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FEBS Lett. Author manuscript; available in PMC 2006 October 12. Published in final edited form as: doi: 10.1016/j.febslet.2004.10.079. | PMCID: PMC1599793 NIHMSID: NIHMS2408 |
All-trans retinoic acid inhibits proliferation of intestinal epithelial cells by inhibiting expression of the gene encoding Krüppel-like factor 5 Sengthong Chanchevalap,a Mandayam O. Nandan,a Didier Merlin,a and Vincent W. Yangab* a Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, 201 Whitehead Research Building, 615 Michael Street, Atlanta, GA, USA b Department of Hematology and Oncology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA Retinoids are known inhibitors of epithelial cell proliferation. Previous studies indicate that Krüppel-like factor 5 (KLF5) is a pro-proliferative transcription factor. Here, we examined the effect of all-trans retinoid acid (ATRA) on proliferation of the intestinal epithelial cell line, IEC6. Treatment of IEC6 cells with ATRA inhibited their proliferation due to G1 cell cycle arrest. This inhibition was correlated with a decrease in the levels of KLF5 mRNA and promoter activity. In contrast, constitutive expression of KLF5 in stably transfected IEC6 cells with a KLF5-expressing plasmid driven by a viral promoter abrogated the growth inhibitory effect of ATRA. Moreover, ATRA inhibited proliferation of several human colon cancer cell lines with high levels of KLF5 expression but not those with low levels of KLF5 expression. Our results indicate that KLF5 is a potential mediator for the inhibitory effect of ATRA on intestinal epithelial cell proliferation. Keywords: Cell cycle, Colon cancer, Krüppel-like factor 5, IKLF, Promoter, Pro-proliferative, Stable transfection Abbreviations: APL, acute promyelocytic leukemia; ATRA, all-trans retinoic acid; CMV, cytomegalovirus; DPBS, Dulbecco’s phosphate buffered saline; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin A; IEC, intestinal epithelial cells; KLF5, Krüppel-like factor 5; MAPK, mitogen-activated protein kinase; PI, propidium iodide; PKC, protein kinase C; RAR, retinoic acid receptor; RARE, retinoic acid response element; RXR, retinoic X receptor Krüppel-like factors (KLFs) belong to a class of zinc finger-containing transcription factors that exhibit homology to the general transcription factor Sp1 [ 1– 4]. Numerous studies indicate that KLFs play important roles in regulating cell proliferation and differentiation. Two KLFs are present at high levels in the intestinal epithelium although in different locations – KLF4 is expressed primarily in the terminally differentiated, post-mitotic epithelial cells [ 5, 6] and KLF5 in the proliferating crypt compartment [ 7, 8]. The two proteins also exhibit different biological properties – KLF4 is an inhibitor of cell proliferation [ 5, 9, 10] but KLF5 stimulates cell proliferation when overexpressed [ 11]. Thus, the two KLFs may function to coordinate proliferation of the intestinal epithelial cells (IEC). Retinoids, and other derivatives of vitamin A, are known to have important functions in regulating differentiation and proliferation. The effects of retinoids are mediated by nuclear receptors, such as retinoic acid receptors (RARs) and retinoic X receptors (RXRs) [ 12]. Because of their potent growth-suppressive effects, retinoids are used as a chemotherapeutic or chemopreventive agent in the treatment of a variety of disorders in preclinical and clinical settings [ 13, 14]. All- trans retinoic acid (ATRA) is one type of retinoid that binds preferentially to RARs, which form homodimers or heterodimers with RXRs and interact with retinoic acid response elements (RAREs) to mediate cellular response [ 15– 17]. ATRA is an effective chemotherapeutic drug for the treatment of acute promyelocytic leukemia (APL) [ 18]. In addition, ATRA exhibits an inhibitory effect in a number of colon cancer cell lines [ 19– 21]. Therefore, ATRA may potentially serve as an anti-proliferative agent in the treatment of cancerous cells. Previous studies have shown that overexpression of KLF5 stimulates proliferation of intestinal epithelial cells, IEC-18, and NIH 3T3 fibroblasts [ 11, 22]. Moreover, RARα interacts with KLF5 and RAR ligands modulate KLF5 transcriptional activity and consequent cellular proliferation [ 23]. In the current study, we aimed at determining whether ATRA may modulate proliferation of intestinal epithelial cells, IEC6, and whether such modulation may be dependent on KLF5. 2.1. Cell lines Non-transformed rat intestinal epithelial cells, IEC6, and the human colon cancer cell lines, DLD1, HT29 and RKO, were purchased from the American Type Culture Collection (Manassas, VA). The colon cancer cell line, Caco2-BBE, was previously described [ 24]. Stable transfection of IEC6 cells was performed by lipofection using pBK-CMV-KLF5-HA [ 7, 11]. Transfected cells were selected with 1 mg/ml G418 and pooled for experimentation. Cells were treated with media containing 10 μM ATRA or ethanol control for the stated periods. 2.2. Cell proliferation assay Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum (FBS), 0.1 U/ml insulin, and 1% streptomycin. Cells were seeded at a density of 100000 cells/6-well dish and treated with ATRA or ethanol for the stated periods of time. Each day, cells were collected, washed with Dulbecco’s phosphate-buffered saline (DPBS), trypsinized, and counted using a hemocytometer. 2.3. Luciferase assay The mouse −1.5 kb KLF5 promoter linked to a luciferase reporter in the pGL2-Basic vector was provided by Dr. A. Levine [ 25] and transfected into IEC6 cells by lipofection along with a Renilla-luciferase reporter as a control. Transfected cells were treated with ethanol or 10 μM ATRA for 3 days before being harvested for reporter assays using the Dual-Luciferase Reporter Assay protocol (Promega, Madison, WI). 2.4. Fluorescence-activated cell sorting analysis Cell cycle analysis was performed using fluorescence-activated cell sorting (FACS) as previously described [ 26, 27]. Cells were trypsinized, washed with DPBS, and cooled to −20 °C overnight in 70% ethanol. Cells were then collected by centrifugation and stained with propidium iodide (PI) solution (50 μg/ml PI, 50 μg/ml RNase A, 0.1% Triton X-100, and 0.1 mM EDTA) for 25 min. Cells were analyzed using FAC-SCalibur cytometer (Becton–Dickinson). 2.5. Western blot analysis Cells were washed with DPBS and collected by centrifugation. The cells were re-suspended in 1× lysis buffer (Cell Signaling, Beverly, MA). The suspended cells were subjected to sonication and centrifugation. Western blot analysis was then performed using a rabbit polyclonal antibody against KLF5, hemagglutinin A (HA) (Santa Cruz Biotech.) or β-actin (Oncogene Research). 2.6. Northern blot analysis RNA was extracted using the Trizol protocol (Invitrogen, Carlsbad, CA). Twenty micrograms of RNA was loaded onto a denaturing agarose gel containing 10× MOPS buffer and 37% formaldehyde, and then transferred to a nylon membrane (Hybond, Amersham, Piscataway, NJ). Radioactive [α-32P] dATP was used to label cDNA probes for KLF5 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The membrane was hybridized in Quikhyb solution (Stratagene, La Jolla, CA), washed under high-stringency conditions, and scanned using a phosphorimager (Amersham). We first determined whether ATRA affects growth and proliferation of IEC6, a non-transformed rat intestinal epithelial cell line. As shown in , treatment of IEC6 cells with 10 μM ATRA resulted in a significant reduction in their rate of proliferation between days 2 and 4 after seeding. This inhibitory effect was the consequence of cell cycle arrest at the G1/S boundary as demonstrated by the significant increase in the G1/S ratios of cells treated with ATRA as compared to control (). We next determined whether ATRA treatment affects expression of the gene encoding KLF5, previously shown to be a pro-proliferative factor in IEC18 and NIH 3T3 cells [ 11, 22]. As shown in and B, treatment of IEC6 cells with ATRA from 5 to 50 μM for 3 days resulted in a dose-dependent reduction in the levels of KLF5 mRNA. Treatment of cells with lower doses of ATRA, between 1 and 1000 nM, resulted in a less prominent and non-linear reduction in the levels of KLF5 mRNA (). For this reason, we used a concentration of 10 μM throughout the present study. We then performed a time-course study in IEC6 cells treated with ATRA or ethanol control. In the presence of ethanol control, the levels of KLF5 mRNA increased over time (). In contrast, the levels of KLF5 mRNA in cells treated with 10 μM ATRA remained relatively unchanged for up to 4 days (). The relative abundance of KLF5 mRNA in treated and control cells () paralleled that of their rates of proliferation as illustrated in . These results indicate that ATRA inhibits expression of KLF5 in a dose-dependent and time-dependent fashion, and that inhibition of KLF5 expression is correlated with a reduction in the rate of proliferation. We then investigated the mechanism by which ATRA inhibits the expression of KLF5. We performed transfection studies in IEC6 cells using a plasmid that contained −1.5 kb of the KLF5 promoter linked to a luciferase reporter and treated them with ATRA or ethanol control. shows that treatment of transfected IEC6 cells with 10 μM ATRA for 3 days resulted in a statistically significant reduction in the activity of the reporter construct when compared to ethanol control. These findings suggest that the effect of ATRA on KLF5 expression is mediated at the level of gene transcription. To further demonstrate that KLF5 mediates the inhibitory effect of ATRA on proliferation of IEC6 cells, we established a stably transfected IEC6 cell line that constitutively expressed KLF5 under the control of the cytomegalovirus (CMV) viral promoter, which is not subject to regulation by ATRA. This cell line, called IEC6-KLF5, contained a significantly higher level of KLF5 mRNA when compared to untransfected IEC6 cells (). In addition, when probed with an antibody against HA, IEC6-KLF5 cells contained HA-tagged KLF5 protein, which was absent from untransfected cells (). As a consequence of stable and constitutive expression of KLF5, IEC-KLF5 cells exhibited a higher rate of proliferation when compared to the untransfected IEC6 cells (). Importantly, treatment of IEC6-KLF5 with 10 μM ATRA failed to reduce the levels of KLF5 mRNA and protein as shown in , respectively. In comparison, the levels of KLF5 mRNA and protein were reduced in untransfected IEC6 cells that were treated with ATRA (). Moreover, constitutive expression of KLF5 in IEC6 cells resulted in resistance to ATRA as demonstrated by the lack of effect of ATRA on the rate of proliferation and cell cycle profile, illustrated in , respectively. These results suggest that the inhibitory effect of ATRA on IEC6 cells is primarily mediated by the ability of ATRA to inhibit endogenous KLF5 expression. To determine whether ATRA may have an effect on proliferation of human colon cancer cell lines, we cultured several established colon cancer cell lines in the presence or absence of 10 μM ATRA. The growth rates of three cell lines, DLD1, Caco2-BBE and HT29, were sensitive to ATRA treatment (, respectively). However, the rate of proliferation of RKO cells was indifferent to ATRA (). A difference between the first three cell lines and RKO cells is that the basal level of KLF5 mRNA is significantly lower in RKO cells as compared to the first three cell lines (, insets). Importantly, the ability of ATRA to reduce cell proliferation is correlated with a reduction in the levels of KLF5 mRNA in DLD1, Caco2-BBE and HT29 cells (, insets). In RKO cells, the level of KLF5 mRNA was unchanged with ATRA treatment (, inset). These results further suggest that KLF5 mediates the inhibitory effect of ATRA on IEC proliferation. Retinoids are known to play a vital role in various physiological processes such as embryogenesis, growth, differentiation and reproduction [ 28]. Studies also showed that retinoic acid can inhibit proliferation of many tumor cells grown in vitro [ 29]. These findings led to the successful clinical application of retinoids in the treatment or prevention of various human cancers, such as APL [ 18]. In the case of APL, ATRA is able to induce complete remission in almost all patients by inducing in vivo differentiation of blast cells in APL [ 18]. Numerous studies have demonstrated that the cellular effects of retinoids are mediated by nuclear receptors, including RARs and RXRs [ 12]. For example, the inhibitory effect of ATRA on proliferation of the human colon cancer cells, HT29, is mediated through RARα [ 19]. Also, induction of RAR in the retinoid-resistant colon cancer cells, LoVo, restored retinoid sensitivity [ 30]. Despite these findings, the mechanisms by which ATRA inhibits tumor cell proliferation remain poorly established. In the current study, we characterized the anti-proliferative effect of ATRA on a non-transformed rat IEC line, IEC6. Our results showed that ATRA inhibits cell proliferation in IEC6 (). This inhibitory effect is primarily due to an arrest in the G 1 to S transition of the cell cycle (). A similar effect of ATRA on the cell cycle has previously been demonstrated in myeloid cells [ 31] and ovarian carcinoma cells [ 32]. In myeloid cells, ATRA-induced cell cycle arrest is accompanied by downregulation of c-Myc and cyclin E [ 33], the latter an essential component for G 1 to S transition. ATRA has also been shown to reduce expression of protein kinase C α (PKCα) and activity of the mitogen-activated protein kinase (MAPK) pathway [ 34]. The Krüppel transcription factor, KLF5, has been shown to positively regulate cell proliferation in NIH 3T3 fibroblasts and IEC [ 11, 22]. Results of our present study indicate that ATRA inhibits expression of KLF5 in IEC6 cells ( and ) by inhibiting the promoter activity of KLF5 (). The reduction in KLF5 mRNA levels due to ATRA treatment is correlated with a reduction in the rate of proliferation ( and ). The dosage of ATRA that we chose was relatively high at 10 μM, based on the result of the dose-response experiment in . Several other studies have used lower dosages [ 35, 36]. However, numerous studies have also used higher concentrations of ATRA similar to ours in order to accomplish the inhibitory effects of ATRA in various cell lines [ 37– 40]. Therefore, the different sensitivity of different cell lines to ATRA is probably a cell type-specific event. IEC6 cells stably transfected with a viral promoter-driven KLF5-expression vector contained a higher level of KLF5 mRNA and protein and a higher rate of proliferation as compared to untransfected IEC6 cells (). When treated with ATRA, the levels of KLF5 mRNA and protein were not decreased in IEC6-KLF5 cells as opposed to untransfected IEC6 cells (). This is primarily due to the constitutive expression of KLF5 from a viral promoter that is not affected by ATRA. This constitutive expression resulted in the IEC-KLF5 cells to resist the inhibitory effect of ATRA as demonstrated in . These results suggest that KLF5 is a mediator for the inhibitory effect of ATRA on proliferation of IEC6 cells, although we could not rule out KLF5-independent mechanisms. It is of interest to note that KLF5 expression is stimulated by the phorbol ester, PMA, a potent activator of PKCα [ 11, 41]. Moreover, studies indicate that KLF5 expression is stimulated by MAPK activation [ 41, 42]. The fact that ATRA inhibits both PKCα and MAPK [ 34] may be a mechanism by which it inhibits KLF5 expression. It should be noted that cyclin D1 has been shown to be a target of regulation by KLF5 [ 42]. The effect of ATRA on G 1 progression can therefore be a consequence of its ability to inhibit cyclin D1 expression through KLF5. ATRA has previously been shown to inhibit proliferation of some, but not all, colon cancer cells [ 19– 21, 30]. Our study demonstrated that 3 colon cancer cell lines, including DLD1, Caco2-BBE and HT29, are sensitive to the inhibitory effect of ATRA but RKO cells are resistant (). Previous studies suggest that the presence or absence of RARs in cells appears to play a role in determining whether cells are sensitive or resistant to ATRA treatment [ 21, 30]. However, all of the cells that we examined contain RARα (data not shown). Instead, the ability of ATRA to inhibit cell proliferation is directly correlated with a reduction in KLF5 mRNA levels in the 3 sensitive cell lines (). The lone cell line, RKO, that is resistant to ATRA action, contains little KLF5 mRNA at baseline, which is not changed with ATRA treatment (). These results further support the role of KLF5 in mediating the inhibitory effect of ATRA on cell proliferation. Furthermore, whether a tumor cell is sensitive to ATRA may in part be determined by the degree of KLF5 expression. The relationship between KLF5 and retinoids in regulating cell proliferation has also been demonstrated in vivo [ 23]. This was shown by the ability of RAR ligands to modulate KLF5 transcriptional activity. Moreover, RAR and KLF5 physically interact. A consequence of this relationship is the ability of a RAR agonist, Am80, to inhibit vascular remodeling in vivo, a process that is dependent on KLF5 [ 23]. Whether RAR and KLF5 physically interact in IEC is unclear at this time. In conclusion, our study demonstrated an important role for KLF5 in mediating the inhibitory effect of ATRA on proliferation of IEC. We also showed that differential sensitivity of colon cancer cells to ATRA may be due to differential levels of KLF5 expression. Additional correlation between KLF5 expression and ATRA sensitivity in other cell lines may further substantiate the role of KLF5 in modulating IEC proliferation. We thank Drs. J. Lingrel and A. 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