for hybridizationwith both sense and antisense probes. All of these controlsconsistently generated a low background.

BrdU immunostaining and labeling index. BrdU labelingwas used as an index of cell proliferation in the intestinaltissues. Immunostaining of BrdU was performed on 4-µmparaffin sections of the proximal jejunum. Deparaffinizedsections were incubated for 20 min in ice-cold 0.3% H2O2 inmethanol to quench any endogenous peroxidase activity.Sections were then washed and treated with 1 M HCl for8 min at 60°C to partially denature the double strand DNA.After sections were blocked with 10% normal rabbit serum for40 min in TBS, sections were then incubated at room tempera-ture for 1.5 h with a mouse anti-BrdU monoclonal IgG (Dako)at 1:100 dilution. Labeling was visualized with a rabbitanti-mouse biotinylated IgG and avidin/biotinylated horserad-ish peroxidase reagents (Dako) and with DAB substrate(Sigma). A normal mouse monoclonal IgG (Dako) was used asa negative control.

BrdU labeling index was derived from counting the num-bers of positively labeled cells and the total number of cryptepithelial cells. For each animal, 50 well-orientated fullcrypts were selected for analysis under a light microscopewith a 340 objective. The number of BrdU-labeled cells andthe total number of crypt epithelial cells on the left half of thecrypts were counted and were used to calculate the meanBrdU labeling index (%) for that animal. Although methotrex-ate caused significant mucosal damage in the proximal smallintestine, at least 50% of mucosal area remained sufficientlyintact for reliable measurements of BrdU labeling index, evenin tissues collected on day 5, when crypt disruption wasmaximal.

Statistical analysis. Results of the RNase protection assays(integrated optical density ratios between TFF3 and GAPDH),goblet cell density (goblet cell number/mm2 crypt area orvillus epithelium), and BrdU labeling (%) between the groupsof animals treated with methotrexate and untreated normalcontrols were compared with one-way ANOVA.

RESULTS

Acute features of methotrexate-induced small boweldamage and repair. The current study aimed to exam-ine the expression of TFF3 during small intestinaldamage and subsequent repair and to correlate theexpression with histopathological changes. Methotrex-ate injections induced considerable histopathologicalchanges (Fig. 1, A–D) in the proximal small intestine,the most sensitive region of rat gut. Damage wasapparent on day 3 (Fig. 1B) and reached maximalseverity on day 5 (Fig. 1C), with histological features ofcrypt loss, villous atrophy, fusion or shortening, andinflammatory cell infiltrate. Intestinal damage thendeclined in severity on day 6, followed by a rapidrecovery on day 7 (Fig. 1D). In the repair phase, thecrypts and villi were elongated on day 7 compared withday 0 normal controls (Fig. 1D), followed by a gradualnormalization over the next 3–4 days (not shown).Semiquantitative scoring of damage severity over thetime course (Fig. 2A) illustrates that the methotrexate-induced damage is acute, similar to findings reportedpreviously (13, 31). Pair-fed controls (receiving nomethotrexate injections but a similar food intake totheir methotrexate-injected counterparts) revealed nosignificant changes in villus height and crypt depthcompared with normal controls (not shown), indicatingthat the histological changes apparent in methotrexate-injected animals did not result from a reduction in foodintake.

The methotrexate-induced mucositis was also charac-terized by an acute time course of depletion and repop-ulation of goblet cells in the intestinal mucosa. Gobletcells in the proximal jejunum were identified by PASstaining (Fig. 1, E–H), and the density of goblet cells inboth villi and crypts was measured (Fig. 2B). Over 98%of all goblet cells were PAS stained during the intesti-nal injury/recovery time course, indicating that thechange in PAS-stained cell measurement reflected thechange in goblet cell number rather than an alterationin mucin synthesis itself. Quantitative analysis of thegoblet cell population in both crypts and villi through-out the methotrexate-induced damage and repair timecourse revealed a time-dependent change in goblet celldensity in both the villus epithelium and crypt mucosa(Fig. 2B). On days 1 and 2 after methotrexate injection,there was no reduction in goblet cell population ineither crypts or villi compared with normal controls(Fig. 2B). Compared with normal controls (Fig. 1E), thegoblet cell numbers in the villi began to decline on day 3(Figs. 1F and 2B), reaching the lowest level on day 5(Figs. 1G and 2B). In the crypts, the loss of goblet cellswas more rapid, with the number dramatically reducedon day 3 to near the lowest level observed on day 5(Figs. 1, F and G, and 2B). In both crypts and villi, thegoblet cell numbers began to increase on day 6, rapidlyreturning to normal levels on day 7 (Figs. 1H and 2B)and then were maintained on day 8 (Fig. 2B).

Changes in TFF3 peptide expression closely associ-ated with intestinal damage and repair. ITF or TFF3,known to be involved in mucosal defense and ulcer

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repair, is produced and secreted into the intestinallumen by goblet cells in association with mucin glycopro-teins. To examine a potential involvement of changes inTFF3 expression in methotrexate-induced intestinaldamage and subsequent regeneration, protein andmRNA expression of TFF3 was examined in jejunaltissue from the damage/repair time course. Westernblotting analysis of TFF3 immunoreactivity in samplesof total protein extracted from the jejunal tissuesreveals a drastic reduction in TFF3 levels duringhistological damage and a rapid recovery during intes-tinal repair. Figure 3A illustrates a representative blotwith TFF3 bands of 7 kDa (and also a 14 kDa dimer

in TFF3 standard) of proximal jejunal protein samples(200 µg each) from the damage/repair time course.Densitometric analysis (Fig. 3B) of the blot revealedlittle change in the TFF3 protein level on day 2compared with the day 0 control. However, comparedwith day 0, TFF3 levels were reduced to 46% on day 3,showed a near total absence on days 4–5, and returnedto 75% on day 6 and 86% on day 7, with the level stableup to day 10. This indicates that the level of TFF3immunoreactivity was closely associated with the histo-logical changes apparent in the intestine.

TFF3 immunoperoxidase staining was used to local-ize the changes in its immunoreactivity over the dam-age/repair time course. Consistent with previous stud-ies (22, 30, 32), TFF3 immunoreactivity was localizedmainly to goblet cells and the luminal surface in bothcrypts and villi of the day 0 normal controls (Fig. 4A).In accordance with the profile of histopathologicalchanges (Figs. 1, A–D) and changes in goblet cellpopulation (Fig. 1, E–H), this staining pattern wasmaintained on days 1 and 2 after the commencement ofmethotrexate injections (data not shown). However, thestaining was diminished on day 3 (Fig. 4B) whenintestinal damage was considerable (Fig. 1B), and thegoblet cell number was significantly reduced (Fig. 1F)and absent on days 4 (data not shown) and 5 (Fig. 4C)when the histological damage severity was maximal(Fig. 1C) and the goblet cell number was the lowest(Fig. 1G). TFF3 staining was again visible on day 6 (notshown) when damage severity declined, and goblet cellsreappeared and returned to normal levels on day 7 (Fig.4D) when the intestinal structure (Fig. 1D) and gobletcell population (Fig. 1H) appeared normal and thenremained unaltered on days 8–10 (not shown). Thisresult indicates that depletion of TFF3 accompaniedthe intestinal damage and particularly the disappear-ance of goblet cells induced by methotrexate, and,conversely, normalization of TFF3 peptide correlatedwith subsequent mucosal repair, particularly the repop-ulation of goblet cells.

TFF3 mRNA marginal upregulation during intesti-nal damage and expanded expression before goblet cellrepopulation. To examine whether the changes in TFF3were also apparent at the mRNA level, a RNase protec-tion assay was employed to measure the changes inTFF3 gene transcript over the time course of intestinaldamage and repair. These measurements were con-ducted on total RNA samples from the tissue specimensused to extract the total protein for the Western blotanalysis. Figure 3C illustrates the result of a represen-tative protection assay for TFF3 compared with theexpression of the GAPDH gene, which was employed asan internal loading control. Densitometric analysis(Fig. 3D) of TFF3 mRNA expression, standardized with

Fig. 1. Features of hematoxylin and eosin staining (A–D) and goblet cell periodic acid-Schiff (PAS) staining (E–H)in proximal jejunum of rats during methotrexate-induced intestinal damage and repair. A and E: normal jejunalhistology and goblet cells (as highlighted by PAS staining, magenta in color) in the normal intestines. B and F:partial histological damage of the jejunum and a significant loss of goblet cells in the intestine of day 3 rats. C and G:severe mucosal histological damage (crypt loss, villus shortening, and fusion) and a near depletion of goblet cells inthe damaged jejunum in day 5 rats. D and H: near normal histology and repopulation goblet cells with mucinproduction in the recovered intestine of day 7 rats. Scale bar in H 5 100 µm (applies to A–H).

Fig. 2. Semiquantitative histopathological damage severity profileand goblet cell count in the proximal jejunum in rats from themethotrexate damage/repair time course. A: scoring of damageseverity based on the various histopathological criteria, showingseverity scores for 4 animals at each time point. B: goblet cell counts(means 6 SE, n 5 4) were expressed as numbers of PAS positivelystained cells per unit area (mm2) of traced epithelium in the villi or oftraced total crypt mucosa. *Goblet cell counts in the villi that aresignificantly different from day 0 control (P , 0.01). a or bCell counts inthe crypts that are significantly different from day 0 control at therespective P , 0.001 and P , 0.05 levels.

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GAPDH expression, revealed a pattern of change recip-rocal to that demonstrated in TFF3 protein as de-scribed above. Although the changes in TFF3 geneexpression did not reach a statistically significant levelon any day after methotrexate injection compared withday 0 control (P . 0.05), the mRNA level tended todecrease initially on day 1, returning to normal levelson day 2, and tended to be upregulated from days 3 to 5during the damage phase and before the goblet cellrepopulation; levels then declined starting from day 6to near or below normal levels on days 8 and 10 afterthe normalization of the goblet cell population.

TFF3 mRNA in situ hybridization was used to local-ize the changes in its level over the damage/repair timecourse. Negative controls, including the use of a senseprobe and the predigestion of RNA in tissue sections byincubating with RNase A (at 50 µg/ml for 30 min at37°C), consistently gave negative staining (not shown).In normal intestine, TFF3 mRNA expression was con-fined to goblet cells in both villi and crypts (Fig. 4E). On

days 1 and 2 during methotrexate treatment, no obvi-ous changes were observed in TFF3 mRNA staining inthe intestine (not shown). On day 3 (Fig. 4F), day 4 (notshown), and particularly day 5 (Fig. 4G), however, theintensity of mRNA staining appeared higher in theremaining goblet cells compared with that of the day 0controls. Furthermore, on day 5, TFF3 mRNA stainingwas also present in some non-goblet-featured epithelialcells (Fig. 4G). From day 6 to day 10, TFF3 mRNAstaining returned to normal (Fig. 4H showing day 7only). These results from the in situ hybridization aswell as from the RNase protection assays indicate thatthere was a marginal increase in TFF3 mRNA expres-sion during the intestinal damage and an expansion inthe cell population expressing TFF3 mRNA just beforethe goblet cell repopulation.

Lack of association between changes in TFF3 peptidelevel and cell proliferation. To examine any potentialmitogenic role for TFF3 in regeneration of the cryptepithelium, the time course of crypt cell production was

Fig. 4. Features of TFF3 immunostaining (A–D) and mRNA in situ hybridization (E–H) in proximal jejunum of ratsduring methotrexate-induced intestinal damage and repair. A and E: TFF3 immunostaining (A; dark staining) andmRNA expression (E; dark staining) predominantly in goblet cells in the normal intestine (day 0 rats). B and F:significant loss of TFF3 immunoreactivity (B) but a more intense mRNA staining (F) in the intestine in day 3 rats. Cand G: depletion of TFF3 immunostaining (C) but a more intense mRNA staining (G) that was expressed also insome nongoblet cells in the damaged jejunum in day 5 rats. D and H: normal TFF3 immunostaining (D) and mRNAexpression (H) in day 7 rats. Scale bar in H 5 100 µm (applies to A–H).

Fig. 3. Changes in TFF3 immunoreactivity and mRNA levels in proximal jejunum during methotrexate-induceddamage and repair. A: representative Western blot showing TFF3 bands from 200-ng recombinant peptide (7 and 14kDa) and from jejunal protein samples (200 µg each) from a normal rat (day 0) and rats 2–8 days after the firstmethotrexate injection. B: densitometry analysis of the Western blot, showing a decrease or depletion of TFF3during days 3–5 and reappearance or normalization of TFF3 during days 6–8. C: representative TFF3 mRNARNase protection assay, showing a blot from 2-µg proximal jejunal RNA from a day 0 normal control rat or rats 1–10days after the first methotrexate injection. RNA was hybridized with TFF3 riboprobe and a riboprobe forglyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (used as an internal loading control), and hybrids weredigested with RNases and visualized by autoradiography after being separated on a PAGE gel. D: densitometryanalysis of the protection assay autoradiographs, expressed as TFF3-to-GAPDH ratios from the protected TFF3 andGAPDH bands (means 6 SE, n 5 4).

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examined in concert with the analysis of TFF3 proteinexpression. Analysis of proliferating cell BrdU labelingin the proximal jejunum reveals that methotrexateinduced a significant decrease in crypt cell proliferationon day 3 (Figs. 5, A and B, and 6) but an upregulationon days 5 and 6 (Figs. 5C and 6) followed by anormalization on day 7 (Figs. 5D and 6). Comparisonbetween the histopathological (Fig. 2A) and cell prolif-eration (Fig. 6) time courses indicated that a decreasein crypt cell proliferation (on day 3) preceded thehistological damage (which was maximal on day 5),and, conversely, crypt cell hyperproliferation (on days 5and 6) preceded epithelial regeneration. Comparisonbetween the time courses of crypt cell proliferation (Fig.6) and intestinal TFF3 protein production (Fig. 3, A andB) shows that the overshoot in crypt cell proliferationwas not preceded by an upregulation of TFF3 peptide(on and before day 5); conversely, no increase in cellproliferation was followed after the normalization ofTFF3 peptide production (on and beyond day 7). On day5, although the TFF3 peptide was absent, crypt cellproliferation was upregulated. On day 6, crypt cellproliferation was upregulated, although the TFF3 levelhad recovered. These data indicate that TFF3 wasunlikely to be acting as a mitogen during the regenera-

Fig. 5. Bromodeoxyuridine (BrdU) labeling in proximal jejunum in normal and methotrexate-damaged rats.Epithelial BrdU labeling (darkly labeled nuclei), confined in crypts in the normal intestine (A), was dramaticallyreduced on day 3 (B), upregulated on day 5 but now with some distribution also in lower villus epithelium (C), andnormalized on day 7 (D). Scale bar in D 5 100 µm (applies to A–D).

Fig. 6. Cell count measurements of BrdU labeling in the proximaljejunum in rats from the methotrexate damage/repair time course.BrdU labeling was expressed as percentage of the number of posi-tively labeled cells over the total numbers of epithelial cells of thecrypts (means 6 SE, n 5 4 animals). *Significantly different from day0 control (P , 0.001).

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tion of the intestine following methotrexate-induceddamage.

DISCUSSION

We have demonstrated that the small intestinalmanifestations of the toxicity of the DNA synthesis-inhibiting chemotherapy agent methotrexate (3, 38) inthe rat reflect an acute and transient process of muco-sal damage and regeneration, confirming previous find-ings (31). This process was characterized by an initialdecrease in crypt cell proliferation, which precedescrypt loss, villus shortening and atrophy, and goblet celldepletion. The repair phase commenced when crypt cellproliferation had returned to normal levels and wasaccelerated during the period of marked upregulationin crypt cell production. The overshoot of cell prolifera-tion resulted in crypt elongation, an increase in villusheight, and goblet cell repopulation. Intestinal regenera-tion continued after day 7 with normalization of cryptdepth and villus height to basal levels over the subse-quent 4–5 days.

We have shown that a depletion of TFF3 proteincoincided with the severity of histopathological dam-age, with the TFF3 peptide absent during the timeperiod of maximal histological damage. Mirroring theTFF3 protein depletion more closely, however, was thetime course of change in the goblet cell population inthe proximal small intestine. TFF3 is produced andsecreted into the intestinal lumen by goblet cells inassociation with mucin glycoproteins (27). During thedamage phase, goblet cells were reduced in number orcompletely depleted, as was TFF3 peptide. During therepair phase, the repopulation of goblet cells wasaccompanied by a return of TFF3 immunoreactivity.Localization of TFF3 peptide to goblet cells and theclose association between the changes of TFF3 peptideand goblet cell number during the damage/repair timecourse indicate that the change in TFF3 peptide may besecondary to the changes in goblet cell population.Alternatively, the depletion of TFF3 peptide may havebeen responsible for the lack of mucin production.However, the latter possibility can be eliminated, sinceit has been shown that, in TFF3 knockout mice, mucinproduction by goblet cells is still preserved (19), indicat-ing that TFF3 is not essential for mucin production.Because the reduction of TFF3 protein production wasmost likely secondary to the depletion of goblet cells,the depletion of TFF3 may have been a late rather thanan early event of methotrexate-induced damage. Ourtime course measurements suggest that methotrexate-induced intestinal damage and the depletion of gobletcells occur as consequences of a decrease in cell prolif-eration earlier in the time course. However, whetherthe diminished expression of mucin glycoproteins andTFF3 in the intestine may have contributed to theintestinal damage awaits further study. The close spa-tial and temporal association between TFF3 peptideproduction and goblet cell number suggests that TFF3and mucins may act in concert to maintain intestinalintegrity. Intestinal goblet cells secrete TFF3 and mu-cin glycoproteins to form a continuous gel that covers

the intestinal epithelial layer and contributes to muco-sal protection (2, 27, 33). In support of this, Kindon etal. (16) demonstrated that TFF3 and intestinal mucinglycoproteins interacted to protect colonic epithelialcell monolayers against toxin- or bile acid-induceddamage.

In the current study, we have demonstrated that theexpression of TFF3 is not positively correlated withcrypt cell proliferation, suggesting that TFF3 is not amitogen for intestinal crypt cells. Instead, our resultsdemonstrate that cell proliferation was high whenepithelial TFF3 expression was low, indicating that thepresence of TFF3 peptide could even be inhibitory tocell proliferation. This notion is consistent with aprevious study in TFF3 knockout mice showing thatthe crypt cell proliferative compartments were ex-panded compared with wild-type mice (19). This findingis also in accordance with previous findings that re-vealed that recombinant TFF3 does not stimulate theproliferation of nontransformed gastrointestinal epithe-lial cells (10).

Observations in TFF3 gene knockout mice havedemonstrated that TFF3 has a role in normal crypt cellmigration and maturation of the intestinal epithelium(19). Furthermore, in vitro and in vivo evidence sug-gests that trefoil peptides, including TFF3, play animportant role in reestablishing mucosal integrity afterinjury by stimulating epithelial cell restitution (10, 20).Elucidating a possible role for TFF3 in stimulatingrepair after methotrexate-induced damage requiresfurther studies, which could include application ofrecombinant peptide either luminally or parenterally.In this context, we have recently shown that luminalapplication of TFF2 in the colon accelerates epithelialhealing after induction of hapten-induced colitis (37).However, the coincident timing of normalization inTFF3 immunoreactivity and crypt/villus regenerationafter the proliferative overshoots on days 5 and 6indicates that the reappearance of TFF3 peptide mayplay a role in the stimulation of cell migration duringmucosal repair. Consistent with the current study, wehave previously demonstrated a decrease in TFF3protein production during acetic acid-induced ulcer-ative damage to the gastric mucosa, with TFF3 produc-tion returning to basal levels only at the time when themacroscopic healing was complete (9). This latter studyalso revealed an upregulated expression of TFF3 in andaround ulcer sites in the gastric tissues in the laterstages of repair after the completion of reepithelization(9), suggesting that TFF3 has an ongoing role inreparative events, such as maturation and positioning,which occur well after the initial reepithelization phasefollowing the initial insult. Similarly, in an aceticacid-induced rat model of colitis, TFF3 expression wasshown to be downregulated during the acute phase ofcolitis and then upregulated during the recovery phase,suggesting its possible involvement in cell differentia-tion such as the formation of goblet cells (14).

In this study, we have described a disparity betweenTFF3 peptide and mRNA expression. Although thelevel of jejunal TFF3 peptide localized in the goblet

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cells was decreased in association with the depletion ofgoblet cells and histological damage, the TFF3 mRNAtended to be upregulated particularly at the time ofmaximal intestinal damage, preceding the upregula-tion of TFF3 peptide during the normalization of themucosa. The explanation for this discrepancy awaitsinvestigation. Although it is possible that the modestincrease in synthesis of TFF3 mRNA during the dam-age process produces TFF3 peptide that is rapidlysecreted in a constitutive manner without storage, thispossibility is unlikely, since, during the TFF3 mRNAupregulation with tissue damage apparent, the gobletcell number was low, suggesting a low cellular massthat can produce TFF3 peptide. A more likely possibil-ity for this discrepancy could be that the transcriptionand translation of TFF3 are not parallel, with transla-tion occurring later than transcription. Supporting thispossibility was the observation that the appearance ofmRNA of trefoil peptides was 2 days earlier than that oftheir peptides in the developing rat intestine (11). By insitu hybridization, we have shown that TFF3 mRNAexpression was confined to goblet cells in normal intes-tine, consistent with previous studies (7, 22, 30), andthat the remaining goblet cells in the damaged intesti-nal mucosa particularly on day 5 displayed more in-tense mRNA staining. Furthermore, on day 5 before therepopulation of goblet cells, the cell population express-ing TFF3 mRNA was expanded to include some non-goblet-featured epithelial cells. Although it has beendocumented that both TFF3 mRNA and peptide aremainly confined to the goblet cells in the mature villusepithelium and the crypts (7, 22, 30), differential expres-sion of TFF3 mRNA and peptide has also been reportedin the crypts of the small bowel. It has been shown thata high level of TFF3 mRNA was also expressed by someundifferentiated epithelial cells in the crypt despite theabsence of its translation into peptide in these non-goblet-featured immature cells (22). Consistent withour current finding that some non-goblet-featured epi-thelial cells also expressed TFF3 mRNA before gobletcell repopulation in this methotrexate-induced mucosi-tis model, TFF3 mRNA has been reported to be presentin poorly differentiated epithelium migrating over le-sions after intestinal resection surgery (25), during therecovery phase in acetic acid-induced colitis (36), or ingastric mucosal damage (1) in the rat and in chronicinflammation in man (24). Perhaps it is this lack ordelay of translation of TFF3 mRNA into TFF3 peptidein these undifferentiated cells to replenish the depletedTFF3 pool during intestinal damage that could explainthe discordance between TFF3 peptide and mRNAexpression described in this study.

It has been suggested that the early expression ofTFF3 mRNA in the immature cells may be an earlymarker of commitment to differentiate into goblet cells(22). Indeed, in the developing rat gut, mRNA expres-sion of trefoil peptides including TFF3 precedes mu-cous cell differentiation (11). It is therefore possiblethat the dramatic reduction in TFF3 peptide in associa-tion with intestinal damage and the depletion of goblet

cells in this methotrexate-induced mucositis could pro-vide a positive feedback signal to the regeneratingcrypt to upregulate TFF3 mRNA in some undifferenti-ated cells. These undifferentiated cells could then usethe upregulated TFF3 mRNA as an early marker forthe commitment of goblet cell differentiation to replen-ish its population and to produce TFF3 peptide. Indeed,repopulation of goblet cells coincided with the reappear-ance of TFF3 peptide following the initiation of recov-ery from damage.

In summary, methotrexate-induced small bowel mu-cositis in the rat is an acute and transient process ofmucosal damage and repair, characterized by an initialdecrease in crypt cell production, leading to crypt loss,villus atrophy, and depletion of goblet cells, followed bycrypt cell hyperproliferation. The upregulated prolifera-tion of crypt epithelial cells precedes crypt and villusregeneration, repopulation of mucous cells, and normal-ization of mucosal structure. This study has shown thatthe TFF3 mRNA expression level marginally increasesduring mucosal damage and cell population expressingTFF3 mRNA, including nongoblet cells before the gob-let cell repopulation, expands; however, this study alsoclearly demonstrated a depletion of TFF3 peptide dur-ing the phase of intestinal damage, particularly gobletcell depletion, and its normalization during the phaseof mucosal regeneration and goblet cell repopulation.We have also revealed a lack of temporal correlationbetween TFF3 peptide expression and crypt cell hyper-proliferation during the regenerative phase, confirmingthe nonmitogenic nature of TFF3. However, the upreg-ulated and expanded expression of TFF3 mRNA preced-ing the goblet cell repopulation and the coincidentalnormalization of TFF3 peptide levels with that of gobletcell repopulation and mucin production after the cryptcell proliferative overshoot or during the remodelingphase suggest that TFF3 may play a role in later eventsof intestinal repair. The current study has highlighteda potentially important role for TFF3 in the mainte-nance of intestinal integrity and in intestinal repair;further understanding of epithelial responses (includ-ing regulation of gene expression of intestinal peptidesor growth factor systems) to intestinal mucosal injuryis needed. Further studies, which could include the useof recombinant TFF3 peptide or an antagonist in vivo,need to be conducted to test whether TFF3 plays anessential role in preventing mucosal damage inducedby methotrexate and in enhancing intestinal repairafter damage.

We thank Kerry Penning, Leanne Srpek, and Ben Edwards forassistance in conducting the animal trials and Annamaria Mercorellafor histological processing of intestinal tissues.

This project was funded in part by a Cooperative Research Centregrant from the Australian Government and project grants (to C. J.Xian, L. C. Read, and A. S. Giraud) from the National Health andMedical Research Council of Australia.

Address for reprint requests and other correspondence: C. J. Xian,Child Health Research Institute, 72 King William Rd., North Ad-elaide 5006, South Australia, Australia.

Received 10 November 1998; accepted in final form 22 June 1999.

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