KLF4 regulation in intestinal epithelial cell maturation

Abstract

The Krüppel-like factor 4 (KLF4) transcription factor suppresses tumorigenesis in gastrointestinal epithelium. Thus, its expression is decreased in gastric and colon cancers. Moreover, KLF4 regulates both differentiation and growth that is likely fundamental to its tumor suppressor activity. We dissected the expression of Klf4 in the normal mouse intestinal epithelium along the crypt-villus and cephalo-caudal axes. Klf4 reached its highest level in differentiated cells of the villus, with levels in the duodenum>jejunum>ileum, in inverse relation to the representation of goblet cells in these regions, the lineage previously linked to KLF4. In parallel, in vitro studies using HT29cl.16E and Caco2 colon cancer cell lines clarified that KLF4 increased coincident with differentiation along both the goblet and absorptive cell lineages, respectively, and that KLF4 levels also increased during differentiation induced by the short chain fatty acid butyrate, independently of cell fate. Moreover, we determined that lower levels of KLF4 expression in the proliferative compartment of the intestinal epithelium are regulated by the transcription factors TCF4 and SOX9, an effector and a target, respectively, of beta-catenin/Tcf signaling, and independently of CDX2. Thus, reduced levels of KLF4 tumor suppressor activity in colon tumors may be driven by elevated beta-catenin/Tcf signaling.

Figure 1. Klf4 is expressed in the villus of the small intestine and in the surface epithelium in large intestine in mouse

(A) Immunohistochemical staining for Klf4 was performed with the same antibody used in panels C and D (dilution 1:1000) in paraffin-embedded sections of mouse small and large intestines. (B) Schematic representation of the crypt-villus axis in the mature small intestine showing the fractionation of the epithelial cells used from villus (fraction 1), where the proliferating cells are found, to crypt (fraction 4), occupied by differentiated cells. (C) Western blot analysis of Sox9 and Klf4 in mouse intestinal epithelial samples from duodenum, jejunum and ileum fractionated as described above. PCNA and L-Fabp expression was used to monitor the cellular fractionation. Tubulin was used as loading control. (D) Western blot analysis of Sox9 and Klf4 in mouse intestinal epithelial samples from colon fractionated as above. PCNA expression was used to monitor the cellular fractionation. Actin was used as loading control.

Figure 2. KLF4 expression increases in non-proliferating transformed colonic epithelial cells after their spontaneous or butyrate induced differentiation

Quantification by real-time RT-PCR of KLF4 mRNA levels, and detection of KLF4 and SOX9 proteins by western-blotting, during spontaneous differentiation in HT29cl.16E (A) and Caco2 (B) cells. Data were expressed as fold increased in KLF4/GAPDH mRNA levels of each time point versus non-differentiated cells (day 0). Actin was used as loading control. Columns, mean of at least three different experiments; bars, SE. (C) y (D) A Klf4-luciferase reporter plasmid where Luciferase expression is driven by 1kb of the 5’-flanking region and 550 bp of the 5’-untranslated region of Klf4 was transfected into HT29cl.16E (C) and Caco2 (D) cell lines at the time points indicated in the figures. Firefly and Renilla luciferase activities were determined 48 hours after transfection. Columns, mean of at least three different experiments; bars, SE. (E) Quantification by real-time RT-PCR of KLF4 mRNA levels, and detection of KLF4 protein by western-blotting, after butyrate treatment for 72 hours to induce differentiation in HT29cl.16E and Caco2 cells. Data were expressed as KLF4/GAPDH mRNA ratios. Columns, mean of at least three different experiments; bars, SE.

Figure 3. KLF4 expression is regulated by TCF4 in Ls174T human colon cancer cell line

(A) Quantification of KLF4 mRNA levels in Ls174T cell line stably transfected with a doxycycline inducible dnTCF4 construct. Real time RT-PCR analysis was performed to quantify KLF4 mRNA levels after 7, 24 and 48 hours of dnTCF4 induction. Data were expressed as fold increase in KLF4/GAPDH mRNA levels of doxycycline treated (dox) versus untreated (ctrl) cells. Time of doxycycline induction is indicated at the bottom of the graph. Columns, mean of duplicates from three different experiments; bars, SE. (B) Detection of the KLF4 protein by western blot in the dnTCF4 stably transfected Ls174T cells with or without doxycycline induction at the indicated time points. Actin served as a loading control.

Figure 4. KLF4 expression is regulated by the transcription factor SOX9

(A) Western blot analysis of KLF4 in the SOX9 stably transfected HT29cl.16E cells with or without doxycycline induction at the indicated time points. Flag served as a monitor of SOX9 over-expression and actin served as a loading control. (B) Effect of induced SOX9 expression on the Klf4-luciferase reporter activity. A KLF4-luciferase reporter plasmid (described in Figure 2) was transfected into HT29cl.16E-SOX9 for this experiment. Firefly and Renilla luciferase activities were determined 48 hours after transfection and culture with or without doxycycline. Columns, mean of five different experiments; bars, SE. ***, p<0.001. (C) Detection of the KLF4 protein by western blot analysis in the dnSOX9 stably transfected HT29cl.16E cells with or without doxycycline induction at the indicated time points. Flag served as a monitor of the dnSOX9 overexpression and actin served as a loading control. (D) Effect of induced dnSOX9 expression on the Klf4-luciferase reporter activity (Klf4-luciferase reporter plasmid described above). Firefly and Renilla luciferase activities were determined 48 hours after transfection and culture with or without doxycycline. Columns, mean of five different experiments; bars, SE. *, p<0.05. (E) Quantification of KLF4 mRNA levels in HT29cl.16E-SOX9 cell line. Real time RT-PCR analysis was performed to quantify KLF4 mRNA levels after 6 and 24 hours of SOX9 induction. Data were expressed as fold increase in KLF4/GAPDH mRNA levels of doxycycline treated (dox) versus untreated (ctrl) cells. Time of doxycycline induction is indicated at the bottom of the graph. Columns, mean of at least three different experiments; bars, SE. (F) Effect of induced SOX9 expression on Klf4-luciferase reporter activity in Caco2 cells. Caco2 cells were cotransfected with an empty vector (ctrl) or the dnSOX9 expression construct, together with Klf4-luciferase reporter (described above). Firefly and Renilla luciferase activities were determined 48 hours after transfection. Columns, mean of three different experiments; bars, SE. **, p<0.01.

Figure 5. TCF4 and SOX9 have an additive effect on KLF4 regulation

(A) Quantification of SOX9 mRNA levels in Ls174T-dnTCF4 cell line transfected with either non-targeting (nt) or SOX9 specific (siSOX9) siRNA and grown 48 or 72 hours. (B) Real time RT-PCR analysis was performed to quantify KLF4 mRNA levels in the same experiment described above. Data were expressed as fold increase in SOX9/GAPDH or KLF4/GAPDH mRNA levels of non-targeting versus specific SOX9 siRNA transfected cells. Time is indicated at the bottom of the graph. Columns, mean of duplicates from three different experiments; bars, SE. (C) Detection of KLF4 and SOX9 proteins by western blot in Ls174T-dnTCF4 cells transfected with either non-targeting (nt) -1,2- or SOX9 specific (siSOX9) -3,4- siRNA and grown with -2,4- or without -1,3- doxycycline for 48 hours. (D) Background-adjusted densitometric data (presented as mean values with standard error bar) from 3 independent experiments. (E) Linear regression of densitometric data for KLF4 as a function of SOX9 levels, obtained from the experiments shown in D.

Figure 6. KLF4 regulation by the transcription factor SOX9 is independent of CDX2 in human colon cancer cells

(A) KLF4-luciferase reporter activity of KLF4-2200 and KLF4-515 in HT29cl.16E cells stably transfected with the doxycycline inducible SOX9 construct. Firefly and Renilla luciferase activities were determined 48 hours after transfection with or without doxycycline (ctrl). Columns, mean of four different experiments; bars, SE. (B) Effect of induced dnSOX9 expression on KLF4- luciferase reporter activity in RKO cells that carry an inactive CDX2 form. RKO cells were co-transfected with an empty vector (ctrl) or the dnSOX9 construct, together with Klf4-luciferase reporter (described in Figure 2). Firefly and Renilla luciferase activities were determined 48 hours after transfection. Columns, mean of three different experiment; bars, SE. **, p<0.01.