KLF4 gene expression is inhibited by the notch signaling pathway that controls goblet cell differentiation in mouse gastrointestinal tract

Abstract

In Kruppel-like factor (KLF)-4-deficient mice, colonic goblet cell numbers are significantly reduced. Goblet cell development is regulated by the Notch signaling pathway. The aim of this study was to examine whether Notch represses KLF4 expression to regulate goblet cell differentiation. We first detected that KLF4 gene expression was upregulated in a human progastrin-overexpressing mouse model where goblet cell hyperplasia has been observed. We then found that mice treated with a gamma-secretase inhibitor (compound E, 10 micromol/kg) for 24 h, which inhibits the Notch signaling pathway, had significantly increased KLF4 mRNA levels in small intestine and colon, accompanied by an increased number of KLF4-expressing cells at the bottom of crypts in small intestine and colon. In a colon cancer cell line (HCT116 cells), KLF4 promoter activity was inhibited by a constitutively active form of Notch1 (ICN1) by transient cotransfection assays. This inhibition was significantly compromised by a dominant-negative RBPjk, a repressive mediator of the Notch signaling pathway. An ICN1-responsive element was then mapped in the human KLF4 promoter between -151 and -122 nucleotides upstream of the transcriptional start site. It was also found that an intact ICN1-responsive element is required for ICN1 to inhibit KLF4 promoter activity by transient cotransfection assays. Our findings thus reveal a possible mechanism by which KLF4 is inhibited by Notch, which controls goblet cell differentiation in mouse gastrointestinal tract.

Fig. 1.

Increased colonic goblet cell numbers correlate with upregulation of Kruppel-like factor (KLF)-4 gene expression in hGAS mice. A: cell positional distribution of goblet cells. B: mean no. of goblet cells in 50 half-crypts of the distal colon of hGAS and FVB/N mice (n = 6 male mice/experimental group). Significant differences were found at cell position 4–38 by the modified median test in A and P < 0.01 by Student's t-test in B. Total RNA and protein were extracted from the colon of adult hGAS mice as described in materials and methods. Quantitative RT-PCR (C) and Western blotting analysis (D) were performed. Three control mice and three hGAS mice were used for RT-PCR analysis, and a representative Western blotting result from 3 independent experiments was shown.

Fig. 2.

Inhibition of Notch converted cryptal cells into goblet cells. The Notch signaling pathway was inhibited by administration of a γ-secretase inhibitor [Compound E (CompE)] for 5 days by daily ip injection (10 μmol·kg⁻¹·day⁻¹) of 6- to 8-wk-old C57BL/6 mice. Frozen sections from mouse small intestine with (B and D) and without (A and C) treatment of CompE were stained with hematoxylin and eosin (HE; A and B) and Periodic acid-Schiff (PAS; C and D), a staining marker of goblet cells.

Fig. 3.

Increased KLF4 mRNA levels in mouse small intestine and colon upon inhibition of Notch. Total RNA from mouse colon and small intestine was extracted, and quantitative RT-PCR was performed as described in materials and methods. A and B: Notch signaling was inhibited by 5-day CompE treatment, and KLF4 expression in mouse small intestine (A) and colon (B) was measured by RT-PCR analysis. C and D: similar to A and B, except that mice were treated with CompE for 24 h. Note that y-axis indicates relative KLF4 mRNA levels after normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Fig. 4.

Increased KLF4 cells in mouse small intestine upon inhibition of Notch. C57BL/6 mice were treated with CompE or control drug for 24 h followed by immunostaining with an anti-KLF4 antibody, and KLF4 positive cells were counted as described in materials and methods. A–C: KLF4 is highly expressed in the villi of mouse intestine, and its expression is also detected in the intestinal crypts. A: frozen section from mouse small intestine was stained with anti-KLF4 antibody followed by observation under a fluorescence microscope. D–F: similar to A–C except the bottom crypt of a control-treated mouse small intestine was shown. G–I: similar to D–F except the bottom crypt of a CompE-treated mouse small intestine was shown. After treatment, KLF4 positive cells increased from 6 cells (inside the white box in D) to 17 cells (inside the white box in G). Note that all white boxes have the same size in very similar areas.

Fig. 5.

Increased KLF4 cells in mouse colon upon inhibition of Notch. Similar to Fig. 4 except mouse colon was examined instead of mouse small intestine. After CompE treatment, KLF4 positive cells increased from 10 cells (inside the white box in A) to 19 cells (inside the white box in D). Note that all white boxes have the same size in very similar areas.

Fig. 6.

ICN1 inhibited the promoter activity of KLF4. Human KLF4 (hKLF4) promoter activity is inhibited by a constitutively active version of human Notch1 (ICN1) by transient transfection and dual luciferase assays using a colon cancer cell line (HCT116). Vector control (PCS2), a constitutively active Notch 1 (ICN1), dominant-negative RBPjk plasmid (dnRBPjk), and combination of ICN1 and dnRBPjk expression constructs were transfected in HCT116 cells with a 2.0-kb hKLF4 promoter reporter construct. After transfection (48 h), dual Luciferase assays were then performed to measure KLF4 promoter activities as described in materials and methods.

Fig. 7.

Identification of an ICN1-responsive element in the hKLF4 promoter. A: diagram of a series of mutant hKLF4 promoters with different 5′-truncations used in the transient cotransfection assays. B: identification of an ICN1-responsive element in hKLF4 promoter. Vector and ICN1 were cotransfected with different hKLF4 promoter reporters, and the relative promoter activities were determined as described in A. C: similar to C, and an ICN1-responsive element was mapped between the −151 and −122 position at the promoter. D: an intact ICN1-responsive element in the hKLF4 promoter is required for ICN1-induced KLF4 inhibition. A wild-type and mutant KLF4 promoter with mutation in the core of the underlined region were used for transient transfection assays. Dual luciferase assays were performed as described previously.