Departments of 1 Pediatrics, 2 Cell and Developmental Biology, and 3 Medicine, Division of Gastroenterology and Nutrition, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2576
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ABSTRACT |
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Altered mucosal integrity and increased cytokine production, including tumor necrosis factor (TNF), are the hallmarks of inflammatory bowel disease (IBD). In this study, we addressed the role of TNF receptors (TNFR) on intestinal epithelial cell migration in an in vitro wound closure model. With mouse TNFR1 or TNFR2 knockout intestinal epithelial cells, gene transfection, and pharmacological inhibitors, we show a concentration-dependent receptor-mediated regulation of intestinal cell migration by TNF. A physiological TNF level (1 ng/ml) enhances migration through TNFR2, whereas a pathological level (100 ng/ml) inhibits wound closure through TNFR1. Increased rate of wound closure by TNFR2 or inhibition by TNFR1 cannot be explained by either increased proliferation or apoptosis, respectively. Furthermore, inhibiting Src tyrosine kinase decreases TNF-induced focal adhesion kinase (FAK) tyrosine phosphorylation and cellular migration. We therefore conclude that TNFR2 activates a novel Src-regulated pathway involving FAK tyrosine phosphorylation that enhances migration of intestinal epithelial cells.
intestinal restitution; Src; focal adhesion kinase
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INTRODUCTION |
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THE SINGLE LAYER of epithelial cells lining the intestine forms an important barrier to a broad spectrum of noxious and immunogenic substances within the lumen. Continued cellular migration along the intestinal crypt-villus axis is necessary for maintenance of this layer through normal cell turnover, mucosal wound healing, and prevention of disease (18). This process is highly regulated by growth factors, cytokines, and matrix metalloproteinases (36, 51). An imbalance between mediators of the inflammatory process with subsequent altered mucosal reepithelialization has been described in individuals with inflammatory bowel disease (IBD), specifically an excess of proinflammatory mediators such as tumor necrosis factor (TNF), interleukin (IL)-8, and IL-12 and/or deficit of anti-inflammatory mediators such as IL-10 (3, 9, 25, 33, 34, 45). Conversely, animals in a germ-free environment (19) or on an elemental diet (10, 30) develop intestinal atrophy. Therefore, we (22) and others (19) have hypothesized that interactions between the intestinal epithelial cell and the contents of the gastrointestinal tract including commensal bacteria, pathogenic bacteria in low numbers, or other antigens may promote a healthy mucosal barrier through production of physiological cytokine levels.
TNF is a proinflammatory cytokine known to play a key role in the pathogenesis of IBD (7, 40, 48). TNF is expressed as a 26-kDa transmembrane precursor that is cleaved into an active soluble 17-kDa polypeptide, which aggregates into homotrimeric complexes (4). It exerts biological action through two distinct cell surface receptors, TNF receptor (TNFR)1 (55 kDa) and TNFR2 (75 kDa) (17, 28). TNF has a fivefold higher affinity for TNFR2 than TNFR1 in mouse cells, suggesting preferential ligation of TNFR2 at physiological TNF concentrations (49, 52). The two receptors mediate opposing effects on intestinal epithelial cell proliferation (22). TNFR1 induces growth arrest through sustained mitogen-activated protein kinase activity (23), whereas TNF stimulates proliferation via TNFR2 (22). Other cell systems show similar receptor-dependent opposing biological effects of TNF. For example, TNFR1 and TNFR2 mediate differing effects on osteoclastogenesis (1) and endothelial permeability (11).
Although there is clear evidence linking TNF to the pathogenesis of IBD, little is known about the effects of TNF on mucosal injury and repair. We have hypothesized that physiological levels of TNF play a role in maintaining an intact intestinal mucosal barrier. For instance, mice lacking TNFR1 receptor do not develop Peyer's patches and have defective germinal center formation (29, 32). Absence of TNFR2 correlates with increased sensitivity to bacterial pathogens (39) and decreased sensitivity to polysaccharide antigens in the intestine and colon (27). However, we are unaware of studies regarding the functional role of TNFR1 or TNFR2 on intestinal epithelial restitution. Therefore, we studied the effect of TNF on intestinal cell migration in a wound closure model. We describe concentration-dependent receptor-mediated regulation of intestinal cell migration by TNF. These findings are consistent with a model in which physiological levels of TNF enhance intestinal cell migration through TNFR2 and pathological levels inhibit epithelial wound closure through TNFR1. These data have potential implications for understanding altered reepithelialization of mucosal ulceration in IBD.
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MATERIALS AND METHODS |
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Materials.
5-Bromo-2'-deoxyuridine (BrdU) Labeling and Detection Kit I was
purchased from Roche Applied Science (Indianapolis, IN). Anti-focal adhesion kinase (FAK) antibody was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Anti-FAK-PY576 antibody was a gift of
Steven Hanks (Vanderbilt University, Nashville, TN). Anti-phospho
Y416-Src antibody was from Cell Signaling Technology (Beverly, MA).
Anti-Src antibody was from Upstate Biotechnology (Lake Placid, NY).
Goat anti-human TNFR1 agonist antibody was purchased from R&D Systems (Minneapolis, MN). ApopTag Fluorescein In Situ Apoptosis
Detection Kit was from Intergen (Purchase, NY). Human recombinant
epidermal growth factor (EGF) was provided by Carlos George-Nascimento
(Chiron, Emeryville, CA). Human recombinant hepatocyte growth factor
(HGF) was purchased from Collaborative Medical Products (Bedford, MA). Monoclonal hamster anti-mouse TNFR1 antagonist was obtained from Genzyme Diagnostics (Cambridge, MA). Murine recombinant TNF was purchased from Pepro Tech (Rocky Hill, NJ). Rabbit anti-human TNFR2
polyclonal antibodies BGL 74935 (directed against both extracellular and intracellular domains including the carboxy terminus) and SMA 73932 (directed against the extracellular domain) were gifts of Renu Heller
(Stanford University, Stanford, CA). Rat tail collagen type I was
purchased from Collaborative Medical Products. Recombinant murine
interferon (IFN)- and murine fibronectin were from Life Technologies
(Gaithersburg, MD). The Src tyrosine kinase-specific inhibitor
PD-161430 was a gift of David Fry (Parke-Davis Pharmaceutical Research,
Ann Arbor, MI). PP1 and PP2 were from Biomol (Plymouth Meeting, PA).
Cell line preparation.
The conditionally immortalized young adult mouse colon (YAMC) and mouse
small intestine epithelial (MSIE) cell lines were established from the
colonic and small intestinal mucosa of the H-2Kb-tsA58
transgenic mouse (Immortomouse) expressing a heat-labile simian virus
(SV)40 large T antigen with an IFN--inducible promoter as previously
described (22, 43, 56). TNFR1
/
and
TNFR2
/
mouse colon epithelial (MCER1
/
and MCER2
/
, respectively) cell lines were prepared by
mating the transgenic knockout mice with the Immortomouse (Charles
River Laboratories, Wilmington, MA). Mice carrying the SV40 gene were
identified by PCR as previously described (56). The mice
that were SV40 positive were then tested for the presence of the
transgene. Mice carrying both genes were mated to regain the homozygous
deletion of the transgene.
Cell culture.
Cells were maintained in RPMI 1640 medium with 5% FBS and 5 U/ml
murine IFN- and grown under permissive conditions at 33°C with 5%
CO2 (22). Before all experiments, cells were
plated onto fibronectin-coated (1 µg/ml) surfaces (43)
and then transferred to 37°C under nonpermissive conditions with
0.5% FBS IFN-
-free medium for 24 h.
Migration assays.
Confluent cells were trypsinized and plated (1.5 × 105 cells) on 35-mm culture dishes coated with fibronectin.
After cell attachment (2 h), the medium was changed to 0.5% FBS
IFN--free medium and incubated overnight at 37°C. The medium was
then aspirated, and seven to nine circular 1-mm cell-free areas were
created with a stabilized rotating silicone tip, using a method similar
to that described by Watanabe and colleagues (54).
Immediately after wounding, the cells were washed twice with PBS, with
culture medium replaced with the indicated factors in the presence or absence of agonist, antagonist antibodies, or pharmacological inhibitors, and then cultured up to 24 h. The surface areas of the
initial wound (set at 100%) and at the end of treatment were recorded
by time-lapse video microscopy and measured by BioQuant Image Analysis
software (Nashville, TN).
Cellular transfections.
Transient transfection of pRK7-p75 or empty vector (gift of Mike Rothe,
Tularik, South San Francisco, CA) was performed on p75/
YAMC cells with 1.5 µg of DNA and reagents provided in the
Lipofectamine Plus Reagent kit (GIBCO BRL, Grand Island, NY) following
manufacturer's guidelines. After transfection for 24 h, cells
were incubated in RPMI medium with 0.5% FBS at 37°C for 6 h
before experiments. p75 expression was detected with the PCR method.
The forward primer sequence and its reverse complement were
5'-ATGGCGCCCGTCGCCGTCTGGGCCGCG-3' and 5'-CAGGCGAGTGAGGCACCTTGGCTTC-3',
respectively. PCR products were resolved and visualized on 2% agarose gel.
Proliferation assay. Wounded and nonwounded YAMC cells cultured on fibronectin-coated chamber slides were treated with TNF or EGF for 8 or 24 h. At the end of treatment, cell proliferation was detected by BrdU labeling. Cells were incubated with 10 µM BrdU in cell culture medium for 30 min at 37°C, and then BrdU-incorporated cells were detected by mouse anti-BrdU antibody and fluorescein-conjugated anti-mouse IgG and observed by fluorescence microscopy. Proliferation rate was determined by counting at least 200 cells in randomly chosen fields and expressing BrdU-positive cells as a percentage of the total number of cells counted. Proliferation around wound sites was determined by counting all cells in the circumference to ~20 cell layers from the wound edge.
Apoptosis assay. YAMC cells were cultured on fibronectin-coated chamber slides and then wounded or not and treated with TNF or EGF for 24 h. At the end of treatment, cells were fixed with 1% paraformaldehyde in PBS. Apoptotic cells were detected by using terminal deoxynucleotidyl transferase [TdT; also termed TdT-mediated dUTP nick end labeling (TUNEL)] and labeled with FITC-conjugated anti-digoxigenin. Slides were then dehydrated and mounted with Vectashield mounting medium. Slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI) by using 1 µg/ml DAPI in mounting medium. The cells were observed by fluorescence microscopy. The percentage of cells undergoing apoptosis was then determined as described for proliferation assays.
Preparation of cellular lysates. After cells were treated with EGF, TNF, or HGF in the presence or absence of TNFR1 agonist, TNFR1 antagonist, or TNFR2 antagonist antibodies for indicated times, cells were rinsed twice with ice-cold PBS and then scraped into cell lysis buffer containing 1% Triton X-100, 20 mM HEPES (pH 7.5), 1 mM orthovanadate, 50 mM glycerol phosphate, 1 mM sodium pyrophosphate, 10 mg/ml leupeptin, 10 mg/ml aprotinin, 18 mg/ml PMSF, and incubated for 30 min on ice. The lysates were centrifuged (14,000 g, 15 min) at 4°C, and the protein concentration of the Triton-soluble fraction was determined with a DC protein assay (Bio-Rad Laboratories, Hercules, CA). The Triton-soluble fraction was collected for immunoprecipitation or mixed with Laemmli sample buffer for SDS-PAGE and Western blot analysis with anti-phospho-Src or anti-Src antibodies.
Immunoprecipitation. Triton-soluble lysates were incubated with anti-FAK antibody at 4°C for 2 h. Recombinant protein A Sepharose conjugate was then added to the mixture and incubated overnight at 4°C. The immunoprecipitates were recovered by centrifugation (14,000 g, 1 min) and washed three times with ice-cold modified RIPA buffer (1% Triton X-100, 1% deoxycholate, 0.1% SDS, 1 mM EDTA, 10 mM Tris, pH 8.5, and 150 mM NaCl), resuspended in Laemmli sample buffer, and denatured for 5 min at 95°C for Western blot analysis with anti-FAK-PY576 or anti-FAK antibodies.
Statistical analysis. The statistical significance of differences between mean values was assessed with paired Student's t-test analysis. The level of statistical significance was set at P < 0.05.
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RESULTS |
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TNF regulates intestinal cell migration in a
concentration-dependent manner.
Two of the hallmarks of IBD include altered mucosal integrity and
increased production of cytokines such as TNF. Therefore, we tested the
effect of TNF on migration in an intestinal epithelial wound closure
model. Migration assays were performed on cells plated as a monolayer
on mouse fibronectin and then treated with EGF or TNF after wounding.
As expected (41), EGF increases the rate of wound closure
(Fig. 1A).
Interestingly, TNF at lower concentrations (1 ng/ml) increases wound
closure at rates comparable to EGF. However, higher concentrations of
TNF (10 and 100 ng/ml) block migration and completely inhibit
EGF-stimulated wound closure. Representative wounds are shown for YAMC
cells at 8 h in Fig. 1B. With Giemsa staining we
observed that the rotating silicone disk removes cells without
removing the underlying extracellular matrix (data not shown).
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TNF inhibition of cellular migration is mediated by TNFR1.
Because we have shown (22) expression of two TNF receptors
on intestinal epithelial cells, we asked whether these
concentration-dependent effects of TNF on cell migration might be
differentially mediated by the two receptors. With the wound closure
model, cells were studied in the presence or absence of antagonist
antibodies to TNFR1 or TNFR2. The TNFR1 agonist antibody causes
inhibition of EGF-stimulated migration, similar to higher
concentrations of TNF (Fig.
2A), whereas antagonist
antibodies to TNFR1 inhibit this TNF effect. Furthermore, antagonist
antibody to TNFR2 does not alter the inhibition of EGF-stimulated
migration by TNF. Wounds in YAMC or MSIE cells treated with either EGF
or TNF (1 ng/ml) are closed by 24 h, preventing relative
comparison between treatments at this time point in either cell line.
To further test our hypothesis that activation of TNFR1 inhibits
EGF-stimulated cell migration we developed mouse colon cell lines
lacking TNFR1 (MCER1/
). These cells show loss of the
inhibitory effect of higher TNF concentrations on wound closure (Fig.
2B). However, they retain stimulation of migration by lower
TNF concentrations, EGF, or HGF.
|
Enhanced migration is mediated through TNFR2.
Our finding that loss of TNFR1 does not prevent TNF-stimulated
migration (Fig. 2B) suggests that this effect is mediated
through TNFR2. To test this hypothesis we first used antagonist TNFR2 antibodies in the presence of low-dose TNF. Blocking antibody to TNFR2
completely inhibits TNF-stimulated migration but not EGF-stimulated
wound closure (Fig. 3A). In
contrast, blocking TNFR1 had no effect on TNF promoting intestinal cell
migration. We developed TNFR2 /
mouse colon epithelial
(MCER2
/
) cell lines to verify the role of TNFR2 in
stimulating cell migration. MCER2
/
cells do not migrate
in response to low levels of TNF. Higher concentrations of TNF block
migration in this cell line and completely inhibit EGF-stimulated
MCER2
/
migration (Fig. 3B). However, low
levels of TNF stimulate migration in MCER2
/
cells
transfected with vector containing full-length TNFR2 but not empty
vector (Fig. 3C). Interestingly, both MCER2
/
and MCER1
/
cells migrate more slowly than either YAMC
or MSIE cells. Therefore, data from these null colon cells are shown at
24 h after wounding. These findings support a model in which TNFR2
mediates TNF-stimulated intestinal epithelial wound closure and
ligation of TNFR1 inhibits growth factor-mediated cell migration.
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Stimulation of cell migration by TNFR2 is associated with catalytic
activities of FAK and Src.
We (42) and others (38) have previously shown
a requirement for tyrosine kinase activity during the process of
cellular migration. However, TNFR2 does not contain an intrinsic
tyrosine kinase (52). The cytoplasmic tyrosine kinase Src
is important for migration of a number of cell types (21, 38,
55). To determine whether TNFR2-mediated migration
requires Src tyrosine kinase activity, we studied YAMC cells in the
presence or absence of the Src tyrosine kinase-specific inhibitors
PD-161430, PP1, or PP2. As shown in Fig.
4, the Src inhibitors block the ability of low-dose TNF to stimulate migration. Molecular dissection of focal
adhesion tyrosine phosphorylation sites reveals tyrosine phosphorylation increases on nonreceptor protein tyrosine kinase FAK
residue 576 during cell adhesion to fibronectin (14, 15). FAK Y576 is a substrate of activated Src kinase (15). To
investigate the effect of TNFR2 stimulation on FAK Y576
phosphorylation, we added low-dose TNF (1 ng/ml) to YAMC cell
monolayers in the presence of antagonist antibodies to either TNFR2 or
TNFR1. Low-dose TNF induces tyrosine phosphorylation of FAK on residue
576 (Fig. 5A). Blocking TNFR2
inhibits this effect; however, TNFR1 does not alter FAK Y576
phosphorylation. Isolated TNFR2 activation with an agonist antibody is
sufficient to induce this increased phosphorylation (Fig.
5A). We further studied the effect of the Src tyrosine
kinase-specific inhibitor on TNF-induced phosphorylation on FAK Y576.
Preincubation of cells with the Src inhibitor reduces TNF-stimulated
FAK Y576 phosphorylation (Fig. 5B).
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DISCUSSION |
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We have shown that TNFR differentially regulate intestinal epithelial cell migration by studying a wound closure assay with three different approaches. First, we observed a concentration-dependent effect with low TNF concentrations enhancing migration and higher concentrations inhibiting migration. An immunoregulatory approach using agonist and antagonist antibodies shows that TNFR2 promotes cellular migration and TNFR1 inhibits this response. Second, loss of TNFR2 results in intestinal cells that no longer migrate in response to TNF. Furthermore, the loss of TNFR1 prevents the antimigratory effect of high TNF levels. Third, loss of TNFR2 results in inhibition of TNF-stimulated Src activation. Src is a necessary kinase for TNF-induced migration and is restored by forced TNFR2 expression. These data suggest that cell migration may be regulated by local TNF levels, with implications for altered wound healing in IBD.
Studies in cell culture and in vivo suggest that the two TNF receptors
mediate distinct biological responses from proliferation to
inflammation, bone resorption, and vascular permeability (1, 11,
22, 39, 50). Because TNF is pathogenic in IBD, we initiated
studies to address the role of TNFR in an in vitro epithelial cell
wound closure model. We found that ligation of TNFR1 or TNFR2 mediates
opposing effects on wound closure. The development of receptor-deficient mice underlines the potential importance of understanding the individual receptor role in mediating TNF responses. For example, increasing TNF levels by deleting TNF AU-rich elements (ARE) is sufficient to induce IBD in a mouse (24).
However, no intestinal inflammation occurred with expression of
ARE
in TNFR1
/
mice. When expressed on the
TNFR2
/
background there was attenuation of
gastrointestinal inflammation. Differential regulation of cellular
migration by TNFR has been shown to occur in nonepithelial cells of the
epidermis as well. TNF promotes migration of cutaneous dendritic cells
through the selective activation of TNFR2 (47). Whereas
the migration of epidermal Langerhans cells is markedly depressed in
p75-deficient mice, it is normal in p55-deficient mice
(53). Likewise, in hematopoietic cells TNF enhances the
chemotaxis of B cells through TNFR2 (8).
Through development of intestinal epithelial cells expressing only one of the two receptors, we are clearly able to assign distinct biological function to each receptor. In similar studies Abu-Amer and colleagues (1) showed that TNFR1 enhances osteoclastogenesis whereas TNFR2 suppresses this response. Our findings in this report further support a role for low levels of TNF as beneficial to intestinal epithelial integrity, because TNFR2 also stimulates proliferation (22). Although the intracellular events that control cell migration are highly complex and remain incompletely understood, a number of intracellular molecules have been implicated in regulating the initial response to cell injury and/or the resultant migration (57). Interestingly, in cells lacking TNFR1 we find reduced migratory responsiveness by endogenous TNFR2 (Fig. 2B), suggesting additional mechanisms of regulation. One possibility is the induction of TNFR2 shedding reported with high levels of TNF (44). Rapid shedding of TNFR2 consequently reduces cell sensitivity to TNF (2), supporting a role for endogenously produced soluble p75 in downregulating TNF-driven responses (39). Another possibility is the so-called ligand passing hypothesis (49). This model suggests enhanced receptor responsiveness of TNFR1 by ligand passing from TNFR2 and therefore seems less likely to explain our observation than does receptor shedding.
Tyrosine kinase activity is necessary for intestinal epithelial cell migration (5, 6, 38, 41, 46). Therefore, in the absence of any endogenous TNFR2 catalytic activity (52) we speculated that intracellular nonreceptor protein kinases mediate TNF-stimulated cell migration. A screen for intermediates involved in signal transduction pathways suggested Src family members as candidate tyrosine kinases regulating TNF-stimulated migration. These cytoplasmic tyrosine kinases participate in a wide variety of cellular responses, including adhesion and motility (38). We found that inhibiting Src tyrosine kinase decreases TNF-induced FAK tyrosine 576 phosphorylation, a Src phosphorylation site on FAK (15). This finding suggests a potential role for Src kinase in low-dose TNF-stimulated migration through activation of FAK. Clearly, FAK-null cells have reduced motility (20). Furthermore, the ability of FAK to regulate migration is dependent on site-specific tyrosine phosphorylation including tyrosine 576 (37). It is likely that phosphorylation on these sites determines FAK interaction with several intracellular signaling molecules including Src family kinases (26). Given this background the data reported here suggest that TNFR2 activates a novel Src-regulated pathway enhancing migration of intestinal epithelial cells involving FAK tyrosine phosphorylation.
The role of Src activation by high TNF levels through TNFR1 in reducing cell migration rate remains unclear. Expression of dominant-negative Src in YAMC cells alters cellular morphology, and the pharmacological inhibition of Src kinase activity in the presence of high TNF induced near complete apoptosis of YAMC cells (data not shown). Although little information is available regarding Src as an antiapoptotic molecule, loss of Src kinase activity enhances osteoclast apoptosis through TNFR1 (58). Furthermore, the loss of Src activity in the acid sphingomyelinase-null mouse enhances TNF family member Fas-induced apoptosis (35). Therefore, the activation of Src via TNFR1 may represent a novel antiapoptotic pathway in intestinal epithelial cells. Clearly, this observation warrants further study.
In conclusion, we have used several approaches to examine the migration of intestinal epithelial cells to a range of TNF levels. We report that TNFR2 stimulates intestinal cell migration, whereas TNFR1 inhibits migration in an in vitro wound closure assay. Receptor-dependent TNF-regulated responses have been reported in other tissues; however, the novel observations of these studies are the TNFR2-stimulated activation of Src and the direct regulation of epithelial cell migration by TNF. The potential roles for these receptors in regulating epithelial monolayer homeostasis in an environment of high TNF levels warrant further investigation with implications for mucosal healing and IBD.
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ACKNOWLEDGEMENTS |
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We thank Peter Dempsey and Steve Hanks for helpful discussions and for critically reviewing the manuscript.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-56008 (D. B. Polk) and DK-58404 (the Vanderbilt University Digestive Disease Research Center).
Address for reprint requests and other correspondence: D. B. Polk, Dept. of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, 21st and Garland Ave., S-4322 MCN, Nashville, TN 37232-2576 (E-mail: d-brent.polk{at}vanderbilt.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published December 4, 2002;10.1152/ajpcell.00309.2002
Received 2 July 2002; accepted in final form 27 November 2002.
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