Intestinal inflammation reduces expression of DRA, a transporter responsible for congenital chloride diarrhea

Hongyun Yang1, Wen Jiang1, Emma E. Furth2, Xiaoming Wen1, Jonathan P. Katz1, Rance K. Sellon3, Debra G. Silberg1, Toni M. Antalis4, Clifford W. Schweinfest5, and Gary D. Wu1

Departments of 1 Internal Medicine and 2 Pathology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; 3 University of North Carolina and Center for Gastrointestinal Biology and Disease, Chapel Hill, North Carolina 27599; 4 Molecular Oncology Laboratory, Queensland Cancer Fund Experimental Oncology Program, Queensland Institute of Medical Research, Brisbane, Queensland 4029, Australia; and 5 Center for Molecular and Structural Biology, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The pathogenesis of diarrhea in intestinal inflammatory states is a multifactorial process involving the effects of inflammatory mediators on epithelial transport function. The effect of colonic inflammation on the gene expression of DRA (downregulated in adenoma), a chloride-sulfate anion transporter that is mutated in patients with congenital chloridorrhea, was examined in vivo as well as in an intestinal epithelial cell line. DRA mRNA expression was diminished five- to sevenfold in the HLA-B27/beta 2m transgenic rat compared with control. In situ hybridization showed that DRA, which is normally expressed in the upper crypt and surface epithelium of the colon, was dramatically reduced in the surface epithelium of the HLA-B27/beta 2m transgenic rat, the interleukin-10 (IL-10) knockout mouse with spontaneous colitis, and in patients with ulcerative colitis. Immunohistochemistry demonstrated that mRNA expression of DRA reflected that of protein expression in vivo. IL-1beta reduced DRA mRNA expression in vitro by inhibiting gene transcription. The loss of transport function in the surface epithelium of the colon by attenuation of transporter gene expression, perhaps inhibited at the level of gene transcription by proinflammatory cytokines, may play a role in the pathogenesis of diarrhea in colitis.

electrolyte; Caco-2; colitis; transcription

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE INTESTINAL EPITHELIUM responds to inflammatory stimulation in numerous ways. In addition to inducing epithelial cells to produce a variety of immune modulating substances (25), cytokines can also alter epithelial tight junctions, resulting in an increase in transepithelial permeability (30). Furthermore, intestinal inflammation alters patterns of epithelial proliferation and differentiation with an increase in the undifferentiated proliferative cell compartment and a decrease in the nonproliferative differentiated cell compartment. Investigators have recently shown that inflammation of the small intestine results in a marked decrease in sucrase-isomaltase gene expression (48), providing evidence that differentiated gene expression can be altered in small intestinal inflammatory states.

There is, however, currently no information regarding the effect of intestinal inflammation on the regulation of genes associated with either differentiation or electrolyte transport in the colonic epithelium. It has been well documented that intestinal inflammatory states result in abnormalities in electrolyte transport characterized by decreased absorption of Na+ and Cl- with stimulation of Cl- secretion (1, 3, 8, 46). A recent advance in the understanding of the role that electrolyte transporters play in the pathogenesis of diarrhea resulted from the identification, by positional cloning, of DRA (downregulated in adenoma) as the gene that is mutated in congenital chloride diarrhea (20). A disorder inherited in an autosomal recessive fashion, congenital chloride diarrhea results in profound watery diarrhea with high chloride content beginning at birth associated with metabolic alkalosis (12, 16). DRA is expressed at high levels primarily in the colonic epithelium, at low levels in the small intestinal epithelium (20, 43), and in the epithelium of the intraprostatic seminal vesicle (C. Schweinfest and M. Willingham, unpublished observations). This gene encodes a Cl-/OH-(HCO3) exchanger that is expressed highly in the apical membranes of the surface epithelium in the colon (6, 20, 33). DRA is also capable of transporting sulfate ions (5, 43).

There is significant functional similarity between DRA and the anion exchange proteins AE1 and AE2, both of which are chloride-bicarbonate transporters also capable of sulfate ion transport (40, 41). Although AE1 is erythroid specific, AE2 is expressed in a variety of tissues, including the biliary epithelium (31). Interestingly, AE2 mRNA expression has been shown to be decreased in liver biopsy specimens of patients with primary biliary cirrhosis compared with controls (35), demonstrating that the expression of this transporter is attenuated in this chronic inflammatory state. Furthermore, studies using the T84 colon cancer cell line have shown that stimulation with interleukin-4 (IL-4) diminishes both chloride secretion and cystic fibrosis transmembrane conductor regulator (CFTR) expression in vitro (49).

In this study we examine the mRNA expression of DRA as a model to determine how colonic transporter gene expression is altered by the inflammatory state in two animal models of colitis, the HLA-B27/beta 2m transgenic rat (19) and the IL-10 knockout mouse (28), as well as in patients with ulcerative colitis. Furthermore, we explore the molecular mechanism by which cytokine stimulation attenuates the expression of DRA in Caco-2 cells, a cell culture model of intestinal epithelial cell differentiation.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isolation of RNA and Northern blots. A HLA-B27/beta 2M transgenic female rat derived from Fisher 344 background, as well as an age-matched female Fisher 344 control rat, 24 wk of age, were purchased from GenPharm International (Mountain View, CA). The rats were killed by CO2 narcosis followed by cervical dislocation. The colon of each animal was then removed, and the cecum was isolated. Bisection of the remaining colon led to two additional segments labeled proximal colon and distal colon. Total RNA for Northern blots were isolated by the guanidinium thiocyanate-CsCl gradient method (27) for each segment following the removal of two 2-mm sections from the distal end of each segment for frozen sectioning. Ten micrograms of total RNA for each sample were electrophoretically separated, transferred to a nylon membrane, and ultraviolet cross-linked as previously described (47). Hybridization of the Northern blots was performed using conditions previously described (23).

Caco-2 cells, obtained from the American Type Culture Collection, were plated at a density of 4 × 104 cells/cm2 in 10-cm dishes containing complete medium. Seventeen days after plating, the postconfluent cells were stimulated with complete medium containing 5 ng/ml of recombinant human IL-1beta , 100 ng/ml of recombinant human IL-6 (both cytokines from R & D Systems, Minneapolis, MN), or both IL-1beta and IL-6. Twenty-four hours later total RNA was isolated as previously described. Total RNA was also isolated from postconfluent Caco-2 cells (day 18 after plating), which were not stimulated with cytokines as well as from preconfluent Caco-2 cells day 5 after plating. The Northern blots were performed as previously described.

The Northern blots were hybridized to a mouse cDNA probe (43) for DRA labeled with 32P using a Random Primers DNA Labeling System (GIBCO BRL, Gaithersburg, MD). Northern blots of RNA extracted from rat colonic tissue and from Caco-2 cells were stripped and rehybridized to a human cDNA probe for villin (Hind III-EcoR I 1883-bp fragment) and 7S ribosomal RNA (24), respectively. Quantification of the hybridization signals was performed by PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA).

Human specimens were obtained from the Cooperative Human Tissue Network. Sample pair 1 came from the Western Division at Case Western University and sample pairs 2 and 3 came from the Midwestern Division at Ohio State University. All patients had a confirmed diagnosis of ulcerative colitis. Total RNA was isolated from frozen tissue specimens as described previously (9), and Northern blots of this RNA were probed with a 32P-radiolabeled EcoR I fragment encoding a portion of the human DRA cDNA (nucleotides 1062-1881). The membranes were hybridized and washed to a final stringency of 0.1× saline sodium citrate (SSC) and 0.1% SDS at 65°C. Each Northern blot was stripped and rehybridized with a radiolabeled oligonucleotide complementary to 18S rRNA as a measure of total RNA loaded in each lane. To control for RNA loading, the mRNA signal intensity was measured relative to 18S rRNA in each sample.

In situ hybridization. In situ hybridization was performed using a modified version of the digoxigenin-labeled method described by Panoskaltsis-Mortari and Bucy (34). In brief, colonic tissue was snap frozen in OCT and stored at -80°C. Sections (5 µm) were cut and placed on Probe-On slides (Fisher Scientific, Pittsburgh, PA). Tissue was obtained from the HLA-B27/beta 2M transgenic rats described previously, from patients with ulcerative colitis and from female IL-10 knockout mice, 28 wk of age, maintained in either a germ-free environment or under specific pathogen-free conditions. The tissue sections were thawed at room temperature for 1-2 min and fixed in 3% paraformaldehyde for 1 min at room temperature. After two washes with 2× SSC, the slides were incubated for 8 min in 0.2 M HCl and rinsed with 0.1 M triethanolamine, pH 8.0. The slides were then incubated in 0.25% acetic anhydride in 0.1 M triethanolamine, pH 8.0, for 15 min followed by a rinse with 2× SSC. Prehybridization was performed at 50°C for 15 min in a solution containing 50% formamide, 4× SSC, 1× Denhardt's, 10% dextran sulfate, 500 µg/ml of heat denatured SS DNA, and 250 µg/ml of yeast tRNA. The slides were then hybridized in prehybridization solution containing heat-denatured (80°C) RNA probe for DRA at 50°C overnight. After hybridization, the slides were rinsed in 2× SSC for 5 min and STE (500 mM NaCl, 20 mM Tris-HCl, pH 7.5, and 1 mM EDTA) for 1 min. After incubation in RNase A (40 µg/ml in STE) for 30 min at 37°C, the slides were washed with 2× SSC containing 50% formamide for 5 min at 50°C followed by washes with 1× SSC and 0.5× SSC, each for 5 min. Immunologic detection of digoxigenin using a sheep-anti-digoxigenin antibody (Boehringer Mannheim, Indianapolis, IN) and NBT/BCIP (Boehringer Mannheim) was performed as previously described (34).

Digoxigenin-labeled RNA probes for rat, mouse, and human DRA (both sense and antisense) were transcribed from the T3 and T7 promoters of pBluescript KS(-) (Stratagene, La Jolla, CA) using the Riboprobe Gemini System 2 kit (Promega) as per the manufacturer's instructions. cDNAs for rat and human DRA were amplified by reverse transcription PCR of total RNA isolated from rat colon or Caco-2 cells, respectively, and cloned into pBluescript KS(-). The primers used for PCR were the same as those described for the cloning of the mouse cDNA (43).

Nuclear run-ons. The DRA gene transcription rate was determined by nuclear run-on assay. Briefly, nuclei were isolated by NP-40 lysis and Dounce homogenization (18) from preconfluent Caco-2 cells (day 5 after plating) or from postconfluent Caco-2 cells (days 14-17) with or without 24 h of stimulation with IL-1beta (5 ng/ml). The transcription reaction was performed at 30°C for 30 min using 5 × 107 nuclei in 5 mM Tris · HCl, pH 8.0, 2.5 mM MgCl2, 150 mM KCl, 0.25 mM ATP, 0.25 mM GTP, 0.25 mM CTP, and 250 mCi [32P]UTP (3,000 Ci/mmol, ICN Biomedicals, Irvine, CA). After digestion of the reaction mixture with DNase I and proteinase K, the labeled RNA transcripts were recovered by the guanidinium thiocyanate-phenol-chloroform method (9) and purified by two precipitations with isopropanol. Hybridization was then performed to excess amounts (5 µg) of DNA targets immobilized on Nytran (Schleicher and Schuell, Keene, NH) membranes for 48 h at 60°C in a hybridization solution containing 10 mM TES (pH 7.4), 0.2% SDS, 10 mM EDTA, 0.3 M NaCl, 1× Denhardt's solution, and 5 µg of Escherichia coli RNA (Calbiochem, La Jolla, CA). The DNA plasmids used were the human cDNA for DRA cloned into pKS- (43), pKS- (Stratagene), and pGAPDH. The final stringency of the subsequent membrane wash was 0.1× SSC and 0.1% SDS at 60°C for 30 min.

Immunohistochemistry. Human colonic tissue, fixed in Formalin and embedded in paraffin, or frozen tissue embedded in OCT, were cut into 5-µm sections. Paraffin-embedded sections were heated to 60°C for 15 min, deparaffinized with xylene, and sequentially rehydrated in 100, 95, and 70% ethanol. Frozen sections for DRA staining were dried and fixed in 10% formaldehyde. After a rinse in 1× PBS buffer containing 0.05% Tween and 0.1% nonfat dry milk (NFDM), both paraffin and frozen sections were treated with 0.3% H2O2 in methanol for 10 min at room temperature. Paraffin-embedded slides, to be stained for DRA, were incubated in 10 mM citric acid buffer, pH 6.7, for 15 min at 90°C and rinsed in 1× PBS buffer. Paraffin-embedded slides stained with anti-NHE3 (Na+/H+ exchanger) antibody were blocked in 1× PBS containing 1% NFDM and 10% normal goat serum for 25 min at 37°C, washed with 1× PBS, and incubated for 45 min in a 1:200 dilution of a previously characterized anti-NHE3 antibody (22). Both frozen and paraffin-embedded slides stained with anti-DRA antibody were blocked in 1× PBS containing 0.1% saponin and 5% normal goat serum for 10 min at room temperature, rinsed in 1× PBS, and incubated for 30 min in 1:100 dilution of anti-DRA antibody (6). After having been washed with 1× PBS, the slides were incubated with a biotinylated goat anti-rabbit second antibody (Vector Laboratories) at a dilution of 1:200 for 40 min at room temperature. The staining was detected by using the horseradish peroxidase Vectastain Elite ABC kit (Vector Laboratories) and 3,3'-diaminobenzidine (Sigma). Immunoperoxidase detection was performed for exactly 1 min for all paraffin-embedded sections. Color development of the frozen anti-DRA stained section was for 6 min. The slides were counterstained in hematoxylin, dehydrated in ethanol and xylene, and mounted with mounting media.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

DRA mRNA is diminished in HLA-B27/beta 2M transgenic rat and in patients with ulcerative colitis. HLA-B27/beta 2M transgenic rats were chosen as a model because the inflammation is relatively quiescent, thus preserving much of the surface epithelium. A Northern blot of RNA isolated from the cecum, and proximal and distal colon from either a HLA-B27/beta 2M transgenic rat or a normal age- and sex-matched control animal showed that DRA expression was dramatically reduced in the transgenic rat (Fig. 1A). To estimate the relative contribution of RNA from epithelial cells with and without the presence of an inflammatory infiltrate, this Northern blot was rehybridized with a probe for villin (Fig. 1A). Villin, a major actin-binding protein of the intestinal epithelial brush border, is expressed by both differentiated and undifferentiated intestinal epithelial cells (4, 37).


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Fig. 1.   Colonic expression of DRA (downregulated in adenoma) mRNA in HLA-B27/beta 2M rat compared with normal control animal, as well as in patients with ulcerative colitis. A: Northern blots of total RNA isolated from the cecum (C) and proximal (P) and distal (D) colon of both HLA-B27/beta 2M transgenic rat and normal control animal were hybridized sequentially to probes for DRA and villin. Ethidium bromide (EtBr)-stained Northern gel is shown below autoradiographs. B: after normalization to villin expression, fold-reduction of DRA mRNA expression in each colonic segment in transgenic rats compared with control animal was determined. C: Northern blot of DRA using RNA isolated from 3 patients with ulcerative colitis. Paired samples of tissue were obtained from each patient, one from area of active colitis (UC) and one from region with normal histology (N). Blot was reprobed for 18S RNA, and abundance of DRA mRNA, normalized to that of 18S, was quantitated and depicted at bottom.

Although there was a dramatic decrease in DRA mRNA in the cecum of the transgenic rat compared with that of the normal animal, there was also a large decrease in villin mRNA (Fig. 1A). Indeed, histologically, the inflammatory process was much more pronounced in the cecum with a very prominent inflammatory infiltrate associated with a significant loss of epithelial cells due to erosions and ulceration (data not shown). This was in striking contrast to both the proximal (Fig. 2C) and distal (data not shown) colon where the inflammatory process was less severe with the preservation of the colonic epithelium. Consistent with this histology are the similar levels of villin expression in the proximal and distal colon of the HLA-B27/beta 2M transgenic rat compared with that of the normal animal. These similar levels also suggest that villin mRNA expression in colonocytes is itself not dramatically altered by intestinal inflammation. The expression of DRA, normalized to that of villin for each of the colonic segments, shows that the expression of DRA in each segment was diminished by five- to sevenfold in the HLA-B27/beta 2M transgenic rat compared with the control animal (Fig. 1B). DRA mRNA expression was also significantly reduced in actively inflamed colon compared with uninvolved tissue in pair-matched specimens from three patients with ulcerative colitis (Fig. 1C).

mRNA expression of DRA in surface epithelium is dramatically diminished in colitis. To determine which epithelial cell population was responsible for the decrease in DRA mRNA expression in the HLA-B27/beta 2M transgenic rat determined by Northern blot (Fig. 1, A and B), in situ hybridization was performed to assess the expression of DRA mRNA histologically (Fig. 2). DRA mRNA was expressed in the upper crypt and surface colonic epithelium of the normal animal (Fig. 2A). In contrast, there was a dramatic reduction in the expression of DRA mRNA specifically in the surface epithelium of the HLA-B27/beta 2M transgenic rat (Fig. 2C). Interestingly, expression of DRA mRNA remains preserved in the upper crypt region of this animal, which is roughly equivalent to that seen in the sections from the normal animal. Hybridization using a sense probe for DRA demonstrates the lack of nonspecific staining in either the colon of the normal rat or the HLA-B27/beta 2M transgenic rat (Fig. 2, B and D, respectively). The loss of DRA mRNA expression in the surface epithelium was also observed in a specific pathogen-free-housed IL-10 knockout mouse, which developed colonic inflammation (Fig. 2F). The upper crypt staining in the sections from tissues demonstrating colitis may be viewed as an internal control, which demonstrates the effectiveness of DRA mRNA detection by in situ hybridization on these samples. As an additional control, DRA expression was also studied in an IL-10 knockout mouse grown in a germ-free environment (Fig. 2E). Consistent with other animal models of inflammatory bowel disease (14), the IL-10 knockout mice grown in the absence of bacteria do not develop intestinal inflammation. The normal expression of DRA on the surface epithelium of this animal demonstrates that the loss of DRA expression is associated with the inflammatory state and not the absence of IL-10 expression.


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Fig. 2.   In situ hybridization for DRA in proximal colon of normal rat, HLA-B27/beta 2M transgenic rat, and interleukin-10 (IL-10) knockout mouse. A: normal rat, antisense probe. B: normal rat, sense probe. C: HLA-B27/beta 2M transgenic rat, antisense probe. D: HLA-B27/beta 2M transgenic rat, sense probe. E: IL-10 knockout mouse maintained in germ-free environment, antisense probe. F: IL-10 knockout mouse maintained in specific pathogen-free conditions, antisense probe. Magnification, ×64.

Immunohistochemistry shows that DRA protein expression is also attenuated in intestinal inflammation. Using immunoperoxidase-based immunohistochemistry, we compared the protein expression of DRA with the expression of a second epithelial ion transport protein that is also expressed on the surface epithelium of the colon, the Na+/H+ exchanger NHE3 (22), in either the normal colon or in a patient with ulcerative colitis.

Both DRA and NHE3 showed intense brush-border staining along with a similar degree of cytoplasmic staining in normal colonic tissue (Fig. 3, A and B, respectively). In contrast, although NHE3 staining remains unchanged in quiescent ulcerative colitis (Fig. 3D), immunostaining for DRA is diminished significantly in the same tissue sample (Fig. 3C). In moderate inflammation, DRA protein expression is completely absent on the surface epithelium (Fig. 3E). Although trace levels of DRA protein expression are detected in the upper crypt (see arrows), brush-border staining is absent. This pattern of DRA protein expression correlates well with the pattern of DRA mRNA expression detected in the same tissue sample by in situ hybridization (Fig. 3F).


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Fig. 3.   Expression of DRA and Na+/H+ exchanger (NHE3) in normal colonic tissue and from patients with ulcerative colitis. A: DRA expression in normal colon, paraffin-embedded section. B: NHE3 expression in normal colon, paraffin-embedded section. C: DRA expression in quiescent ulcerative colitis, paraffin-embedded section. D: NHE3 expression in quiescent ulcerative colitis (serial section of C), paraffin-embedded section. E: DRA expression in moderately active ulcerative colitis, frozen section. Although immunostaining of surface epithelium is absent, a small amount of cytoplasmic staining is noted in upper crypt (see arrows). F: DRA mRNA expression, by digoxigenin in situ hybridization, in same tissue sample as in E, frozen section. Magnification, ×64.

IL-1beta reduces DRA mRNA expression in postconfluent Caco-2 cells by transcriptional mechanism. Proinflammatory cytokines play an important role in regulating the inflammatory state. To elucidate the molecular mechanism by which inflammation may alter DRA gene expression, the effect of the proinflammatory cytokines IL-1beta and IL-6 on DRA gene expression was studied in Caco-2 cells. Silberg et al. (43) have previously shown that DRA mRNA is induced dramatically in Caco-2 cells on growth to a postconfluent state. The magnitude of this induction is further demonstrated when the mRNA for DRA in preconfluent Caco-2 cells is compared with that in the postconfluent state (Fig. 4B). The transcriptional activity of the DRA gene increases somewhat in postconfluent cells compared with the preconfluent state (Fig. 4B), suggesting that the upregulation of DRA in postconfluent cells occurs at least in part at the level of gene transcription. The lack of a dramatic increase in transcriptional activation, however, suggests that additional posttranscriptional mechanisms may contribute to the increase in DRA gene expression in postconfluent Caco-2 cells, such as has been noted for the regulation of another transport gene, CFTR (45).


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Fig. 4.   Effect of cytokine stimulation on DRA mRNA expression and gene transcription in postconfluent Caco-2 cells. A: Northern blot of total RNA isolated from postconfluent Caco-2 cells treated with either IL-1beta , IL-6, or both for 24 h, hybridized sequentially to probes for DRA and 7S. B: nuclear run-ons performed using nuclei isolated from preconfluent (Pre), postconfluent (Post), or postconfluent Caco-2 cells treated with IL-1beta (Post, IL-1beta ). As control, nuclear run-ons were also performed with nuclei isolated from Hep G2 cells, a cell line that does not express DRA. Position of DNA targets is identified to left of nuclear run-ons. Northern blot of DRA in pre- and postconfluent Caco-2 cells is shown above nuclear run-ons.

The effect of cytokine stimulation on the expression of DRA in postconfluent Caco-2 cells is shown in Fig. 4A. IL-1beta reduces DRA mRNA levels by approximately fourfold, whereas stimulation with IL-6 does not have an effect. Stimulation with both cytokines together did not augment the effect of IL-1beta alone on DRA expression. Nuclear run-ons show that IL-1beta decreases the transcriptional activation of the DRA gene in postconfluent Caco-2 cells by approximately threefold (Fig. 4B). Indeed, hybridization of newly transcribed transcripts to the DNA targets for DRA synthesized by nuclei isolated from IL-1beta -stimulated postconfluent Caco-2 cells compared with a cell line that does not express DRA, Hep G2 cells (43), shows nearly equivalent levels of expression (Fig. 4B). It is possible that cross-hybridization of other mRNA species that have a level of similarity to DRA may account for the hybridization to the DNA target for DRA in Hep G2 cells. This would suggest that the transcriptional activity of the DRA gene in postconfluent Caco-2 cells treated with IL-1beta may be very minimal.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Diarrhea, a prominent symptom of intestinal inflammatory states such as ulcerative colitis, is the result of a complex interaction of multiple inflammatory mediators and their effects on the intestinal epithelium. Several of these substances have been shown to stimulate intestinal secretion (11), whereas others have been shown to decrease transepithelial resistance by disrupting epithelial barrier function (30). Investigators have also shown that intestinal epithelial cells may respond to various cytokines by producing acute phase proteins such as alpha -1-antitrypsin (32), as well as decreasing the expression of sucrase-isomaltase, a marker of differentiated epithelial cells in the small intestine (48). We hypothesized therefore that these inflammatory mediators may also alter the gene expression of ion transporters in the intestinal epithelium.

Northern analysis (Fig. 1A) clearly shows that the mRNA expression of DRA is diminished throughout the colon of HLA-B27/beta 2M transgenic rats. Although epithelial expression of DRA may be decreased due to inflammatory stimulation, a proportional decrease in DRA mRNA may be observed secondary to the presence of an inflammatory infiltrate, expansion of the undifferentiated crypt compartment, or the loss of surface epithelial cells by erosion and ulceration of the colonic mucosa. In an attempt to control for these additional variables, DRA mRNA expression was normalized to the expression of villin mRNA (Fig. 1B). After normalization there was a consistent five- to sevenfold decrease in DRA expression in all three colonic segments of the HLA-B27/beta 2M transgenic rat compared with control (Fig. 1B). This result is consistent with the striking reduction in DRA mRNA expression also observed in regions of active colitis compared with normal colon in pair-matched samples from patients with ulcerative colitis (Fig. 1C).

In situ hybridization showed that DRA mRNA expression was localized to the differentiated, nonproliferating, surface, and upper crypt epithelium of the normal rat colon (Fig. 2A) as has been described in humans (6, 20). The pattern of mRNA expression therefore correlates well with DRA protein expression (Fig. 3A). In contrast to normal rats the expression of DRA in the HLA-B27/beta 2M transgenic rat was significantly diminished specifically in the surface colonocytes (Fig. 2C). The loss of DRA expression in the surface epithelium in the IL-10 knockout mouse with inflammation and in patients with ulcerative colitis (Figs. 2F and 3F, respectively) demonstrates that the attenuation of DRA expression is not unique to a specific disease process but rather appears to be a general phenomenon of colonic inflammation. Interestingly, DRA expression was maintained in the upper crypt region.

It has been observed that the proliferative zone of the colonic crypt, normally located in the lower two-thirds of the crypt, expands upward to the upper third and on to the surface epithelium of patients with colitis (2, 42). This suggests that inflammation may lead to a decrease in the differentiated cell compartment in the colon similar to the villus atrophy noted in small intestinal inflammatory states (29). Although this hyperproliferative state was also observed in the colonic specimens used in this study (data not shown), the persistence of DRA expression in the upper crypt is not consistent with the notion that there is a loss of cellular differentiation. Instead, our results simply demonstrate that the surface epithelium responds differently to intestinal inflammation than does the epithelium located in the upper crypt region.

Protein expression for DRA, as assessed by immunohistochemistry in Fig. 3, was also diminished in ulcerative colitis. Although DRA expression is decreased significantly in mildly inflamed colon (Fig. 3C) compared with normal colon (Fig. 3A), a small degree of brush-border staining is still present. The preservation of NHE3 expression in the same tissue sample (Fig. 3D), however, demonstrates that the observed reduction in DRA protein is not a result of a global dysfunction of transporter gene expression induced by intestinal inflammation. Instead, this result suggests that it is a specific process that may be restricted to a subset of genes expressed by the intestinal epithelium. Additional studies will be required to further characterize the relationship between the activity of inflammation and the degree to which DRA expression is attenuated. Although DRA protein expression is selectively altered in mild inflammation, it is likely that a more general alteration of epithelial gene expression may occur with severe inflammation where more significant damage of the surface epithelium occurs.

Although further investigation will be required to elucidate the mechanism by which DRA expression is diminished by the inflammatory state in vivo, the inhibition of DRA expression in postconfluent Caco-2 cells by IL-1beta (Fig. 4A) suggests that cytokines may play a role. Indeed, IL-1beta is thought to play a significant role in the pathogenesis of intestinal inflammatory diseases such as ulcerative colitis (7) and has been shown to be expressed by rat colonocytes after induction of inflammation (36). Although IL-6 has been shown to induce the expression of inflammatory mediators in Caco-2 cells as well as to inhibit the expression of a marker in intestinal differentiation, sucrase-isomaltase (48), it does not have an effect on DRA expression (Fig. 4A). This suggests that the effects of cytokines on the expression of genes which help to identify a differentiated phenotype in the intestinal epithelium are gene specific. Nuclear run-on assays show that the attenuation of DRA mRNA expression in postconfluent Caco-2 cells by IL-1beta appears to be regulated at least in part by the inhibition of gene transcription (Fig. 4B).

The clinical relevance of attenuated DRA expression in the pathogenesis of diarrhea was not addressed by our studies. It is generally believed that the surface epithelium of the colon plays a major role in electrolyte absorption (26, 44). Our results show that the loss of mRNA expression for DRA in ulcerative colitis is reflective of diminished protein expression (Fig. 3, E and F). It is therefore likely that the loss of DRA mRNA expression on the surface epithelium of both the HLA-B27/beta 2M transgenic rat and the IL-10 knockout mouse is also indicative of reduced or absent protein expression. In light of the lack of detectable brush-border staining in moderately active ulcerative colitis (Fig. 3E) it is tempting to speculate that the loss of DRA expression may be a contributing factor in the disruption of electrolyte absorption observed in the intestinal inflammatory state (39). Results of colonic perfusion studies in patients with congenital chloridorrhea compared with normal controls suggest Cl- transport is critical to maintain electroneutral Na+ and Cl- absorption in the proximal colon when coupled with Na+/H+ exchange (21). Indeed, mutations of the DRA gene have been identified in patients with congenital chloride diarrhea that eliminate not only the chloride but also the sulfate transport properties of this protein (33).

The colon is responsible for a significant amount of sulfate absorption (15). Increased levels of sulfate in the feces, which is the critical substrate for sulfate-reducing bacteria (10, 17), may play a role in the observed increase in colonization of patients with ulcerative colitis with sulfate-reducing bacteria (17). In turn, it has been hypothesized that the resultant hydrogen sulfide production by these bacteria may be toxic to colonocytes perhaps by disrupting the energy metabolism of these cells (38). It has been suggested that the digestion of sulfated mucins by bacterial fermentation may be one mechanism by which sulfate ions may be released in the colon (17). Based on the data presented in this paper, a decrease in the expression of a colonic sulfate transporter such as DRA may also play a role in perpetuating the colonization of ulcerative colitis patients with sulfate-reducing bacteria.

Very little is currently known about the regulation of electrolyte transporter gene expression in inflammatory states in vivo. In one of the only reports to study gene regulation from this perspective, Dupuit et al. (13) showed that CFTR protein expression was either lost or aberrantly expressed in the cytoplasm of ciliated epithelium associated with severe inflammation. In contrast to our studies, however, there was no relationship between the levels of CFTR mRNA transcripts and CFTR protein expression, suggesting that inflammation alters the expression of CFTR at a posttranscriptional and/or posttranslational level. Therefore, it is unlikely that the mechanisms responsible for the loss of CFTR expression are similar to those responsible for attenuation of DRA expression in the presence of colonic inflammation.

Ultimately, further investigation will be required to determine if the attenuation of DRA expression participates in the pathogenesis of diarrhea in patients with colitis. Our studies, however, provide the first evidence that transporter gene expression in the superficial colonic epithelium is altered by inflammation. In addition to altering intestinal barrier function and acting as secretagogues, proinflammatory cytokines may also promote diarrhea by inhibiting the transcription of genes involved in the transport function of the colonic epithelium.

    ACKNOWLEDGEMENTS

The authors thank Drs. Sue A. Keilbaugh and Richard H. Moseley for the critical review of this manuscript, and Drs. Deborah Gumucio and Juanita Merchant for the gifts of cDNAs for villin and GAPDH, respectively. We would also like to thank Dr. Mark Donowitz for the generous gift of antibodies to NHE.

    FOOTNOTES

This work was supported by National Institutes of Health Grants DK-47709, AI-39368, DK-50306, DK-34987, and AI-01122.

Present address of R. K. Sellon: Dept. of Veterinary Clinical Sciences, Washington State Univ., Pullman, WA 99164.

Address for reprint requests: G. D. Wu, 600 Clinical Research Bldg., 415 Curie Blvd., Philadelphia, PA 19104-6144.

Received 13 August 1997; accepted in final form 27 August 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 275(6):G1445-G1453
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