Cooperation between HNF-1alpha, Cdx2, and GATA-4 in initiating an enterocytic differentiation program in a normal human intestinal epithelial progenitor cell line

Abstract

In the intestinal epithelium, the Cdx, GATA, and HNF transcription factor families are responsible for the expression of differentiation markers such as sucrase-isomaltase. Although previous studies have shown that Cdx2 can induce differentiation in rat intestinal IEC-6 cells, no data are available concerning the direct implication of transcription factors on differentiation in human normal intestinal epithelial cell types. We investigated the role of Cdx2, GATA-4, and HNF-1alpha using the undifferentiated human intestinal epithelial crypt cell line HIEC. These transcription factors were tested on proliferation and expression of polarization and differentiation markers. Ectopic expression of Cdx2 or HNF-1alpha, alone or in combination, altered cell proliferation abilities through the regulation of cyclin D1 and p27 expression. HNF-1alpha and GATA-4 together induced morphological modifications of the cells toward polarization, resulting in the appearance of functional features such as microvilli. HNF-1alpha was also sufficient to induce the expression of cadherins and dipeptidylpeptidase, whereas in combination with Cdx2 it allowed the expression of the late differentiation marker sucrase-isomaltase. Large-scale analysis of gene expression confirmed the cooperative effect of these factors. Finally, although DcamKL1 and Musashi-1 expression were downregulated in differentiated HIEC, other intestinal stem cell markers, such as Bmi1, were unaffected. These observations show that, in cooperation with Cdx2, HNF-1alpha acts as a key factor on human intestinal cells to trigger the onset of their functional differentiation program whereas GATA-4 appears to promote morphological changes.

Fig. 1.

Undifferentiated human intestinal crypt cells lack Cdx2 and HNF-1α expression. A: representative RT-PCR analysis for detection at the transcript level of the differentiation markers sucrase-isomaltase, dipeptidylpeptidase IV (DPPIV), LI-cadherin and E-cadherin, and the transcription factors HNF-1α and Cdx2 in differentiated Caco-2/15 (5 and 10 days postconfluence) and undifferentiated human intestinal epithelial crypt (HIEC) cells. Although differentiated cells express all of these markers, undifferentiated HIEC cells express only low amounts of DPPIV. β-Actin was used for normalization. B and C: representative immunofluorescent staining on frozen sections of the adult small intestinal mucosa for the detection of HNF-1α in red (B and C) and Cdx2 in green (B′ and C′) showing nuclear expression in all differentiated cells of the villi (B and B′) and at the upper part of the crypts (C and C′); bar in B = 100 μm. D: higher magnification immunofluorescent pictures from the proximal ileum for the detection of HNF-1α in red (D), Cdx2 in green (D′), and merged HNF-1α/Cdx2 (D″). Nuclear staining by 4,6-diamidino-2-phenylindole (DAPI) was in blue; bar in D″ = 20 μm. Both transcription factors were found to be expressed relatively ubiquitously in the nuclei of upper crypt and villus epithelial cells (B and C), whereas a limited number of cells located in the lower portion of the crypts showed weak or negative staining (arrows in D, D′, and D″ denote negative nuclei).

Fig. 2.

Characterization of HIEC cells expressing Cdx2 and HNF-1α individually under induced and uninduced conditions. A: Western-blot analysis of Cdx2 and HNF-1α expression in inducible HIEC cell lines. Cytokeratin-18 was used as a loading control (not shown). B and C: proliferation assay with inducible HIEC cell lines expressing Cdx2 or HNF-1α alone showing that both factors significantly slow down intestinal cell proliferation. Cells were grown up to day 17 after seeding and counted at the indicated times. HIEC^IndEmpty cells [presence or absence of doxycycline (+dox and −dox, respectively)] were grown in parallel with HIEC^IndCdx2 (B) and HIEC^IndHNF-1α (C). Relative amounts of cyclinD1 (D) and p27^Kip1 (E) were evaluated by western blot analyses in HIEC induced for the expression of Cdx2 or HNF-1α by doxycycline. Cytokeratin-18 was used as a loading control (not shown) to determine the relative amounts. *P = 0.023, **P = 0.001.

Fig. 3.

Effect of Cdx2 and HNF-1α combined expression on HIEC cell proliferation. A: proliferation assay with HNF-1α expressing HIEC cells (IndHNF-1α+dox) reinfected for the constitutive expression of Cdx2 (HNF1α+Cdx2) demonstrated an additive effect of the 2 factors on the inhibition of cell proliferation compared with cells that expressed Cdx2 alone (IndCdx2 +dox; Cdx2). A control infection was performed with the same virus containing the empty vector (IndEmpty +dox; Empty). B: histogram illustrating the percentage (%) of cells distributed in each of G1, S, and G2+M cell-cycle phases for HIEC control (IndEmpty +dox; Empty), Cdx2 alone (IndCdx2 +dox; Cdx2), and HNF-1α/Cdx2-expressing cells (IndHNF-1α/Cdx2 +dox; HNF1α/Cdx2) obtained by iCys laser scanning cytometry analysis (*P < 0.03, **P < 0.005).

Fig. 4.

Representative RT-PCR analysis for the characterization of the HNF-1α- and Cdx2-expressing HIEC inducible cell lines. A: detection at the transcript level of differentiation markers in inducible cell populations under uninduced (−) or induced (+) conditions. Postconfluent Caco-2/15 cells were used as differentiated cell control. Cdx2 has little or no effect on HIEC cell differentiation markers when expressed alone (lane 5) as opposed to HNF-1α that induces expression of LI- and E-cadherin as well as DPPIV (lane 7). DPPIV was found to be expressed under a HNF-1α dosage-independent manner since it was similarly detected under +dox conditions (lane 6), which only allow basal HNF-1α expression. B: detection at the transcript level of the differentiation markers in the induced HIEC^IndHNF-1α cells reinfected with an empty virus (lane 2) or virus coding for the constitutive expression of Cdx2 (lane 3). HIEC^IndEmpty cells + doxycycline were used as control (lane 1). Expression of transcription factors was verified in all samples. Primers for β-actin were used for normalization. Combination of HNF-1α with Cdx2 induces expression of the terminal differentiation marker sucrase-isomaltase.

Fig. 5.

Intestinal distribution of GATA-4 and its effect on HIEC cell growth. Representative immunofluorescent staining on frozen sections of adult proximal ileum mucosa for the detection of GATA-4 in the villus (A) and crypt (B) regions (bars = 100 μm). Nuclear staining of GATA-4 was ubiquitously detected throughout the epithelium from the lower part of the crypts to the tip of the villi. C: Western blot analysis of GATA-4 expression in inducible HIEC cell lines. GATA-4 was not detectable in control cells (HIEC^IndEmpty) or in Cdx2- or HNF-1α-expressing cells. GATA-4 was expressed at significant levels in doxycycline-induced HIEC^IndGATA-4 cells. D: proliferation assay with the GATA-4-inducible HIEC cell line demonstrates no significant effect on growth. HIEC^IndEmpty cells (presence or absence of doxycycline) were grown in parallel with HIEC^IndGATA-4 cell populations.

Fig. 6.

Characterization of the double-inducible HIEC^{IndHNF-1α/Cdx2} cell line and impact of the HNF-1α/Cdx2/GATA-4 combination on the expression of intestinal epithelial differentiation markers. A: Western blot analysis exposing leaky expression of HNF-1α, but not of Cdx2, in the absence (−) of doxycycline in HIEC^Teton B: representative RT-PCR analysis for the detection of sucrase-isomaltase, DPPIV, LI-cadherin, and E-cadherin transcripts in the presence (+) or absence (−) of doxycycline in control (IndEmpty) (−) and Hnf-1α/Cdx2 (+) cells. Postconfluent Caco-2/15 cells were used as a well-differentiated control. Combined HNF-1α/Cdx2 expression induced the 4 tested markers (lane 6), whereas the addition of GATA-4 further increased LI-cadherin and reduced E-cadherin expression (lane 8) at significant levels (C), an observation further confirmed by real-time PCR (**P < 0.01). D: representative Western blot confirming the expression of the 3 factors at the protein level. A reduction of E-cadherin in response to the constitutive expression of GATA-4 in the induced HIEC^{IndHNF-1α/Cdx2} cells was also apparent.

Fig. 7.

Phase-contrast microscopy of induced HIEC^IndHNF-1α cells infected with either empty (A) or GATA-4 expressing vector (C) or doxycycline-induced HIEC^{IndHNF-1α/Cdx2} cells infected with either empty (B) or GATA-4 expressing vector (D). GATA-4 expression induces the reorganization of the monolayer into latticelike structures (arrows in C and D).

Fig. 8.

Ultrastructural characterization of the effects of HNF-1α, Cdx2, and GATA-4 expression in HIEC cells by transmission (A, C, D, F) and scanning (B, E, G) electron microscopy. Doxycycline-induced HIEC^IndHNF-1α cells infected with either empty (A and B) or GATA-4 expressing vector (C–E) or doxycycline-induced HIEC^{IndHNF-1α/Cdx2} cells infected with the GATA-4 expressing vector (F and G) were characterized. Cells showed poorly differentiated features typical to the original HIEC cells except upon forced expression of GATA-4. Bars = 2 μm in A and C; 10 μm in B, E, and G; 500 nm in D and F.

Fig. 9.

Hierarchical clustering of the genes differentially expressed in response to HNF-1α/Cdx2 expression in the absence or presence of GATA-4. A: common expression patterns were determined by nonbiased clustering analysis, revealing several groups of genes whose expression was modulated in response to HNF-1α/Cdx2 and HNF-1α/Cdx2+GATA-4 in HIEC cells. B: modulation of the differentially expressed genes in HIEC^{IndHNF-1α/Cdx2}+empty vector or HIEC^{IndHNF-1α/Cdx2}+GATA-4 cells, compared with HIEC^IndEmpty cells, was determined for each main biological process (P < 0.05).

Fig. 10.

Combined effects of GATA-4 with HNF-1α and Cdx2 on cell polarity marker expression in HIEC cells. Increased expression of zonula occludens-2, cingulin, villin-2, claudin-11, and calbindin-2 detected in microarrays (Table 2) was confirmed by real-time PCR in HIEC^{IndHNF-1α/Cdx2}+GATA-4 compared with HIEC^{IndHNF-1α/Cdx2}+empty vector (*P < 0.05; **P < 0.005).

Fig. 11.

Effect of prodifferentiation transcription factors on the expression of intestinal stem/progenitor cell markers in HIEC. Expression levels of Bmi1, DcamKL1, Musashi-1, and Lgr5 were quantified in HIEC^IndEmpty and HNF-1α/Cdx2/GATA-4 expression HIEC by real-time PCR. Total epithelial fractions were used as control. Significant decreases of DcamKL1 and Musashi-1 were observed in differentiating HIEC compared with HIEC^IndEmpty (*P < 0.05).