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15-Deoxy-^12,14 Prostaglandin J₂ Up-Regulates Krüppel-Like Factor 4 Expression Independently of Peroxisome Proliferator-Activated Receptor by Activating the Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Signal Transduction Pathway in HT-29 Colon Cancer Cells

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

Received May 19, 2005; accepted August 2, 2005

	Abstract

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15-Deoxy-^12,14 prostaglandin J₂ (15d-PGJ₂) is a natural ligand for the peroxisome proliferator-activated receptor (PPAR) that exhibits antiproliferative activity in colon cancer cells, but its mechanism of action is still poorly understood. In this study, we showed that Krüppel-like factor 4 (KLF4) is one of the downstream effectors of 15d-PGJ₂. Treatment of HT-29 cells with 15d-PGJ₂ resulted in up-regulation of both KLF4 mRNA and protein expression, and these increases were also observed in other colon cancer cell lines. Down-regulation of KLF4 expression by small interfering RNA (siRNA) targeting KLF4 reduced 15d-PGJ₂-mediated G₁ phase arrest, suggesting that KLF4-mediated function of 15d-PGJ₂. The effect of 15d-PGJ₂ on KLF4 expression seems not to involve its nuclear receptor PPAR, in that our data show that:1) KLF4 gene promoter does not contain putative PPRE sequence, 2) 15d-PGJ₂ rapidly activates extracellular signal-regulated kinase (ERK) and induces KLF4 mRNA expression, 3) KLF4 is induced by 15d-PGJ₂ but not by rosiglitazone, a synthetic PPAR ligand, and 4) 15d-PGJ₂ is unable to stimulate PPAR-dependent promoter activity in the absence of cotransfected PPAR. Moreover, 15d-PGJ₂-mediated KLF4 mRNA expression was blocked by 2'-amino-3'-methoxyflavone (PD98059) or 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126), two ERK kinase MAP inhibitors, whereas the phosphoinositol-3 kinase inhibitors wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) had no such effect. Furthermore, KLF4 induction by 15d-PGJ₂ occurred only in signal transducer and activator of transcription 1 (STAT1)-expressing, not in STAT1-knockout cells. Together, these results suggest that 15d-PGJ₂-induced growth inhibition of colon cancer cells is mediated, at least in part, through up-regulation of KLF4 expression. This induction is unlikely to be mediated through the PPAR receptor but may involve the mitogen-activated protein kinase kinase/ERK pathway and is STAT1-dependent.

KLF4, also known as gut-enriched Krüppel-like factor (GKLF), is a member of the Krüppel-like zinc finger transcription factor family and expresses extensively in the epithelial cells of the gastrointestinal tract (Dang et al., 2000; Shie et al., 2000b; Bieker, 2001). In cultured fibroblasts, the levels of KLF4 mRNA expression were associated with the growth state of cells. Serum deprivation or contact inhibition increases KLF4 expression, and its level decreased in response to serum stimulation (Shields et al., 1996). Forced expression of KLF4 in colon cancer cells resulted in inhibition of DNA synthesis and cell growth (Chen et al., 2000; Dang et al., 2003). These effects seem to be mediated through activating of p21^WAF1/Cip1 expression and/or suppressing of cyclin D1 or ODC (ornithine decarboxylase) gene promoter activity (Shie et al., 2000a; Zhang et al., 2000; Chen et al., 2002a). More recently, the importance of KLF4 in controlling the G₁/S cell cycle checkpoint and preventing mitotic entry after DNA damage is further demonstrated by using small interfering RNA (siRNA) (Yoon et al., 2003). Altogether, these results indicate that KLF4 and 15d-PGJ₂ possess similar antiproliferative activity and that KLF4 could be one of the mediators of 15d-PGJ₂ in colon cancer cells.

In this study, we examined the effect of 15d-PGJ₂ on KLF4 expression in HT-29 colon cancer cells. Our results show that that 15d-PGJ₂ inhibits proliferation of HT-29 cells and induces up-regulation of KLF4 mRNA and protein levels. The induction of KLF4 by 15d-PGJ₂ seems not to involve in nuclear receptor PPAR but is dependent on the activation of MEK/ERK pathway. These data may provide a novel mechanism governing the antiproliferative property of 15d-PGJ₂ in colon cancer cells.

	Materials and Methods

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Reagents. 15d-PGJ₂ and rosiglitazone were purchased from Cayman Chemicals (Ann Arbor, MI). PD98059, U0126, wortmannin, and LY294002 were obtained form Calbiochem (La Jolla, CA). [-³²P]dCTP and [³H]thymidine were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Other chemical reagents were obtained from Sigma (St. Louis, MO) unless mentioned specifically.

Cell Culture and Treatment. The human colon carcinoma cell lines HT-29, DLD-1, Caco-2, RKO, and HCT116 were obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained in McCoy's growth medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 µg/ml streptomycin, and 100 U/ml penicillin (Invitrogen, Carlsbad, CA) in an atmosphere of 95% air and 5% CO₂ at 37°C. Cells in exponential growth were plated before serum starvation for 24 h. Serum-starved cells were then exposed to 15d-PGJ₂ for the indicated times in serum-free media. Mouse fibroblasts CD+ and CD-cell lines, derived from wild-type and STAT1 knockout mice, respectively, were kindly provided by Dr. David Levy (New York University Medical Center, New York, NY).

Cell Viability Assay and Measurement of DNA Synthesis. The cells were treated with 15d-PGJ₂ (1–10 µM) in serum-free media after starving for 18 h. Cell viability was determined by MTT assay, and was presented as percentage of control. For the assay, 20 µl of MTT dye was directly added to cell culture. After 2 h, media was removed and cells were lysed with 800 µl of dimethyl sulfoxide. The absorbance at 540 nm was read with a spectrophotometer. For DNA synthesis assay, the cells were labeled with [³H]thymidine during the final 2 h. After labeling, the cells were washed with ice-cold phosphate-buffered saline, fixed in 10% trichloroacetic acid. The cells were lysed in 0.5 N NaOH, and incorporated [³H]thymidine was measured by a liquid scintillation counter and presented as percentage of control.

Western Blot Analysis. After treatment, cells were washed twice with ice-cold phosphate-buffered saline, scraped, and pelleted by centrifugation (200g). Cell pellets were then lysed in the standard radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing protease inhibitors. Protein concentrations were determined by Bio-Rad assays, and 25 to 50 µg of protein from each sample was separated on the 10% SDS-polyacrylamide gel. After electrophoresis, the proteins were transferred to nitrocellulose membranes. The membrane was probed with antibodies against phosphor-ERK1/2, ERK1/2, phosphor-Akt, Akt, and MKP1. Polyclonal KLF4 antibody was used at 1:500 dilution. Protein levels were detected using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence following the manufacturer's instruction (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The concentration of each protein was quantified by scanning blot images, using Scion Image software (Scion Corporation, Frederick, MD), and expressed as density (-fold increase) relative to that of the control (designated as 1.0). The expression level of ERK1/2 kinase was calculated from at least five independent experiments.

RNA Isolation and Northern Blot Analysis. Total RNA was isolated by the STAT-60 method following the manufacturer's instructions (Leedo Medical Laboratories, Inc., Houston, TX). RNA samples (20 µg) were denatured and size-fractionated by electrophoresis on 1.1% agarose-formaldehyde gels and transferred onto Hybond-N nylon membranes (GE Healthcare). Hybridization was then performed overnight at 42°C using a 450-base piar Apa-PstI fragment of the human KLF4 DNA that was radiolabeled with [³²P]dCTP (Random primer labeling kit was from Roche Diagnostics, Indianapolis, IN). Blots were washed with 2x sodium chloride/sodium phosphate/EDTA and 0.1% SDS, followed by 0.1x sodium chloride/sodium phosphate/EDTA and 0.1% SDS. All blots were stripped and reprobed with PPAR or PPAR cDNA probe. PPAR and PPAR expression plasmids were kindly provided by Dr. Bert Vogelstein (The Johns Hopkins University, Baltimore, MD). The blots were also stained with ethidium bromide to verify RNA loading. The level of mRNA expression was quantified by scanning blot images, using Scion Image software and expressed as density (-fold increase) relative to that of the control (designated as 1.0). The results were calculated from four to five independent experiments.

Plasmids, Transfections, and Luciferase Assays. The reporter plasmids pAOXPPREluc, containing the PPRE of the rat acyl-CoA oxidase gene, and pAOXBluc, containing only the basal promoter of the gene, were kindly provided by Dr. Takashi Osumi (Himeji Institute of Technology, Department of Life Science, Graduate School of Science, Hyogo, Japan). All transfection experiments were performed using LipofectAMINE reagent (Invitrogen) according to the manufacturer's instructions.

For 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 -galactosidase activity for each sample.

Preparation of siRNA and Transfection. KLF4 siRNA was synthesized by Dharmacon (Boulder, CO) and targeted the coding region 86–92 relative to the start codon of human KLF4 gene (GenBank accession number XM_047517). The sequences of the 21-nucleotide sense and antisense RNA are as follows: 5'-GACCGAGGAGUUCAACGAUUU-3' (sense) and 5'-AUCGUUGAACUCCUCGGUCUU-3' (antisense). The control siRNA duplex was also generated based on the sequence of an unrelated protein. For transient transfections, HT-29 cells were seeded at a density of 50 to 70% on plates with a 60-mm diameter in McCoy's medium containing 10% FBS. On the following day, transfections were performed with the use of LipofectAMINE Plus Reagent (Invitrogen), according to the manufacturer's recommended protocol. Cells were harvested 48 h after transfection by manual scraping in lysis buffer. The relative expression of endogenous KLF4 was monitored by Western blot analysis using a polyclonal antibody against KLF4, as described previously.

Statistical Methods. Results are expressed as means ± S.E.M., and a two-way analysis of variance with Dunnett's post-test (InSat Software; GraphPad Software, San Diego, CA) was performed. Differences between group means are analyzed by Student's t test and are considered significant at P < 0.05.

	Results

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15d-PGJ₂ Inhibits Cell Growth and Reduces DNA Synthesis in HT-29 Cells. HT-29 cells are a well-characterized cell culture model for human colon cancer. Thus, in this study, we used this cell line to examine the effect of 15d-PGJ₂ on cell growth and DNA synthesis. HT-29 cells were cultured in the medium containing 10% FBS in the presence or absence of 15d-PGJ₂ for the indicated intervals, and cell growth was measured by MTT assay. For comparison, rosiglitazone, a synthetic thiazolidinedione PPAR ligand, was also used in this experiment. Figure 1A shows that the growth of HT-29 cells was inhibited by 15d-PGJ₂ in a time-dependent manner, whereas rosiglitazone had no significant effect. To confirm that this inhibitory effect was specific for 15d-PGJ₂, we examined the effect of 15d-PGJ₂ on cell growth and DNA synthesis under the serum-free condition by MTT and [³H]thymidine incorporation assays. As shown in Fig. 1, B–E, treatment of HT-29 cells with 15d-PGJ₂, but not rosiglitazone, resulted in marked inhibition of both cell growth and DNA synthesis. The growth-inhibitory effect of 15d-PGJ₂ was also dose-dependent (Fig. 1, D and E). These results indicated that the effect of 15d-PGJ₂ on cell growth was not mediated through other serum factors.

View larger version (22K): [in this window] [in a new window]   Fig. 1. Effect of PPAR ligand 15d-PGJ2 or rosiglitazone on the growth of HT-29 cells. A, a time course of the effect of 15d-PGJ2 or rosiglitazone on the growth of HT-29 cells in the presence of serum. Cells were seeded at a density of 5 x 104 cell/well (12-well plates) in McCoy's medium with 10% FBS. After 24 h, the medium was replaced with fresh medium, and 15d-PGJ2 (10 µM) or rosiglitazone (10 µM) was added to cells. At the indicated times, cell viability was determined by MTT assay as described under Materials and Methods. Each value represents the mean ± S.E.M. of triplicate measurements, and the same experiment has been repeated three times with similar results. B–E, the effect of 15d-PGJ2 or rosiglitazone on cell growth and DNA synthesis in HT-29 cells in the absence of serum. Cells were seeded at a density of 5 x 104 cell/well (12-well plates) and starved for 18 h in serum-free medium. The serum-deprived cells were then treated with vehicle (as a control), 15d-PGJ2 (10 µM), or rosiglitazone (10 µM). After 24 h, cell viability and DNA synthesis were measured by MTT and [3H]thymidine incorporation assay as described under Materials and Methods. Each value represents the mean ± S.E.M. of triplicate measurements, and the same experiment has been repeated at least three times with similar results. *, P < 0.05 compared with control.

15d-PGJ₂ Up-Regulates Levels of KLF4 mRNA and Protein Expression in HT-29 and Other Colon Cancer Cells. KLF4 has recently been shown to function as a negative regulator of cell proliferation. To explore whether KLF4 is involved in the 15d-PGJ₂-mediated growth inhibition, KLF4 mRNA and protein levels were examined in 15d-PGJ₂-treated or untreated cells by Northern and Western blot analysis. As shown in Fig. 2, A and B, expression of KLF4 mRNA was induced rapidly by 15d-PGJ₂ in time- and dose-dependent manners. The increase in KLF4 mRNA levels was first detected as early as 1 h after treatment, the maximal effect was observed at 4 h, and then the KLF4 concentration was gradually decreased. To determine whether the effects of 15d-PGJ₂ on KLF4 expression could be observed in other colon cancer cells, KLF4 mRNA levels in additional cell lines were examined. As illustrated in Fig. 2C, increases in KLF4 mRNA levels were observed not only in HT-29 but also in DLD1, CaCo-2, RKO, and HCT116 cells after 15d-PGJ₂ treatment. Moreover, treatment with 15d-PGJ₂ also led to a time-dependent accumulation of KLF4 protein, the level of which peaked at 4 h (Fig. 2D).

View larger version (55K): [in this window] [in a new window]   Fig. 2. 15d-PGJ2 up-regulates levels of KLF4 mRNA and protein expression in HT-29 cells. A, HT-29 cells were grown in the absence of serum for 18 h and then incubated with vehicle alone (C) or with 10 µM 15d-PGJ2 (P) for the indicated time (0–24 h). Total RNA was prepared, and the expression of KLF4, PPAR, and PPAR mRNA was determined by Northern blot analysis. B, HT-29 cells were grown in the absence of serum for 18 h and then incubated with increasing concentrations (0–20 µM) of 15d-PGJ2 for 4 h. The expression of KLF4, PPAR, and PPAR mRNA was determined by Northern blot analysis. The blots stained with ethidium bromide, showing the presence of 18S and 28S RNA, were included to confirm equal RNA loading. C, Northern blot analysis of KLF4 mRNA expression in five human colon cancer cell lines in the presence (P) or absence (C) of 15d-PGJ2. D, HT-29 cells were incubated with either vehicle alone (-) or with 10 µM 15d-PGJ2 (+) for 2 to 24 h. The KLF4 and -tubulin protein levels were examined by Western blot analysis as described under Materials and Methods. The level of mRNA and protein expression was quantified, expressed as relative density to the control (-fold increase over control), and shown on the bottom of each gel.

The Induction of KLF4 mRNA by 15d-PGJ₂ Is Independent of PPAR in HT-29 Cells. By analyzing its promoter sequence (1200 base pairs upstream from the transcription starting site), KLF4 does not seem to contain a potential conserved consensus PPRE in this region (Biology Workbench, version 3.2; http://workbench.sdsc.edu). These data suggest that 15d-PGJ₂-induced KLF4 up-regulation is most likely to be mediated through a PPAR-independent mechanism. To test this idea, we examined the effect of cycloheximide (CHX), an inhibitor of de novo protein synthesis, on the expression of KLF4 mRNA in presence of 15d-PGJ₂. HT-29 cells were preincubated with CHX (100 µg/ml) for 30 min and then treated with 15d-PGJ₂. As shown in Fig. 3A, CHX alone has no effect on KLF4 mRNA level. In contrast, the combination of 15d-PGJ₂ and CHX resulted in a greater increase in KLF4 mRNA level than that observed after treatment with 15d-PGJ₂ alone. In addition, these increases are also time-dependent (Fig. 3B, top and bottom). These data indicate that early de novo protein synthesis may not be required for 15d-PGJ₂-induced KLF4 up-regulation. Next, we compared the effect of 15d-PGJ₂, rosiglitazone, and Wy-14,643, a ligand of PPAR, on KLF4 mRNA induction. HT-29 cells were treated with these ligands for 4 h, and then KLF4 mRNA levels were determined by Northern Blot analysis. As shown in Fig. 3C, KLF4 mRNA levels increased significantly by treatment with 15d-PGJ₂, but not with either rosiglitazone or Wy-14,643, suggesting a different signaling mechanism involved in these ligands.

View larger version (41K): [in this window] [in a new window]   Fig. 3. The induction of KLF4 mRNA by 15d-PGJ2 is independent of PPAR in HT-29 cells. A and B, induction of 15d-PGJ2-mediated KLF4 mRNA expression is independent of new protein synthesis. HT-29 cells were preincubated with (+) or without (-) cycloheximide (CHX) for 30 min and then treated with or without (-) 15d-PGJ2 for 4 h. Total RNA was isolated, and expression of KLF4 mRNA was determined by Northern blot analysis (A). HT-29 cells were preincubated with CHX for 30 min and then treated with vehicle or 15d-PGJ2 (10 µm) for the previous periods as indicated. The levels of KLF4 mRNA expression were examined by Northern blot analysis of 20 µg of total RNA with 32P-labeled KLF4 probe (B). C, 15d-PGJ2 specifically induced expression of KLF4 mRNA in HT-29 cells. Cells were serum-starved for 18 h and treated with vehicle (Me2SO, 0.2%), 15d-PGJ2 (10 µM), rosiglitazone (10 µM), or Wy-14,643 (10 µM) for 4 h. Total RNA was isolated and subjected to Northern blot analysis. D, blocking of PPAR receptor does not antagonize induction of KLF4 mRNA by 15d-PGJ2. HT-29 cells were preincubated with various concentrations of GW9662, as indicated, for 30 min and then treated with 15d-PGJ2 for 4 h. Total RNA was isolated and levels of KLF4 RNA expression were determined by Northern blot analysis. D, cotransfection with PPAR expression vector is necessary to observe pAOXPPREluc activity in 15d-PGJ2-stimulated HT-29 cells. HT-29 cells were transfected with pAOXBluc or pAOXPPREluc, or together with PPAR expression vector, as shown. 15d-PGJ2 (10 µM) was then added to cells 24 h after transfection. Luciferase activity was measured 48 h after transfection. All values reported above are the average of three transfections carried in duplicate ± S.E.M. and were normalized to untreated cells transfected with the respective reporter gene construct alone.

"Loss-of-function" mutations in PPAR have recently been identified in a subset of colorectal tumors (Sarraf et al., 1999). To examine whether the endogenous PPAR in HT-29 cells is responsive to 15d-PGJ₂, we performed transient transfection experiments. A report construct containing the PPRE of the rat acyl-CoA oxidase gene (pAOXPPREluc) or a control plasmid (pAOXBluc) was transfected into HT-29 cells. As illustrated in Fig. 3D, in the absence of PPAR, treatment with 15d-PGJ₂ has no effect on PPRE reporter activity. However, when pAOXPPREluc was cotransfected with PPAR expression vector into HT29 cells, a significant increase in PPRE reporter activity was detected, and the activity was increased further after treatment with 15d-PGJ₂ (Fig. 3D). These results suggest that endogenous PPAR in HT-29 cells may not be functional or is transcriptionally inactive, even though high level of its expression in these cancer cells has been reported (Sarraf et al., 1998). These data further support our hypothesis that 15d-PGJ₂-induced KLF4 expression is probably mediated through a PPAR-independent pathway in HT-29 cells.

15d-PGJ₂ Induces a Rapid and Transient Activation of ERK1/2 in HT-29 Cells. Activation of the ERK1/2 is generally related to growth stimulating actions of many growth factors. However, studies have indicated that growth inhibition can also result from ERK1/2 activation (Tsukada et al., 2001; Lahlou et al., 2003). To understand the signaling mechanisms involving in 15d-PGJ₂-induced KLF4 expression, we examined the effect of 15d-PGJ₂ on the activity and expression ERK1/2, the downstream substrates of MEK1/2, in HT-29 cells. Cells were treated with 15d-PGJ₂ (10 µM) in a serum-free medium for 2, 5, and 60 min, the levels of phosphorylated ERK1/2 and total ERK1/2 proteins were determined by using antibodies specific to the phosphorylated ERK1/2 (activated forms) and to total ERK1/2. As shown in Fig. 4, A and B, 15d-PGJ₂, but not rosiglitazone, markedly induced a transient activation of p-ERK1/2. The increase in phosphorylated ERK1/2 was first observed at 2.5 min after addition of 15d-PGJ₂ and reached a peak (a 3.5-fold increase) at 5 min before returning to basal levels at 60 min. Treatment of HT-29 cells with increasing concentrations (1.0–10 µM) of 15d-PGJ₂ for 5 min also resulted in a dose-dependent activation of p-ERK1/2 (Fig. 5C). No change in total ERK1/2 protein level was detected in these studies.

View larger version (39K): [in this window] [in a new window]   Fig. 4. 15d-PGJ2 induces a rapid and transient activation of ERK1/2 in HT-29 cells. HT-29 cells were grown in the absence of serum for 18 h and then stimulated with 15d-PGJ2 (A) or rosiglitazone (B) for different times (2.5–60 min) or with increasing concentrations of 15d-PGJ2 (1.0–10 µM) for 5 min (C). The cell lysates were isolated, and phosphorylated ERK1/2 and total ERK1/2 were determined by Western blot analysis using antibodies specific to phosphorylated ERK1/2 and total ERK1/2. D, HT-29 cells were treated with 10 µM 15d-PGJ2 for the indicated times. The cell lysates were isolated, and MKP1 protein levels were determined by Western blot analysis. Left, a representative result from three independent experiments is shown. Right, the density of specific band was scanned and quantified. The ratio of phosphorylated ERK1/2 to total ERK1/2 or MKP1 to -tubulin protein expression level is shown. Quantitative data shown on the right, represented means ± S.E.M. for three replicate determinations, and expressed as -fold induction over control. *, P < 0.05 versus control.

View larger version (42K): [in this window] [in a new window]   Fig. 5. MEK/ERK pathway is involved in 15d-PGJ2-mediated KLF4 mRNA expression. The serum-starved HT-29 cells were preincubated with increasing concentrations of PD98059 or U0126, as indicated, for 30 min. The cells were then stimulated with 15d-PGJ2 (10 µM) for 5 min. The cell lysates were isolated and phosphorylated ERK1/2 (p-ERK1/2) and total ERK1/2 (ERK1/2) was determined by Western blot analysis (A and C). The serum-starved HT-29 cells were precultured in the presence of various concentrations of PD98059 or U0126, as indicated, for 30 min. The cells were then treated with 15d-PGJ2 (10 µM) for 4 h. The total RNA was isolated and subjected to Northern blot analysis of KLF4 mRNA expression (B and D). Quantitative data shown on the right represent means ± S.E.M. of five replicate determinations and are expressed as -fold induction over control. In A and B, *, P < 0.05 compared with cells treated with 15d-PGJ2 but without PD98059. In C and D, *, P < 0.05; **, P < 0.01 compared with cells treated with 15d-PGJ2 but without U0126.

The activity of ERK1/2 is tightly regulated by phosphorylation and dephosphorylation processes. MKP1, a dual specific phosphatase, is induced by many stimuli that activate ERK1/2 and subsequently inactivate ERK1/2. To confirm the findings above, the level of MKP1 expression in HT-29 cells was evaluated after treatment with 15d-PGJ₂. As illustrated in Fig. 4D, the MKP1 was rapidly induced by15d-PGJ₂ in a time-dependent manner, supporting the involvement of ERK1/2 activation and inactivation in 15d-PGJ₂-mediated effects.

MEK/ERK, but Not PI-3 Kinase Pathway, Is Involved in 15d-PGJ₂-Mediated KLF4 mRNA Expression. To determine whether the MEK/ERK signaling pathway is involved in 15d-PGJ₂-induced KLF4 mRNA expression, the effects of specific inhibitors (PD98059 and U0126) of MEK, an upstream kinase of ERK, on KLF4 mRNA levels were further tested. HT-29 cells were pretreated with increasing concentrations of PD98059 (0.3–30 µM) or U0126 (0.2–20 µM) for 30 min followed by 4 h of 15d-PGJ₂ stimulation. ERK1/2 activities and KLF4 mRNA levels were determined by Western and Northern blot analysis. As shown in Fig. 5, A–D, pretreatment with PD98059 or U0126 dose-dependently inhibited 15d-PGJ₂-induced ERK1/2 phosphorylation and KLF4 mRNA up-regulation. These results suggest that the MEK/ERK pathway is engaged in the induction of KLF4 mRNA by 15d-PGJ₂.

It has been reported that the activation of the MEK/ERK signaling pathway is regulated by PI-3 kinase (Pandey et al., 1999). To examine the involvement of PI-3 kinase, we examined the expression level of phosphorylated Akt, one of the downstream targets of PI3-kinase, in response to 15d-PGJ₂ treatment. As shown in Fig. 6A, Akt was activated by 15d-PGJ₂ in a time-dependent manner; the highest level was observed at 5 min. However, pretreatment of HT-29 cells with increasing concentrations of a PI-3 kinase inhibitor, wortmannin (10–1000 nM) or LY294002 (0.25–25 µM), had no effect on 15d-PGJ₂-mediated KLF4 mRNA expression (Fig. 6, B and C). These data indicate that 15d-PGJ₂ activates PI-3 kinase in HT-29 cells, but the activation may not involve 15d-PGJ₂-induced KLF4 mRNA expression.

View larger version (46K): [in this window] [in a new window]   Fig. 6. PI3-kinase is not responsible for 15d-PGJ2-induced KLF mRNA expression. A, HT-29 cells were serum-starved for 18 h and stimulated with 15d-PGJ2 (10 µM) for the indicated times. Total lysates were isolated and phosphorylated Akt (p-Akt) and total Akt (Akt) were determined by Western blot analysis. B and C, the serum-starved cells were preincubated with increasing concentrations of wortmannin (10–1000 nM) or LY294002 (0.25–25 µM) for 30 min and then treated with 15d-PGJ2 (10 µM) for 4 h. The total RNA was isolated and expression of KLF4 mRNA was determined by Northern blot analysis. Quantitative data shown on the right represent means ± S.E.M. for five replicate determinations and are expressed as -fold induction over control. *, P < 0.05 versus control.

15d-PGJ₂-Mediated KLF4 mRNA Expression Requires STAT1 and Its Phosphorylation. STAT1, a member of the signal transducers and activators of transcription (STAT) family, has been shown to play a role in interferon--induced KLF4 expression (Chen et al., 2002b). To define the role of STAT1 in 15d-PGJ₂-mediated KLF4 activation, we examined KLF4 mRNA levels in two mouse fibrosarcoma cell lines, CD+ (STAT1^+/+) and CD-(STAT1^-/-), derived from wild-type and STAT1 knockout mice, respectively. As shown in Fig. 7A, 15d-PGJ₂ induced a time-dependent increase in KLF4 mRNA levels in the STAT1-expressing (STAT1^+/+) but not in STAT1-knockout (STAT1^-/-) cells. These data suggest that STAT1 is necessary for 15d-PGJ₂-mediated KLF4 mRNA up-regulation. Moreover, the effect of 15d-PGJ₂ on STAT1 tyrosine phosphorylation was examined by Western blot analysis. HT-29 cells were treated with 15d-PGJ₂ (10 µM) for various periods of times (0–60 min), and cell lysates were collected and analyzed for the presence of phosphorylated STAT1 using a mouse monoclonal antibody that specifically recognized Tyr-701-phosphorylated STAT1 (p-STAT1). As demonstrated in Fig. 7, B and C, 15d-PGJ₂ also induced tyrosine phosphorylation of STAT1 in HT-29 cells.

View larger version (39K): [in this window] [in a new window]   Fig. 7. 15d-PGJ2-mediated KLF4 mRNA expression requires STAT1 and its phosphorylation. A, STAT1 is necessary for 15d-PGJ2-mediated KLF4 mRNA up-regulation. STAT1+/+/+ and STAT1-/- cells, derived from the wild-type and the STAT1 knockout mice, respectively, were incubated with 15d-PGJ2 (10 µM) for the indicated times and levels of KLF4 mRNA expression were determined by Northern blot analysis. B and C, 15d-PGJ2 induces STAT1 phosphorylation on tyrosine. HT-29 cells were grown in the absence of serum for 18 h and stimulated with 15d-PGJ2 (10 µM) for the indicated times. The levels of STAT1 phosphorylated on Tyr-701 (p-STAT1) and total STAT1 (STAT1) were detected by Western blot analysis using antibody specifically recognizing STAT1 phosphorylated on Tyr-701 or total STAT1. Quantitative data of relative phosphorylation level as presented on blot A was shown on B represent means ± S.E.M. for five replicate determinations and are expressed as -fold induction over control. *, P < 0.05 versus control.

View larger version (37K): [in this window] [in a new window]   Fig. 8. Down-regulation of KLF4 expression by siRNA reduces 15d-PGJ2-mediated G1 phase arrest in HT-29 cells. Effect of KLF4 siRNA on the expression of KLF4 protein in HT-29 cells. Cells were transfected with KLF4 siRNA or control siRNA, and cell lysates were analyzed for the level of KLF4 protein by Western blot analysis as described under Materials and Methods. A, a representative result from three independent experiments is shown. B, the density of each band was scanned and quantified as indicated. Results are means ± S.E.M. for three replicate determinations. *, P < 0.05 compared with control. C, effect of KLF4 siRNA on 15d-PGJ2-mediated cell cycle progression in HT-29 cells. HT-29 cells were treated with Me2SO (control) or with 15d-PGJ2, or transfected with KLF4 siRNA followed by treatment with 15d-PGJ2, and the percentage of distribution of cells in G1, S, and G2/M were determined by fluorescence-activated cell sorting analysis as described under Materials and Methods. Similar results were observed in a duplicate analysis.

	Discussion

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The MAP kinase superfamily is composed of several subfamilies, including ERK, c-Jun N-terminal kinase, and p38. Their activation is regulated by two upstream protein kinases: a MAP kinase kinase kinase, such as Raf, and a MAP kinase kinase. The MAP kinase kinase that regulates ERK is called MEK or ERK kinase (Chang and Karin, 2001). Many different stimuli can activate the protein kinase cascade that activates ERK. Although the activation of the ERK signaling pathway is linked to cell proliferation and tumorigenic activity, several recent studies have indicated that ERK activation can also lead to growth arrest by induction of p21^WAF/Cip1 or p27^kip1 cyclin-dependent kinase inhibitor (Hu et al., 1999; Pages et al., 1999). In the present study, 15d-PGJ₂ induces rapid and transient activation of ERK1/2 and triggers expression of MKP1, which may form an inhibitory feedback loop to fine-tune the activity of ERK1/2, resulting in a net transient activation of the ERK1/2 signal. Moreover, activation of ERK1/2 signal seems to be essential for upregulation of KLF4 mRNA expression, because two specific MEK inhibitors, PD98059 and U0126, attenuate KLF4 mRNA induction in a dose-dependent manner. Although PI3-kinase has also been reported to activate the ERK pathway by several stimuli, our data show that the induction of KLF4 by 15d-PGJ₂ is not affected by wortmannin and LY294002, suggesting that the PI3-kinase pathway may not be involved in up-regulation of KLF4 by 15d-PGJ₂. Further studies are required to identify the exact molecular mechanism by which ERK activation induces the expression of KLF4 mRNA.

The transcription factor STAT1 plays an important role in growth arrest. It has been reported that STAT1 can directly induce expression of many key proteins, including p21^WAF/Cip1, involved in controlling the cellular processes of growth arrest (Chin et al., 1996). Our previous studies have demonstrated that up-regulation of KLF4 expression by interferon- is dependent on STAT1 and its phosphorylation (Chen et al., 2002b). In this study, we find that 15d-PGJ₂ induces rapid tyrosine phosphorylation of STAT1 in HT-29 cells and that induction of KLF4 expression by 15d-PGJ₂ is observed only in STAT1-expressing (STAT1^+/+) but not in STAT1 knockout (STAT1^-/-) cells. These data suggest that STAT1 and its phosphorylation are essential for 15d-PGJ₂-mediated effects, although detailed mechanisms remain to be addressed.

PPAR ligands regulate the expression of several genes associated with cell growth and differentiation in a ligand- and cell type-specific manner. Both PPAR-dependent and -independent mechanisms have been reported. For example, troglitazone, but not other PPAR ligands, induces early growth response-1 (Erg-1) gene expression independently of PPAR in HCT116 colon cells (Baek et al., 2003). In the present study, our data also indicate that PPAR may not be required in 15d-PGJ₂-induced KLF4 expression. These data include: 1) KLF4 does not contain putative PPRE sequences in its promoter region; 2) 15d-PGJ₂ rapidly activated ERK and induced KLF4 mRNA expression, which does not require new protein synthesis (Clay et al., 2001; Gupta et al., 2003); 3) KLF4 expression is selectively induced by 15d-PGJ₂ but not rosiglitazone, another specific ligand for PPAR; and 4) 15d-PGJ₂ cannot stimulate PPAR-dependent promoter activity in the absence of cotransfected PPAR. These results are consistent with report from Takeda et al. (2001) showing that the effect of 15d-PGJ₂ on c-fos gene expression in vascular smooth cells is mediated through a nongenomic or PPAR-independent mechanism. Although the possibility of a PPAR-dependent induction of KLF4 by 15d-PGJ₂ in other cells cannot be excluded, our current study indicates that, in HT-29 cells at least, the effect of 15d-PGJ₂ on KLF4 expression does not involve its nuclear receptor PPAR.

The interaction between KLF4 and PPAR has not been explored previously. As illustrated in Fig. 2, the induction of KLF4 by 15d-PGJ₂ peaked at 4 h, and the effect was then gradually decreased. In contrast, 15d-PGJ₂ treatment in HT-29 cells resulted in transient decreases in PPAR levels between 2 and 8 h, and these decreases in PPAR expression correspond to the increases in KLF4 concentration in these cells. These data suggest that KLF4 may inhibit PPAR expression. It is noteworthy that the level of PPAR mRNA also increased when the expression of PPAR was inhibited (Fig. 2, A and B). PPAR has been shown to function as a potent inhibitor of transcriptional activity of PPAR gene (Shi et al., 2002). More studies will be required to elucidate the association between KLF4 and the family of PPAR receptors.

In conclusion, we have demonstrated that KLF4 is one of the down-stream effectors of 15d-PGJ₂ in HT-29 colon cancer cells. 15d-PGJ₂ induces the expression of KLF4 in both time- and dose-dependent manners and through a PPAR-independent mechanism that requires ERK signaling, and it is STAT1-dependent. Several studies have reported that cyclopentenone prostaglandins, especially 15d-PGJ₂, are not formed in a biologically relevant level in vivo (Bell-Parikh et al., 2003; Powell, 2003). In the present study, the amount of 15d-PGJ₂ required to induce growth inhibition and KLF4 expression in colon cancer cells was significantly higher than the concentration of intracellular 15d-PGJ₂ formed under physiological condition, as reported by Bell-Parikh et al. (2003). However, the level of 15d-PGJ₂ in the microenvironment of the colonic mucosa has not yet been determined. The physiological relevance of our current observation, therefore, will warrant further evaluation. Nevertheless, our data may provide a novel mechanism for the antitumorigenic properties of 15d-PGJ₂.

	Footnotes

 
This work was supported in part by United States Public Health Services grants DK061376 (to Z.Y.C.) and CA82593 (to C.-C.T.).

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.014944.

ABBREVIATIONS: PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator-activated receptor response element; 15d-PGJ₂, 15-deoxy-^12,14 prostaglandin J₂; PD98059, methoxyflavone; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; KLF4, Krüppel-like factor 4; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide; CHX, cycloheximide; Wy-14,643, pirinixic acid; ERK, extracellular signal-regulated kinase; PI-3, phosphoinositide-3; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; STAT, signal transducer and activator of transcription; MAP, mitogen-activated protein; GW9662, 2-chloro-5-nitro-N-phenylbenzamide.

Address correspondence to: Dr. Zhi Yi Chen, Section of Gastroenterology, Boston University School of medicine EBRC X-513, 650 Albany Street, Boston, MA 02118. E-mail: zhiyi.chen{at}bmc.org

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