ARTICLE |
Correspondence to: Jan Dekker, Laboratory of Pediatrics, Room Ee1571A, Erasmus University, Dr Molewaterplein 50, 3015GE Rotterdam, The Netherlands. E-mail: dekker@kgk.fgg.eur.nl
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Summary |
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The rapidly dividing small intestinal epithelium is very sensitive to the cytostatic drug methotrexate. We investigated the regulation of epithelial gene expression in rat jejunum during methotrexate-induced damage and regeneration. Ten differentiation markers were localized on tissue sections and quantified at mRNA and protein levels relative to control levels. We analyzed correlations in temporal expression patterns between markers. mRNA expression of enterocyte and goblet cell markers decreased significantly during damage for a specific period. Of these, sucrase-isomaltase (-62%) and CPS (-82%) were correlated. Correlations were also found between lactase (-76%) and SGLT1 (-77%) and between I-FABP (-52%) and L-FABP (-45%). Decreases in GLUT5 (-53%), MUC2 (-43%), and TFF3 (-54%) mRNAs occurred independently of any of the other markers. In contrast, lysozyme mRNA present in Paneth cells increased (+76%). At the protein level, qualitative and quantitative changes were in agreement with mRNA expression, except for Muc2 (+115%) and TFF3 (+81%), which increased significantly during damage, following independent patterns. During regeneration, expression of each marker returned to control levels. The enhanced expression of cytoprotective molecules (Muc2, TFF3, lysozyme) during damage represents maintenance of goblet cell and Paneth cell functions, most likely to protect the epithelium. Decreased expression of enterocyte-specific markers represents decreased enterocyte function, of which fatty acid transporters were least affected.
(J Histochem Cytochem 50:15251536, 2002)
Key Words: methotrexate, intestinal function, malabsorption, diarrhea
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Introduction |
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THE USE of the cytostatic drug methotrexate (MTX) in anti-cancer treatments severely impairs intestinal epithelial function, which constitutes a major dose-limiting factor in treatment schedules (-irradiation, this drug induces diarrhea and anorexia, which is associated with malabsorption, malnutrition, and dehydration (
As a folic acid analogue, the action of MTX primarily inhibits DNA synthesis by binding to the enzyme dihydrofolate reductase (
In our rat model of chemotherapeutic damage and regeneration in the small intestinal epithelium, four phases could be discriminated after the two-dose administration (IV) of 20 mg/kg plus 10 mg/kg body weight MTX (
Studies have been performed with the use of particular growth factors, such as TGF (
The functions of intestinal epithelial cells are reflected by the expression of cell type-specific genes. Very little is known about the absorptive and protective functions of epithelial cells during damage and regeneration induced by chemotherapy. Sucraseisomaltase and lactase, responsible for intestinal disaccharidase activities, were decreased during MTX-induced damage in rat small intestine (
In the present study, the functional capacities of the epithelial cells were investigated during MTX-induced damage and repair, through analysis of the expression of a range of differentiation markers. The following markers were used for enterocytes: carbamoylphosphate synthase (CPS), sucraseisomaltase (SI), lactase, glucose transporter-5 (GLUT5), sodium glucose co-transporter 1 (SGLT1), intestinal- and liver fatty acid-binding protein (I-FABP and L-FABP, respectively). For goblet cells the markers mucin (MUC2) and trefoil factor 3 (TFF3) were investigated and lysozyme was used to study Paneth cell function. The jejunum was chosen as a representative for the small intestine, because morphology and proliferation were similarly affected along the small intestinal axis in this model (
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Materials and Methods |
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Animals
Six-week-old male Wag/Rij rats (Broekman; Utrecht, The Netherlands) were kept under specific pathogen-free conditions with free access to water and defined semi-synthetic chow (Hope Farms; Woerden, The Netherlands). Glucose, starch, and cellulose constitute the carbohydrates in the food: 424 g/kg, 140 g/kg, and 45 g/kg, respectively. On analysis, no other carbohydrates were found. The folic acid content was 0.98 mg/kg. The dosage schedule of MTX treatment was defined during pilot studies. At day -1, 20 mg/kg body weight methotrexate (MTX, Ledertrexate SP forte; Cyanamid Benelux, Etten-Leur, The Netherlands) was injected IV under light anesthesia. At 24 hr later (day 0), a second injection of 10 mg/kg body weight MTX was given. Control animals received equivalent volumes of 0.9% (w/v) sodium chloride solution both times. At days 1, 2, 3, 4, 5, 6, 8, and 10 after the second dose of MTX, three rats were sacrificed by decapitation after anesthesia. Three control animals were sacrificed at day 8. The experiments were performed with permission of the Animal Ethics Committee of our institution. Jejunal segments (anatomic middle of the small intestine) were rinsed in PBS, fixed in 4% (w/v) paraformaldehyde (Merck; Darmstadt, Germany) in PBS, and processed for immunohistochemistry according to standard procedures (
Immunohistochemistry
Expression and localization of protein were detected using immunohistochemistry according to standard procedures (
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mRNA Quantitation
Total RNA was isolated from frozen tissue using Trizol reagent (Gibco-BRL; Gaithersburg, MD). Ten µg of total RNA was run on 1% or 1.2% (w/v) agarose gels containing 0.5 M glyoxal (Boehringer Mannheim; Mannheim, Germany) and blotted overnight onto Hybond-N+ membranes (Amersham; Poole, UK). Serial dilutions of each RNA sample were analyzed to ensure that the mRNA quantification, of each marker mRNA, using its cognate cDNA probe, was performed within the linear range of this technique. All blots were baked for 2 hr at 80C and hybridized using rat-specific, [32P]-dATP-labeled cDNA probes. The following rat-specific cDNA probes were used: CPS (
Protein Quantitation
Changes in epithelial protein expression were determined in tissue homogenates using quantitative Western blotting. Tissue was homogenized in HEPES buffer [20 mM HEPES (Merck), pH 7.5, 20 mM NaCl (Boehringer), 0.5% (w/v) Triton X-100 (BDH; Poole, UK)] containing 10 µg/ml pepstatin A (Sigma; St Louis, MO), 10 µg/ml leupeptin (Sigma), 1 mM phenylmethylsulfonyl fluoride (Sigma), and 0.38 U/ml aprotinin (Sigma) as protease inhibitors, using a Polytron (Kinematica AG; Basel, Switzerland). Ten µg of total protein was separated on 7% or 15% (w/v) SDS-PAGE and blotted for 1 hr onto Hybond+ membranes. Serial dilution series of the protein samples were analyzed to ensure that the quantification of each protein by its cognate antibody was performed in the linear range of this technique. Routine staining of the SDS-PAGE gels for protein after blotting showed complete transfer of the proteins to the membrane. After incubation for 1 hr in blocking buffer [Tris pH 7.8 (Merck), 2 mM CaCl2 (Merck), 5% (w/v) low-fat milk powder (Nutricia; Wageningen, The Netherlands), 0.05% (w/v) Triton X-100 (BDH), and 0.05% (v/v) Antifoam (Sigma)], the blots were incubated overnight at 4C with one of the primary antibodies (Table 1). After rinsing in blocking buffer, the blots were incubated for 2 hr in blocking buffer, containing 0.1 µCi/ml 125I-labeled protein A (Amersham). After rinsing three times in Tris buffer (pH 7.8), the blots were dried and exposed to PhosphorImager plates for 3 days. Each individual protein marker yielded clear and reproducible protein bands at the expected molecular weight as described in the original publications (Table 1). Signals were quantified as described above.
Statistical Analysis
Changes in mRNA and protein expression levels during damage and regeneration were statistically analyzed using the MannWhitney U-test. The statistical correlation coefficient (rho) between mRNA and protein expression and between morphological parameters and mRNA/protein expression was analyzed using Spearman's test, based on data from the individual rats. Similarly, correlation coefficients between the various mRNAs/proteins were used to study coordinate gene expression. In all tests, p<0.05 was considered statistically significant.
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Results |
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The Model
Using this animal model, we have shown previously that we could induce characteristic cytotoxic damage to the intestinal epithelium of rats by a 2-day treatment with MTX (
Basically, the response to MTX was similar in each region of the small intestine (
Enterocytes
Immunohistochemistry was performed to study the fate of enterocytes during the experiment and to localize enterocyte-specific protein expression. Because a very sensitive but non-linear detection method was used to visualize binding of antibodies, staining intensities were not quantitatively interpreted throughout the study. In control rats, CPS protein was expressed in a mosaic pattern and was localized in series of adjacent enterocytes both in crypt and in the villous epithelium (Fig 1A). On days 12 most of the crypts were not stained for CPS, but the protein was still present in the villous region (Fig 1B). On days 34, CPS protein expression was less abundant on the villi compared to controls and rats of days 12. Part of the enterocytes on the villous tips showed CPS immunoreactivity during this period of severe villous damage, but the crypt and villous enterocytes were mostly CPS-negative (Fig 1C). On days 56, the mosaic pattern of CPS had returned on part of the villi, and the expression pattern was fully restored on days 810 (Fig 1D).
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SI was found in the brush border and in the Golgi region of villous enterocytes in control rats (Fig 1E). The estimated number of SI-positive villous cells decreased according to the length of the villi, and during the phase of damage on days 1-4 the intracellular Golgi staining was absent (Fig 1F and Fig 1G). Very few SI-positive cells were seen on days 34, during maximal villous damage on the tips of the rudimentary villi (Fig 1G). As the villi increased in length during regeneration, the localization of SI-positive cells returned to normal (Fig 1H).
Like SI, GLUT5 and SGLT1 proteins were localized in the brush border of villous enterocytes in control tissue (Fig 1I and Fig 1M). The estimated numbers of GLUT5- and SGLT1-positive villous enterocytes decreased gradually towards the tips of the shortening villi during days 14 until both markers were undetectable on day 4 (Fig 1K and Fig 1O). From day 6 onwards, expression patterns of SGLT1 and GLUT5 were similar to those found in controls (Fig 1L and Fig 1P).
I-FABP and L-FABP expression was localized in the cytoplasm of all villous enterocytes in controls (Fig 1Q and Fig 1U). MTX-induced changes in expression were similar for both FABP proteins. These changes were much less pronounced than for the brush border markers SI, GLUT5, and SGLT1. Despite the increasing villous cell damage up to 4 days after MTX treatment, I-FABP and L-FABP immunostaining remained present in enterocytes of the remaining villous enterocytes (Fig 1R, Fig 1S, Fig 1V, and Fig 1W). As in controls, I-FABP and L-FABP were expressed in all villous enterocytes on days 510 (Fig 1T and Fig 1X).
Lactase mRNA levels could be detected as an enterocyte marker, but lactase protein could not be localized using the available antibodies because of its very low expression levels in these adult rats (
Goblet and Paneth Cells
The fate of goblet and Paneth cells was studied by immunohistochemistry for secretory products produced specifically by these cells. In contrast to the enterocyte markers, the markers for these cell types appeared to largely persist throughout the course of damage and regeneration. MUC2 was stained in the goblet cells at all time points during the experiment, and MUC2-positive goblet cells were also expressing TFF3 (Fig 2). The estimated number of MUC2- and TFF3-positive cells was decreased compared with controls during days 12 along the cryptvillous axis during the increasing villous atrophy (Fig 2B and Fig 2F). This was followed around day 4 by a depletion of MUC2- and TFF3-positive cells in the lower villous region and an accumulation of MUC2- and TFF3-positive cells in the villous tip region (Fig 2C and Fig 2G). During regeneration, MUC2 and TFF3 expression became comparable with controls (Fig 2D and Fig 2H).
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Paneth cells in the crypts were lysozyme-positive at all days analyzed. A transient induction of lysozyme immunostaining was seen during crypt damage on days 12 (Fig 2I and Fig 2J), which gradually returned to normal staining patterns on days 34 and beyond (Fig 2K and Fig 2L).
Expression of Enterocyte-specific mRNAs
The expression of cell type-specific mRNA was quantified by Northern blotting for each individual rat, as shown in Fig 3A. Pooling of quantitative data from two consecutive time points was performed to minimize intra-animal variations and to accentuate the differences between markers during the course of damage and regeneration (Fig 4).
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The changes in mRNA expression of each enterocyte marker had a characteristic pattern in time (Fig 4A). However, the level of each enterocyte mRNA returned to levels that were not significantly different from control levels at days 810 after MTX treatment. We performed correlation analysis on changes in the mRNA levels of these markers on the data from each individual animal. Four types of patterns could be distinguished, based on temporal differences in sensitivity to MTX treatment. Within each type, the expression patterns of the markers were highly correlated, whereas there were no statistically significant correlations with the expression patterns of the markers from any other type. We could distinguish type 1, CPS and SI; type 2, SGLT1 and lactase; type 3, I-FABP and L-FABP; and type 4, GLUT5 (Fig 4). Of these, CPS and SI mRNAs were most affected at an early stage. During minimal crypt depth on days 12, levels of CPS and SI mRNA expression were 82% and 62% decreased, respectively, compared with controls (Fig 4A). During maximal villous damage on days 34, CPS and SI were still significantly decreased, but the levels recovered relatively rapidly compared to the other markers.
SGLT1 and lactase mRNA levels were the most affected at a later stage, at maximal villous damage on days 34 and recovered more slowly compared to CPS and SI (Fig 4A). The expression of SGLT1 and lactase mRNA was already significantly decreased on days 12 compared with control (-50%). This decrease progressed to days 34, when SGLT1 and lactase mRNAs were hardly detectable (-77% and -76%, respectively). In contrast to CPS and SI mRNAs, SGLT1 and lactase mRNAs were still significantly decreased on days 56.
I-FABP and L-FABP mRNA levels were markedly less affected than the previous four markers and were first significantly affected at maximal villous damage, whereas the mRNA levels recovered relatively late and more slowly compared to CPS, SI, SGLT1, and lactase (Fig 4A). Expression levels of I-FABP and L-FABP mRNAs decreased significantly on days 34 at 52% and 45%, respectively (Fig 4A). As for SGLT1 and lactase, regeneration within the tissue was not immediately associated with a normalization of I-FABP and L-FABP mRNA expression, because both markers were still significantly decreased on days 5-6.
In contrast to all other enterocyte markers, the mRNA levels of GLUT5 were only affected significantly during one specific phase of disease, i.e., at maximal villous damage (days 34, -53%), and recovered rapidly during regeneration (Fig 4A).
We analyzed the relations between the level of expression of each marker and the crypt depth and villous atrophy, measured as described previously (
Expression of Goblet and Paneth Cell-specific mRNAs
The response of each of the markers for the goblet and Paneth cells was very specific and was different from any of the changes seen for the enterocyte markers (Fig 3A and Fig 4A). MUC2 mRNA expression was significantly but inversely correlated to crypt depth (rho=-0.610; p=0.001). During early damage on days 12, MUC2 mRNA levels remained stable. Thereafter, MUC2 mRNA levels were significantly decreased and remained below control levels up to days 810 (Fig 4B). MUC2 and TFF3 protein co-localized to the same cells (Fig 2). Interestingly, though, TFF3 mRNA levels were not significantly correlated to MUC2 mRNA levels. TFF3 mRNA levels were decreased only on days 34 relative to controls (Fig 4B).
Lysozyme mRNA levels were significantly increased (+76%) at early stages of damage on days 12 (Fig 4B), in agreement with the immunohistochemical localization of the protein (Fig 2J), and were rapidly normalized on days 36. There was no significant correlation with any of the morphological parameters. Surprisingly, a second induction of lysozyme mRNA levels occurred on days 810 (Fig 4B) despite the seemingly normal lysozyme protein expression at the immunohistological level (Fig 2L).
Expression Levels of Epithelial Proteins
Temporal expression patterns of epithelial proteins were quantified on Western blots, as illustrated in Fig 3B, and compared with expression patterns of the encoding mRNAs. Despite our efforts we were not able to quantify the protein levels of all markers. Therefore, we were not able to quantify SGLT1, lactase, and lysozyme protein due to lack of antibodies that were able to reproducibly detect these antigens on Western blotting.
The protein expression level of each enterocyte marker showed a specific pattern in time. Correlation analysis, determined from the data of the individual rats, revealed significant correlations between protein expression levels and the cognate mRNA levels of CPS (rho=0.520; p=0.01), SI (rho=0.900; p= 0.037), I-FABP (rho=0.482; p=0.015), and L-FABP (rho=0.591; p=0.001), but not of GLUT5.
Two isoforms of the CPS protein were detected using these polyclonal antibodies, as described earlier (Fig 3B;
I-FABP and L-FABP protein levels tended to decrease compared with controls from day 3 onwards, but only L-FABP protein decreased significantly on days 56 (Fig 4C). On days 810, expression of both FABP protein levels was not statistically different from controls. As shown in Fig 4C, GLUT5 protein levels tended to increase during the early phase of damage on days 12, but levels were dramatically decreased on days 34 compared with controls. During regeneration, GLUT5 protein expression was rapidly increased, as it was in the range of control levels on days 510, in line with GLUT5 mRNA expression (Fig 4A and Fig 4C).
In goblet cells, protein levels were not significantly correlated with the levels of the encoding mRNAs. MUC2 as well as TFF3 protein levels were significantly increased during all phases of damage despite the reduced mRNA levels (Fig 4B and Fig 4D). In contrast to MUC2, TFF3 protein remained significantly elevated on days 810. Despite the overall similarity in response to MTX, there was no significant correlation between Muc2 and TFF3 protein levels, based on the analysis of the data from the individual rats.
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Discussion |
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The loss of small intestinal epithelial cells and the occurrence of villous atrophy caused by MTX has been well described and has been appreciated since the 1970s (
There are strong indications from our results and from previously published data (
Decreased expression of SGLT1 mRNA during damage is in agreement with previous findings using 5-FU as cytostatic drug (
Glycohydrolase expression, measured by SI and lactase mRNA and SI protein, was significantly correlated with villous height. The decreased expression of these brush border proteins during villous damage confirms previous findings (
GLUT5 is responsible for the uptake of fructose. Fructose, which was absent from the diet in the present study, induces GLUT5 mRNA and protein expression (
The strong correlation between crypt depth and CPS mRNA expression was in line with the fact that CPS mRNA is expressed exclusively by crypt enterocytes, whereas the protein is also found in villous enterocytes (
Several functions have been reported for the cytosolic fatty acid-binding proteins in epithelial cells, as reviewed by
In general, the presence and functionality of goblet cells and Paneth cells were preserved during the MTX-induced pathology. The expression patterns of MUC2, TFF3, and lysozyme indicated a generally enhanced secretion of cytoprotective molecules by goblet cells and Paneth cells during MTX-induced damage. Secretion of mucin and trefoil factor molecules is known to contribute to protection of the epithelium via a mucous layer (MUC2) and by promoting epithelial restitution (TFF3) (
Despite the overall similarities in MUC2 and TFF3 protein and mRNA levels, no significant correlations were found in their levels of gene expression during MTX-induced damage and regeneration. This suggests that both genes were not co-ordinately regulated in goblet cells in response to chemotherapeutic damage, confirming recent work of others (
The defense provided by Paneth cells is likely to act locally in the crypt micro-environment, e.g., in the area of the stem cells, via the secretion of a variety of crypt defensins and peptides that enhance innate mucosal immunity (
In our study, an unexpected diversity in responses to MTX was observed between the 10 epithelial gene products. In particular, the remaining villous enterocytes during villous atrophy show a markedly specific response regarding their gene expression. These moribund cells appear to go through a programmed set of events that may be destined to maintain optimal epithelial performance during this stressed situation. Clinically, it may be possible to take advantage of these pre-programmed decisions by the absorptive epithelial cells to maintain optimal nutritional status in patients treated with cytostatic drugs. The central importance of the epithelial barrier function and the ability to regenerate after serious insults is reflected by the response of goblet cells and Paneth cells, in producing mucus, trefoil peptides and bactericidal peptides. The intestine thus reacts very specifically and diversely to severe damage or noxes and it appears very important to understand the different mechanisms involved in order to tailor enteral nutrition for patients treated with cytostatic drugs.
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Acknowledgments |
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Supported by Numico BV, Wageningen, the Netherlands.
We thank Dr W.H. Lamers for the CPS probe and antibodies, and Dr S. Krasinsky for providing the SI probe. Dr K.Y. Yeh and Dr A. Quaroni are gratefully acknowledged for supplying anti-sucrase antibodies. Furthermore, we thank Dr C.F. Burant for kindly providing SGLT1 and GLUT5 probes, and Dr B. Hirayama for his valued SGLT1 antibodies. Dr D. Yver kindly provided anti-GLUT5 antibodies. The I-FABP and L-FABP probes and antibodies were a generous gift from Dr J.I. Gordon. Dr D.K. Podolsky is acknowledged for providing both the TFF3 probe and the anti-TFF3 antibodies and Dr J.H. Power for providing the lysozyme probe. We also thank Dr K. Redekop for statistical advice and support.
Received for publication November 21, 2001; accepted June 5, 2002.
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