Short-chain fatty acids inhibit intestinal trefoil factor gene expression in colon cancer cells

Chau P. Tran1, Mary Familari1, Lorraine M. Parker1, Robert H. Whitehead2, and Andrew S. Giraud1

1 Department of Medicine at Western Hospital, University of Melbourne, 3011 Melbourne; and 2 Ludwig Institute for Cancer Research, Royal Melbourne Hospital, 3052 Victoria, Australia

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -thioglycerol, 50 U/ml penicillin, and 50 µg/ml streptomycin.

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 -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.

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 -20°C until assay.

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.

For goblet cell quantitation, LIM 1215 cells grown on coverslips for 2 days were treated with or without butyrate for 8, 24, and 96 h, fixed in 4% paraformaldehyde, and stored in 70% ethanol. Mucin-containing cells were visualized by periodic acid-Schiff (PAS) staining. Cell viability was checked by trypan blue exclusion.

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 [alpha -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 [alpha -32P]UTP-labeled riboprobes by in vitro transcription (Promega).

A human mucin 2 (MUC2) antisense oligonucleotide probe of 48 nucleotides, a gift from Drs. Tony Corfield and Neil Myerscough (Dept. of Medicine Laboratory, Bristol Royal Infirmary, UK), was used in Northern analysis. For the mobility shift assay, an [alpha -32P]dATP-labeled double-stranded oligonucleotide probe of 36 nucleotides encompassing the butyrate response element (BRE) was generated from the promoter region of the hITF gene between -142 and -107 bp by the following oligonucleotides: 5'-GAGAACAGGAGC<UNL>AGCCACAGCCA</UNL>GGAGGGAGA-3' (sense) and 3'-TGTCCTCG<UNL>TCGGTGTCGGT</UNL>CCTCCCTCTCGGA-5' (antisense).

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'-GAGAACAGGAGC<UNL><B>AT</B></UNL>CA<UNL><B>A</B></UNL>AGTACGGAGGGAGA-3' (sense) and 3′-TGTCCTCG<UNL><B>TA</B></UNL>GT<UNL><B>T</B></UNL>TC<UNL>ATG</UNL>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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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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Table 1.   ITF expression by colon cancer cell lines

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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Fig. 1.   Immunohistochemical localization of intestinal trefoil factor (ITF) in LIM 1215 (A) and LIM 1863 (B). ITF immunoreactivity is present throughout cytoplasm (arrows). LIM 1215 grows as monolayer (×400), whereas LIM 1863 cells formed crypt-like organoids with central lumen (×150).

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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Fig. 2.   A: time course of release of ITF from LIM 1215 cells (secreted ITF expressed in pmol/well, total pool expressed as pmol/mg cellular protein). B: lack of stimulation of secretion by calcium ionophore A-23187.

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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Fig. 3.   Northern blot analysis of human ITF (hITF) message. Total RNA (20 µg/lane) from LIM 1215 (lane 1), LIM 1863 (lane 2), human colon (lane 3), and stomach (lane 4) were separated by gel electrophoresis (A), transferred to a solid support, and hybridized with labeled hITF cDNA (B).

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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Fig. 4.   Effect of various concentrations of short-chain fatty acids (SCFA) on hITF release in medium and cell content in LIM 1215 after 96 h of incubation. Butyrate (A), propionate (B), and acetate (C) all inhibited hITF dose dependently.

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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Fig. 5.   Ribonuclease protection analysis of hITF message. Total RNA (5 µg/well) from LIM 1215 cells exposed to SCFA (0-30 mM) for 96 h was hybridized to both hITF and human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) riboprobes. hITF mRNA level was quantified as %signal intensity (control 100%) after normalization to hGAPDH signal. A: butyrate. B: propionate. C: acetate.

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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Fig. 6.   Time course of inhibition of hITF expression and induction of alkaline phosphatase by 3 mM butyrate. A: ribonuclease protection assay of hITF mRNA (5 µg/well), quantified as signal intensity after hGAPDH normalization (at 8 h, control n = 5, butyrate n = 6, P < 0.01). B: determination of alkaline phosphatase activity in control and butyrate-treated LIM 1215 cells.


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Fig. 7.   Temporal effect of butyrate on number of periodic acid Schiff (PAS)-positive mucus cells. A: proportion of PAS-positive cells per microscopic field. B: absolute number of PAS-positive cells counted per field. * P < 0.01. Three separate cultures were counted for each treatment at each time.

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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Table 2.   Effects of actinomycin D on butyrate inhibition of hITF expression

                              
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Table 3.   Nucleotide consensus sequences of putative butyrate response elements from the 5' promoter region of the cyclin D1, calbindin-D28k, metallothionein IIA, and human ITF genes


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Fig. 8.   Electrophoretic mobility shift assay of LIM 1215 nuclear extract (3 µg/lane), using a butyrate response element (BRE)-containing 32P-labeled oligonucleotide probe. Control, untreated cells; butyrate, 2 mM treatment for 24 h. N, no competitor; B, BRE; M, mutated BRE. 1 and 2, competitor concentrations at, respectively, 50 (10 ng) and 500 (100 ng) times the labeled probe.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    ACKNOWLEDGEMENTS

We acknowledge the provision of a hITF cDNA clone by Dr. D. K. Podolsky, Harvard Medical School, Boston, MA.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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