From the Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
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ABSTRACT |
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Intestinal trefoil factor (ITF) is selectively
expressed in goblet cells of the small and large intestinal mucosa.
Detailed analysis of the rat ITF (RITF) promoter was undertaken by
transient transfection and gel mobility shift assays (GMSAs) using the
goblet cell-like LS174T colon cancer-derived cell line. Various lengths of wild-type or mutant constructs of the 5-flanking region were linked
to the pXP2 reporter gene luciferase. Expression of
118 RITF was
significantly decreased compared with
154 RITF, and transfection with
an 18-base pair construct (
141 to
124) resulted in more than 5-fold
greater expression than transfection with the promoterless pXP2 gene
construct alone. Using various synthetic oligonucleotide mutants, GMSAs
revealed that only a 9-base pair sequence (CCCCTCCCC) in this element
was required for specific binding, overlapping but distinct from a
Sp1-like element. GMSA demonstrated that this element was specifically
bound by nuclear proteins from intestinal cells with a goblet cell-like
phenotype. These studies demonstrate that a 9-base pair element
(goblet cell response element) between
154 and
118 in the RITF
promoter gene is a cis-active element bound by a distinct nuclear
transcription factor and is capable of directing intestine and goblet
cell-specific expression.
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INTRODUCTION |
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Trefoil factors are a family of small peptides expressed at various sites throughout the gastrointestinal tract. The members of this family share an array of structural features including a distinctive motif of six cysteine residues termed a trefoil or a P domain. Thim (1) postulated that the six cysteine residues could contribute to the formation of three intrachain loops via the formation of disulfide bonds; the resultant predicted three-looped structure prompted the trefoil designation. A recent nuclear magnetic resonance analysis as well as x-ray crystallography of one of the trefoil peptides supported the presence of a distinctive secondary structure consistent with the putative three-intrachain loop formation (2). Members of the family identified in mammals possess one or two P domains (3-5). Amphibians have been found to express trefoil proteins with as many as four P domains (6).
Members of the trefoil peptide family appear to be expressed in a region-specific fashion along the length of the gastrointestinal tract. Human spasmolytic polypeptide bears two trefoil motifs and is expressed primarily in the stomach (5), although the porcine homologue was originally isolated from pancreas (7). pS2, bearing a single trefoil motif and initially cloned as the product of an estrogen-responsive gene from a breast cancer cell line (8), is normally expressed only in the gastric antrum in man (9). Cloning of the rodent homologues of pS2 and spasmolytic polypeptide confirmed that expression is site-specific along the longitudinal axis of the upper gastrointestinal tract in a pattern that has been conserved in evolution (10, 11).
Intestinal trefoil factor (ITF)1 is a third member of the trefoil peptide family identified in humans (12, 13), rats (14, 15), and mice (16, 17) that contains a single P domain. In contrast to pS2 and human spasmolytic polypeptide, ITF is normally selectively expressed in the normal small and large intestinal mucosa, complementing the pattern of expression of the other members of the family in the normal gastrointestinal tract. More specifically, ITF expression is normally confined to the goblet cell population within the intestinal epithelium (14).
Goblet cells of the small and large intestine secrete a complex mixture of mucin glycoprotein onto the cell surface, but their functional importance in gastrointestinal tract mucosa has not been well defined. Moreover the basis of selected gene expression responsible for the distinctive goblet cell phenotype has not been defined. Genes encoding the apomucin peptide backbones of mucin glycoproteins are enormous in size and highly complex, hampering progress in efforts to define the regulatory effect conferring goblet cell-specific expression. The selective expression of ITF in intestinal goblet cells suggests that characterization of the gene encoding this peptide may provide insight into regulatory elements responsible for goblet cell-specific gene expression. Among the trefoil family members, only the promoter of pS2 gene normally expressed in gastric mucosa has been partially characterized (18, 19). Although several genes expressed in the intestinal epithelium including fatty acid-binding protein (20), sucrase-isomaltase (21), and lactase (22) have been cloned and their regulatory elements studied, none are products of goblet cells.
Our previous report showed relatively high levels of specific
expression in transient transfection studies using promoters as short
as 154 bp of the 5-flanking region of the rat ITF (RITF) gene. The
presence of goblet cell-specific promoter element(s) within close
proximity to the transcriptional start site was suggested by those
preliminary efforts (15). Further investigation of the RITF promoter
was undertaken, and a cis-active element capable of directing goblet
cell-specific expression was identified within this region. Moreover,
this element appears to be bound by a distinct nuclear transcription
factor.
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MATERIALS AND METHODS |
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Plasmid DNA Constructs--
The promoterless luciferase gene
construct pXP2 (23) was a gift from Dr. Lee Kaplan. The 5-flanking
region of the RITF gene, the 1671-bp fragment, was subcloned from
pRITF20C containing the entire RITF gene into pXP2 vector to form the
construct
1671 RITF-luc (1.7WT-luc) and transformed into competent
E. coli DH5
1 cells (CLONTECH
Laboratories; Palo Alto, CA) as described previously (15). To confirm
correct orientation and preservation of the start codon of luciferase,
plasmid DNA was subjected to restriction mapping and sequencing of the
insertion junctions.
Cell Cultures-- Human colon cancer cell lines LS174T and Caco-2, rat intestinal epithelial cell line IEC-6, human hepatocellular carcinoma cell line HepG2, and human cervix epithelioid cancer cell line HeLa obtained from the American Type Culture Collection (ATCC, Rockville, MD) were grown in Eagle's minimum essential medium except IEC-6 in Dulbecco's modified Eagle's medium supplemented with 4.5 g/liter D-glucose, 10% fetal calf serum, Eagle's balanced salt solution, nonessential amino acids, sodium pyruvate, 4 mM L-glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin. The H2 subclone of human colon cancer cell line HT-29 originally obtained from Dr. Daniel Louvard was grown in either Dulbecco's modified Eagle's medium with glucose (undifferentiating conditions) or in the presence of galactose as the sole source of carbohydrate to induce goblet cell-like differentiation as described previously (24, 25). All cell lines were grown in 5% CO2 at 37 °C.
Transient Transfection Promoter Analysis-- Transient transfection was accomplished by the calcium phosphate precipitation method. Sixteen hours prior to transfection, 8 × 105 cells were plated out in triplicate in 35-mm wells of a six-well cell culture plate. Complete media was refreshed 2 h prior to transfection. Efficiency of transfection was standardized by co-precipitation of the construct of interest with pTK-GH, consisting of the minimal thymidine kinase promoter driving the human growth hormone gene as a reporter gene (26), and adjusting for the amount of human growth hormone expressed, as determined by a commercially available radioimmunoassay (hGH Allegro Kit, Nichols Institute Diagnostics, San Juan Capistrano, CA). Calcium phosphate-precipitated plasmid DNA was added to each well and incubated at constant 5% CO2 for 4 h before a 2-min exposure to 15% glycerol. Cells were subsequently cultured for 48 h prior to assay for reporter gene expression. For determination of luciferase activity, cells were lysed and assayed immediately using a commercial luciferase assay system (Promega) measured in a luminometer (Analytical Luminescence Laboratory, Monolite 2010). Luciferase activity was adjusted for transfection efficiency reflected in the level of growth hormone, expressed as nanograms of hGH/ml of medium. Where noted, promoter activity was expressed as a percentage of the expression of the maximal promoter construct RSV-luc (a gift from Dr. Loyal Tillotson), consisting of the RSV promoter joined to the luciferase gene, or a -fold increase of the expression of the pXP2.
Nuclear Extracts and Gel Mobility Shift Assays
(GMSAs)--
Nuclear extracts from cultured intestinal cells were
prepared by Nonidet P-40 detergent lysis and 0.5 M NaCl
extraction as described by Schreiber et al. (27). Nuclear
extracts from other cell lines for GMSA tissue distribution studies
were a kind gift from Drs. Anil K. Rustgi and Timothy C. Wang. Protein
concentration was determined according to the Bradford assay (28).
The wild-type, double-stranded synthetic probes used in this
study were
154TTTTCCTCCCTAACCCTCTCCCCTCCCCCTCGGACTC
118
(WT1) and
141CCCTCTCCCCTCCCCCTC
124
(WT2) and were labeled by Klenow fill-in reaction in a buffer consisting of 10 mM Tris-HCl; 5 mM
MgCl2; 7.5 mM dithiothreitol; 33 µM concentration of dATP, dGTP, and dTTP; 0.33 µM [
-32P]dCTP; and 1 unit of DNA
polymerase I Klenow fragment and then polyacrylamide gel-purified. The
Sp1 oligonucleotide was used as a control probe and was labeled by the
same reaction. GMSAs were carried out by incubating 10 µg of nuclear
extract with 5 fmol of probe (20,000 cpm) in a 20-µl binding reaction
containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl,
1 mM ZnCl2, 1 mM dithiothreitol, 1 mM EDTA, 10% glycerol, and 1 µg of poly(dA-dT). In
experiments comparing binding by nuclear extracts between Sp1 and
goblet cell-response element (GCRE) probes, 1 mM
ZnCl2 was added to both reaction mixtures. After incubation
at room temperature for 30 min, samples were loaded onto 6%
polyacrylamide, 0.25 × Tris borate gels and electrophoresed at 10 V/cm for 2 h. Competition experiments were carried out by preincubating the nuclear extracts with a 100-fold excess of unlabeled wild-type (WT1 or WT2) or mutant (M1-8) competitor oligonucleotides prior to the addition of the probe. The consensus motif Sp1
(TAAGCTAGCCCCGCCCCGCTCG) oligonucleotide used as a competitor
corresponds to the binding site found in the simian virus 40 early
promoter (29). The antibodies against Sp1 (
Sp1) and Sp3 (
Sp3),
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), were
incubated with nuclear extracts for 30 min at room temperature prior to
GMSAs. The gels were dried and exposed to Kodak X-AR film for 6-24 h
at
80 °C.
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RESULTS |
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Expression of the Reporter Gene Luciferase under the Control of
Deletions of Rat ITF 5-Flanking Region in LS174T
Cells--
Constructs in which various lengths of wild-type and mutant
5
-flanking region of the RITF gene were ligated to the reporter luciferase were transfected into LS174T cells, a goblet cell-like intestinal cell line. The level of expression of the various deletion constructs containing from
1671 to
154 of the 5
-flanking region (1.7WT-, 1.0WT-, 0.7WT-, 0.3WT-, and 0.2WT-luc) was between 8 and 11%
of the expression observed for the maximal promoter-reporter construct,
RSV-luc, in the LS174T cells. In contrast, the gene expression of the
construct containing
117 of the 5
-flanking region (0.1WT-luc) was
significantly decreased to 2% (Fig. 1). Decreased gene expression of the mutant construct deleted between
154
and
118 (1.7
1-luc) was also observed compared with expression of
wild-type full-length construct 1.7WT-luc. The level of expression in
Caco2 cells, representing a columnar absorptive-like enterocyte phenotype, was consistently less than 2% that of RSV-luc, consistent with previous observations (15). These results suggest that element(s)
between
154 and
118 of the RITF 5
-flanking sequences are able to
enhance expression in a goblet cell-specific manner.
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Characterization of Nuclear Protein from LS174T Cells Binding to
GCRE in RITF Gene Promoter--
Two synthetic oligonucleotides of
different length and corresponding mutants were generated as probes or
competitors for use in GMSA experiments to assess nuclear extracts from
the goblet cell line for the presence of proteins interacting with the
5-flanking region implicated in transient transfection studies.
Wild-type probe 1 (WT1) spanning
154 to
118 and wild-type probe 2 (WT2) spanning
141 to
124 were radiolabeled. Mutations of WT1
probe, both upstream (M1) and downstream (M2) of WT2 sequences, were also prepared (Fig. 2A).
DNA-binding protein complexes derived from nuclear extracts of the
LS174T cells appeared to be similar between the GMSA using WT1 and WT2
probe. Two major bands closely similar in size were present as
demonstrated in Fig. 2B. Furthermore, competition studies
from the GMSA using WT1 probe showed that the M1, M2, and WT2
oligonucleotides competed for binding to those proteins equally with
the unlabeled WT1 probe. These data suggested that a DNA-binding site
for the two proteins is present within the 18 bp (
141 to
124)
corresponding to the WT2 probe. Therefore, subsequent GMSA experiments
were undertaken with WT2 as a probe.
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Gene Expression Analysis of the Mutated RITF Gene Promoter--
To
determine whether binding of GCRE in GMSA correlates with enhancer
activity of this element, the effects of various mutations in 154 to
118 nucleotides of the RITF promoter gene expression were assessed by
transient transfection assay. Minimal DNA constructs containing the
wild-type 37-bp element (WT1,
154 to
118) or 18-bp element (WT2,
141 to
124) and the mutant 18-bp element (M3 to M8,
141 to
124)
were generated using synthetic oligonucleotides subcloned into pXP2,
and the resulting constructs were designated WT1-luc, WT2-luc, or M3-
to M8-luc, respectively (Fig.
5A). Constructs were
transiently transfected into LS174T cells, and gene expression was
analyzed by measuring relative luciferase activity and represented as
-fold increase of the activity of pXP2 (Fig. 5A).
Transfections with WT1-luc and WT2-luc resulted in more than 5-fold
greater (5.8-fold with WT1-luc and 5.2-fold with WT2-luc) expression
than transfection with pXP2 alone. There was no significant difference in expression between WT1-luc and WT2-luc (Fig. 5A).
Although mutations M3, M4, and M8 had no effect on wild-type WT2-luc
expression, mutant constructs (M5-, M6-, and M7-luc) reduced expression
nearly to the base line observed with the minimal construct pXP2 (Fig. 5A). The mutated sequences of M5 to M7 corresponded to the
same 9-bp (
135 to
127) element of GCRE identified through GMSA
(Fig. 4).
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Characterization of Nuclear Proteins from Intestinal Epithelial Cell Lines Binding to GCRE-- Using nuclear extracts from various kinds of intestinal cell lines, GMSA was performed to characterize the protein complexes bound to GCRE. To ensure the quality of nuclear protein extracts, GMSA using a consensus Sp1 site as a probe was also performed (Fig. 6B). Crude nuclear protein extracts were prepared from LS174T (5 µg), HT29 (5 µg), undifferentiated (7.5 µg) or differentiated H2 (2.5 µg) subclone of HT29, and Caco2 (2.5 µg) cells, and binding reactions were carried out with the Sp1 or WT2 probe. As sufficient Zn2+ was chelated by the EDTA present in the nuclear extract isolation buffer to prevent Sp1 from binding (30), 1 mM ZnCl2 was added to the GMSA reactions. All of the nuclear proteins bound the Sp1 consensus sequence (Fig. 6A). The identity of the Sp1-binding protein was confirmed by supershift after the addition of anti-Sp1 antibody. These data demonstrated the presence of equal Sp1 binding activity, confirming the adequacy of nuclear extracts from the different cell lines. Using the same reaction conditions, the nuclear proteins from HT29 and Caco2 cells did not appear to bind the GCRE, but strong binding to GCRE was observed by nuclear proteins from LS174T and differentiated H2 cells (Fig. 6B). While little binding of GCRE was observed with nuclear proteins from undifferentiated H2 cells, binding to GCRE was significantly greater by nuclear proteins prepared from H2 cells after goblet cell-like differentiation. These results suggest that expression of nuclear factors that bind to GCRE is associated with differentiation to a goblet cell-like phenotype.
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Characterization of Tissue Specificity of the GCRE and Its Binding Protein-- GMSA was carried out using nuclear extracts made from cell lines derived from a variety of tissues to further characterize the specificity of expression of the GCRE-binding proteins. As shown in Fig. 7, the GCRE-binding nuclear factor was largely absent in nongoblet cell lines with the exception of HepG2 (liver) and HeLa (cervix) cells. A very small amount of binding protein was also observed in lung (LX-1) cells. Of interest, little binding to GCRE was observed by nuclear proteins from IEC6 cells, a nontransformed rat intestinal crypt cell line. Binding to Sp1 was observed by all of the nuclear proteins in the same fashion as shown in Fig. 6A (data not shown).
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DISCUSSION |
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Goblet cells are abundant constituents of the surface epithelium within the small and large intestine, but characterization of the molecular basis of goblet cell differentiation and function has been quite limited. Differentiation of epithelial cells in the gastrointestinal tract is a complex and dynamic process. In normal mucosa, not only is the tissue-specific phenotype maintained along the longitudinal axis from esophagus to large bowel, but vertical differentiation from crypt to villus is sustained as well. Moreover, differentiation into several region-specific subpopulations is observed within the epithelium along the length of the gastrointestinal tract. Among the growing list of cloned genes whose products are intestine-specific, ITF represents the first within the gastrointestinal tract exclusively expressed by goblet cells. Following earlier initial studies of molecular cloning of the RITF gene (15), in this paper we report the identification of a goblet cell-specific enhancer element in the RITF gene promoter bound by a goblet cell nuclear protein.
Trefoil proteins, including ITF, are secreted onto the mucosal surface and appear to function to preserve mucosal integrity, protecting the epithelium from injury by a variety of noxious agents (31-33). Furthermore, these factors facilitate rapid healing after injury by promoting restitution, the initial phase of epithelial migration that reestablishes surface continuity (7, 31). Mice rendered deficient in ITF through targeted gene deletion are exquisitely sensitive to injury by standard agents (e.g. dextran sodium sulfate) due to impaired restitution (34). An ulcer-associated cell lineage has been reported to appear adjacent to areas of gastrointestinal ulceration, with cells containing EGF-immunostaining material in the base of the newly budding cell lineage and trefoil protein-producing cells appearing more distally along the developing ductule (35).
There has been limited past characterization of the trefoil gene
regulatory elements and as yet no delineation of the regulatory elements that are responsible for the regional selective expression of
the different trefoil peptides. The presence of an EGF-responsive element in the 5-flanking region of the pS2 gene (19) has led to
speculation about the role of EGF in inducing expression of trefoil
proteins in response to mucosal injury (36). However, scrutiny of the
5
-flanking region of the ITF gene demonstrated no significant homology
to known EGF response elements (15).
Transient expression of deletion constructs containing various lengths
of RITF gene 5-flanking region ligated to a luciferase reporter gene
indicate that an element present between
154 and
118 of RITF
5
-flanking sequences is able to enhance expression in a goblet
cell-specific manner (Fig. 1). Subsequent mutational analysis indicates
that this regulatory potential is conferred by a 9-bp element.
Moreover, GMSA demonstrates the presence of two proteins that bind
specifically to the same sequence (CCCCTCCCC, named GCRE) in nuclear
extracts of goblet cells (Figs. 2 and 3). A search of this 5
-flanking
region of the RITF gene reveals none of the known regulatory elements
that have been demonstrated to play a role in intestine-specific
expression. Thus, with the exception of some AT repeats, no areas of
significant homology appear to exist within the full-length RITF
promoter and the reported 5
-flanking regions of the genes from human
intestinal alkaline phosphatase (37), human intestinal fatty
acid-binding protein (20), porcine aminopeptidase N (38), human and
mouse sucrase-isomaltase (21, 39), or human lactase-phlorizin hydrolase
(22). However, there is an Sp1-like motif (CCTCCCC) in the GCRE, and
one of the two specific binding proteins appears to be one of the Sp
binding proteins, a family of zinc finger proteins (Fig. 4). The Sp1
motif is present in the enhancer regions of diverse genes (40, 41). While it appears that one of the proteins present in nuclear extract from goblet cell-like lines binds in an Sp1-like fashion, it is also
apparent that these extracts contain a protein that is distinct from an
Sp1 element that binds the GCRE.
The GMSA using nuclear extracts from various undifferentiated H2 cells grown in standard conditions contains little GCRE-binding protein (Fig. 6). In contrast, differentiated H2 cells, which exhibit a goblet cell-like phenotype, contain much greater nuclear protein bound to GCRE than undifferentiated H2 cells. No specific binding reaction to GCRE is observed with the nuclear proteins from Caco2 cells, which exhibit a columnar enterocyte phenotype (Fig. 6). Although the DNA-protein complexes observed in experiments using the nuclear proteins from LS174T and H2 cells are the same in size and although their binding appears to be specific as indicated by competition assays, it remains possible that these GCRE-binding proteins are different. At a minimum, these studies indicate that intestinal cells that have differentiated to goblet cell-like phenotype possess nuclear binding proteins that recognize the GCRE.
The analysis of cell specificity of the GCRE and its binding protein demonstrate that the latter is specifically associated with intestinal goblet-like cells. No GCRE was observed in the nuclear proteins from esophagus, stomach, pancreas, kidney, or breast. While the GCRE-binding proteins are present in liver and cervix cell lines, it appears that this reflects the Sp1-binding protein and another factor that is distinct from that in goblet-like cells because transient transfection assays suggest that the binding of the GCRE sequence in these cells does not promote ITF transcription. Interestingly, IEC6 cells, a nontransformed intestinal epithelial cell line established in this laboratory from neonatal rat intestinal crypt cells (42), do not possess the nuclear factors that bind to the GCRE. Previous studies have shown that IEC6 cells do not have detectable RITF mRNA by Northern blot analysis (14). These findings suggest that some unknown regulatory factor exists for vertical differentiation from crypt to villus in small and large intestine. Transient transfection experiments comparing LS174T cells and cells derived from other organs suggest that the GCRE of the RITF gene promoter is capable of directing intestine and goblet cell-specific expression.
In summary, we have identified a goblet cell-specific enhancer element in RITF gene promoter bound by a goblet cell nuclear protein. Further characterization of this enhancer element may provide insight into the molecular basis of the goblet cell phenotype. Future studies may also identify the genetic elements responsible for "ectopic" expression of ITF in pathologic conditions.
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ACKNOWLEDGEMENTS |
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We thank Drs. T. C. Wang and H. Nakagawa at Massachusetts General Hospital for helpful discussions.
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FOOTNOTES |
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* These studies were supported by National Institutes of Health Grants DK46906 and DK43351.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.
To whom correspondence should be addressed: Gastrointestinal Unit,
Massachusetts General Hospital, Fruit St., Boston, MA 02114. Fax:
617-724-2136; E-mail: Podolsky.Daniel{at}mgh.harvard.edu.
1 The abbreviations used are: ITF, intestinal trefoil factor; RITF, rat intestinal trefoil factor; GCRE, goblet cell-response element; GMSA, gel mobility shift assay; EGF, epidermal growth factor; bp, base pair(s); WT, wild type; RSV, Rous sarcoma virus.
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REFERENCES |
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