Division of Pediatric Gastroenterology and Nutrition, Children's Hospital Medical Center and the University of Cincinnati, Cincinnati, Ohio 45229
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ABSTRACT |
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To study the
molecular mechanisms controlling guanylin expression, we have cloned
the mouse guanylin gene, including 2.7 kb of upstream sequence. We show
that the first 133 base pairs (bp) of the upstream guanylin promoter
are sufficient to drive near maximal (6-fold over basal) luciferase
reporter gene expression in Caco-2 intestinal cells; at least 300 bp of
upstream promoter are required for reporter gene expression in HT-29
intestinal cell lines. Using electromobility shift assays, we
demonstrate that nuclear proteins bind to the hepatocyte nuclear
factor-1 (HNF-1) consensus sequence in the guanylin promoter. The HNF-1 consensus sequence, located in the immediate 5' flanking region, is required for transcriptional activation of the guanylin gene in both
intestinal cell lines. Mutagenesis of the HNF-1 consensus sequence
abolishes transcriptional activation of guanylin promoter-luciferase reporter gene constructs. Cotransfection of these constructs with HNF-1 augments transcriptional initiation of the reporter gene. In
contrast, HNF-1
has no significant effect on transcription of the
reporter gene. These experiments demonstrate that HNF-1
is an
important regulatory element in the transcriptional activation of
guanylin.
Caco-2 cells; HT-29 cells; guanylate cyclase
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INTRODUCTION |
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THE IDENTIFICATION of guanylin (9), which is 50% homologous to the heat-stable enterotoxin (STa) elaborated by enterotoxigenic Escherichia coli, provides a potential explanation for the endogenous function of the intestinal STa receptor, guanylate cyclase C (GCC). Guanylin is thought to be involved in fluid and electrolyte balance in the intestine through the same intracellular pathway as STa; that is, guanylin, like STa, binds to GCC (9). This binding results in increased guanosine 3',5'-cyclic monophosphate production, which activates the cystic fibrosis transmembrane conductance regulator (CFTR) via a protein kinase intermediary (12, 26, 35, 46). Activation of the CFTR, in turn, results in increased chloride secretion, decreased sodium and chloride absorption, and possibly bicarbonate secretion (13).
Guanylin mRNA has been shown to be distributed in a complex, region-specific pattern within the human intestine (15), a pattern resembling that of its receptor GCC (18). Signal is present in the superficial epithelial cells of the human colon, villous cells of the human ileum, and crypt cells throughout the small intestine (15). Based on the cellular localization of guanylin mRNA, we tested the hypothesis that cis-active elements were present in the mouse guanylin promoter that could direct expression of the guanylin gene. Previous analysis of the guanylin gene (Guca2) revealed a number of similarities between the mouse and human gene; both genes are ~1.7 kb and are composed of three exons (14, 42). To determine the molecular mechanisms governing guanylin expression in the intestine, we have cloned additional upstream sequence of the mouse guanylin gene. Using various human colonic cell lines that express guanylin, we now show that the mouse guanylin promoter is transcriptionally active in these cell lines.
A minimal (133 bp) promoter is transcriptionally active in
Caco-2 cells. This immediate 5' flanking sequence is an
evolutionarily conserved region of the guanylin gene with 76% homology
to the human promoter region. The mouse promoter contains a putative binding site for hepatocyte nuclear factor-1 (HNF-1) at
53 to
41, an element that is identical between human and mouse
guanylin genes.
HNF-1 proteins, including HNF-1 and HNF-1
, belong to a class of
transcription factors that are important in the transcriptional activation of many genes, including albumin and
-1-antitrypsin (8,
28). HNF-1
and HNF-1
are also known as HNF-1 and vHNF-1 or LF-B1
and LF-B3 (29, 38). Therefore, based on our initial data and our
analysis of the promoter region, we hypothesized that the HNF-1 binding
site is necessary for the transcriptional activation of the guanylin
gene.
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EXPERIMENTAL PROCEDURES |
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Tissue culture cell lines. The Caco-2 human colon carcinoma cell line was cultured as previously described (6, 25). HT-29 cell lines used included HT-29-18-N2 and HT-29-18-C1 subclones (gifts of Cynthia Sears), which are committed to differentiate to specific cell types (16, 20). HT-29-18-N2 cells are mucin-secreting, goblet cell-like, whereas the HT-29-18-C1 cells are enterocyte-like. In addition, we used the undifferentiated cell line HT-29-CP [gift of John Barnard (3)]. All cell lines were routinely grown in Dulbecco's modified Eagle's medium containing 25 mM glucose, 50 IU/ml streptomycin, 10 µg/ml penicillin, and 10% fetal calf serum. In addition, HT-29 cell lines were supplemented with 10 µg/ml of human transferrin and 4 mM glutamine (16, 32). The 293 human embryonic kidney cells were cultured according to the recommendations of the American Type Culture Collection (Rockville, MD).
Isolation of promoter sequence.
Inverse polymerase chain reaction (PCR) (33) was initially used to
obtain additional upstream sequence. Briefly, mouse genomic DNA (strain
129/Sv) was digested with BamH I. The DNA fragments were ligated to themselves via the BamH I compatible
ends, and PCR was performed using oligonucleotides F3 MSP PCR INV
(5'-CTGTTGAGCCCCATCAGATAAGC-3') and B16 MSP PCR INV
(5'-CCAGCCAGCCATATTTTCCC-3'). A 1,100-bp fragment was
generated and ligated into pCRII (Invitrogen). Four separate clones
were sequenced to discern any infidelity in the PCR product. Using this
inverse PCR product (890 to
78 and 232 to 557) of the
mouse guanylin gene, we screened a
DASH II
Sau3A partial 129/Sv mouse genomic
library (gift of Marcia Shull) by hybridization as previously described
(42). Two clones,
2mgg2 and
2mgg5, were isolated, and restriction
fragment digests of DNA from these clones yielded fragments of the
predicted molecular weight based on genomic Southern blot analysis. The
clone
2mgg5 was identified as containing the most upstream sequence
by Southern hybridization of an EcoR I, BamH
I, or Hind III digest when hybridized with the inverse
PCR product. DNA from
2mgg5 was digested with either EcoR
I or BamH I and cloned into the respective
EcoR I or BamH I site of pTZ18U (gift of
Charlotte Paquin). Subclones containing the appropriately sized
fragments as determined by restriction digestion (not shown) were
further verified by sequencing with the oligonucleotide P1
(5'-CAATGTGAATACCTCCCTG-3'). Additional sequence beyond
890 bp and confirmation of the inverse PCR sequence were
performed using sequential oligonucleotides as primers. Sequence analysis was performed using MacVector (Kodak).
Cloning of promoter constructs into luciferase reporter gene
vector.
The initial reporter gene construct consisted of 133 bp of upstream
promoter sequence linked to the 5' untranslated sequence cloned
into the pGL3-basic (Promega). To accomplish this a 2.7-kb EcoR I fragment containing 133 bp of upstream sequence and
the entire Guca2 gene were gel
purified from the lambda clone mgg10 (42). This EcoR I
fragment was digested with BamH I, and a 689-bp fragment containing 133 bp of upstream sequence was cloned into EcoR I/BamH I digested pTZ18U. A fragment
containing 133 bp of upstream sequence and 5' untranslated region
was isolated by digestion of the 689-bp EcoR
I/BamH I fragment with
Sau3A and
Nla III. The largest fragment (172 bp)
of the digest containing the sequences from
133 to +39 was gel
purified and cloned into pIC20R (27) using a
Sau3A compatible
Bgl II site (reconstructing the
Bgl II site) and an
Nla III compatible
Sph I site. The
Nla
III/Sph I fusion in pIC20R was
converted to an Nco I site by PCR. The
fragment containing this minimal promoter region was digested with
Bgl II/Nco I and cloned into the
Bgl
II/Nco I site of pGL3-basic. The
fidelity of this construct was confirmed by sequencing. Thus the intact
relationship between upstream promoter sequence and the 5'
untranslated sequence of guanylin is retained in the luciferase reporter construct. Convenient restriction sites were used to subclone
increasing lengths of genomic fragments into the reporter construct. To
fuse the
839 bp guanylin promoter to luciferase, a
Bgl
II/Spe I (
133 to
2)
digest of the pGL3/
133 plasmid was excised and replaced with a
BamH I/Spe I
(
839 to
2) fragment of the
2mgg5 subclone. To fuse the
1,886 bp guanylin promoter to luciferase, an
Xho
I/Spe I (vector to
2) fragment
of
2mgg5 was subcloned and inserted into
Xho
I/Spe I prepared pGL3/
133. To
fuse the
300 bp guanylin promoter to luciferase, a
Bst
XI/Sma I (
300 to
839)
fragment of the pGL3/
839 construct was removed; the remaining
plasmid was ligated to yield the pGL3/
300 construct. The
fidelity of these constructs was confirmed by restriction digest
analysis.
HNF-1 site-directed mutagenesis.
Two vectors were constructed that contained a 3-bp substitution in the
consensus HNF-1 sequence. To construct the pGL3/133
HNF-1 vector, a 175-bp PCR fragment of the murine guanylin promoter from
133 to +42 was directionally subcloned into a
Xba I/Hind III site of
pAlterI (Promega). A mutagenic oligonucleotide
(5'-CAAGGCCCCAGG
TA
TGAGTAACCCC-3'), which altered the HNF-1 consensus site, was used. The altered bases are
underlined. Mutagenesis was performed according to the manufacturer's
instructions (Promega). Screening of plasmid colonies for incorporation
of the desired mutation employed restriction analysis, taking advantage
of the elimination of a Dde I site at
the mutation. After the fidelity of the construct was confirmed by
sequencing, the 175-bp fragment was subcloned into the
Nco I/Bgl II site of pGL3-basic (Promega),
yielding the pGL3/
133
HNF-1 construct. The
pGL3/
300
HNF-1 construct was made by isolating an
Xcm
I/Xcm I fragment of the
pGL3/
133
HNF-1 construct and subcloning this into the
Xcm
I/Xcm I site of the pGL3/
300
construct. Restriction digest analysis confirmed proper orientation and
size of the construct. Mutagenesis of this site did not affect the AP1
site (GTAACC), which overlaps the HNF-1 site on the 3' end. The
bases modified from the wild-type HNF-1 site to yield the altered
HNF-1 site are underlined:
5'-
TA
TGAGTAAC-3'.
Northern analysis.
Total RNA was extracted from tissue culture cells and from human
intestinal tissues using acid guanidine
isothiocyanate-phenol-chloroform extraction (5). Total RNA (20 µg)
was fractionated by electrophoresis in a 1.5% agarose-1.9%
formaldehyde gel, transferred to a nylon membrane (MagnaGraph, MSI,
Westboro, MA) by capillary action, and crosslinked to the membrane
using a Stratalinker (Stratagene, La Jolla, CA). The following cDNA
probes, radiolabeled with
[32P]CTP by random
primer DNA synthesis, were used: a human guanylin fragment isolated
from pMON 22305 (47), an HNF-1 fragment isolated from pBJ5 (gift of
Gerald Crabtree), an HNF-1
fragment isolated from pBJ5 (gift of
Gerald Crabtree), and a human GCC receptor fragment (23). The blots
were hybridized under stringent conditions as previously described (19,
21). Northern blots previously hybridized with these probes were
rehybridized with a labeled oligonucleotide complementary to 18S
ribosomal RNA (25) to quantitate relative amounts of total RNA loaded
in each lane of the gels as previously described (19). Visualization
and quantitation of positive signals were accomplished with the
PhosphorImager system (Molecular Dynamics, Sunnyvale, CA).
Nuclear extract preparation.
Nuclear proteins were isolated by a modification of the method
described by Schreiber et al. (41). Briefly, confluent cells in 75 cm2 flasks were trypsinized and
suspended in 10 ml of freshly warmed media. After pelleting by
centrifugation at 1,500 g for 5 min, the cells were washed with 10 ml of
tris(hydroxymethyl)aminomethane-buffered saline (TBS) and then
repelleted. The washed cells were resuspended in 1 ml of TBS,
transferred to a 1.5-ml Eppendorf tube, and repelleted by spinning for
15 s in a microcentrifuge. The pellet was resuspended in ice-cold
buffer A [(in mM) 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.9, 10 KCl, 0.1 EDTA, 0.1 ethylene
glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA), 1 dithiothreitol (DTT), and 0.5 phenylmethylsulfonyl
fluoride (PMSF)]. After incubation on ice for 15 min, 25 µl of
10% Nonidet P-40 (Sigma) were added. The mixture was vortexed for 10 s
and centrifuged for 30 s (14,000 g).
The resulting nuclear pellet was resuspended in 50 µl of cold buffer B [(in mM) 20 HEPES, pH
7.9, 0.4 NaCl, 1 EDTA, 1 EGTA, 1 DTT, and 1 PMSF]. After vigorous
rocking on a shaking platform for 15 min at 4°C, the extract was
centrifuged for 5 min in a Microfuge (14,000 g). Ten microliters (~5 µg) of
this supernate were used for electromobility shift assays (EMSAs).
EMSAs.
For EMSAs, ~100,000 counts/min of a
32P-labeled guanylin promoter
oligonucleotide (64 to
30) containing the HNF-1 consensus sequence was added into four reaction mixtures. The first reaction, a
negative control, contained a 1× gel shift binding buffer
(Promega) without nuclear extract. In the remaining three
reactions, nuclear extract was added with or without
unlabeled competitor DNA oligonucleotides (~100-fold excess). The
reactions were allowed to incubate for 20 min before electrophoresis on
a 4% nondenaturing acrylamide gel in 0.5× TBE buffer (45 mM
Tris-borate, 1 mM EDTA).
Transient transfection assays.
Cells were transfected 24 h after subculture into six-well plates at
~50-60% confluency. For Caco-2 and 293 cells, DNA (0.36 nM pGL3
vector with or without insert and 0.4 µg pSV40gal to normalize for
transfection efficiency) was suspended in 100 µl OPTI-MEM
(GIBCO-BRL). For cotransfection experiments, 0.2-0.8 µg of
either HNF-1
or HNF-1
in pBJ5 was added to the DNA suspension. Then 10 µl of lipofectin (GIBCO-BRL) were diluted in 100 µl
OPTI-MEM, added to the DNA suspension, and incubated at room
temperature for 10 min. This suspension was diluted to 1 ml with
OPTI-MEM and added to the six-well plates. After 24 and 48 h, fresh
media replaced the previous suspension. For HT-29 cell lines, a similar protocol was followed except cellfectin (GIBCO-BRL) was substituted for
lipofectin and pCMV
gal or pRSV
gal was used to control for transfection efficiency. A 20-µl cell lysate was analyzed for both
luciferase (Promega) and
-galactosidase (Tropix) according to the
manufacturer's instructions. Both enzyme activities were measured in a
Berthold Lumat LB9501 luminometer. Reporter gene expression was
calculated after a minimum of three transfection experiments with each
construct.
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RESULTS |
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Isolation of promoter sequence.
A 4.8-kb EcoR I fragment from the mgg5 clone, whose size
was predicted from Southern blots of mouse genomic DNA probed with guanylin, was subcloned into pTZ18U. The sequence of the promoter contained within this fragment is shown in Fig.
1; also shown are the corresponding
sequences of the human guanylin promoter up to
331 (14, 34).
Matches to the consensus sequences of known regulatory elements (10)
are also shown in Fig. 1. We had previously demonstrated by primer
extension analysis the presence of a transcriptional start site
spanning 3 bp at the beginning of exon 1 of the murine guanylin gene
(42). There is a conserved element between
31 and
25
(TTTAAAA) between mouse and human, which was suggested as a TATA box
(34). Also, sequence analysis reveals the presence of a hepatocyte
nuclear factor (HNF-1) site at nucleotides
53 to
41. This
sequence is also present in the human guanylin promoter (Fig. 1). In
both human and mouse, this binding site has 11 of 13 (underlined)
nucleotide matches with the HNF-1 consensus binding sequence
(5'-
A
A/T
-3').
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Northern analysis. We evaluated several human intestinal epithelial cell lines for the presence of guanylin mRNA. As shown in Fig. 2, the 650-bp guanylin mRNA is detectable by day 2 after Caco-2 cell subculture. Also, levels of guanylin mRNA increase after subculture, paralleling the increase previously reported in Caco-2 cellular differentiation and GCC expression (23). In addition, guanylin mRNA expression was seen in those HT-29 cell subclones committed to differentiate to either an enterocyte-like (HT-29-18-C1) or goblet cell-like (HT-29-18-N2) phenotype but not in the undifferentiated HT-29-CP cell line (Fig. 2). In contrast, guanylin mRNA was not detectable in poly(A)+ RNA isolated from 293 cells (Fig. 2).
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EMSAs.
The ability of the nuclear proteins to bind to the guanylin promoter
oligonucleotide containing the HNF-1 consensus sequence was examined by
EMSAs. As shown in Fig. 3, extracts from
two guanylin-expressing intestinal cell lines, Caco-2 and HT-29-18-C1,
contained factors that bound the HNF-1-guanylin promoter
oligonucleotide probe. The nuclear protein-probe complex binding was
specifically inhibited by addition of 100-fold excess of unlabeled
HNF-1 competitor DNA but not by the addition of 100-fold excess of
nonspecific oligonucleotide DNA (T cell receptor-). In contrast,
extracts from the non-guanylin-expressing 293 cell line did not bind to
the HNF-1 probe. Minor nonspecific binding of the oligonucleotide probe
occurred in all three EMSAs.
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Transient transfection assays. The transcriptional activity of the guanylin promoter in three guanylin mRNA-expressing intestinal cell lines was examined by transient transfection as shown in Fig. 4. Transfection results are expressed relative to the luciferase activity seen with the pGL3-SV40 promoter, which encodes the luciferase gene under the control of the SV40 promoter. Results of basal activity for each cell line, obtained by transfection with pGL3-basic, are also shown.
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Cotransfection with HNF-1 or HNF-1
.
Having demonstrated that the 5' flanking sequence of the guanylin
gene was transcriptionally active in a number of cell lines, we wished
to determine the functional significance of the HNF-1 binding site. The
effects of cotransfection with HNF-1
or HNF-1
are shown in Figs.
5 and 6. In the top panels
of Fig. 5, A-D, the effect on reporter
gene expression of a 3-bp substitution in the HNF-1 site is displayed.
In the Caco-2 cell line, mutagenesis of the HNF-1 site reduced reporter
gene expression to basal levels with the
133
HNF-1 construct.
In the 293 kidney cell line, no increase over basal reporter gene
expression was seen with the wild-type or the
133
HNF-1
constructs. In the two HT-29 cell lines, which require a longer
promoter construct for transcriptional activation, alteration of the
HNF-1 sequence in the
300 bp construct abolished reporter gene
expression.
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DISCUSSION |
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Analysis of an HNF-1 site common to both mouse and human guanylin
promoters demonstrates the importance of this consensus sequence in the
transcription of the guanylin gene. Nuclear proteins from several
intestinal cell lines bind the HNF-1 consensus sequence located in the
immediate 5' flanking region of the mouse guanylin gene. By
demonstrating that mutagenesis of this consensus sequence abolishes
reporter gene expression, we show that the HNF-1 binding site is
required for reporter gene expression in Caco-2 cells, which have near
maximal expression with the minimal promoter, and that the HNF-1
binding site is also necessary in HT-29 cell lines, which require an
additional length of promoter for reporter gene expression.
Furthermore, we have identified HNF-1 as a potent activator of
transcriptional activation through cotransfection experiments. In
addition to augmenting reporter gene expression in guanylin-expressing
intestinal cell lines, HNF-1
enables acquisition of reporter gene
expression in 293 kidney cells that express neither endogenous guanylin
nor HNF-1
mRNA.
Expression of guanylin mRNA in intestinal cell lines. We have identified several intestinal cell lines that may be useful for the study of transcriptional control of guanylin expression. Caco-2 cells are a human colon carcinoma cell line that spontaneously differentiates after achieving confluence into cells that closely resemble small intestinal enterocytes (37). HT-29 cells are a family of cell lines originally derived from a human colon adenocarcinoma. These cells differentiate into multiple epithelial cell types (2, 11, 16), and a number of subclones have been characterized. HT-29-18-C1 and HT-29-18-N2 cell types are committed to develop into either an enterocyte-like or mucin-secreting phenotype, respectively (16, 36). The enterocyte-like cell line is phenotypically distinguishable from Caco-2 cells. Unlike Caco-2 cells (6), this cell line does not express GCC (data not shown). More recently, Caco-2 cells have been shown to be competent for transcriptional activity of the GCC promoter (24). Previous studies, from our laboratory and others, have demonstrated that GCC mRNA or GCC protein (ligand binding) is expressed in villous epithelial cells of the small intestine (7) and in some species in crypt epithelial cells as well (1, 18, 45). GCC mRNA is also robustly expressed throughout the superficial and deep epithelial cells of the colonic glands (22). Thus, as is seen in the Caco-2 cell line, it is likely that GCC and guanylin mRNA are expressed in vivo in the same cell types, consistent with a paracrine or an autocrine function for guanylin.
Role of HNF-1 in transcriptional control of the guanylin gene.
A notable difference between the transcriptional activation of guanylin
in the Caco-2 and the HT-29 cell lines is the ability of the short
133 bp promoter to be fully active in the Caco-2 cell line but
not in the HT-29 cell lines. This promoter fragment contains a
consensus binding site for HNF-1. In the human guanylin promoter, a
minimal promoter (
157 to
5) construct containing the
HNF-1 site also demonstrated near maximal reporter gene expression in
T84 intestinal cells (34). Although the HNF-1 site alone is not
sufficient to direct transcriptional initiation in HT-29 cells as
indicated by the inability of the short promoter to be transcriptionally active, the alteration of the HNF-1 site in the
transcriptionally active
300 construct abolishes reporter gene
expression. Therefore, the HNF-1 site appears to be required for
guanylin expression in HT-29 cell lines as well as Caco-2 cells.
Role of HNF-1 in transcriptional activity of hepatic and nonhepatic
genes.
HNF-1 was first found in the liver as a transcription factor binding to
the -fibrinogen promoter and since that time has been shown to
regulate many genes, including albumin and
-1-antitrypsin (8, 28).
Based on the patterns of expression of genes regulated by HNF-1,
several authors initially concluded that the expression of HNF-1 in
tissues other than the liver functioned in the regulation of genes that
are expressed in both the liver and in other tissues (28). There is new
evidence to dispute this conclusion, as several nonhepatic genes,
including sucrase-isomaltase and villin, have been shown to be
regulated by HNF-1 (40, 48). Guanylin is another example of a
nonhepatic gene that is highly expressed in the intestine and is
regulated by HNF-1.
HNF-1 in vivo.
An HNF-1 gene-targeted mouse has recently been described (38). Mice
lacking HNF-1 develop failure to thrive, die around weaning, and
have marked liver enlargement (38). The transcription rates of genes
such as albumin and
-1-antitrypsin are reduced; the absence of any
transcriptional activity for phenylalanine hydroxylase gives rise to a
phenotype of phenylketonuria. No intestinal disorder has been
identified macroscopically or histologically, but these authors have
not specifically evaluated the effect of targeted disruption of
HNF-1
on intestinal function or gene expression. Further
investigation of these mice may allow characterization of the in vivo
effects of HNF-1
on guanylin gene regulation.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-47318 and DK-07727-02.
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FOOTNOTES |
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Address for reprint requests: M. B. Cohen, Div. of Pediatric Gastroenterology and Nutrition, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229.
Received 4 September 1996; accepted in final form 23 June 1997.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Almenoff, J. S.,
S. I. Williams,
L. A. Scheving,
A. K. Judd,
and
G. K. Schoolnik.
Ligand-based histochemical localization and capture of cells expressing heat-stable enterotoxin receptors.
Mol. Microbiol.
8:
865-873,
1993[Medline].
2.
Augeron, C.,
and
C. L. Laboisse.
Emergence of permanently differentiated cell clones in a human colonic cancer cell line after treatment with sodium butyrate.
Cancer Res.
44:
3961-3969,
1984[Abstract].
3.
Barnard, J. A.,
and
G. Warwick.
Butyrate rapidly induces growth inhibition and differentiation in HT-29 cells.
Cell Growth Differ.
4:
495-501,
1993[Abstract].
4.
Barrera-Hernandez, G.,
I. E. Wanke,
and
N. C. Wong.
Effects of diabetes mellitus on hepatocyte nuclear factor 1 decrease albumin gene transcription.
J. Biol. Chem.
271:
9969-9975,
1996
5.
Chomczynski, P.,
and
N. Sacchi.
Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction.
Anal. Biochem.
162:
156-159,
1987[Medline].
6.
Cohen, M. B.,
N. J. Jensen,
J. A. Hawkins,
M. R. Thompson,
M. J. Lentze,
and
R. A. Gianella.
Receptors for Escherichia coli heat-stable enterotoxin in human intestine and in a human intestinal cell line (Caco-2).
J. Cell. Physiol.
156:
138-144,
1993[Medline].
7.
Cohen, M. B.,
E. A. Mann,
C. Lau,
S. Henning,
and
R. A. Giannella.
A gradient in expression of the Escherichia coli heat-stable enterotoxin receptor exists along the crypt-to-villous axis in rat small intestine.
Biochem. Biophys. Res. Commun.
186:
483-490,
1992[Medline].
8.
Courtois, G.,
J. G. Morgan,
L. A. Campbell,
G. Fourel,
and
G. R. Crabtree.
Interaction of a liver-specific nuclear factor with the fibrinogen and 1-antitrypsin promoters.
Science
238:
688-692,
1987[Medline].
9.
Currie, M. G.,
K. F. Fok,
J. Kato,
R. J. Moore,
F. K. Hamra,
K. L. Duffin,
and
C. E. Smith.
Guanylin: an endogenous activator of intestinal guanylate cyclase.
Proc. Natl. Acad. Sci. USA
89:
947-951,
1992[Abstract].
10.
Faisst, S.,
and
S. Meyer.
Compilation of vertebrate-encoded transcription factors.
Nucleic Acids Res.
20:
3-26,
1992[Medline].
11.
Fantini, J.,
B. Abadie,
A. Tirard,
L. Remy,
J. P. Ripert,
A. El Battari,
and
J. Marvaldi.
Spontaneous and induced dome formation by two clonal populations derived from a human adenocarcinoma cell line, HT29.
J. Cell Sci.
83:
235-249,
1986[Abstract].
12.
Forte, L. R.,
P. K. Thorne,
S. L. Eber,
W. J. Krause,
R. H. Freeman,
S. H. Francis,
and
J. D. Corbin.
Stimulation of intestinal Cl transport by heat-stable enterotoxin activation of cAMP-dependent protein kinase by cGMP.
Am. J. Physiol.
263 (Cell Physiol. 32):
C607-C615,
1992
13.
Guba, M.,
M. Kuhn,
W.-G. Forssmann,
M. Classen,
M. Gregor,
and
U. Seidler.
Guanylin strongly stimulates rat duodenal secretion: proposed mechanism and comparison with other secretagogues.
Gastroenterology
111:
1558-1568,
1996[Medline].
14.
Hill, O.,
M. Kuhn,
H. D. Zucht,
Y. Cetin,
H. Kulaksiz,
K. Aderman,
G. Klock,
G. Rechkemmer,
W. G. Forssman,
and
H. J. Magert.
Analysis of the human guanylin gene and the processing and cellular localization of the peptide.
Proc. Natl. Acad. Sci. USA
92:
2046-2050,
1995[Abstract].
15.
Hochman, J. A.,
D. Sciaky,
D. Witte,
T. Whitaker,
and
M. B. Cohen.
The guanylin promoter confers intestinal epithelial cell-specific expression (Abstract).
Gastroenterology
110:
805A,
1996.
16.
Huet, C.,
C. Sahuquillo-Merino,
E. Coudreier,
and
D. Louvard.
Absorptive and mucus-secreting subclones isolated from a multipotent intestinal cell line (HT-29) provide new models for cell polarity and terminal differentiation.
J. Cell Biol.
105:
345-357,
1987[Abstract].
17.
Krause, W. J.,
G. L. Cullingford,
R. H. Freeman,
S. L. Eber,
K. C. Richardson,
K. F. Fok,
M. G. Currie,
and
L. R. Forte.
Distribution of heat-stable enterotoxin/guanylin receptors in the intestinal tract of man and other mammals.
J. Anat.
184:
407-417,
1994[Medline].
18.
Krause, W. J.,
R. H. Freeman,
and
L. R. Forte.
Autoradiographic demonstration of specific binding sites for E. coli enterotoxin in various epithelia of the North American opossum.
Cell Tissue Res.
260:
387-394,
1990[Medline].
19.
Laney, D. W.,
E. A. Mann,
S. C. Dellon,
D. R. Perkins,
R. A. Giannella,
and
M. B. Cohen.
Novel sites for expression of an Escherichia coli heat-stable enterotoxin receptor in the developing rat.
Am. J. Physiol.
263 (Gastrointest. Liver Physiol. 26):
G816-G821,
1992
20.
Lesuffleur, T.,
A. Barbat,
C. Luccioni,
J. Beumatin,
M. Clair,
A. Kornowski,
E. Dussaulx,
B. Dutrilaux,
and
A. Zweibaum.
Dihidrofolate reductase gene amplification-associated shift of differentiation in methotrexate-adapted HT-29 cells.
J. Cell Biol.
115:
1409-1418,
1991[Abstract].
21.
Lewis, G. L.,
D. P. Witte,
D. W. Laney,
M. G. Currie,
and
M. B. Cohen.
Guanylin mRNA is expressed in villous enterocytes of the rat small intestine and superficial epithelia of the rat colon.
Biochem. Biophys. Res. Commun.
196:
553-560,
1993[Medline].
22.
Li, Z.,
and
M. F. Goy.
Peptide-regulated guanylate cyclase pathways in rat colon: in situ localization of GCA, GCC, and guanylin mRNA.
Am. J. Physiol.
265 (Gastrointest. Liver Physiol. 28):
G394-G402,
1993
23.
Mann, E. A.,
M. B. Cohen,
and
R. A. Giannella.
Comparison of receptors for E. coli heat-stable enterotoxin: novel receptor present in IEC-6 cells.
Am. J. Physiol.
264 (Gastrointest. Liver Physiol. 27):
G172-G178,
1993
24.
Mann, E. A.,
L. Jump,
and
R. A. Giannella.
Cell line-specific transcriptional activation of the promoter of the guanylyl cyclase C/heat-stable enterotoxin receptor gene.
Biochim. Biophys. Acta
1305:
7-10,
1996[Medline].
25.
Mann, E. A.,
and
J. B. Lingrel.
Developmental and tissue-specific expression of rat T-kininogen.
Biochem. Biophys. Res. Commun.
174:
417-423,
1991[Medline].
26.
Markert, T.,
A. B. Vaandrager,
S. Gambaryan,
D. Pohler,
C. Hausler,
U. Walter,
H. R. DeJong,
T. Jarchau,
and
S. M. Lohmann.
Endogenous expression of type II cGMP-dependent protein kinase mRNA and protein in rat intestine.
J. Clin. Invest.
96:
822-830,
1995[Medline].
27.
Marsh, J. L.,
M. Erfel,
and
E. J. Wykes.
The pIC plasmid and phage vectors with versatile cloning sites for recombination selection by insertional activation.
Gene
32:
481-485,
1984[Medline].
28.
Mendel, D. B.,
and
G. R. Crabtree.
HNF-1, a member of a novel class of dimerizing homeodomain proteins.
J. Biol. Chem.
266:
677-680,
1991
29.
Mendel, D. B.,
L. P. Hansen,
M. K. Graves,
P. B. Conley,
and
G. R. Crabtree.
HNF1 and HNF1
(vHNF-1) share dimerization and homeodomains, but not activation domains, and form heterodimers in vitro.
Genes Dev.
10:
1042-1056,
1991.
30.
Mendel, D. B.,
P. A. Khavari,
P. B. Conley,
M. K. Graves,
L. P. Hansen,
A. Admon,
and
G. R. Crabtree.
Characterization of a cofactor that regulates dimerization of a mammalian homeodomain protein.
Science
254:
1762-1767,
1991[Medline].
31.
Miura, N.,
K. Iwai,
and
I. Miyamoto.
Analysis of the rat hepatocyte nuclear factor (HNF) 1 gene promoter: synergistic activation by HNF4 and HNF1 proteins.
Eur. J. Cell Biol.
60:
376-382,
1993[Medline].
32.
Montrose-Rafizadeh, C.,
W. B. Guggino,
and
M. H. Montrose.
Cellular differentiation regulates expression of Cl transport and cystic fibrosis transmembrane conductance regulator mRNA in human intestinal cells.
J. Biol. Chem.
266:
4495-4499,
1991
33.
Ochman, H.,
F. J. Ayala,
and
D. L. Hartl.
Use of polymerase chain reaction to amplify segments outside boundaries of known sequence.
Methods Enzymol.
218:
309-321,
1993[Medline].
34.
Pardigol, A.,
H. Mager,
O. Hill,
and
W. Forssmann.
Functional analysis of the human guanylin gene promoter.
Biochem. Biophys. Res. Commun.
224:
638-644,
1996[Medline].
35.
Pfeifer, A.,
A. Aszodi,
U. Seidler,
P. Ruth,
F. Hofmann,
and
R. Fassler.
Intestinal secretory defects and dwarfism in mice lacking cGMP-dependent protein kinase II.
Science
274:
2082-2086,
1996
36.
Phillips, T. E.,
C. Huet,
P. R. Bilbo,
D. K. Podolsky,
D. Louvard,
and
M. R. Neutra.
Human intestinal goblet cells in monolayer culture: characterization of a mucus-secreting subclone derived from the HT29 colon adenocarcinoma cell line.
Gastroenterology
94:
1390-1403,
1988[Medline].
37.
Pinto, M.,
S. Robine-Leon,
M. D. Appay,
M. Kedinger,
N. Triadou,
E. Dussaulx,
B. Lacroix,
P. Simon-Assmann,
K. Haffen,
J. Fogh,
and
A. Zweibaum.
Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture.
Biol. Cell
47:
323-330,
1983.
38.
Pontoglio, M.,
J. Barra,
M. Hadchouel,
A. Doyen,
C. Kress,
J. P. Bach,
C. Babinet,
and
M. Yaniv.
Hepatocyte nuclear factor 1 inactivation results in hepatic dysfunction, phenylketonuria, and renal Fanconi syndrome.
Cell
84:
575-585,
1996[Medline].
39.
Rey-Campos, J.,
T. Chouard,
M. Yaniv,
and
S. Cereghini.
vHNF1 is a homeoprotein that activates transcription and forms heterodimers with HNF1.
EMBO J.
10:
1445-1457,
1991[Abstract].
40.
Robine, S.,
C. Sahuquillo-Merino,
D. Louvard,
and
E. Pringault.
Regulatory sequences on the human villin gene trigger the expression of a reporter gene in a differentiating HT29 intestinal cell line.
J. Biol. Chem.
268:
11426-11434,
1993
41.
Schreiber, E.,
P. Matthias,
M. M. Muller,
and
W. Schaffner.
Rapid detection of octamer binding proteins with "mini-extracts", prepared from a small number of cells.
Nucleic Acids Res.
17:
6419,
1989[Medline].
42.
Sciaky, D.,
J. L. Kosiba,
and
M. B. Cohen.
Genomic sequence of the murine guanylin gene.
Genomics
24:
583-587,
1994[Medline].
43.
Serfas, M. S.,
and
A. L. Tyner.
HNF1 and HNF1
expression in mouse intestinal crypts.
Am. J. Physiol.
265 (Gastrointest. Liver Physiol. 28):
G506-G513,
1993
44.
Suh, E.,
L. Chen,
J. Taylor,
and
P. G. Traber.
A homeodomain protein related to caudal regulates intestine-specific gene transcription.
Mol. Cell. Biol.
14:
7340-7351,
1994[Abstract].
45.
Swenson, E. S.,
E. A. Mann,
M. L. Jump,
D. P. Witte,
and
R. A. Giannella.
The guanylin/STa receptor is expressed in crypts and apical epithelium throughout the mouse intestine.
Biochem. Biophys. Res. Commun.
225:
1009-1014,
1996[Medline].
46.
Vaandrager, A. B.,
A. G. M. Bot,
and
H. R. DeJonge.
Guanosine 3',5'-cyclic monophosphate-dependent protein kinase II mediates heat-stable enterotoxin-provoked chloride secretion in rat intestine.
Gastroenterology
112:
437-443,
1997[Medline].
47.
Wiegand, R. C.,
J. Kato,
M. D. Huang,
K. F. Fok,
J. F. Kachur,
and
M. G. Currie.
Human guanylin: cDNA isolation, structure and activity.
FEBS Lett.
311:
150-154,
1992[Medline].
48.
Wu, G. D.,
L. Chen,
K. Forslund,
and
P. G. Traber.
HNF1 and HNF1
regulate transcription via two elements in an intestine-specific promoter.
J. Biol. Chem.
269:
17080-17085,
1994