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© 2004 The American Society for Nutritional Sciences J. Nutr. 134:792-798, April 2004

Nutrient-Gene Interactions

Krüppel-Like Factor 4 Is Transactivated by Butyrate in Colon Cancer Cells1

Zhi Yi Chen2, Sybille Rex and Chi-Chuan Tseng

Section of Gastroenterology, VA Boston Healthcare System and Boston University School of Medicine, Boston, MA 02118

2To whom correspondence should be addressed. E-mail: zhiyi.chen{at}bmc.org.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
High-fiber diets decrease the incidence of colorectal cancers, and SCFA, derived from dietary fiber, are involved in the regulation of cell growth, differentiation, and apoptosis of the colonic epithelium. The mediators of these effects remain poorly defined. Krüppel-like factor-4 (KLF4/GKLF) is a zinc-finger transcription factor that exhibits some physiologic properties similar to those of SCFA in the colon. The present study was undertaken to examine the role of KLF4 in the butyrate-mediated effect in colon cancer HT-29 cells. Butyrate induced KLF4 mRNA expression and stimulated KLF4 promoter activity in a dose- and time-dependent manner in HT-29 cells. Similar effects were observed in SCFA possessing different carbon lengths (C3–C7), but not in branched isobutyric acid, indicating that the stimulatory properties of SCFA were related to fatty acid structure. Transfection studies using 5' deletion and mutant constructs of the KLF4 promoter demonstrated that the butyrate-responsive element was located at a putative stimulatory protein (Sp)1-binding site. Electrophoretic mobility shift assays using an oligonucleotide containing a consensus Sp1-binding element revealed a DNA-protein complex that was enhanced by butyrate treatment and supershifted by the Sp1 antiserum. Furthermore, the effects of butyrate on cell growth and KLF4 mRNA expression were the same as those of trichostatin A (TSA), a specific inhibitor of histone deacetylase (HDAC1). Overexpression of HDAC1 significantly attenuated transcriptional activation of the KLF4 promoter by butyrate or TSA. These results suggest that KLF4 may function as one of the downstream effectors of butyrate that mediates its growth arrest effect in the colon. Moreover, transactivation of KLF4 by butyrate appears to be mediated through interaction with a Sp1-binding domain on the promoter and is also likely to involve histone acetylation.

KEY WORDS: • GKLF/KLF4 • butyrate • histone acetylation • short chain fatty acids

Although numerous studies have suggested an important role of genetic predisposition to the development of colorectal cancer, there is strong epidemiologic evidence illustrating the involvement of dietary factors in colonic carcinogenesis as well. This notion is supported by the fact that the colorectal epithelium is in direct contact with the luminal contents, and that a high-fiber diet is associated with a decrease in the incidence and the growth of colon cancers (1,2). SCFA are produced in the large intestine by bacterial fermentation of dietary fibers and the 4-carbon fatty acid, butyrate, has attracted most of the research interest. Dietary supplementation of fiber increased intestinal butyrate concentration and reduced colonic cell proliferation and tumor mass in vivo in a rat colon cancer model (3). Furthermore, butyrate was also shown to inhibit cell growth and promote differentiation and apoptosis in vitro in colorectal cancer cell lines (4,5). The mediators governing butyrate effects in the gastrointestinal tract, however, remain poorly defined.

Krüppel-like factor 4 (KLF4/GKLF)3 is a zinc-finger transcription factor, expressed extensively in the epithelial cells of the gastrointestinal tract (6). Several studies demonstrated a potential role of KLF4 in modulating growth and differentiation of the colonic epithelia (7,8). In colon cancer cells, constitutive overexpression of KLF4 induces cell cycle arrest at the G1 phase and inhibits DNA synthesis and cell proliferation (9,10). These effects appear to be mediated through activation of the p21WAF1/Cip1 (11), and/or suppression of the cyclin D1 promoter (12). Recently, our laboratory showed that KLF4 transactivated the human intestine alkaline phosphatase gene and promoted sodium butyrate–induced differentiation and apoptosis in colon cancer cells (unpublished data). These data indicate that KLF4 and butyrate possess similar physiologic properties.

The human colonic adenocarcinoma cell line (HT-29) has been used extensively as a model for studying intestinal epithelial cell differentiation in vitro. Under standard growth conditions, HT-29 cells display an undifferentiated phenotype. When they are treated with butyrate, HT-29 cells differentiate into different lineages that resemble those found in the normal intestinal epithelium. Therefore, we utilized HT-29 cells to examine the role of KLF4 in butyrate-mediated effects in the colon; our results suggest that KLF4 might function as a downstream effector of butyrate.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Cell culture.

The HT-29 cell line was obtained from the American Type Culture Collection. Cells were maintained in McCoy’s 5A medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 mg/L streptomycin, and 1 x 105 U/L penicillin in an atmosphere of 95% air and 5% CO2 at 37°C. All fatty acids and trichostatin A (TSA) were purchased from Sigma Chemical.

Cell growth assay.

The rate of cell growth was measured by a semiautomatic tetrazolium-based colorimetric assay (MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), as described previously (13). Briefly, HT-29 cells were seeded into a 24-well plate at a density of 2.5 x 104 cells/well and incubated overnight at 37°C. Cells were treated with butyrate (4 mmol/L) or TSA (50 µg/L) for various periods of time and 20 µL of MTT (2.5 g/L) was then added to each well. Cells were incubated for an additional 2 h to allow the dye and mitochondrial dehydrogenase to interact in the viable cells, and the absorbance at 540 nm was measured with a spectrophotometer.

Cell cycle analysis.

Cell cycle distribution of HT-29 cells was analyzed using flow cytometry as described previously (14). Briefly, cells treated with butyrate or TSA were trypsinized, washed with PBS, and fixed in 70% ethanol (v:v). Fixed cells were then incubated in PBS containing 1.0 mg/L RNase A at 37°C for 30 min, stained with propidium iodide (5.0 mg/L), and analyzed on a FACScan flow cytometer.

RNA isolation and Northern blot analysis.

Total RNA was isolated by the STAT-60 method according to the manufacturer’s instructions (Leedo Medical Laboratories). RNA samples (20 µg) were size fractionated by electrophoresis in the 1.2% (wt:v) agarose/6% formaldehyde (v:v) gel, as described previously (9). Hybridization was then performed using a 450-bp Apa-Pst1 fragment of human KLF4 DNA that was radiolabeled with [32p]-dCTP. The blots were washed and developed after exposure to an X-ray film at -70°C using a Cronex intensifying screen (DuPont) and quantified with a Phosphor Imager (Molecular Dynamics). All blots were stripped and reprobed with a DNA fragment encoding the constitutively expressed glyceradehyde-3-phosphate dehydrogenase gene (GAPDH; Clontech) to verify equal loading of RNA.

Plasmid constructions.

To examine transcriptional regulation of the KLF4 promoter by butyrate, the mouse KLF4 gene was cloned as described previously (15). Various truncated promoter constructs were generated from a full-length KLF4 promoter by restriction endonuclease digestion or by PCR using appropriate primers. The pKLF4–515, pKLF4–250, pKLF4–145, and pKLF4–51 constructs contain a 5'-flanking sequence of the KLF4 gene upstream from the transcription-starting site at 515, 250, 145, and 51 bp, respectively. All constructs were sequenced and ligated to the pGL3-Luc plasmid containing a firefly luciferase reporter gene. Three putative stimulatory protein (Sp)1-binding sites (Sp1–1, Sp1–2, and Sp1–3) on the pKLF4–145 construct were mutated using the QuikChange Mutagenesis Kit (Stratagene) and the following primers: 5'-GGGGGCTGCGGGAAGTTTTGGAGAAAGGCAG-3' (pKLF4–145-mtSp1–1; mutation of the Sp1–1 site), 5'-AAGAAAGGCAGGGGTTTTGGCCTGGCGGCGGAG-3' (pKLF4–145-mtSp1–2; mutation of the Sp1–2 site), and 5'-CGCCACAGGGAGGAGTTTTGGAGCAAGCGAGCGA-3' (pKLF4–145-mtSp1–3; mutation of the Sp1–3 site). These constructs were confirmed by sequence analysis.

Transient transfection and luciferase assay.

Cells were transfected by using LipofectAMINE PLUS reagents according to the manufacturer’s instructions (GIBCO BRL). Briefly, HT-29 cells were seeded into a 12-well plate (1 x 105 cells/well). The next day, cells were transfected with 0.5–1.0 µg/well of reporter and pCMV ß-gal plasmid DNA for 5 h; 24 h after the transfection, the medium was changed, and butyrate or TSA was added. Cell lysates were collected for luciferase assay 48 h later.

For the luciferase assay, the cell lysate (100 µL) was first mixed with the luciferase substrate solution, and luciferase activity was measured using a luminometer with automatic injection. With each experiment, luciferase activity was determined in triplicate and normalized with ß-galactosidase activity for each sample.

Electrophoretic mobility shift assay (EMSA).

Nuclear extracts were prepared as described previously (12). A double-stranded oligonucleotide probe corresponding to the sequence (-109 to -91) of the KLF4 promoter (5'-AGGCAGGGGCGGGGCCTG-3') was end labeled with [{gamma}-32P] by T4 polynucleotide kinase. Assays were performed by incubating 5 µg of nuclear extract in the binding buffer (Promega) containing 200,000 dpm of labeled probe at room temperature for 20 min. To confirm the binding specificity, the reaction was competed with a 50-fold molar excess of unlabeled wild-type or mutant oligonucleotide. For the supershift experiments, the Sp1 antibody (2 µL of 200 mg/L; Santa Cruz Biotechnology) was incubated with nuclear extracts on ice for 30 min before being added to the binding reaction. The samples were electrophoresed on 4% nondenatured polyacrylamide gels (wt:v) in the 0.5X TBE buffer, and gels were dried and subjected to autoradiography.

Statistical methods.

Results are expressed as means ± SEM. A 2-way ANOVA with Dunnett’s post-test (InSat software, GraphPad Software) was performed to determine the dose- and time-response effects. Differences between group means were analyzed by Student’s t test. Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Butyrate inhibits cell growth and induces G1 cell cycle arrest of HT-29 cells.

Butyrate treatment of HT-29 cells resulted in growth retardation. The inhibitory effect first occurred on d 2 (P = 0.15) and was significant from d 3 through d 5 (Fig. 1A; P < 0.05).



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  		FIGURE 1 Effects of butyrate on cell growth and cell cycle distribution of HT-29 cells. (A) HT-29 cells were cultured with medium alone (control) or butyrate (4 mmol/L) for 0–5 d. Values are means ± SEM, n = 3. *Different from control, P < 0.05. Fluorescence-activated cell sorting analysis of HT-29 cells cultured with medium alone (B) or with 4 mmol/L butyrate (C) for 24 h. The percentages of cells in each phase are shown in the right upper corner (n = 4).

 
The cell cycle distribution of HT-29 cells was also altered by butyrate treatment. Butyrate decreased the percentage of cells arrested at the S-phase (30 ± 5% to 4 ± 2%, P < 0.05) and increased cells at the G1 phase (44 ± 10 to 84 ± 5%, P < 0.05) (Fig. 1B). No change in the fraction of apoptotic cells was observed in the current studies because only adherent cells were collected for analysis.

Butyrate increases KLF4 mRNA levels and transactivates the KLF4 promoter.

The effect of butyrate on KLF4 mRNA expression was examined in HT-29 cells. KLF4 mRNA levels were significantly induced by butyrate; these effects first appeared at 30 min and reached a maximum at 8 h (Fig. 2A). The induction of KLF4 mRNA expression by butyrate was also dose dependent from 0.5 to 4.0 mmol/L (Fig. 2B). Moreover, coincubation with cycloheximide (CHX) resulted in an additional increase in KLF4 mRNA expression (Fig. 2C), indicating that KLF4 is a butyrate-induced immediate-early gene.



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  		FIGURE 2 Butyrate induces KLF4 mRNA expression in HT29 cells. (A) HT-29 cells were treated with 4 mmol/L butyrate from 0 min to 24 h; (B) cells were treated with different concentrations of butyrate, as indicated, for 8 h; and (C) HT-29 cells were (+) or were not (-) pretreated with CHX (10 mg/L) for 2 h and then incubated with (+) or without (-) 4 mmol/L butyrate for 8 h. Total cellular KLF4 and GAPDH mRNA levels were determined by Northern blot analysis.

 
In reporter assays, butyrate stimulated KLF4 promoter activity in a dose-dependent manner [promoter activity in response to different concentrations of butyrate (mmol/L): 4.0 > 2.0 > 1.0 > 0.5 > control]. A 14-fold increase occurred in cells treated with 4.0 mmol/L butyrate (Fig. 3A; P < 0.05). Furthermore, transactivation of the KLF4 promoter by butyrate was also time dependent. Activation of the KLF4 promoter by butyrate was higher at 24 than at 8 h (Fig. 3B; P < 0.05). These results suggest that the induction of KLF4 expression by butyrate is mediated, at least in part, through transcriptional activation of the KLF4 gene.



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  		FIGURE 3 Transactivation of the KLF4 promoter by butyrate in HT-29 cells. (A) Cells were transiently transfected with pKLF4–515, and luciferase activity was measured after incubation with medium alone (0) or with various concentrations of butyrate (0.5–4.0 mmol/L) for 24 h. *Different from control without butyrate, P < 0.05. (B) HT-29 cells were transfected with pKLF4–515 and incubated without (-) or with (+) 4 mmol/L butyrate for 4–24 h. Values are means ± SEM, n = 3. Asterisks indicate different from control without butyrate at each time point: *P < 0.05; **P < 0.01.

 
Effects of different SCFA on KLF4 expression.

To assess the effects of various SCFA on KLF4 expression, HT-29 cell were treated with different SCFA. Butyrate (C4) significantly increased KLF4 mRNA level (1.7-fold; Fig. 4A), whereas the branched iso-butyrate (iso-C4) had no such effect (Fig. 4B). In contrast, other SCFA (C3, C5, C6, and C7) had stimulatory effects on KLF4 mRNA levels, although with different intensities. The effects of SCFA on KLF4 promoter activity were similar to those on the mRNA level (Fig. 4C). These data indicate that the stimulatory effects of SCFA on KLF4 gene expression are likely related to fatty acid structure.



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  		FIGURE 4 Effects of different SCFA on KLF4 mRNA levels and promoter activities in HT-29 cells. (A) Cells were treated with 4 mmol/L unbranched (C3, propionate; C4, butyrate; C5, valeric acid; C6, caproate; or C7, heptanoic acid) or branched-chain fatty acid, iso-C4 for 8 h. The levels of KLF4 and GAPDH mRNA were determined by Northern blot analysis. (B) Densitometric measurements of KLF4 mRNA levels after SCFA treatment. The data are expressed as fold increases over control. Values are means ± SEM, n = 4. *Different from control, P < 0.05. (C) HT-29 cells were transiently transfected with pKLF4–515, and luciferase activity was measured after incubation with different SCFA for 24 h. The promoter activity is expressed as fold increase over control without butyrate treatment. Values are means ± SEM, n = 3. *Different from control, P < 0.05.

 
Butyrate transactivates the KLF4 promoter through Sp1-binding domains.

To characterize the region on the KLF4 promoter involved in the interaction with butyrate, a series of truncated KLF4 promoter constructs were generated and transiently transfected into HT-29 cells. Butyrate treatment induced similar increases in the promoter activity of pKLF4–515, pKLF4–250, and pKLF4–145 constructs (Fig. 5A). This induction was significantly reduced, however, in the pKLF4–51 construct, indicating that the region between -145 and -51 on the promoter contained a putative butyrate-interaction domain.



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  		FIGURE 5 The butyrate response element on the KLF4 promoter in HT-29 cells. (A) HT-29 cells were transfected with the KLF4 promoter plasmid, and luciferase activity was analyzed after incubation with 4 mmol/L butyrate for 24 h. The luciferase activity of butyrate-treated cells is expressed as fold increase over control cells. Values are means ± SEM, n = 3. Asterisks indicate different from pGL3-Basic control: *P < 0.05; **P < 001. (B) The sequence of KLF4 promoter, located between -150 and +10 bp from the transcription starting site. Three putative Sp1-binding elements are underlined and labeled as Sp1–1, Sp1–2, and Sp1–3. The TATA box is shown in bold. (C) Relative luciferase activities of HT-29 cells transfected with KLF4 promoter constructs containing wild-type pKLF4–145 (W) or mutated pKLF4–145 (M1), pKLF4–145 (M2), or pKLF4–145 (M3) constructs. The partial sequences of these constructs are shown on the left with the mutated region underlined. Values are means ± SEM, n = 3. Asterisks indicate different from wild-type pKLF4–145 treated with butyrate, *P < 0.05; **P < 0.01.

 
Three putative Sp1-binding elements were identified on the proximal portion of the KLF4 promoter and were designated as Sp1–1, Sp1–2, and Sp1–3 in this report (Fig. 5B). To determine the involvement of each individual Sp1 binding site in butyrate-stimulated KLF4 gene transcription, three mutated KLF4 constructs were generated from pKLF4–145, and were designated as pKLF4–145-mtSp1–1 (M1), pKLF4–145-mtSp1–2 (M2), and pKLF4–145-mtSp1–3 (M3) (Fig. 5C). Mutation of the Sp1–1 or Sp1–3 site (M1 or M3) significantly decreased butyrate-stimulated KLF4 promoter activity by 54 ± 11 or 50 ± 8%, respectively. In contrast, KLF4 promoter activity induced by butyrate was almost completely abolished (80 ± 12% reduction) when the Sp1–2 site was mutated (M2) [Fig. 5C, P < 0.05, P < 0.01 compared with butyrate-treated cells possessing pKLF4–145 (w) DNA]. No additional reduction of promoter activities was observed in the double mutants, in which either Sp1–1 and Sp1–2, Sp1–2 and Sp1–3, or Sp1–1 and Sp1–3 were mutated, or in the triple mutant in which all three Sp1 sites were altered (data not shown). These results suggest that the Sp1–2 site on the KLF4 promoter is likely a putative butyrate-responsive domain.

Electrophoretic mobility shift assays (EMSA) were performed to determine whether this consensus Sp1-binding domain is the butyrate-response element on the KLF4 promoter. A synthetic double-stranded DNA containing the wild-type Sp1–2 site was used as a probe. Nuclear protein was extracted from HT-29 cells treated with or without butyrate. Four DNA-protein binding complexes (C1–C4) were detected when the wild-type Sp1–2 oligonucleotide probe was incubated with the nuclear extract (Fig. 6, lanes 2 and 7). These complexes were completely abolished by an excess amount of unlabeled oligonucleotide (lanes 3 and 8), but not by the mutated oligonucleotide (lanes 4 and 9), indicating the specificity of the DNA-protein interaction. The intensity of the C1 band appeared to be increased after butyrate treatment, and it was supershifted by the Sp1 antibody (lanes 5 and 10), suggesting that the C1 complex contained Sp1. These data indicate that the consensus Sp1-binding domain, located between -109 and -91 of the KLF4 promoter, contains a butyrate-response element.



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  		FIGURE 6 Electrophoretic mobility shift assay of DNA and protein interaction in HT-29 cells. Nuclear extracts from butyrate-untreated (lanes 1–5) or -treated (lanes 6–10) HT-29 cells were incubated with an end-labeled double-stranded oligonucleotide probe corresponding to the Sp1–2-binding element on the KLF4 promoter. Competition experiments were carried out in the absence (lanes 2 and7) or presence of a 50-fold molar excess of unlabeled wild-type (lanes 3 and 8), or mutated oligonucleotide (lanes 4 and 9). The DNA-protein binding complexes were indicated as C1, C2, C3, or C4. The DNA-protein complex (C1) was supershifted by the addition of Sp1 antibody in the reaction (SS, lanes 5 and 10).

 
Histone hyperacetylation is involved in transactivation of KLF4 gene by butyrate.

To determine the involvement of histone hyperacetylation in butyrate-mediated KLF4 expression, a specific histone deacetylase inhibitor (TSA) was examined for its ability to modulate cell growth and KLF4 expression in HT-29 cells. TSA exhibited physiologic properties similar to those of butyrate not only in inducing growth arrest of HT-29 cells (P < 0.05; Fig. 7A and B), but also in upregulating KLF4 mRNA expression (Fig. 7C, D, and E). The effects of TSA on cell growth were similar to those published previously (14).



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  		FIGURE 7 Effects of TSA on cell growth, cell cycle distribution, and KLF4 mRNA levels in HT-29 cells. (A) Cells were cultured with medium alone (control) or TSA (50 µg/L) for 0–5 d. Values are means ± SEM, n = 3. *Different from control, P < 0.05. HT-29 cells were cultured in the presence of medium alone (B) or 50 µg/L TSA (C) for 24 h, and the DNA content was determined by FACS analysis. The percentage (%) of cells in each cell cycle phase is shown. Values are means ± SEM, n = 4. The levels of KLF4 and GAPDH mRNA were determined by Northern blot analysis in (C) HT-29 cells treated with 50 µg/L TSA from 0 to 24 h; (D) cells treated with different concentrations of TSA, as indicated, for 8 h; and (E) cells were (+) or were not (-) pretreated with CHX (10 mg/L) for 2 h and then incubated with (+) or without (-) 50 µg/L TSA for 8 h.

 
The role of histone hyperacetylation in butyrate- or TSA-induced KLF4 gene expression was further examined in transfection studies. HT-29 cells were cotransfected with pKLF4–515 with or without histone deacetylase (HDAC1) DNAs. Butyrate and TSA transactivated pKLF4–515 activities, and these effects were attenuated by cotransfection with HDAC1 (Fig. 8). The inhibition of butyrate- or TSA-stimulated KLF promoter activity by HDAC was dose dependent; the highest reduction occurred with 2.0 µg HDAC1 DNA (P < 0.05 compared with cells without HDAC). These data are consistent with the effects of histone hyperacetylation in butyrate-induced KLF4 gene expression.



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  		FIGURE 8 Overexpression of HDAC1 attenuates transactivation of the KLF4 promoter by butyrate or TSA in HT-29 cells. HT-29 cells were transiently transfected with pKLF4–515 plasmid alone or cotransfected with increasing concentrations of HDAC1 (0.5–2.0 µg). Cells were then treated with 4 mmol/L butyrate or 100 µg/L TSA. The results are expressed as fold increases over control. Values are means ± SEM, n = 3. Asterisks indicate different from cells without HDAC1 in each group, *P < 0.05, **P < 0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
It has been documented both in vitro and in vivo that butyrate is capable of inducing growth arrest, differentiation, and apoptosis in a variety of colorectal cancer cells. The molecular mechanisms by which butyrate mediates these functions are not well understood. In the present study, we showed that butyrate treatment in HT-29 cells resulted in cell cycle arrest and growth inhibition. These results are consistent with those reported by other laboratories (4,5). Previously, we demonstrated that overexpression of KLF4 in HT-29 cells led to growth inhibition and cell cycle arrest at the G1 phase (9,15). These data indicated that KLF4 and butyrate had similar physiologic properties. Furthermore, our current study showed that butyrate stimulated KLF4 mRNA expression in a time- and dose-dependent manner. These results are similar to those reported by Hinnebusch et al. (16). Recently, our laboratory also showed that KLF4 promoted butyrate-induced differentiation and apoptosis in HT-29 cells, suggesting a potential role of KLF4 in the maturation process of the colonic epithelium (unpublished observation). Together, these studies indicate that KLF4 may function as a downstream target of butyrate that mediates its biological properties in the colon. Previously, we reported that the expression of KLF4 is decreased in colon cancer tissue and that the adenomatous polyposis coli (APC) gene regulates KLF4 gene expression (9,17). It is possible that the effects of butyrate on KLF4 expression in the normal colonic mucosa may be different from these observed in HT 29 cells, possessing mutated p53, ras, and APC genes. This possibility is currently under investigation in our laboratory.

In recent years, considerable effort was made to identify the genes that mediate butyrate-induced growth arrest in the gastrointestinal tract. Butyrate upregulates p21WAF1/Cip1 mRNA expression in a p53-independent manner and induces growth inhibition of colon cancer cells (18,19). In the current study, we showed that butyrate treatment in HT-29 cells increased KLF4 mRNA levels and transactivated KLF4 promoter activities. The induction of KLF4 gene expression by butyrate appears to be independent of p53 and occurs in an immediate-early fashion, because HT-29 cells do not possess wild-type p53 protein, and cycloheximide pretreatment has no effect on butyrate-induced KLF4 mRNA expression. Previously, Zhang et al. (11) reported that KLF4 transactivated p21WAF1/Cip1 gene expression in NIH 3T3 cells during growth arrest. Together, these data suggest that butyrate may induce KLF4 gene expression which then upregulates p21WAF1/Cip1 levels and results in growth arrest.

Although butyrate (C4) is the most abundant SCFA in the gastrointestinal tract, a variety of other SCFA are also present in the colonic lumen, including the branched and unbranched chain fatty acids with different carbon lengths. The effect of different SCFA on KLF4 mRNA expression and its promoter activity was also evaluated in this report. Our data show that unbranched SCFA (C3-C7) exhibited properties similar to those of C4 in inducing KLF4 expression, although butyrate appears to have the greatest effect among them. In contrast, iso-C4 had no effect on KLF4 gene expression. These data are consistent with previous studies showing that C4, but not iso-C4 induced the expression of genes encoding subunits of cytochrome c oxidase to promote differentiation and apoptosis of HT-29 cells (20). Our studies also support the hypothesis that the effect of SCFA on KLF4 gene expression is related to their structure, but not to their chain length. As stated above, SCFA is the primary and the preferred energy source of normal colon epithelial cells, and its concentration in the colonic lumen may be affected by different dietary components. Further work examining the effect of dietary fiber on KLF4 mRNA expression in vivo is warranted.

The molecular mechanism by which butyrate modulates KLF4 gene expression has not been defined previously. Transfection studies and the EMSA demonstrated that one of three consensus Sp1 binding domains on the proximal portion of the KLF4 promoter contained a butyrate-response element. However, mutation of the Sp1–2 site on the KLF4 promoter attenuated but did not completely abolish the effect of butyrate, indicating that butyrate may also interact with other domains on the promoter. Alternatively, butyrate treatment may elicit other regulatory mechanisms, in addition to the direct interaction with DNA cis elements, to transactivate the KLF4 promoter.

Post-translational modification of nucleosomal histone was shown recently to play an important role in the regulation of eukaryotic gene expression (21). The most extensively studied post-translational modifications of nucleosomal histones are acetylation and deacetylation, and reported data have suggested a potential role of histone deacetylases in the development of human cancers (22). In this report, we showed that trichostatin (TSA), a highly specific and sensitive inhibitor of histone deacetylase, exhibited physiologic properties similar to those of butyrate in inducing cell cycle arrest and growth inhibition of HT-29 cells. Furthermore, TSA upregulated KLF4 mRNA levels to the same extent as butyrate, and cotransfection with HDAC1 significantly reduced transcriptional activation of the KLF4 promoter by butyrate or TSA. These data are consistent with the potential involvement of histone acetylation in butyrate-induced KLF4 expression. This conclusion is supported by previous reports demonstrating that sodium butyrate induced hyperacetylation of histone through inhibition of the histone deacetylase (23,24). Whether other mechanisms contribute to butyrate-promoted KLF4 gene expression awaits further investigation.

The colonic epithelium undergoes rapid turnover in which cell production is usually balanced by cell extrusion. Within a few days, colonic epithelial cells derived from a stem cell population at the base of the crypt migrate from a region of proliferation in the basal three quarters of the crypt to the surface and assume a more differentiated phenotype. During malignant transformation of colonic mucosa, the region of proliferation expands, and differentiation is abnormal or disrupted. We showed previously that KLF4 inhibited proliferation of colonic epithelial cells (10,25). In the current study, we demonstrated that KLF4 also played a role in butyrate-promoted differentiation of the colonic mucosa. In a previous report, we showed that KLF4 mRNA levels are significantly decreased in the colon cancer tissue (9). It is plausible that induction of KLF4 gene expression (as occurs with butyrate stimulation) leads to cell cycle arrest and differentiation of the colonic epithelium. The downregulation of KLF4 allows cells to enter the cell cycle and leads to uncontrolled cell growth and tumor development.

In summary, results from the current study suggest that KLF4 may function as a downstream target of butyrate that mediates its growth arrest, differentiation, and apoptosis in the colon. Furthermore, transactivation of the KLF4 promoter by butyrate appears to be mediated through interaction with a Sp1-binding domain, but this process may also involve histone acetylation.


   FOOTNOTES
 
1 Supported in part by United States Public Health Services grants DK061376 to Z.Y.C., and CA82593 to C.-C.T. Back

3 Abbreviations used: C4, butyrate; CHX, cycloheximide; EMSA, electromobility shift assay; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GKLF/KLF4, gut-enriched Krüppel-like factor/Krüppel-like factor 4; HDAC1, histone deacetylase 1; iso-C4, iso-butyric acid; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Sp1, stimulatory protein 1; TSA, trichostatin A. Back

Manuscript received 4 November 2003. Initial review completed 2 December 2003. Revision accepted 21 January 2004.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

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