1 Department of Medicine at
Western Hospital, Intestinal trefoil factor (ITF) gene expression
was detected in five colon cancer cell lines. ITF was synthesized by
mucous cells of LIM 1215 and LIM 1863 lines, from which it is secreted constitutively. The ITF mRNA transcript was estimated to be 0.6 kb. In
LIM 1215 cells, the expression of ITF was potently and dose-dependently
inhibited by short-chain fatty acids (butyrate > propionate > acetate) within 8 h of application. The inhibitory effect of butyrate
was ablated by actinomycin D and preceded its effects on
differentiation of LIM 1215 cells as indicated by induction of alkaline
phosphatase activity and counting of periodic acid-Schiff-positive cells. The human ITF promoter contained an 11-residue consensus sequence with high homology to the butyrate response element of the
cyclin D1 gene. Mobility shift assays show specific binding of this
response element to nuclear protein extracts of LIM 1215 cells. We
conclude that butyrate inhibits ITF expression in colon cancer cells
and that this effect may be mediated transcriptionally and
independently of its effects on differentiation.
TFF; transcriptional regulation
THE TREFOIL PEPTIDES are a small family of polypeptides
characterized by one or more triple-looped, cysteine-rich regions called trefoil domains (37). There are three mammalian members of the
group: 1) spasmolytic polypeptide
(SP or TFF2, see Ref. 46), a 12-kDa molecule with two trefoil domains,
and 2) intestinal trefoil factor (ITF or TFF3) and
3) pS2 (TFF1), which have molecular weights of 6-7 kDa
and one trefoil domain. Whereas SP and pS2 are expressed in the stomach
(39), ITF is predominantly found throughout the intestine and colon
(34).
The trefoil peptides are major secretory products of many mucous
epithelia (particularly of the gastrointestinal tract), and they are
strongly upregulated in response to drug- or disease-induced epithelial
damage and inflammation (30, 47). In addition, trefoil peptides, in
particular pS2, have been found in secretory granules of neuroendocrine
cells close to sites of mucosal damage in cases of Crohn's disease
(48), and therefore it is possible that they subserve multiple
functions as luminal and circulating or local regulatory factors.
Current evidence for specific physiological roles for trefoil peptides
is sparse, although they are able to stimulate cell migration (6, 12)
and have cytoprotective activity (28), making it likely that they are
involved in some aspects of cellular repair. This conclusion has been
strengthened by the results of recent studies in which impaired
gastrointestinal repair was observed in mice in which either the ITF
(23) or the pS2 gene (22) was disrupted by homologous recombination.
To date little is known about the regulation of trefoil peptide gene
expression. A recent report suggests that modulators of mucus secretion
such as carbachol, vasoactive intestinal polypeptide, and somatostatin
are able to increase the steady-state expression of ITF mRNA and
protein (24), and ITF itself appears able to upregulate SP gene
expression (8). Local gastrointestinal inhibitors of trefoil gene
expression have not been identified; however, short-chain fatty acids
(SCFA), formed in the gastrointestinal tract as a result of bacterial
fermentation of undigested dietary components (45), may be important in
this respect. The majority of SCFA in the colon is accounted for by the
presence of butyrate, propionate, and acetate in an approximate molar
ratio of 15:25:60 (10). These SCFA are actively absorbed by the colonic
epithelium, with butyrate being the preferred substrate as a metabolic
fuel (31). It has been well established that butyrate is an inhibitor of proliferation and an inducer of cell differentiation in numerous tumor cell lines (3, 19, 43). Butyrate likely acts through the
inhibition of histone deacetylase or DNA hypermethylation to alter the
expression of numerous genes (4, 27). Significantly, inhibition of
expression involves a number of genes involved in differentiation and
growth regulation such as cyclin D1 (21), urokinase type plasminogen
activator (2), as well as the oncogene c-myc (32).
Because the normal gut epithelium cannot be maintained for extended
periods in cell culture, we sought alternative model systems for
studying trefoil gene regulation as well as possible receptor-mediated functions of the peptides themselves. We chose colon cancer cell lines,
particularly the LIM 1215 line, because it has been established that
several colon cancer cell lines express the ITF gene (29), and the LIM
1215 cell line has been particularly well characterized with respect to
its constituent cell phenotypes and its response to mitogens (9, 44).
In addition, it has been shown that LIM 1215 cells are responsive to
SCFA both in terms of proliferation and differentiation (43). During
the course of our investigations we have established that several colon
cancer cell lines of mixed phenotype both synthesize and secrete ITF
and that SCFA potently inhibit ITF synthesis and secretion before their
effects on cell differentiation. We also report for the first time that
the transcriptional effects of the most potent SCFA butyrate are
probably mediated by a specific regulatory element situated in the
5' flanking region of the human ITF (hITF) gene.
Cell lines and cultures.
The colon cancer cell lines LIM 1215, 1863, 1899, 2412, and 2537 were
screened for trefoil immunoreactivity. Cell lines were maintained in
RPMI 1640 medium (ICN) with 10% FCS, 100 nM insulin, 2 µM
hydrocortisone, 10 µM ITF release.
For release experiments, cells were either seeded into 25-ml Falcon
flasks at 2 × 105 cells/ml
in 10 ml of medium and maintained for 4 days in an atmosphere of 5%
CO2 in air at 37°C, or grown
in six-well culture plates at 2 × 105 cells/ml in 2 ml of
medium. For time-course experiments cells were grown as
above in six-well plates for periods between 1 and 7 days, media were
collected, and cells were harvested by centrifugation or scraping from
wells or flasks, lysed by boiling in PBS for 5 min, and stored at
SCFA treatments.
LIM 1215 cells were seeded at 5 or 2 × 105 cells/well into 6- or 12-well
plates and cultured to 80% confluency before treatment with SCFA
(Sigma) at various concentrations in medium with 1% FCS for 3-4
days. After media were collected, cells were lysed in 50 mM
Tris · HCl containing 0.5% Nonidet P-40, and both
media and cell lysates were frozen at Immunohistochemistry and histology.
Pellets of LIM 1863 cells and monolayer cultures of LIM 1215 cells
grown on glass coverslips were washed in PBS and fixed for 1 h in
buffered Formalin. The former were embedded in paraffin, and 4-µm
sections were cut and glued onto slides. Slides were incubated with
normal goat serum (1:20) for 30 min, primary antibody (rITF) at 1:300
to 1:600 for 2 h, and biotinylated goat anti-rabbit IgG (1:200) for 30 min and then visualized by the avidin-biotin method (Vector) followed
by color development with diaminobenzidine/peroxide. The rat ITF (rITF)
antibody has been characterized previously (35). It stains secretory
granules of cells with a goblet (mucin) phenotype in normal and
neoplastic tissue.
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-thioglycerol, 50 U/ml penicillin, and 50 µg/ml streptomycin.
20°C until use. For experiments designed to test the effect
of raising intracellular calcium on ITF secretion, cells grown in
six-well plates at 2 × 105
cells/ml in 2 ml of medium containing 5 mM calcium were treated with
the calcium ionophore A-23187 (1 µM) for 60 min and then treated as
for the time-course experiments before assay.
20°C until assay.
RIA. The COOH-terminal nonapeptide of rITF with the addition of an NH2-terminal tyrosine (COOH-terminal decapeptide or CD) was used as an immunogen after coupling to keyhole limpet hemocyanin, to raise high-titer antisera in New Zealand White rabbits as previously described (36). Assays were performed with antisera at a final dilution of 1:20,000. Diluent was ammonium acetate buffer (0.05 M, pH 6.8, containing 0.1% EDTA, 0.2% BSA, 0.05% sodium azide). Total assay volume was 1 ml; 5,000 counts/min of 125I-labeled peptide were added to each tube. RIA standards were the CD of rITF at 7.8-10,000 fmol/tube. Tubes were incubated at 4°C for 24-48 h, and antibody-bound label was separated from unbound label by the addition of a mixture of goat anti-rabbit IgG (100 µl, 1:150) and normal rabbit serum (100 µl, 1:600) in phosphate buffer and polyethylene glycol (100 µl, 10% wt/vol) for 24 h at 4°C or donkey anti-rabbit cellulose suspension (Sac-Cel, IDS) for 30 min at 4°C (or 60 min at room temperature). Nonspecific binding for the ITF assay was <1.5% (n = 20). Assay sensitivity was <10 fmol/tube; IC50 of label binding was 353 ± 14 fmol/ml (n = 12), whereas intra- and interassay variation was <15%.
Alkaline phosphatase and protein assays.
LIM 1215 monolayers at 80% confluency were treated with 3 mM butyrate
for 8, 12, or 27 h, washed in PBS, harvested in mannitol buffer (50 mM
D-mannitol, 2 mM Trizma base, pH
7.4), and stored at 20°C. Cells were homogenized, and after
addition of Triton X-100 to a final concentration of 0.1%, assayed for
alkaline phosphatase activity using
p-nitrophenyl phosphate as substrate
(49). Protein content was quantified after a modified Bradford protocol
(5) using BSA as standard.
Probes and recombinant plasmids.
hITF cDNA of 400 bp (1-400 nt) in PCR 1000 vector (Invitrogen)
(38) was excised with EcoR
I/Hind III and labeled with
[-32P]dATP by
random priming (Amersham). For antisense RNA probes, hITF cDNA was
subcloned into Bluescript pKSII (Stratagene) after removal of the
poly(A)+ tail. RT-PCR (Promega)
was used to generate a 200-bp glyceraldehyde-3-phosphate dehydrogenase
(hGAPDH) fragment (351-551 nt) from human colon total RNA using
the following primers: 5'-ATGGATCCGTGTCTTCACCACCATGGAGAA-3' (sense) and 5'-CAGAATTCGCTTGTCATGGATGACCTTGGC-3'
(antisense) and cloned into Bluescript pSKII (Stratagene).
Both hITF- and hGAPDH-containing plasmids were linearized and used as
templates for generating [
-32P]UTP-labeled
riboprobes by in vitro transcription (Promega).
Northern blot analysis. Total RNA extracted from cells according to the protocol of Chomczynski and Sacchi (7) was electrophoresed (5-20 µg) in 1-1.2% agarose-1.2% formaldehyde gel-1× MOPS and transferred to a nylon membrane (Hybond N, Amersham). Blots were hybridized overnight at 42°C in 50% formamide with the random-primed 32P-labeled hITF cDNA fragment or in 20% formamide with MUC2 antisense oligonucleotide and washed at high stringency (65°C, 0.2× saline-sodium citrate) before autoradiography.
RNA. Antisense 32P-labeled hITF and hGAPDH riboprobes were incubated with total ribonuclease protection assay (2-20 µg) and hybridized overnight at 42°C essentially as described by Kreig and Melton (20), except that all hybrids were digested at 20 µg/ml RNase A and 1 µg/ml RNase T1 (Sigma) at 37°C for 30 min. Protected fragments were separated on a 5% polyacrylamide-8 M urea gel.
Electrophoretic mobility shift assay.
LIM 1215 cells were treated with or without 2 mM butyrate for 24 h, and
nuclear extracts were prepared as described previously (13) with minor
modifications. Electrophoretic mobility shift assay was performed by
mixing 3 µg nuclear protein with 0.2 ng of the BRE oligonucleotide
probe (5,000-10,000 counts/min) in a total volume of 15 µl
binding buffer (10 mM Tris · HCl, pH 8, 1 mM EDTA, 50 mM NaCl, 5 mM MgCl2, 1 mM
dithiothreitol, 5% glycerol) containing 1 µg poly(dI-dC). After
20-min incubation on ice, the complexes were resolved by
electrophoresis on a 6% polyacrylamide gel and visualized by exposing
to X-ray film. In competition experiments, both the probe and unlabeled
competitor fragments were premixed in binding buffer before the
addition of extract. Competitors included BRE oligonucleotide, as well
as its mutated form (MBRE) in which only the bases within the 11-bp
region were altered, as indicated by the bold type:
5'-TACGGAGGGAGA-3' (sense) and
CCTCCCTCTCGGA-5' (antisense).
Statistical analysis. Results were expressed as means ± SE, n = 3-6, with P value determined by paired t-test after ANOVA. A P value <0.05 was considered significant.
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RESULTS |
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Five colon cancer cell lines (LIM 1215, 1863, 1899, 2412, 2537) were grown in cell culture, and the conditioned medium and cell content from each were assayed for ITF immunoreactivity (Table 1). It is clear that all colon cancer cell lines express ITF to variable extents. Cell content of ITF ranged from 1.2 to 24.1 pmol/mg protein, which is comparable to the concentration range of ITF found previously in our laboratory in a series of low- to well-differentiated colon carcinomas (10-200 pmol/g wet wt or 1-20 pmol/mg protein; Ref. 35). In each case basal release of ITF into media was at least 17% of the total pool (secreted and cell content) and varied between 17 and 78%, suggesting heterogeneity in the regulation of basal ITF secretion from different lines. LIM 1215 and 1863 cells were chosen for further study because they have been well characterized, low passage number clones are available, and they have a relatively high basal expression of ITF.
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Immunohistochemistry showed that cells with a mucous phenotype stained strongly with anti-ITF antisera in both LIM 1215 and 1863 cell lines (Fig. 1). Not all cells contained immunoreactivity (enterocyte-like cell phenotype); however, immunopositive mucous cells showed strong granular staining throughout the cell cytoplasm.
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The temporal changes in ITF expression by LIM 1215 cells grown in monolayer culture are shown in Fig. 2A. The release of ITF into cell culture medium increased in a linear fashion for 5 days, suggesting active secretion derived from an expanding cellular pool. The total immunoreactivity (cell content plus released peptide) expressed per milligram of cellular protein also increased in a similar fashion over the culture period, suggesting that trefoil synthesis was taking place. Similar data were obtained from the time-course studies on the floating cell line LIM 1863 (data not shown).
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To investigate the mode of ITF secretion from LIM 1215 cells, near-confluent monolayers were incubated with 0, 0.01, or 1 µM of the calcium ionophore A-23187 for 1 h (Fig. 2B). Gastric endocrine cells, which secrete their peptide products in a regulated fashion, show a marked increase in secretion in response to A-23187 (17); however, ITF secretion from LIM 1215 cells was not augmented even at 1 µM, indicating that secretion is near maximally stimulated and likely to be constitutive for this cell line.
ITF synthesis in LIM 1215 and 1863 cell lines was confirmed by Northern hybridization (Fig. 3). The LIM 1863 cell line had a higher concentration of ITF mRNA compared with LIM 1215 cells. The intense signal in human colon served as a positive control, whereas a less intense band in human stomach confirmed previous reports that low-level ITF expression occurs in the gastric mucosa (40). The hITF transcript was estimated to be ~0.6 kb in length, in accordance with previous findings (29). Subsequent studies were performed exclusively on LIM 1215 cells because this adherent line was easier to manipulate in quantitative studies of secretion than the floating LIM 1863 cell aggregates.
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Preliminary studies indicated that butyrate inhibited ITF secretion into cell culture medium. To explore the effect of butyrate in terms of its concentration, a dose-response curve was established (Fig. 4A). ITF release was inhibited by butyrate at concentrations as low as 0.1 mM (P < 0.05, n = 6), whereas at 1 mM the inhibition was at least 60% (P < 0.05, n = 6). There was also a comparable dose-dependent decrease in cellular ITF concentrations (expressed per milligram protein), indicating that ITF synthesis was inhibited by the application of butyrate. Cell viability in the presence of 2 mM butyrate, as determined by trypan blue exclusion, was over 90% at 24 h but decreased slightly to 85% by 96 h.
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Based on the results of butyrate treatment, two other major SCFA, namely propionate and acetate, were also tested for their effects on ITF synthesis. Both of these SCFA inhibited ITF synthesis and release in a dose-dependent manner, with maximal inhibition achieved at 4 mM for propionate and 30 mM for acetate (Fig. 4, B and C), compared with 1-2 mM for butyrate. This effect was also evident in the steady-state synthesis of ITF (Fig. 5) as quantified by RNase protection analysis of SCFA-treated LIM 1215 cells, which showed a dose-dependent decrease in ITF mRNA levels with increasing concentrations of SCFA, and potency correlated with carbon chain length. The levels of hGAPDH mRNA were uniform in both control and treated groups (Fig. 5, A-C).
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To distinguish between the possibilities that butyrate mediates its effects either directly or indirectly due to the induction of an enterocyte cell phenotype, ITF mRNA (Fig. 6A) and the enterocyte brush border and differentiation marker alkaline phosphatase (Fig. 6B) were quantified 8, 12, and 20 or 27 h after application of butyrate. Medium containing no butyrate was used as a control at each time point. As shown in Fig. 6, the effect of butyrate on ITF synthesis is evident as early as 8 h after application, whereas the induction of alkaline phosphatase is not observed until 27 h. To assess the effects of butyrate on the goblet cell population, the number of PAS-positive cells at 8, 24, and 96 h in both control and butyrate-treated cultures was counted (Fig.7A). There was no significant difference in the proportion of goblet cells in control and butyrate-treated groups, although there was a trend to a reduction after butyrate treatment at 96 h (P = 0.16). However, as shown in Fig. 7B, there was an ~70% decrease in the absolute number of goblet cells after 96 h of butyrate treatment compared with controls, confirming that butyrate is a potent antiproliferative agent.
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|
The time course of expression of the MUC2 gene was also examined after butyrate treatment by Northern blotting (gel not shown) and quantified by gel densitometry. There was no difference between groups either at 8 h (control 6.0 ± 1.1, butyrate 4.5 ± 0.3, P = 0.27, n = 3) or at 24 h (control 5.8 ± 0.6, butyrate 4.9 ± 0.3, P = 0.43, n = 3).
In an attempt to investigate whether the butyrate-induced inhibition of
hITF gene expression may be mediated transcriptionally, cells were
exposed to butyrate alone or butyrate and actinomycin D (10 ng/ml), and
ITF mRNA was quantified by RNase protection analysis. The results
(Table 2) showed that actinomycin D completely ablated
the inhibition of ITF expression caused by butyrate, suggesting that
butyrate may exert its effects transcriptionally. A subsequent search
of the 5'-flanking region of the hITF gene (33) revealed a
sequence of 11 nucleotides situated between 130 and
120
bp upstream of the transcription initiation site, which displays strong
homology to three other previously reported butyrate-response elements
(Table 3), namely cyclin D1 (21), calbindin-D28k (16), and metallothionein II (1). Whereas butyrate induces gene expression of
calbindin-D28k and metallothionein II, it inhibits cyclin D1 and ITF
expression. To test this putative BRE, an oligonucleotide probe
encompassing this region of the hITF promoter was incubated with LIM
1215 nuclear extracts (Fig.
8). A DNA-protein complex was
observed (lane N) and confirmed to
be specific by competition study. Unlabeled BRE-containing
oligonucleotide at increasing concentrations (lanes
B1 and B2)
successfully competed with the probe, as shown by the reduced density
of the DNA-protein complex. Furthermore, a mutated BRE oligonucleotide
(MBRE) at the same concentration failed to compete for this complex
(lanes M1 and M2), indicating that the altered
bases were necessary for binding activity. There was no apparent
difference in the intensity of the DNA-protein complex between control
and butyrate-treated samples (lane N
in both groups).
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DISCUSSION |
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We have shown that five colon cancer cell lines with different adherent ability are able to synthesize and secrete appreciable amounts of ITF. All of these cell lines exhibit a mixed phenotype of mainly enterocytes (absorptive) or goblet (mucous) cells (42, 44), indicating a conserved pattern of ITF expression associated with mucous secretion. The localization of ITF in colon cancer cells with morphological characteristics consistent with mucus secretion is in accord with previous reports from our laboratory and from others, which have demonstrated the presence of ITF peptide and mRNA expression in goblet cells of normal and neoplastic human colon (29, 35), and further underscores a likely role for the trefoil peptides in some aspects of normal mucus physiology, possibly in maintaining the barrier function of mucus.
It is clear from the studies presented here that different colon cancer cell lines secrete ITF to variable extents. LIM 2412 and 2537 have relatively low ITF secretion rates (<20% total pool), indicative of regulated secretion, whereas for LIM 1863, 1215, and 1899, basal ITF secretion is 42, 78, and 90% of the total pool, respectively, suggesting constitutive release; the insensitivity of ITF secretion from LIM 1215 cells to raised intracellular calcium concentrations strengthens this likelihood.
The inhibitory effect of the major SCFA, in particular butyrate, on ITF expression has been clearly demonstrated in this study. The effects observed are not due to butyrate toxicity because it has been previously shown that LIM 1215 cell viability, as measured by the rate of glycoprotein synthesis and lactate dehydrogenase release, is not reduced by butyrate at concentrations <4 mM (14).
The inhibition of ITF synthesis and secretion by butyrate presents an apparent paradox because both have beneficial effects in terms of colonic physiology: butyrate is antitumorigenic yet promotes maturation and proliferation patterns of the normal colon, whereas ITF, like other members of the trefoil peptide family, is involved in injury repair and is cytoprotective. An explanation may lie in the ability of butyrate to inhibit molecules that promote cell invasiveness. Thus butyrate potently inhibits urokinase (u-PA) expression by both normal and neoplastic colonocytes (and conversely enhances the expression of PAI-1, which inhibits u-PA; 15). Similarly ITF has been shown to stimulate cell migration of both nontransformed intestinal epithelial cells and colon cancer cell lines (12, 28), and therefore the downregulation of ITF expression by butyrate may reflect a common inhibition of molecules potentially associated with cell migration irrespective of their cellular targets.
Previous studies in several colon cancer cell lines have also established that butyrate and, to a lesser extent, propionate inhibit growth as well as induce cell differentiation (3, 43). An implication of this is that ITF may be involved in cell proliferation, hence its downregulation of expression on exposure to butyrate. However, there have been conflicting reports on the proliferative effect of trefoil peptides. Porcine SP appears to be mitogenic for two different neoplastic cell lines (18), but this mitogenicity requires glutathione (26). Conversely, it has been shown that both SP and ITF are without proliferative effects on numerous human colon cancer cell lines, as well as nontransformed rat intestinal epithelial cell lines, even at concentrations as high as 80 µM (12).
A more likely explanation for the inhibition of ITF expression by butyrate lies in the ability of this SCFA to induce differentiation of colon cancer cells. Whitehead et al. (43) found that butyrate inhibited growth and induced enterocyte differentiation in LIM 1215 cells after at least 2 days, marked by a large increase in alkaline phosphatase activity. Moreover, the number of mucus-containing cells decreased after 5 days of culture in the presence of 1 mM butyrate, indicative of differentiation away from the mucous cell and toward the enterocyte cell phenotype. Because it has been confirmed in this study that ITF expression is associated with goblet (mucous) cells, the decrease in ITF expression over 4 days of culture in butyrate-containing medium could be due in part to a reduction in the number of goblet cells. A caveat to this, however, is that whereas both propionate and acetate were reported to have little differentiating effect on colonocytes (14, 41), we have shown here that at moderate doses both fatty acids can effectively inhibit ITF expression. The potency of butyrate over the other two SCFA can be explained on the basis that butyrate is the preferred oxidative fuel for colonic epithelial cells (31) and may be preferentially utilized after uptake.
Whereas a change in phenotype could account for the downregulation of ITF by SCFA over time, it does not explain our observation of the rapid inhibition of ITF expression by butyrate before phenotypic changes indicative of differentiation. Butyrate has been reported to modify gene expression via several mechanisms, including inhibition of histone deacetylase (4), DNA hypermethylation (associated with inhibitory regulation; 27), as well as specific actions on promoter activity (11). DNA sequences conferring the modulation of gene expression by butyrate have been identified in the upstream regulatory region of several genes (Table 2). The strong homology between the putative hITF gene BRE and that of the cyclin D1, calbindin-D28k, and metallothionein IIA genes, suggests its direct involvement in the inhibition of ITF expression. Indeed, the detection of a specific BRE-protein complex indicates that butyrate regulation could occur in this region of the hITF promoter, perhaps by altering the activity of the binding protein(s) but not their binding capacity. Characterization of the hITF promoter and the proteins that interact with it is currently in progress.
The butyrate-induced inhibition of ITF expression in LIM 1215 cells implies a role for ITF in the maintenance of the goblet cell phenotype. The downregulation of ITF might be one of the signals for LIM 1215 cells to alter commitment away from the goblet cell-directed maturation pathway and instead allow enterocyte differentiation. Recently, preliminary evidence for a goblet cell-specific enhancer element, which is different from the BRE, has been determined for the rat ITF gene (25). An inspection of the hITF gene promoter region (33) shows several sequences highly homologous to the goblet cell-specific enhancer element, but their importance in directing goblet cell differentiation has yet to be assessed.
It therefore appears that both positive and negative regulatory elements exist with respect to goblet cell lineage determination in the colon. An understanding of the factors that modulate them may ultimately provide insight into the mechanisms governing differentiation patterns of the constituent cells of the distal gut and their perturbation in neoplasia.
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ACKNOWLEDGEMENTS |
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We acknowledge the provision of a hITF cDNA clone by Dr. D. K. Podolsky, Harvard Medical School, Boston, MA.
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FOOTNOTES |
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This study was supported by a project grant from the National Health and Medical Research Council of Australia (A. S. Giraud) and by a Postgraduate Scholarship from the University of Melbourne (C. P. Tran).
Address for reprint requests: A. S. Giraud, Univ. of Melbourne, Dept. of Medicine at Western Hospital, Footscray 3011, Australia.
Received 16 July 1997; accepted in final form 19 March 1998.
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