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. 2008 Nov;125(3):397–407. doi: 10.1111/j.1365-2567.2008.02847.x

Prostaglandin E2 and Krüppel-like transcription factors synergistically induce the expression of decay-accelerating factor in intestinal epithelial cells

Jinyi Shao 1, Vincent W Yang 2, Hongmiao Sheng 1
PMCID: PMC2669143  PMID: 18435741

Abstract

The decay-accelerating factor (DAF) prevents the intestinal mucosa from bystander killing by complement. Prostaglandin E2 (PGE2) induces the expression of DAF that may protect the tumour environment from complement attack. In the present study, we demonstrate synergistic actions of PGE2 and two Krüppel-like factors (KLFs), which are zinc finger-containing transcription factors, in DAF regulation. Overexpression of KLF4 and KLF5 robustly induced transcriptional activity of the DAF promoter. In combination, PGE2 and either KLF4 or KLF5 increased the expression of DAF in a synergistic fashion. Moreover, cyclooxygenase (COX-1 and COX-2) enzymes, KLF4/5 and DAF protein were coordinately expressed in normal intestinal mucosa as well as in intestinal neoplasm. In radiation-injured mouse intestine, COX-1 was rapidly induced and remained at relatively high levels. While KLF5 was quickly elevated after irradiation, KLF4 exhibited a delayed increase. Interestingly, levels of DAF increased gradually following the induction of COX-1 and KLFs. Mimicking the circumstances in vivo, coexpression of both COX and KLFs resulted in a synergistic or additive induction of DAF transcription in intestinal epithelial cells. Our data suggest that COX-derived PGE2 may collaborate with KLF4/5 to regulate the activation of the complement system and exert diverse effects on the intestinal epithelium.

Keywords: decay-accelerating factor, gut, Krüppel-like factor, transcription

Introduction

Intestinal epithelial cells synthesize and secrete complement components into the lumen, protecting them from invading microorganisms.1–3 Intestinal mucosal damage and inflammation increase complement activation in the gastrointestinal tract.4,5 To avoid bystander killing by complement, it is critical that complement activation on intestinal epithelial cells is precisely controlled by the expression of complement-regulatory proteins. The decay-accelerating factor (DAF/CD55), a glycoprotein, which is anchored to the plasma membrane of the cell by a glycosyl-phosphatidylinositol linkage, prevents the assembly and accelerates the dissociation of C3 convertases; therefore, it blocks the activation of the complement system via both the classic and alternative pathways.6 As a result, DAF protects intestinal epithelium from autologous complement injury. The critical roles of DAF in the gastrointestinal tract are evident by reports that individuals with a genetic DAF deficiency (Inab phenotype) are susceptible to a number of intestinal disorders.7 Expression of DAF is markedly increased at the intestinal mucosa of patients with inflammatory bowel disease.8 In intestinal neoplasia, DAF is thought to protect the tumour environment from complement attack, evading immune surveillance. DAF is strongly expressed on tumour cells and is deposited in large amounts within the tumour matrix; levels of DAF are elevated in at least 75% of colorectal carcinomas.9 Patients with colon cancer who have tumours expressing high levels of DAF have a significantly worse survival than patients whose tumours have low DAF expression.10 It is apparent that DAF plays various roles in the gastrointestinal tract, protecting the intestinal mucosa from injury as well as promoting intestinal neoplasia.

Cyclooxygenase (COX) and its derived prostaglandins exert a wide range of biological and pathological effects on the gastrointestinal tract, including intestinal barrier, electrolyte transportation, motility, inflammation and neoplasia.11,12 Prostaglandin E2 (PGE2)plays critical roles in protecting the intestinal surface from mucosal damage. The underlying mechanism includes mucus synthesis and secretion, mucosal bicarbonate secretion, restitution and epithelial cell survival.11,13–15 On the other hand, PGE2 is a key mediator for the proneoplastic actions of COX-2, promoting proliferation, migration and metastasis of colorectal carcinoma cells.16–18 Cumulative evidence suggests that PGE2 modulates key aspects of immunity.19 PGE2 inhibits the proliferation of T cells, reduces the production of interleukin-2, and induces CD4+ CD8+ thymocytes to undergo apoptosis; therefore, is thought to be immunoinhibitory.20,21 In a recent study, Holla et al.22 have shown that PGE2 induces the expression of DAF in colon cancer cells, suggesting that prostaglandins may inhibit activation of the complement pathway and allow malignant cells to evade complement-mediated cytotoxicity. They have further demonstrated that a cyclic AMP-responsive element (CRE) site within the DAF promoter is responsible for PGE2 induction of DAF transcription. However, PGE2 appears to stimulate the activity of the DAF promoter in spite of the mutation of the CRE site, suggesting that regulation of DAF expression by PGE2 involves additional mechanisms.

Krüppel-like factors (KLFs) are evolutionarily conserved zinc finger-containing transcription factors with diverse regulatory functions in cell proliferation and differentiation.23,24 KLFs can be transcriptional activators or repressors, binding to a similar DNA sequence that contains either a CACCC homology or a GC-rich region.25–28 Two KLFs are highly expressed in the intestinal epithelium: KLF4 (gut-enriched KLF or GKLF) is localized in differentiated villi29 and KLF5 (intestine-enriched KLF or IKLF) is found in proliferative crypt compartments.30–32 Numerous genes that relate to cell proliferation, differentiation, adhesion and tissue homeostasis have been identified as targets of KLF4.33 In the present study, we sought to determine additional mechanisms governing the regulation of DAF by COX/PGE2 in intestinal epithelial cells. We found that PGE2 induction of DAF transcription involves the activity of both KLF4 and KLF5. Moreover, PGE2 and KLFs increase the expression of DAF in a synergistic fashion. COX-1 was colocalized with KLF4 and KLF5 in normal intestinal epithelium, whereas COX-2 and KLF5 were coordinately expressed in colorectal carcinoma. In a total body irradiation mouse model, the induction of DAF closely followed the increase of COX-1 and KLFs. Given the distinct localization and function of KLF4 and KLF5 in the intestinal epithelium; our results suggest that COX-derived PGE2 may collaborate with KLF4 and KLF5 to protect the intestinal mucosa from injury or promote intestinal neoplasia by way of modulating the activation of the complement system.

Materials and methods

Patient samples

Tumour sections of hereditary nonpolyposis colorectal cancer (HNPCC) were obtained from the National Cancer Institute as previously described.34

Animal samples

All animals were treated in a manner which complied with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and was approved by the Institutional Animal Care and Use Committee of Indiana University (Protocol #2833 to Dr Hongmiao Sheng). C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were housed in an animal-holding room under controlled light, temperature and humidity. Whole-body irradiation was carried out in a Gamacel 40 caesium irradiator at 0·85 cGy/min and a total dose of 12 Gy.14 Mice were killed at the indicated time-points after irradiation. Total RNA was extracted from the intestinal mucosa for real-time polymerase chain reaction (PCR) assays.

Cell culture and reagents

LS-174T cells were purchased from the American Type Culture Collection (Manassas, VA) and maintained in McCoy’s 5A medium containing 10% fetal bovine serum. LS-174T-TR4 cells, which express the Tet repressor, were provided by Dr van de Wetering35 (Hubrecht Laboratory, Utrecht, the Netherlands). For establishment of a stable cell line, mouse KLF4 was cloned into the pcDNA4/TO (Tet-on) vector and transfected into LS-174T-TR4 cells. Stable clones were selected with blastcydine (100 μg/ml) and zeocin (150 μg/ml). Inducible expression of mouse KLF4 in LS-174T-i-mKLF4 cells was confirmed using reverse transcription (RT) PCR. The PGE2, 17-phenyl-trinor-PGE2, butaprost, sulprostone and PGE1 alcohol were purchased from Cayman Chemical (Ann Arbor, MI).

RNA extraction and Northern blot analysis

The extraction of total cellular RNA was carried out as previously described.36 RNA samples (20 μg per lane) were separated on formaldehyde–agarose gels and blotted onto nitrocellulose membranes. The blots were hybridized with cDNA probes labeled with [α-32P]dCTP by random primer extension (Stratagene, La Jolla, CA). After hybridization and washes, the blots were subjected to autoradiography.

Real-time RT-PCR

The method for real-time quantitative PCR or TaqMan technique (Applied Biosystems, Foster City, CA) has been described previously.37 Primer/probe sets were designed using Invitrogen D-lux™ Designer and purchased from Invitrogen (Carlsbad, CA). The sequences of the primer/probe sets were based on messenger RNA (mRNA) sequences of the mouse COX-1 (NM_008969), mouse COX-2 (NM_011198), mouse KLF4 (NM_010637) and mouse KLF5 (NM_009769). One-step RT-PCR was performed with 10 ng RNA for both target gene and endogenous controls. Duplicate CT values were analysed in Microsoft Excel using the comparative CT (ΔΔCT) method as described by the manufacturer (Applied Biosystems).

Immunoblot analysis

Cells were lysed for 30 min in lysis buffer (50 mm glycerol phosphate, 10 mm HEPES pH 7·4, 1% Triton X-100, 70 mm NaCl, 1 mm Na3VO4, 25 μg/ml aprotinin, 10 μg/ml leupeptin and 1 μm phenylmethylsulphonyl fluoride) and sonicated for 20 seconds. Clarified whole cell lysates were denatured and fractionated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA), which were incubated with the antibodies indicated and developed by the enhanced chemiluminescence system (Amersham, Arlington Heights, IL). Anti-DAF antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-KLF4 and anti-KLF5 antibodies were purchased from Abcam (Cambridge, MA).

Immunohistochemistry

Human and mouse tissues were fixed in 10% formalin, paraffin-embedded and cut into sections. Endogenous peroxidase activity was quenched by incubating the sections in 0·3% hydrogen peroxide for 20 min at room temperature. After the sections were blocked in 1·5% normal serum in phosphate-buffered saline for 1 h, primary antibody was added to the sections and incubated overnight at 4°. The sections were then incubated with biotinylated secondary antibody and ABC-AP reagent (Vectorstain ABC-AP kit, Vector Laboratories, Burlingame CA). Peroxidase activity was demonstrated by applying 3,3′-diaminobenzidine containing 0·02% hydrogen peroxide for 10 min. The sections were counterstained with toluidine blue O. Anti-COX-1 (1 : 500), anti-COX-2 (1 : 250) and anti-DAF (1 : 50) antibodies were purchased from Santa Cruz Biotechnology. Anti-KLF4 (1 : 100) and anti-KLF5 (1 : 100) antibodies were purchased from Abcam.

Transient transfection and luciferase assay

A fragment of the human DAF promoter (−733 to +22) was kindly provided by Dr Edward Medof of Case Western Reserve University.38 The fragment was inserted into the pGL3 vector (Promega, Madison, WI) and named pGL3-DAFA. The pGL3-DAFA was digested with KpnI plus XhoI, KpnI plus SacI, or KpnI plus SmaI and religated to yield pGL3-DAFB (−424 to +22), pGL3-DAFC (−205 to +22) and pGL3-DAFD (−160 to +22). The human DAF promoter fragment without the CRE site (−66 to +22) was amplified from pGL3-DAFA vector using PCR with primers 5′-AGAGCCCCAGCCCAGACCCCGCCCAAA-3′ (forward) and 5′-AGCGAGTTG CAGTAAGTCAGAA-3′ (reverse) and inserted into pGL3 (pGL3-DAFE). The assay to determine transcriptional activity was described previously.39 Briefly, cells were transfected with 0·5 μg DAF reporter plasmid along with 0·1 μg of the pRL-SV plasmid, containing the Renilla luciferase gene (Promega), using the FuGENE 6 procedure (Roche, Indianapolis, IN) as described in the manufacturer’s protocol. Transfected cells were lysed at the indicated times for luciferase assay. Firefly and Renilla luciferase activities were measured using a Dual-Luciferase Reporter assay system (Promega) and a luminometer. Firefly luciferase values were standardized to the Renilla values. The active protein kinase A (PKA) expression plasmid, pMT3-KLF4 and pMT3-KLF5 expression vectors that were used in cotransfection assays were described previously.37,40,41

Data analysis

All statistical analyses were performed on a personal computer with the statview 5.0.1 software (SAS Institute Inc., Cary, NC). Analyses between two groups were determined using the unpaired Student’s t-test. Differences with a P value of < 0·05 were considered statistically significant.

Results

PGE2 induction of DAF expression

Previous study has demonstrated that PGE2 induces the expression of DAF in colon cancer LS-174T cells.22 Similar results were reproduced here; addition of 0·5 μm PGE2 strongly increased the expression of both DAF mRNA and protein levels (Fig. 1a and b, respectively). To determine the mechanism by which PGE2 induced DAF expression, we investigated the role of PGE2 signalling in DAF transcription. The nucleotide sequence of a 755-base-pair (−733 to +22) DAF 5′-flanking region was cloned into the pGL3 luciferase reporter vector (pGL3-DAFA). After LS-174T cells were transiently transfected with the DAF promoter reporter vector, PGE2 treatment rapidly increased the transcriptional activity of the DAF promoter and resulted in an approximately 15-fold increase in luciferase activity (Fig. 1c). To determine the signalling mechanism by which PGE2 induced DAF transcription, E-prostanoid receptor (EP) agonists were employed to stimulate the DAF promoter. The EP4 signalling pathway appeared to mediate PGE2-induced DAF transcription, because only the EP4 agonist, PGE1 alcohol, mimicked the action of PGE2 to stimulate the DAF promoter. The EP4 acts through the increase in levels of cyclic AMP (cAMP) and activation of PKA. Consequently, ectopic expression of active PKA reproduced the similar effect of PGE2 and increased the activity of the DAF promoter by over 15-fold (Fig. 1c). These results suggest that PGE2-induced DAF transcription was mediated by the EP4/cAMP/PKA pathway in LS-174T cells. The cAMP/PKA signalling pathway stimulates the expression of target genes often through a conserved CRE. A CRE site has been previously mapped at nucleotides −71 to −65 within the DAF promoter.42 To determine the role of the CRE site in PGE2 induction of DAF transcription, a series of deletion mutants of DAF promoter reporter plasmids was constructed (Fig. 1d). Transient transfection assay demonstrated that PGE2 significantly increased the activity of pGL3-DAFA, -B, -C and -D. In contrast, deletion of the CRE site completely abolished PGE2-induced DAF transcription, as was the case with the pGL3-DAFE construct (Fig. 1e). It was also noted that serial deletion of the sequences between nucleotides −733 and −71 gradually reduced the PGE2-induced activation of the DAF promoter, suggesting that additional mechanisms were involved in PGE2 induction of DAF transcription.

Figure 1.

Figure 1

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Prostaglandin E2 (PGE2) induction of decay-accelerating factor (DAF) expression in LS-174T cells. (a) Northern blot analysis of DAF messenger RNA. LS-174T cells were serum-deprived for 48 hr and then treated with 0·5 μm PGE2 for the indicated times. Total RNA was extracted and levels of DAF were detected using Northern blot analysis. (b) Western blot analysis of DAF protein. LS-174T cells were serum-deprived for 48 hr and then treated with 0·5 μm PGE2 for the indicated times. Cellular protein was extracted and levels of DAF were determined using Western blot analysis. Results shown are representative of three separate experiments. (c) PGE2 induction of DAF transcription. LS-174T cells were transiently transfected with pGL3-DAFA reporter vector (see the Materials and methods section). Cells were treated with the stated agents for 6 hr before being harvested for luciferase assays (V = ethanol, E2 = 0·5 μm PGE2, EP1 = 0·5 μm 17-phenyl-trinor-PGE2, EP2 = 0·5 μm butaprost, EP3 = 0·5 μm sulprostone and EP4 = 0·5 μm PGE1 alcohol). In the cotransfection assay, LS-174T cells were transfected with pGL3-DAFA along with empty vector (Ve) or active protein kinase A expression vector (PKA). Cells were lysed 24 hr after the transfection. Firefly and Renilla luciferase activities were measured and standardized. The mean ± SD of Renilla-adjusted luciferase values performed in quadruplicate are plotted. *P<0·05. Luciferase assays in all figures were performed at least three times independently. (d) Construction of deletion mutants of DAF promoter reporters. Deletion mutants of the DAF promoter were cloned into pGL3-basic reporter vector. Inline graphic: CACCC; Inline graphic: CRE; ▪: TATA. (e) Roles of the cAMP-response element (CRE) site in PGE2 induction of DAF transcription. LS-174T cells were transiently transfected with deletion mutants of the DAF promoter pGL3-DAFA, -B, -C, -D and -E. PGE2 (0·5 μm) was added 6 hr before harvest. Firefly and Renilla luciferase activities were measured and standardized. Plotted is the mean ± SD of Renilla-adjusted luciferase values performed in quadruplicate. *P<0·05.

KLF4 induction of DAF transcription

To determine other cis-elements within the DAF promoter that were activated by PGE2, we analysed the nucleotide sequence of the DAF promoter. Eight CACCC motifs were found within the DAF promoter, which are known binding sites for KLFs, suggesting the potential involvement of KLFs in DAF transcription. To test whether KLF4 regulated DAF transcription, mutants of DAF promoter reporter plasmids, in which CACCC motifs were gradually deleted (Fig. 1d), along with a KLF4 expression vector were cotransfected into LS-174T cells. Overexpression of KLF4 robustly increased DAF transcription from the full-length promoter (Fig. 2a). Transcriptional activity of the KLF4 was associated with the presence of CACCC motifs; deletion of CACCC motifs resulted in a gradual reduction of KLF4-induced DAF promoter activity. The induction of DAF transcription by KLF4 was completely attenuated on pGL3/DAFD and E reporter vectors, which contained no CACCC element.

Figure 2.

Figure 2

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Role of Krüppel-like factor 4 (KLF4) in prostaglandin E2 (PGE2) induction of decay-accelerating factor (DAF). (a) KLF4 stimulation of DAF transcription. LS-174T cells were transiently transfected with deletion mutants of the DAF promoters pGL3-DAFA, -B, -C, -D and -E along with empty pMT3 vector (V) or pMT3-KLF4 expression vector (KLF4) for 24 hr. Firefly and Renilla luciferase activities were measured and standardized. The mean ± SD of Renilla-adjusted luciferase values performed in quadruplicate are plotted. *P<0·05. (b) PGE2 increased levels of KLF4 protein. LS-174 cells were serum-deprived for 48 hr and then treated with 0·5 μm PGE2 for the indicated times. Cellular protein was extracted and levels of KLF4 were detected using Western blot analysis. Results shown are representative of three independent experiments. (c) PGE2 activation of the KLF4 promoter. LS-174T cells were transiently transfected with KLF4 promoter reporter plasmid (pGL-KLF4). PGE2 (0·5 μm) was added 6 hr before harvest. Firefly and Renilla luciferase activities were measured and standardized. The mean ± SD of Renilla-adjusted luciferase values performed in quadruplicate are plotted. *P<0·05.

We next assessed whether PGE2 regulated the expression of KLF4 in LS-174T cells. Immunoblot analysis revealed that PGE2 treatment modestly increased the levels of KLF4 protein in LS-174T cells (Fig. 2b). Moreover, PGE2 induced KLF4 expression at the transcriptional level; PGE2 increased the activity of the KLF4 promoter approximately fourfold in LS-174T cells (Fig. 2c). These results suggest that PGE2 may stimulate DAF transcription through increasing the expression of KLF4.

PGE2 and KLF4 induced DAF transcription in a synergistic fashion

The KLF4 is constitutively expressed in the intestinal epithelium. On the other hand, PGE2 is secreted by a variety of cell types in the gut. Therefore, PGE2 and KLF4 may collaboratively regulate DAF transcription in intestinal epithelial cells. LS-174T cells were transiently transfected with DAF promoter reporter vectors along with empty vector or KLF4 expression plasmid. PGE2 was added 24 hr after the transfection. Either exposure to PGE2 or expression of KLF4 robustly increased the activity of the DAF promoter. However, in combination, PGE2 and KLF4 synergistically induced the transcription of the DAF (Fig. 3). The synergy between PGE2 and KLF4 was abrogated when all CACCC elements within the DAF promoter were deleted because only the PGE2 effect was observed in pGL3-DAFD-transfected LS-174T cells. Removal of both CACCC and CRE elements completely abolished the stimulatory effects of KLF4 and PGE2 on DAF transcription; neither PGE2 nor KLF4 stimulated the activity of the pGL3-DAFE promoter.

Figure 3.

Figure 3

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Prostaglandin E2 (PGE2) and Krüppel-like factor 4 (KLF4) synergistically induced decay-accelerating factor (DAF) transcription. LS-174T cells were transiently transfected with deletion mutants of the DAF promoters pGL3-DAFA, -B, -C, -D and -E along with empty vector (V) or KLF4 expression vector (KLF4) for 24 hr. Vehicle (−) or 0·5 μm PGE2 (+) was added 6 hr before harvest. Firefly and Renilla luciferase activities were measured and standardized. The mean ± SD of Renilla-adjusted luciferase values performed in quadruplicate are plotted. *P<0·05.

It was of importance to determine whether induction of DAF transcription by KLF4 truly increased the expression of DAF protein. LS-174T cells were transfected with a Tet-on system.35 Stable clones were selected by blastcydine and zeocin and named LS-174T-i-mKLF4 cells, which expressed doxycycline-inducible mouse KLF4. Addition of doxycycline increased levels of KLF4 mRNA in three LS-174T-i-mKLF4 clones, as noted by RT-PCR. Levels of DAF protein were coordinately elevated when KLF4 was induced by doxycycline (Fig. 4a). As demonstrated in Fig. 4(b), LS-174T-i-mKLF4 clone 3 was treated with both doxycycline and PGE2. Addition of either doxycycline or PGE2 increased the levels of DAF protein; however, the presence of both doxycycline and PGE2 synergistically induced the expression of DAF protein. Furthermore, doxycycline and PGE2 synergistically induced the transcription of the DAF as well, as noted by transient transfection assays (Fig. 4c). As expected, EP4 signalling mediated PGE2-induced DAF transcription as well as the synergy between PGE2 and KLF4 (Fig. 4d).

Figure 4.

Figure 4

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Krüppel-like factor 4 (KLF4) and prostaglandin E2 (PGE2) induction of decay-accelerating factor (DAF). (a) Establishment of LS-174T-i-mKLF4 cell line. Mouse KLF4 was cloned into the pcDNA4/TO (Tet-on) vector and transfected into LS-174T-TR4 cells. Stable clones were selected with blastcydine (100 μg/ml) and zeocin (150 μg/ml). RNA and protein were extracted from three clones (1, 2 and 3) after addition of doxycycline (1 μg/ml) for 24 hr. Levels of mouse KLF4 were evaluated using reverse transcription–polymerase chain reaction, and DAF protein was determined by Western analysis. (b) PGE2 and KLF4 synergistically increased DAF expression. LS-174T-i-mKLF4 clone #3 was grown in serum-free medium for 24 hr and then treated with 1 μg/ml doxycycline (Dox), 0·5 μm PGE2 (E2), or both doxycycline and PGE2 for the indicated times. Levels of DAF and β-actin proteins were analysed by Western blot. Results shown are representative of three separate experiments. (c) PGE2 and KLF4 synergistically increased DAF transcription. LS-174T-i-mKLF4 cells were transfected with pGL3-DAFA for 24 hr and then vehicle (V) or 1 μg/ml doxycycline (Dox) was added. Cells were incubated for an additional 24 hr. PGE2 (+) or vehicle (−) was added into culture media 6 hr before harvest. Firefly and Renilla luciferase activities were measured and standardized. The mean ± SD of Renilla-adjusted luciferase values performed in quadruplicate are plotted. *P<0·05. (d) Roles of EP4 in the synergy of PGE2 and KLF4. LS-174T-i-mKLF4 cells were transiently transfected with pGL3-DAFA reporter vector and treated with vehicle (V) or 1 μg/ml doxycycline (Dox) for 24 hr. Treatment was added 6 hr before harvesting cell lysates (V = ethanol, E2 = 0·5 μm PGE2, EP1 = 0·5 μm 17-phenyl-trinor-PGE2, EP2 = 0·5 μm butaprost, EP3 = 0·5 μm sulprostone and EP4 = 0·5 μm PGE1 alcohol). Firefly and Renilla luciferase activities were measured and standardized. The mean ± SD of Renilla-adjusted luciferase values performed in quadruplicate are plotted. *P<0·05.

PGE2 and KLF5 synergistically induced DAF expression

Since KLF5 is also predominantly expressed in the intestine and shares the binding elements with KLF4, we next examined the roles of KLF5 in DAF expression. Ectopic expression of KLF5 significantly increased the transcription of the full-length DAF promoter (Fig. 5a). A significant reduction of luciferase activity was observed between pGL3-DAFB-transfected and pGL3-DAFC-transfected LS-174T cells, suggesting that the sequence between nucleotides −424 and −205 was critical for KLF5-induced DAF transcription. Exposure to PGE2 increased the levels of KLF5 protein in LS-174T cells, indicating the involvement of KLF5 in PGE2 stimulation of DAF transcription (Fig. 5b). Furthermore, KLF5 and PGE2 synergistically increased the activity of the DAF promoter; the synergy was attenuated in LS-174T cells transfected with pGL3-DAFD, in which all CACCC elements were deleted (Fig. 5c). To confirm that PGE2 and KLF5 regulated DAF expression in a collaborative manner, KLF5 was ectopically expressed in LS-174T cells. Although transient expression of KLF5 did not significantly alter the expression of DAF, a synergistic induction of DAF protein was observed when KLF5-expressing LS-174T cells were exposed to PGE2.

Figure 5.

Figure 5

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Roles of Krüppel-like factor 5 (KLF5) in prostaglandin E2 (PGE2) induction of decay-accelerating factor (DAF). (a) KLF5 stimulation of DAF transcription. LS-174T cells were transiently transfected with deletion mutants of the DAF promoters pGL3-DAFA, -B, -C, -D and -E along with empty pMT3 vector (V) or pMT3-KLF5 expression vector (KLF5) for 24 hr. Firefly and Renilla luciferase activities were measured and standardized. The mean ± SD of Renilla-adjusted luciferase values performed in quadruplicate are plotted. *P<0·05. (b) PGE2 increased KLF5 protein levels. LS-174 cells were serum-deprived for 48 hr and then treated with 0·5 μm PGE2 for the indicated times. Cellular protein was extracted and levels of KLF5 and β-actin were detected using Western blot analysis. (c) PGE2 and KLF5 induced DAF transcription in a synergistic manner. LS-174T cells were transiently transfected with deletion mutants of the DAF promoter pGL3-DAFA, -B, -C, -D and -E along with empty vector (V) or KLF5 expression vector (KLF5) for 24 hr. Vehicle (−) or 0·5 μm PGE2 (+) was added 6 hr before harvest. Firefly and Renilla luciferase activities were measured and standardized. The mean ± SD of Renilla-adjusted luciferase values performed in quadruplicate are plotted. *P<0·05. (d) PGE2 and KLF5 synergistically increased DAF expression. LS-174T cells were transiently transfected with pMT3 vector (−) or pMT3-KLF5 (+). Vehicle (−) or 0·5 μm PGE2 (+) were added 24 or 48 hr after transfection. Cellular protein was extracted 24 hr after the addition of PGE2. Levels of DAF protein were analysed by Western blot. Results shown are representative of three independent experiments.

Coordinated expression of COX, KLFs and DAF in the intestine

It was important to determine the possibility that DAF may be coordinately regulated by PGE2 and KLFs in vivo. The cellular localization of COX enzymes, KLFs and DAF was evaluated using immunohistochemistry. COX-1 is expressed in the normal intestine and localized in both epithelial cells and stromal cells,15,34,43 whereas COX-2 is not expressed in the normal gut but in colon cancers.44,45 Representative tissue sections from normal mouse small intestine were examined for COX-1, KLF4, KLF5 and DAF immunoreactivity. As demonstrated in Fig. 6, COX-1 was expressed in both epithelial cells and stromal cells of the intestine. While COX-1 immunoreactivity was detected in the proliferative crypt zone, strong positive staining was observed in the lamina propria of the intestine (Fig. 6a). The abundance of KLF4 immunoreactivity was less in the crypts and was increased toward the villus tip in the luminal one-half to two-thirds of the intestinal epithelium (Fig. 6b). In contrast, KLF5-positive epithelial cells were present within the lower and middle portions of the glands in normal intestinal mucosa (Fig. 6c). The immunoreactivity of both KLF4 and KLF5 was predominantly localized to the nuclei of the intestinal epithelial cells. The DAF protein was detected in the intestinal epithelium (Fig. 6d). Immunoreactivity of DAF was localized predominantly in the apical membrane of intestinal epithelial cells. Strong DAF staining was also detected in the stromal compartment of the intestine.

Figure 6.

Figure 6

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Immunohistochemistry of cyclooxygenase 1/2 (COX1/2), Krüppel-like factor 4/5 (KLF4/5) and decay-accelerating factor (DAF) in the intestine and colon cancer. Immunoreactivity of COX-1 (a, × 200), KLF4 (b, × 200), KLF5 (c, × 200) and DAF (d, × 200) in mouse small intestine. Immunostaining of COX-2 (e, × 400), KLF4 (f, × 400), KLF5 (g, × 400) and DAF (h, × 400) in hereditary non-polyposis colorectal cancers.

To determine the colocalization of COX-2, KLFs and DAF, tumours from patients with HNPCC were subjected to immunohistochemistry. We have previously demonstrated that COX-2 is overexpressed in HNPCC tumours.34 The COX-2 immunoreactivity was located in the cytoplasm of tumour cells, with a focal supranuclear enhancement staining pattern (Fig. 6e). KLF4 protein was present in carcinoma cells with nuclear localization (Fig. 6f). The expression of KLF5 was more abundant in tumour cells and was localized in both nuclei and cytoplasm (Fig. 6g). Whereas strong immunoreactivity of DAF was detected in the plasma membrane of tumour cells, DAF protein was also present in the stromal compartment (Fig. 6h).

Expression of COX, KLFs and DAF in radiation-injured intestine

To evaluate the expression of COX, KLFs and DAF in intestinal mucosal injury, a whole body irradiation mouse model was employed.46,47 C57BL/6J mice were irradiated at a total dose of 12 Gy. The intestinal mucosa was collected 6, 24, 48 and 72 hr after irradiation. Total RNA was extracted for real-time PCR assays. Expression of COX-1 was rapidly induced by the radiation injury and remained at high levels throughout the entire experiment. Whole body irradiation quickly stimulated KLF5 expression; an approximately fourfold induction of KLF5 mRNA was observed by 6 hr. The expression of KLF5 returned to basal levels by 24 hr after the irradiation. In contrast, induction of KLF4 occurred 48 hr after the radiation injury and continued to the end of the experiment. Interestingly, levels of DAF mRNA were gradually increased after the irradiation; about a sevenfold increase was detected by 72 hr (Fig. 7).

Figure 7.

Figure 7

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Expression of cyclooxygenase 1 (COX-1), Krüppel-like factor 4/5 (KLF4/5) and decay-accelerating factor (DAF) in radiation-injured intestine. C57BL/6J mice received a 12-Gy whole body irradiation. RNA was extracted from the intestinal mucosa at the indicated time-points (n = 3). Messenger RNA levels of COX-1, KLF4, KLF5 and DAF were determined by real-time reverse transcription–polymerase chain reaction and expressed as fold to levels in mice without irradiation (CTR). The expression of COX-1 (▴) and DAF (•) at all time-points was demonstrated as a cell line chart. Levels of KLF4 (dark grey bars) and KLF5 (light grey bar) that were significantly increased from control levels were expressed as a cell bar chart. Results shown are representative of two separate experiments.

To further demonstrate that the colocalization of the COX enzyme and KLFs may truly result in a synergistic induction of DAF, we forced LS-174T cells to express a variety of combinations of COX and KLF isoforms. LS-174T-i-mKLF4 cells were transfected with either COX-1 or COX-2 expression vector along with pGL3-DAFA reporter vector. Ectopic expression of either COX-1 or COX-2 stimulated the activity of the DAF promoter. Interestingly, induction of KLF4 by the addition of doxycycline synergistically enhanced COX-1- or COX-2-induced DAF transcription (Fig. 8a). Moreover, in a transient transfection assay, coexpression of KLF5 and COX-1/2 also induced DAF transcription in an additive or synergistic fashion (Fig. 8b).

Figure 8.

Figure 8

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Synergistic induction of decay-accelerating factor (DAF) transcription by cyclooxygenase (COX) enzymes and Krüppel-like factors (KLFs). (a) Synergy between KLF4 and COX-1/2. LS-174T-i-mKLF4 cells were transfected with pGL3-DAFA along with pcDNA3-COX1 (COX-1) or pcDNA3-COX-2 (COX-2) expression vectors for 24 hr. Vehicle (V) or 1 μg/ml doxycycline (Dox) was added 8 hr before harvest. Firefly and Renilla luciferase activities were measured and standardized. The mean ± SD of Renilla-adjusted luciferase values performed in quadruplicate are plotted. *P<0·05. (b) Synergy between KLF5 and COX-1/2. LS-174T cells were transiently transfected with pGL3-DAFA along with combinations of COX-1/2 and KLF5 expression vectors for 48 hr. Empty pMT3 and pcDNA3 vectors were used as control plasmids. Firefly and Renilla luciferase activities were measured and standardized. The mean ± SD of Renilla-adjusted luciferase values performed in quadruplicate are plotted. *P<0·05.

Discussion

Given the critical roles of DAF in the protection of host tissues from autologous complement injury, understanding of the regulatory mechanisms of DAF expression in the gut is of importance. The present studies demonstrate a novel mechanism by which COX/PGE2 and KLF transcription factors synergistically induce the expression of DAF in intestinal epithelial cells. It was critical to show that the synergistic actions between PGE2 and KLFs on DAF expression occur in vivo. We used immunohistochemistry to demonstrate the coordinated expression of COX-1/2 enzymes, KLF4/5 and DAF protein. It is known that COX-1 is constitutively expressed in normal intestine, while COX-2 is overexpressed in colorectal neoplasms.44 Although the localization of COX enzymes in the intestinal epithelium and neoplasia is inconsistent, COX-1 and COX-2 have been shown to be present in both epithelial cells and stromal cells.15,34,43,48 Since COX enzyme-derived PGE2 serves as both autocrine and paracrine lipid mediators to signal changes within their immediate environment, both epithelial and stromal cells may provide PGE2 to activate EP receptors on intestinal epithelial cells. Our results show that COX-1 was expressed in normal intestinal epithelial cells at the proliferating crypt zone, where KLF5 immunoreactivity was colocalized. COX-1 was also expressed in the stromal compartment of the villus, while KLF4 was localized in villous epithelial cells. In HNPCC, COX-2, KLF4/5 and DAF were expressed and predominantly localized in the epithelial cells of the tumours. These observations suggest that collaborative induction of DAF by COX-1 and KLF5 in crypt cells may have protective effects on intestinal stem cells. Coordinate expression of COX-1 and KLF4 in the villus, on the other hand, defends functionally matured enterocytes against complement attack, because PGE2 generated by stromal cells may enhance KLF4 transcriptional activity and increase DAF production in the epithelial cells of the villus. In colon cancers, simultaneous overexpression of COX-2/PGE2 and KLF5 in tumour cells is a potential mechanism for high levels of DAF in colorectal carcinomas.

Furthermore, we elucidated the dynamic expression of COX-1, KLF4/5 and DAF in radiation-injured mouse intestine. It has been shown that radiation injury increases the expression of COX-1 and the levels of PGE2, which promote crypt stem cell survival and proliferation.43 We found that levels of COX-1 and KLF5 were rapidly elevated in the intestinal mucosa following irradiation. KLF4, however, exhibited a delayed increase, while COX-1 remained at relatively high levels. The expression of DAF elevated gradually following the induction of COX-1 and KLF4/5, suggesting the possibility that induction of COX and KLF4/5 resulted in increased expression of DAF in vivo. Supporting our hypothesis, simultaneous overexpression of COX-1/2 enzymes and KLF4/5 in intestinal epithelial cells synergistically increased DAF transcription. These in vivo and in vitro studies provided physical evidence that endogenous prostaglandins generated by both COX-1 and COX-2 may collaborate with KLF4 or KLF5 to stimulate the expression of DAF in injured or transformed intestinal epithelium.

The PGE2 acts via specific transmembrane G protein-coupled receptors.49 EP1 receptor signals via generation of inositol triphosphate and increased intracellular Ca2+. Both EP2 and EP4 receptors are coupled to stimulatory G (Gs) proteins and signal through increased cAMP, whereas the EP3 receptor is coupled to inhibitory G (Gi) proteins which inhibit the generation of cAMP. Both EP2 and EP4 appear to be the predominant mediators of PGE2 trophic signalling in intestinal epithelial cells, protecting them from cell death and promoting their proliferation.14,17,50 PGE2 stimulates the transcription of a number of genes through the cAMP/PKA pathway where the CRE within the promoter plays critical roles.39,51 A recent study22 has reported that PGE2 induces the transcription of the DAF, and a CRE site mapped between nucleotides −383 and −205 within the DAF promoter is required for PGE2 activation of the DAF promoter. The present studies suggest that PGE2 induction of DAF transcription in LS-174T cells was mediated by EP4 signalling. A typical CRE/TATA structure is not found within the DAF promoter. However, a modified CRE site (TGACGCAG, nucleotides −71 to −65) which is followed by a TATA variant was essential for PGE2 induction of DAF transcription.38,42

The precise roles of DAF in intestinal biology and pathology are not completely understood. Intestinal trefoil factor induces DAF expression and enhances the protective activity against complement activation in intestinal epithelial cells.3 Sodium butyrate enhances complement-mediated cell injury via downregulation of DAF in colonic cancer cells.52 Recent studies demonstrate that genetic disruption of the DAF exacerbates ethanol-induced liver damage, strongly suggesting protective roles for DAF in the digestive system.53 PGE2 is involved in a variety of protective actions on intestinal mucosa, as well as in intestinal neoplasia.54 Our results suggest that induction of DAF expression may be an important mechanism by which COX/PGE2 exerts protective effects on the intestinal epithelium and neoplasia. We have previously demonstrated synergistic actions between PGE2 and a number of signalling pathways. PGE2 signalling collaborates with other oncogenic pathways to regulate the expression of growth factors and oncogenes in a synergistic fashion.55,56 PGE2 and the epidermal growth factor receptor signalling system synergistically stimulate the growth and migration of colon cancer cells.55 The present studies provide further evidence that PGE2 exerts its biological and pathological effects through stimulating other critical signalling pathways in a synergistic fashion.

KLF4 and KLF5 transcription factors are enriched in the epithelial cells of the intestine and play critical roles in proliferation and differentiation of intestinal epithelial cells. KLF4 and KLF5 often exhibit contrasting biological activities on intestinal epithelial growth.24,32 For example, radiation-induced cell cycle arrest at both the G1/S and G2/M transition points is associated with induction of KLF4, that stimulates the transcription of the p21waf1/Cip1, and suppresses the expression of cyclin D1and cyclin B1.57–59 In contrast, expression of KLF5 is associated with proliferation and growth of intestinal epithelial cells, stimulating the expression of cyclin D1, cyclin B1 and Cdc2, accelerating the progression of the cell cycle.41 A large body of studies indicates that KLF4 may have tumour-suppressive effects while KLF5 is pro-oncogenic.24,32 DAF may provide protective effects on both normal intestinal epithelium and intestinal neoplasia so it is not surprising that both KLF4 and KLF5 play similar roles in the regulation of DAF expression.

In summary, the present study links KLF transcription factors to COX/PGE2, presenting the collaborative actions between these two critical signalling systems in the intestinal epithelium. Synergistic induction of DAF by COX/PGE2 and KLF4/5 may provide rapid protection against complement attack upon intestinal mucosal injury. Persistent increase of DAF expression; on the other hand, protects transformed intestinal epithelial cells from bystander killing by complement, evading immune surveillance. These novel findings provide insight into the functional role of the COX/PGE2 system and KLF transcription factors in the gut and may contribute to new therapeutic strategies for a variety of intestinal disorders.

Acknowledgments

This work was supported in part by the National Institutes of Health Grants DK-065615 (H.S.), DK-52230 (V.W.Y.) and CA-84179 (V.W.Y.).

Abbreviations

COX

cyclooxygenase

CRE

cAMP responsive element

DAF

decay-accelerating factor

HNPCC

hereditary non-polyposis colorectal cancer

KLF

Krüppel-like factors

PG

prostaglandin

PKA

protein kinase A

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