Regulation of RELM/FIZZ isoform expression by Cdx2 in response to innate and adaptive immune stimulation in the intestine
Mei-Lun Wang,1
Marcus E. Shin,2
Pamela A. Knight,3
David Artis,4
Debra G. Silberg,2
Eunran Suh,2 and
Gary D. Wu2
1Division of Gastroenterology and Nutrition, The Children's Hospital of Philadelphia, 2Division of Gastroenterology, University of Pennsylvania School of Medicine, and 4Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania; and 3Division of Veterinary Clinical Studies, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Midlothian, United Kingdom
Submitted 29 September 2004
; accepted in final form 24 November 2004
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ABSTRACT
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Host immune responses to commensal flora and enteric pathogens are known to influence gene expression in the intestinal epithelium. Although the Cdx family of caudal-related transcription factors represents critical regulators of gene expression in the intestinal epithelium, the effect of intestinal immune responses on Cdx expression and function has not been determined. We have shown that bacterial colonization and Th2 immune stimulation by intestinal nematode infection induce expression of the intestinal goblet cell-specific gene RELM
. In this study, we investigated the transcriptional regulation of resistin-like molecule/found in inflammatory zone (RELM/FIZZ, RELM
) and its isoforms RELM
and RELM
to ascertain the role of Cdx in modifying intestinal gene expression associated with innate and adaptive immune responses. Analysis of the RELM
promoter showed that Cdx2 plays a critical role in basal gene activation in vitro. This was confirmed in vivo using transgenic mice, where ectopic gastric and hepatic expression of Cdx2 induces expression of RELM
, but not RELM
or RELM
, exclusively in the stomach. Although there was no quantitative change in colonic Cdx2 mRNA expression, protein distribution, or phosphorylation of Cdx2, bacterial colonization induced expression of RELM
, but not RELM
or RELM
. In contrast, parasitic nematode infections activated colonic expression of all three RELM isoforms without alteration in Cdx2 expression. These results demonstrated that Cdx2 participates in directing intestine-specific expression of RELM
in the presence of commensal bacteria and that adaptive Th2 immune responses to intestinal nematode infections can activate intestinal goblet cell-specific gene expression independent of Cdx2.
MUCOSAL IMMUNE RESPONSES can influence gene expression and alter the mucosal phenotype in the intestinal epithelium by affecting cell proliferation, differentiation, cytoprotection, and cytotoxicity (8, 48, 63, 67). Growing evidence demonstrates that innate immune responses, through Toll-like receptors and the transcription factor NF-
B, play an important role in the regulation of genes involved in host intestinal epithelial defense against certain bacterial pathogens (1, 2, 11). Furthermore, bacterial colonization of the gut with commensal organisms has been shown to regulate gene expression in the intestinal epithelium (18, 29, 30). Alternatively, adaptive immune responses, such as those that are mediated by Th2, can have a significant effect on the intestinal epithelial phenotype, resulting in goblet cell hyperplasia and hypertrophy (32, 34).
The Cdx family of caudal-related transcription factors is known to play a critical role in the regulation of intestine-specific gene expression. Although Cdx1 and Cdx2 have distinctive patterns of expression in the intestinal tract (33, 46, 56), they can activate similar sets of genes in vitro. In this regard, the diverse group of target genes for Cdx transcription factors overlaps those associated with host mucosal immunity and includes genes involved in intestinal epithelial proliferation (39, 58), cell adhesion (26, 53), nutrition (61, 65), and cell cycle regulation (14, 40). Furthermore, Cdx overexpression and underexpression have been linked to alterations in intestinal goblet cell phenotype in human diseases such as Barrett's metaplasia and subsets of colonic malignancies, respectively (16, 43, 50). However, the role of Cdx in the setting of host innate and adaptive immune responses has not been reported.
We previously described an intestine-specific gene, resistin-like molecule/found in inflammatory zone (RELM/FIZZ, RELM
), the expression of which is limited to goblet cells in the colonic epithelium. In mice, RELM
gene expression is regulated by host innate immunity in response to commensal bacterial colonization (21). More recently, we showed that this gene is highly induced by host Th2-mediated immune responses, where it is believed to be an immune effector of resistance to parasitic intestinal nematode infections (4). Given its unique pattern of expression, the transcriptional regulation of RELM
might provide important insights into mechanisms by which the innate and acquired immune systems regulate gene expression in the colonic epithelium.
In this study, we use in vitro and in vivo model systems to investigate the transcriptional regulation of RELM
and demonstrate that RELM
is regulated by Cdx2, but not Cdx1. In contrast to RELM
, however, the two other RELM isoforms, RELM
and RELM
, are neither regulated by Cdx2 nor induced by bacterial colonization in the colon. Although Cdx2 expression is not altered by bacterial colonization, these results suggest that this transcription factor is necessary, but not sufficient, for colonization-dependent activation of colonic RELM
expression. By contrast, in the absence of any alteration in Cdx2 expression, all three members of the RELM family are induced to high levels in the intestine through host Th2 immune responses to nematode infection, suggesting that adaptive immune responses can activate goblet cell-specific gene expression through Cdx2-independent mechanisms.
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MATERIALS AND METHODS
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Cell culture and transfections.
Human colon cancer cell lines HT-29, Caco-2, and LS174T (American Type Culture Collection) were maintained at 37°C in 5% CO2 in a humidified incubator. LS174T cells were maintained in MEM supplemented with penicillin-streptomycin (GIBCO, Grand Island, NY) and 10% FBS (Hyclone, Logan, UT). Caco-2 and HT-29 cells were maintained in DMEM supplemented with penicillin-streptomycin and 10% FBS. Cells were seeded at 5 x 105 cells/well in 12-well plates 1 day before transfection. Cells were transfected with 2 µg of the pGL2 construct of interest and 0.5 µg of cytomegalovirus (CMV)-
-galactosidase plasmid using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in Opti-MEM without antibiotics according to the manufacturer's recommendations. At 24 h after transfection, cells were harvested, and lysates were prepared using reporter lysis buffer (Promega, Madison, WI) according to the manufacturer's instructions. Eighty microliters of cell lysate were mixed with 100 µl of luciferase assay reagent (Promega), and luminescence was measured in a luminometer (Dynex, Chantilly, VA). The cell lysate (80 µl) was subjected to
-galactosidase assay with o-nitrophenyl-
-D-galactopyranoside. Transcriptional activation was normalized to
-galactosidase activity. Each plasmid was assayed in triplicate in two separate experiments.
Animals and pathogens.
Balb/c (Jackson Laboratory, Bar Harbor, ME, or B & K Universal, Hull, UK) and Foxa3/Cdx2 transgenic mice were maintained under specific pathogen-free (SPF) conditions at the University of Pennsylvania or a conventional environment at the University of Edinburgh. The maintenance, infection, and recovery of Trichuris muris, Trichinella spiralis, and Nippostrongylus brasiliensis were carried out as previously described (3, 35, 36). All experiments were performed under the guidelines of the University of Pennsylvania Institutional Animal Care and Use Committee (Protocol #s 222600 and 700537) or UK Home Office Animals (Scientific Procedures) Act 1986 (License #PPL 60/3023). Germ-free C.B17.SCID mice were bred and housed in the germ-free facility of the Department of Biology at the University of Pennsylvania under sterile conditions in Trexler isolators (Standard Safety, McHenry, IL). Conventionalization of formerly germ-free C.B17.SCID mice was performed by transfer of germ-free mice to an SPF environment for 35 days, as previously described (21).
Construction of human RELM
/pGL2 reporter genes.
A 870/+50 region of the human RELM
promoter was amplified by PCR with Taq polymerase using a human genomic DNA template. The primer sequence was as follows: 5'-AAGCTTAGGGTTGCTGAAGAACAG-3' (forward) and 5'-CTCGAGGGATAAGGAATTGTGAAG-3' (reverse). The 920-bp amplicon was cloned into the promoterless pGL2 basic reporter plasmid (Promega), which contains a luciferase structural gene immediately downstream of a polylinker, to create the 870 hR
plasmid construct. All deletional reporter plasmid constructs were generated from the 870 hR
plasmid construct. The 754 construct (754 hR
) was prepared by restriction digestion of 870 hR
using restriction sites at 754 (BglII) and +50 (HindIII). The 418 construct (418 hR
) was prepared by restriction digestion of 870 hR
using restriction sites at 418 (ApaI) and +50 (HindIII). After restriction digestion, Klenow (Promega) was used for blunt-end formation, and the preparation was subjected to religation using the Rapid Ligation kit (Roche, Indianapolis, IN). All constructs were amplified using the Maxi Prep kit (Qiagen, Valencia, CA). The fidelity of the PCR amplification was verified by sequence analysis, and plasmid purity was confirmed by an absorption ratio (260/280 nm) >1.6.
Isolation of nuclear extracts and electrophoretic mobility shift assay.
Nuclear extracts were prepared as described previously (68) and stored at 80°C until use. Protein concentration was measured with protein assay reagent (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. The sequences of the synthesized oligonucleotides are listed in Fig. 2A. Single-stranded oligonucleotides were annealed and radiolabeled using Klenow (Roche) and [
-32P]dTTP. Radiolabeled probes were purified on a G-25 Sephadex Quick Spin column (Roche). Nuclear proteins (5 µg) were preincubated at room temperature for 10 min with 1x binding buffer and 1 ng of poly(dI-dC) in a total volume of 19 µl. Radiolabeled oligonucleotide probe (1 µl, 20,000 cpm) was added, and the mixture was incubated at room temperature for 20 min. For competition analysis, a 100-fold molar excess of unlabeled probe was added to the mixture 10 min before addition of the radiolabeled probe. Supershift experiments using polyclonal antibodies against Cdx1 or Cdx2 (CNL) were performed as previously described (60, 62). DNA loading buffer was added to the mixtures, and the samples were loaded onto a 4% nondenaturing polyacrylamide gel. Gels were run in 0.5x Tris-boric acid-EDTA at 20 mA, vacuum-dried, and autoradiographed for 24 h at 80°C.

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Fig. 2. Cdx binds to and transactivates the human RELM promoter in LS174T cells. A: sequence of base pairs 588 to 550 of the human RELM promoter with consensus Cdx binding sites shown in boldface type. WT, wild type. Sequences of oligonucleotides, incorporating 2-bp mutations (underlined) at 5' (5'M) and 3' (3'M), as well as 5' and 3' (2xM), Cdx binding sites are shown. B: EMSA using nuclear proteins isolated from LS174T cells and the 32P-labeled WT oligonucleotide probe. Lanes 26, competition of nuclear protein binding using 100-fold excess unlabeled double-stranded oligonucleotides for the Cdx binding site shown in A as well as a previously defined Cdx consensus sequence, SIF1. Lanes 7, 8, and 9, supershift (SS) EMSA using antibodies specific for Cdx1, Cdx2, and Cdx1/Cdx2, respectively. C: transient transfection of LS174T, Caco-2, and HT-29 cells using luciferase reporter constructs incorporating 5'M [588 hR (5'M)], 3'M [588 hR (3'M)], and 2xM [588 hR (2xM)] mutations into the 588 hR construct. Values are means (n = 3).
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RNA isolation, 5'-rapid amplification of cDNA ends PCR, and primer extension.
Mice were killed, and the colon was removed, gently cleared of stool, and divided in half lengthwise. One-half was immediately frozen in liquid N2, and the other half was coiled to form a Swiss roll, preserved in 10% buffered formalin, and embedded in paraffin for later use in immunohistochemistry. RNA was isolated from murine frozen tissues as well as from colon cancer cell lines by cesium chloride gradients followed by gel electrophoresis to verify RNA integrity. Primer extension analysis and 5'-rapid extension of cDNA ends (RACE) PCR were performed as previously described (69). The oligonucleotide sequence was 5'-AACCACAGCCATAGCCAC-3' for the 3'-RACE primer and SI-CCC for the 5'-RACE primer (69). The sequence of the oligonucleotide used for the primer extension analysis was 5'-AGAGTTTTCCCCTAAGAGCA-3'.
Quantitative RT-PCR.
SuperScript II first-strand synthesis kits (Invitrogen) were used to create cDNA from total RNA. Quantitative RT-PCR was performed on an ABI 7000 (Applied Biosystems, Foster City, CA), with Syber Green used as the fluorescent probe. Primers were designed using Primer Express software (Applied Biosystems). A primer matrix was run on each set of new primers to determine the correct template concentration range and to evaluate whether the analysis was complicated by primer-dimer formation. Primers used in these studies were as follows: 5'-ATGGGTGTCACTGGATGTGCTT-3' (forward) and 5'-AGCACTGGCAGTGGCAAGTA-3' (reverse) for RELM
, 5'-TGGCTTTGCCTGTGGATCTT-3' (forward) and 5'-GCAGTGGTCCAGTCAACGAGTA-3' (reverse) for RELM
, and 5'-ATGGCTGTGGATCTTGGGATAT-3' (forward) and 5'-CGGTGGCCCAGTCCATT-3' (reverse) for RELM
. PCR conditions were as follows: 50°C for 2 min, 95°C for 10 min, and 40 cycles at 95°C for 15 s followed by 60°C for 1 min. A dissociation curve was run with each PCR to ensure that primer-dimer formation did not occur. PCR results were analyzed using the 
Ct analysis (User Bulletin 2, Applied Biosystems), with GAPDH used as the housekeeping gene.
Immunohistochemistry and Alcian blue staining.
For immunohistochemical analysis of RELM
, we used formalin-fixed and paraffin-embedded tissue sections and previously characterized affinity-purified antibodies, as previously described (21). RELM
staining (1:1,000) was performed in a similar fashion using affinity-purified polyclonal antibodies (Novus Biologicals, Littleton, CO). Cdx2 staining was performed as previously described (56) using affinity-purified polyclonal antibodies against the Cdx2 NH2 terminus (CNL, 1:1,500) and Cdx2 COOH terminus (Cterm, 1:1,000). Additional staining was performed using an antibody against Cdx2 with a phosphorylated serine at position 60 (P-Cdx2-S60, 1:1,000) (52). For Alcian blue staining, tissues were fixed in 10% formalin overnight, washed in 1x PBS, and embedded in paraffin. Sections (6 µm) were cut and mounted, and slides were deparaffinized. After application of 3% aqueous acetic acid to the slides, 1% Alcian blue in 3% acetic acid, pH 2.5, was applied. Sections were washed and counterstained with 0.1% nuclear fast red, dehydrated, mounted, and viewed by light microscopy.
Western blot.
Immunoblots were performed (21) using commercially available RELM
and RELM
recombinant proteins (Preprotech, Rocky Hill, NJ) as well as a flag-tagged RELM
fusion protein synthesized using a mammalian expression system (7).
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RESULTS
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A region between 418 and 588 in the RELM
promoter contains two potential Cdx binding sites and transactivates the human RELM
promoter in a goblet cell-specific fashion. The constitutive expression of RELM
specifically in the intestine suggested that this gene might be regulated by nuclear proteins shown previously to regulate intestine-specific gene expression, such as members of the Cdx family of transcriptional activators (60, 64). A 706-bp region of the human RELM
promoter was sequenced, and the start of transcription was identified by 5'-RACE RT-PCR for the murine and human genes (data not shown). Primer extension analysis (Fig. 1A ) identified a single predominant transcriptional start site in the murine RELM
promoter. Potential binding sites for multiple transcription factors, including members of the Cdx family, were identified in the human (21) and murine RELM
promoters (data not shown).
We previously reported endogenous expression of RELM
in the goblet cell-like LS174T cell line, but not in other intestinal cell lines such as Caco-2 and HT-29 (21). Transient transfection of luciferase reporter constructs of the RELM
promoter into these three cell lines revealed that high-level transcriptional activity, specifically in LS174T cells, required a cis-acting region located between base pairs 418 and 706 (Fig. 1B). This indicated that an element within the 418/706 region could activate the human RELM
promoter in a goblet cell-specific manner.
The human RELM
promoter contains two consensus cis elements that bind Cdx1 and Cdx2 and are responsible for promoter transactivation. Sequence analysis of the RELM
promoter revealed the presence of two Cdx consensus binding sites (60) located between base pairs 550 and 588 (Fig. 2A). Electrophorectic mobility shift assays (EMSAs) were performed to characterize the DNA-protein interactions in this region of the promoter (Fig. 2B). With the use of nuclear proteins isolated from LS174T cells and an oligonucleotide spanning base pairs 588 and 550 of the human RELM
promoter, specific nuclear protein-DNA interactions were revealed by successful competition of binding using an excess of the unlabeled wild-type oligonucleotide as well as an oligonucleotide containing the SIF1 element previously shown to bind Cdx proteins (65) (Fig. 2B, lanes 13). Competition using excess unlabeled oligonucleotides containing two base-pair mutations of the 5' (5'M) or 3' (3'M) binding sites (Fig. 2A) led to partial inhibition of complex formation, whereas competition with an oligonucleotide containing mutations at both sites (2xM) did not lead to inhibition of complex formation (lanes 4, 5, and 6, respectively). These results suggested that Cdx family proteins are able to bind independently to two binding sites within a region of the RELM
promoter important for transcriptional activation in LS174T cells.
The nuclear proteins responsible for these DNA-protein interactions were identified by supershift EMSAs using antibodies specific for Cdx1, Cdx2, or Cdx1 and Cdx2 (Fig. 2B, lanes 7, 8, and 9, respectively). These studies identified the faster-mobility complex as Cdx1 and a second, more slowly migrating complex, Cdx2. Although we cannot exclude the possibility that additional proteins may bind to these regions of the RELM
promoter, these results show that Cdx1 and Cdx2 are components of the observed binding complex.
To investigate the functional importance of these two binding sites, the same site-directed mutations of the human RELM
promoter were incorporated into the 588 luciferase reporter construct and transiently transfected into LS174T, Caco-2, and HT-29 cells (Fig, 2C). Disruption of the 5' Cdx binding site (5'M) completely prevented activation of the RELM
promoter in LS174T cells, whereas mutation of the 3' Cdx binding site (3'M), alone or in combination with the 5' site (2xM), resulted in only partial reduction of RELM
promoter activation. These results are representative of three independent transfection studies in which plasmids from three different plasmid isolations were used. Although these results are consistent with the notion that the binding of Cdx1 and/or Cdx2 to the RELM
promoter is important for its activation, the results also suggest two additional features that require further investigation. 1) Although all three transfected cell lines have been shown to express Cdx1 and/or Cdx2 protein (data not shown) (25), site-directed mutations of the two Cdx binding elements altered activation of the RELM
promoter only in the LS174T cell line. 2) Independent mutation of the two Cdx binding sites resulted in a greater reduction of RELM
promoter activation in LS174T cells than in both mutations together (2xM). These results suggest additional DNA-protein interactions between base pairs 418 and 588 that influence the goblet cell-specific and Cdx-dependent activation of the RELM
promoter.
Cdx2, but not Cdx1, is important for transactivation of the RELM
promoter in LS174T cells.
The relative importance of Cdx1 and Cdx2 in the transcriptional activation of RELM
was studied in the goblet cell-like LS174T cell line (Fig. 3). The wild-type 588 hR
construct was cotransfected with empty vector pRC-CMV, an expression vector for Cdx1 (pRC-CMV-Cdx1), or an expression vector for Cdx2 (pRC-CMV-Cdx2). Cotransfection of the 588 hR
construct with pRC-CMV Cdx2 led to a two- to threefold increase in reporter gene activity compared with cotransfection with the empty pRC-CMV vector, whereas reporter gene activity remained unchanged after cotransfection with Cdx1.
To confirm the importance of Cdx2 in the activation of RELM
, cotransfections were performed using various amounts of Cdx1 and Cdx2 with the wild-type 588 hR
construct. Increased reporter gene activity corresponded to increased amounts of pRC-CMV Cdx2, and not Cdx1, confirming the more important role of Cdx2 in RELM
promoter activation.
Ectopic expression of Cdx2 in gastric glandular epithelium leads to expression of RELM
, but not RELM
or RELM
.
Our findings in vitro that Cdx2 plays an important role in the goblet cell-specific activation of RELM
were further investigated in vivo through study of a transgenic Foxa3/Cdx2 mouse, in which the cis-regulatory elements of the Foxa3 (Hnf3
) gene were used to direct ectopic Cdx2 expression to the gastric epithelium (55). Concomitant with a 10,000-fold increase in expression of Cdx2 in the glandular stomach compared with wild-type littermates (Fig. 4A), these transgenic mice develop intestinal metaplasia of the glandular stomach, ectopic development of gastric goblet cells, and ectopic expression of intestine-specific genes (55).
To investigate the possibility that RELM isoforms might also be transcriptionally regulated by Cdx2 in vivo, we used quantitative RT-PCR to compare the relative change in expression of RELM
, RELM
, and RELM
in the glandular stomach of Foxa3/Cdx2 transgenic mice with that in wild-type littermates. Ectopic Cdx2 expression in the glandular epithelium led to a dramatic induction of RELM
, but not RELM
or RELM
, in the stomachs of these transgenic mice (Fig. 4, BD). Immunohistochemistry was performed to confirm RELM
protein expression in the glandular epithelium of the Foxa3/Cdx2 transgenic mouse stomach. Although the gastric epithelium of wild-type controls did not stain for RELM
, RELM
protein expression was localized to goblet-like cells in the gastric epithelium of transgenic mice (Fig. 4, E and F).
The Foxa3/Hnf3
transcription factor plays a role in the regulation of gene expression in early endodermal tissues; thus it is expressed not only in the gastrointestinal tract, but also in the liver and pancreas (47). Because the expression of Cdx2 in Foxa3/Cdx2 transgenic mice is driven by Foxa3 regulatory elements, Cdx2 is ectopically expressed in these endodermally derived tissues. We hypothesized that RELM
expression might be induced by extraintestinal Cdx2, and we used quantitative RT-PCR to explore this possibility by comparing the expression of RELM
, RELM
, and RELM
in the liver of Foxa3/Cdx2 transgenic mice.
Despite a 3,000-fold induction in Cdx2 expression in the liver, hepatic expression of RELM
, RELM
, and RELM
remained unchanged in Foxa3/Cdx2 transgenic mice (Fig. 4, AD). This result suggested that although extraintestinal expression of Cdx2 alone was not sufficient for RELM
or RELM family gene expression, additional factors unique to the gastrointestinal tract, such as bacterial colonization, might be necessary for activation of the RELM
gene by Cdx2.
Bacterial colonization leads to an induction of RELM
, but not Cdx2, RELM
, or RELM
, expression in the colon of SCID mice, where it is a specific marker of goblet cell maturation.
Our recent description of the induction of RELM
expression by commensal bacteria in the colonic goblet cells of Balb/c and SCID mice demonstrates that this process occurs independently of the acquired immune system (21). To determine whether stimulation by the innate immune system was also sufficient to induce expression of other RELM isoforms, we used RT-PCR to compare the expression of RELM
, RELM
, and RELM
in the colons of germ-free C.B17.SCID mice as well as formerly germ-free C.B17.SCID mice conventionalized by transfer to an SPF environment for 35 days (Fig. 5A). Although mRNA expression of RELM
and RELM
remained unchanged in the presence of commensal bacteria, expression of RELM
increased approximately sixfold in the colon of formerly germ-free SCID mice after conventionalization by commensal bacteria. Consistent with previous results in Balb/c mice, the expression of two other goblet cell genes, Muc2 and Tff3, was only modestly induced by bacterial colonization (data not shown) (21).
We and others observed smaller and fewer goblet cells in germ-free (Balb/c) mice than in their conventionally reared counterparts (31, 32). We further investigated the effect of bacterial colonization on goblet cell morphology and goblet cell gene expression in SCID mice. Alcian blue staining of Swiss-rolled colons from germ-free C.B17.SCID mice, as well as from mice conventionalized by transfer to an SPF environment for 35 days, showed that the presence of commensal flora led to crypt hyperplasia and dramatically increased the number and size of goblet cells in the upper crypt and surface epithelium (Fig. 5, B and C). The maturation and proliferation of colonic goblet cells were coincident with an increase in RELM
protein expression (Fig. 5, D and E). Our data indicate that expression of RELM
, but not RELM
or RELM
, is induced by colonization of the gut with commensal flora, where it is a highly specific marker for goblet cell maturation in the cecum of the colon through a process that occurs independently of the acquired immune system.
Given the regulation of RELM
expression by commensal bacteria and Cdx2, we investigated the possibility that Cdx2 could also be upregulated by bacterial colonization. RNA isolated from the colons of germ-free and SPF-colonized SCID mice was used in quantitative RT-PCR for Cdx2. In contrast to the induction of RELM
by commensal bacteria, there was no change in the mRNA expression of Cdx2 after 35 days of SPF association (Fig. 5F). Similarly, Cdx2 protein expression was not affected by bacterial colonization, as demonstrated by immunohistochemistry using antibodies specific for the Cdx2 COOH and NH2 termini in Swiss-rolled colon samples (data not shown). Because posttranslational modifications such as phosphorylation have been shown to modify the transcriptional activity of Cdx2 (52), we performed immunohistochemistry using the P-Cdx2-S60 antibody but found no significant difference in Cdx2 expression patterns between germ-free and SPF colonized mouse colons. Taken together, our data suggest that although bacterial colonization is a key factor in RELM
expression, Cdx2 might play a role in directing the intestine specificity of RELM
in the presence of commensal bacteria.
RELM
and RELM
are activated by Th2-mediated acquired immune responses that are independent of Cdx2.
We recently demonstrated that host intestinal Th2 responses, typified by stimulation by intestinal parasitic nematode infections, lead to a robust and specific induction in RELM
expression (4). To determine whether other RELM isoforms might be similarly induced by Th2 stimulation, we examined the intestinal expression of RELM
and RELM
in response to infection by three different intestinal nematodes. Mice were infected with the small intestinal nematodes N. brasiliensis or T. spiralis or the large bowel pathogen T. muris. The mRNA expression of RELM
and RELM
was determined using quantitative RT-PCR (Fig. 6).
Compared with a 19- to 30-fold increase in colonic RELM
expression (4), there was a 10- to 50-fold increase in small intestinal RELM
expression and an 8- to 10-fold increase in small intestinal RELM
expression 18 days after infection with T. spiralis and N. brasiliensis (Fig. 6A). In the colon, a 33-fold induction in RELM
expression and a 20-fold induction in RELM
was seen after infection with T. muris. With the use of an antibody specific for RELM
(Fig. 6B), immunohistochemistry revealed that nematode infection results in the induction of RELM
specifically in colonic goblet cells (Fig. 6C). These results were particularly notable given the lack of constitutive expression of RELM
and RELM
in the intestine.
We next examined whether the induction of RELM gene expression by intestinal nematode infection might be linked to a change in Cdx2 expression. Using quantitative RT-PCR, we examined the colonic expression of Cdx2 16 and 18 days after T. muris infection, because Th2 cytokine levels and RELM
expression peak at these time points (4). Our results indicate that Cdx2 expression remains unchanged at these time points (Fig. 6D), suggesting that the Th2-mediated activation of these three RELM isoforms occurs through mechanisms that are independent of Cdx2.
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DISCUSSION
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Our observation that Cdx2 plays a critical role in the regulation of RELM
gene expression is consistent with the constitutive intestine-specific expression pattern of this gene in conventionally housed mice. The caudal-related proteins (Cdx) are a gene family with homology to the caudal homeobox gene found in Drosophila. Like all genes in the homeobox family, Cdx proteins are transcription factors with important roles in development and patterning (9, 10, 51, 57, 66). In mice and humans, Cdx1 and Cdx2 are considered to be important regulators of intestine-specific gene expression (20, 38, 60, 61).
In this study, we provide evidence for the importance of Cdx2 in the transcriptional activation of RELM
in vitro and in vivo. The inability of Cdx2 to activate RELM
in the liver of Foxa3/Cdx2 transgenic mice suggests that although it is not sufficient to activate its expression, Cdx2 is important for the intestinal targeting and basal expression of RELM
in the colon. Indeed, the observation that Cdx2 mRNA expression, pattern of protein expression, or posttranslational processing are not altered in the colonic epithelium upon bacterial colonization suggests that commensal bacteria in the colon may play an essential role in the activation of RELM
in the presence of Cdx2. In support of this conclusion, we show that the closely related RELM isoforms RELM
and RELM
are not induced by the ectopic expression of Cdx2 in transgenic mice or by bacterial colonization of the gut. The concept that Cdx2 is required for targeting "basal" transcriptional activation of this gene to the intestinal epithelium, whereas commensal bacteria result in high-level activation of RELM
, is consistent with many other paradigms for tissue-specific gene expression (64). Furthermore, the observation that the expression of other intestinal genes is enhanced by bacterial colonization suggests that the regulation of RELM
may be reflective of a more general process (8, 25).
In contrast to the innate immune response induced in the colon by bacterial colonization, adaptive Th2-mediated immune activation by infection with intestinal nematodes activates all three RELM isoforms. Although RELM
has been shown to be expressed in the lung epithelium, macrophages, and enteric neurons (28), we now show that, like RELM
, RELM
expression can be strongly induced in intestinal goblet cells by nematode infections. Our study now shows that the induction of RELM expression in goblet cells is a generalized feature of Th2 immune responses. Indeed, even the induction of RELM
in the ovalbumin model of pulmonary inflammation occurs in the setting of a Th2-mediated response, in which goblet cell metaplasia of the bronchial epithelium is a prominent finding (28, 37). Despite their similar structures, important differences exist between the different RELM isoforms. For example, RELM
is not able to homo- or heterodimerize with other RELM family members because of the absence of an NH2-terminal cysteine amino acid, which is conserved in the other RELM family members (7). Although we recently showed that RELM
may play an important role as an immune effector of resistance to parasitic nematode infections of the gastrointestinal tract, additional investigation is required to ascertain whether RELM
and/or RELM
have a similar function.
Germane to the activation of RELM isoform expression by Th2-mediated immune responses associated with goblet cell hyperplasia and hypertrophy is the evidence supporting a specific role for Cdx2 in the development of intestinal goblet cell phenotype. Expression of Muc2, a goblet cell-specific gene expressed throughout the small intestine and the colon, is regulated by Cdx2 (45, 70). In vitro, gain-of-function studies in IEC6 cells show that expression of Cdx2 leads to development of goblet cells (61). In vivo, ectopic expression of Cdx2 in the stomachs of transgenic mice results in intestinal metaplasia with ectopic goblet cell development (49, 55), and in humans, aberrant expression of Cdx2 is associated with the intestinal goblet cell phenotype, a diagnostic feature of intestinal metaplasia (12, 19). Evidence for the importance of Cdx2 in the goblet cell phenotype is further supported by studies of Cdx2 loss of function. Mice heterozygous for germ line Cdx2 inactivation develop intestinal hamartomas, in which somatic inactivation of the second Cdx2 allele results in loss of the goblet cells (17, 42). These findings are consistent with Cdx2 silencing and decreased numbers of goblet cells in minimally differentiated colon carcinomas in humans (24, 27).
Despite the evidence for Cdx2 in the development of the goblet cell phenotype, the absence of any alteration in Cdx2 expression in the intestine of nematode-infected mice together with the robust induction of RELM
and RELM
expression, two genes that we show are not activated by Cdx2 in vivo, strongly suggests that the Th2-dependent activation of goblet cell responses in the intestinal tract occurs independently of Cdx2. The activation of this family of genes by Th2 immune stimulation likely involves other pathways. Th2 immune signaling via IL-4 and IL-13 signaling, for example, is clearly linked to host resistance to intestinal nematode infection (5, 6, 13, 15, 23, 44, 59). Further downstream, signal transducer and activator of transcription (STAT6) has not only been linked to goblet cell hypertrophy in the setting of intestinal nematode infection (34, 41) but has also been shown to regulate the expression of RELM
(59). Moreover, it is known that the innate immune system may play a role in regulating adaptive immune responses (2, 22, 54). In this regard, future studies may focus on the role of NF-
B and STAT6 in the regulation of RELM isoforms.
In summary, we have utilized the expression patterns of three closely related members of the RELM gene family to explore the role of Cdx2 in the regulation of intestinal gene expression in response to innate and adaptive immune responses. Although Cdx2 expression levels are not altered by bacterial colonization or by nematode infection of the gut, activation of the RELM
gene by Cdx2 in vitro and in vivo demonstrates an essential role for this transcription factor in the expression of intestinal genes in conventionally housed mice. In contrast, the alterations in intestinal gene expression induced by adaptive immune responses of the intestinal tract are likely to be independent of Cdx2.
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GRANTS
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This work was supported by National Institutes of Health Grants AI-39368 (to G. D. Wu), DK-07066 (to M.-L. Wang), AI-061570 (to D. Artis), and DK-59539 (to D. G. Silberg), University of Pennsylvania Center for Digestive and Liver Diseases Pilot Program Grant DK-50306 (to D. Artis), Wellcome Trust Grants 060312 (to P. A. Knight) and 059967 (to D. Artis), and Molecular Biology and Morphology Cores of National Institute of Diabetes and Digestive and Kidney Diseases Center Grant DK-50306.
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ACKNOWLEDGMENTS
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The authors acknowledge the generous gift of the germ-free and SPF-conventionalized C.B17.SCID mice from Dr. John J. Cebra as well as the kind donation of pRC-CMV-Cdx1 and Cdx2 plasmids from Dr. John P. Lynch.
Present address of D. G. Silberg: AstraZeneca LP, D2C-023, 1800 Concord Pike, PO Box 15437, Wilmington, DE 19850-5437.
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FOOTNOTES
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Address for reprint requests and other correspondence: G. D. Wu, 600 Clinical Research Bldg., 415 Curie Blvd., Philadelphia, PA 19104-6144 (E-mail: gdwu{at}mail.med.upenn.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.
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