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
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ABSTRACT |
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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-B27/2m 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/
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-1
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
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INTRODUCTION |
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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-B27/2m
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.
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MATERIALS AND METHODS |
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Isolation of RNA and Northern blots.
A HLA-B27/2M 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).
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/
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).
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-1 (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.
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RESULTS |
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DRA mRNA is diminished in HLA-B27/2M transgenic rat
and in patients with ulcerative colitis.
HLA-B27/
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/
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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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-B27/2M 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/
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/
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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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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IL-1 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-1
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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DISCUSSION |
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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 -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-B27/2M 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/
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-B27/2M 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-1 (Fig. 4A) suggests
that cytokines may play a role. Indeed, IL-1
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-1
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-B27/2M 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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