Pinin modulates expression of an intestinal homeobox gene, Cdx2, and plays an essential role for small intestinal morphogenesis - PubMed (original) (raw)

Pinin modulates expression of an intestinal homeobox gene, Cdx2, and plays an essential role for small intestinal morphogenesis

Jeong-Hoon Joo et al. Dev Biol. 2010.

Abstract

Pinin (Pnn), a nuclear speckle-associated protein, has been shown to function in maintenance of epithelial integrity through altering expression of several key adhesion molecules. Here we demonstrate that Pnn plays a crucial role in small intestinal development by influencing expression of an intestinal homeobox gene, Cdx2. Conditional inactivation of Pnn within intestinal epithelia resulted in significant downregulation of a caudal type homeobox gene, Cdx2, leading to obvious villus dysmorphogenesis and severely disrupted epithelial differentiation. Additionally, in Pnn-deficient small intestine, we observed upregulated Tcf/Lef reporter activity, as well as misregulated expression/distribution of beta-catenin and Tcf4. Since regulation of Cdx gene expression has been closely linked to Wnt/beta-catenin signaling activity, we explored the possibility of Pnn's interaction with beta-catenin, a major effector of the canonical Wnt signaling pathway. Co-immunoprecipitation assays revealed that Pnn, together with its interaction partner CtBP2, a transcriptional co-repressor, was in a complex with beta-catenin. Moreover, both of these proteins were found to be recruited to the proximal promoter area of Cdx2. Taken together, our results suggest that Pnn is essential for tight regulation of Wnt signaling and Cdx2 expression during small intestinal development.

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Figures

Fig. 1

Deletion of Pnn in developing small intestinal epithelium. (A) Whole-mount X-Gal staining of E15.5 control intestine positive for both Shh-Cre and R26R demonstrates strong Cre activity in full length of developing mouse intestine. (B, C) Section images of X-Gal stained control Shh-Cre;R26R+/−;Pnn+/2f intestine reveal specific Cre activity in epithelial cells of small intestine. (D) Inactivation of Pnn was analyzed by quantitative RT-PCR assays with different parts of small intestine (duodenum, jejunum, and ileum). Pnn transcript level was significantly reduced in all parts of mutant small intestine at E17.5. Expression levels are normalized to Gapdh. Error bars represent standard deviation. All p values are compared to each control intestine. ***: p < 0.001 (two-tailed unpaired Student’s t-tests, n=3). (E–H) Pnn immunostaining (red signal within the nuclei) shows successful and specific inactivation of Pnn in epithelial cells of mutant small intestine (arrows in H). Note similar Pnn immunostaining of stromal and muscle cells of control and mutant intestines. Original magnification: (A) 10X; (B, C) 200X; (E, F) 100X; (G, H) 400X.

Fig. 2

Villus dysmorphogenesis in Pnn mutant small intestine. (A–F) H&E staining of small intestines demonstrates severe villus defect in mutant. At E14.5, mutant small intestine shows slight delay in intestinal epithelial morphogenesis. However, at later stages, villus defect of mutant intestine becomes more apparent, exhibiting much shorter and fewer villi. Insets in E, F show high magnification images of control and mutant villi, respectively. Arrows indicate nuclei. (G, H) BrdU incorporation assay shows a regularly organized pattern of BrdU-positive epithelial cells in E16.5 control intervillus area (arrows in G), however, randomly distributed BrdU-positive epithelial cells (arrows in H) are observed in Pnn mutant small intestine. (I) Quantification of BrdU-positive cells in small intestinal epithelia after 2hr in vivo BrdU labeling reveals increased mislocalization of BrdU-positive epithelial cells in mutant intestine. The number of total epithelial cells and BrdU-positive epithelial cells were determined by counting cells from the same magnification images (n=9) of each control and mutant small intestine (E16.5) and presented as percentage of cell numbers. The position of cells was determined according to the cell at the bottom of intervillus space as 0. *: p < 0.05, **: p < 0.01 (two-tailed unpaired Student’s t-tests). All p values are compared to control samples. Original magnification: (A, B) 200X; (C, D) 100X; (E, F) 50X; (Insets in E, F) 400X; (G, H) 400X.

Fig. 3

Disrupted differentiation of Pnn mutant small intestinal epithelial cells. (A, B) Electron microscopy of E16.5 small intestine reveals disorganized brush border formation in Pnn mutant small intestine (arrows in A, B). (C, D) Alcian blue staining demonstrates differentiation of sialomucin-producing goblet cells in control intestine (blue signal, arrows in C), while the absence of developing goblet cells is obvious in mutant villi at E16.5. (E, F) Representative images of PAS staining on E16.5 control and mutant small intestine also reveal interrupted differentiation of goblet cell lineage in Pnn mutant small intestine. While control small intestine shows numerous PAS stain-positive cells (arrows in E), mutant intestine failed to exhibit staining positive goblet cells. Insets are higher magnification images of an individual villus. (G, H) Immunostaining analysis of chromogranin A reveals impaired differentiation of enteroendocrine cell lineage in Pnn mutant small intestine at E18.5. Chromogranin A-positive cells are shown in red and marked with arrows. Insets in G, H are higher magnification images showing chromogranin A-positive cells. (I, J) Control enterocytes show strong alkaline phosphatase activity (red reaction in I), while mutant villus epithelial cells display marginal IAP activity at E17.5. (K) Semi-quantitative RT-PCR assays reveal significantly reduced expression of cytodifferentiation markers in E17.5 mutant small intestine. The experiment was carried out with three or more mice for each control and mutant, and all mutant intestines examined consistently showed significant reduction in the transcript levels of genes shown. (L) Quantitative real-time RT-PCR analyses of E18.5 control and mutant small intestines confirm downregulation of marker genes. Expression levels for all tested genes are normalized to Gapdh level. Statistical analysis was performed using two-tailed unpaired Student’s t-tests (n=3). **: p < 0.01 vs. control, ***: p < 0.001 vs. control. Error bars represent standard deviation. Original magnification: (A, B) 5300X; (C, D) 400X; (E, F) 200X; (Insets in E, F) 400X; (G, H) 200X; (Insets in G, H) 400X; (I, J) 400X.

Fig. 4

Downregulation of Cdx homeobox genes in Pnn mutant small intestine. (A, B) Immunohistochemical analysis of Cdx2 expression demonstrates homogenous epithelial-specific Cdx2 expression (red signal) in control small intestine. However, significant downregulation of Cdx2 is observed in mutant small intestine at E16.5. (C, D) While control intestine shows a typical Cdx1 expression pattern (brown signal in C) with gradient along the crypt-villus axis, the Pnn mutant intestinal epithelium exhibits undetectable Cdx1 immunoreactivity. (E) Quantitative real-time RT-PCR assays reveal that mRNA levels of Cdx2 and Cdx1 are markedly decreased in Pnn mutant small intestines. Expression levels are normalized to Gapdh. Error bars represent standard deviation. All p values are compared to the control samples. *: p < 0.1, **: p < 0.01, ***: p < 0.001 (two-tailed unpaired Student’s t-tests, n=3). (F) A schematic diagram shows primer location used for ChIP assays on the promoter area of Cdx genes in mouse. Numbers are relative to the transcription start site (+1). Tcf binding elements are shown as red blocks. (G) ChIP assays demonstrate significantly reduced level of H3K4me3, a general marker for active chromatin, on both of Cdx2 and Cdx1 promoters in E17.5 mutant small intestine. The analyses were performed three times and the results shown were seen in all experiments. Original magnification: (A, B) 400X; (C, D) 200X.

Fig. 5

Upregulation of Topgal reporter activity in Pnn mutant small intestine. (A–D) Whole-mount X-Gal staining of E16.5 and E18.5 Topgal-positive small intestines shows distinct rings of β-galactosidase activity only in Pnn mutant small intestine (arrows in B, D). Control and mutant intestines were incubated in X-Gal staining solution for only 1 hour for A, B and for 30 minutes for C, D. (E, F) E16.5 small intestines were Red-Gal stained for 1 hour, sectioned, and counterstained with Alcian Blue. Topgal reporter activity is mainly detected in basal area of mutant villi (arrows in F). Original magnification: (A–D) 50X; (E, F) 400X.

Fig. 6

Misregulation of β-catenin, Tcf4, and Axin2 in mutant intestine. (A, B) Immunohistochemical staining of E17.5 intestines reveals considerable accumulation of β-catenin in Pnn mutant intestinal epithelial cells (arrows in B). Note that the increased β-catenin level is detected mostly in the basal area showing elevated Topgal activity in mutant small intestine (shown in Fig. 5F). (C, D) Aberrant expression pattern of Tcf4 is also detected in Pnn mutant intestinal epithelium. At E16.5, control intestine shows typical intervillus-restricted expression of Tcf4, however, Pnn mutant intestines present nearly uniform Tcf4 expression. (E, F) Axin2 immunostaining shows obvious Axin2 nuclear localization (arrows) in only a few cells of control villus epithelial cells, but the majority of cells of Pnn mutant intestine demonstrate positive nuclear Axin2 staining. (G) Quantitative RT-PCR assays demonstrate increased expression of Axin2 in E14.5 and E17.5 mutant small intestine, while transcript level of β-catenin in mutant small intestine remains similar to that of controls. The demonstrated extent to which Axin2 is increased in the epithelial cells of Pnn mutant intestine may be underestimated due to the inclusion of non-epithelial cells in the sample preparation. *: p < 0.05 vs. control. (two-tailed unpaired Student’s t-tests, n=3). Original magnification: (A, B) 400X; (C, D) 200X; (E, F) 400X.

Fig. 7

Association between PNN and β-catenin. (A) Ponasterone A (4µM) treatment effectively induces robust exogenous PNN expression in EcR293-PNNGFP cells. (B) Co-immunoprecipitation assays reveal that PNN forms a complex with β-catenin in EcR293-PNNGFP cells. While CtBP1, CtBP2, and p68 previously known to interact with PNN are successfully immunoprecipitated with PNN, non-PNN interacting PCNA is not detected in PNN immunoprecipitates. (C) PNN’s association with β-catenin is also observed in OE33 esophageal adenocarcinoma cells. PCNA serves as a negative interaction control. (D) CtBP2 is also in a complex with β-catenin. Co-immunoprecipitation of endogenous CtBP2 shows the presence of β-catenin. While large amount of CtBP1 is co-immunoprecipitated, p68 and PCNA are not detectable in CtBP2 immunoprecipitates. IP: immunoprecipitation; IB: immunoblotting; Input: 4% of total sample.

Fig. 8

Specific recruitment of PNN to the active proximal promoter of CDX2. (A) Semiquantitative RT-PCR analysis shows that OE33 and EcR293 cells are expressing CDX2, but not CDX1. HET-1A (Human esophageal epithelial cell line) and LoVo (human colon adenocarcinoma cell line) cells serve as negative and positive controls, respectively. (B) A schematic illustration shows the location of primers used for ChIP assays on the promoter area of human CDX genes. Numbers are relative to the transcription start site (+1). Tcf binding elements are shown as red blocks. (C) ChIP assay with antibody against H3K4me3 shows active chromatin status of CDX2 promoter in EcR293-PNNGFP cells. (D, E) ChIP analyses demonstrate specific recruitment of PNN to active CDX2 promoter, but not to the inactive CDX1 promoter area in EcR293 (D) and OE33 (E) cells. (F, G) CtBP2 is also recruited to CDX2 promoter in OE33 (F) and mouse ES cells (G) as evidenced by ChIP assay with anti-CtBP2 antibody. As expected, Tcf4 is also shown to be recruited to Cdx2 promoter in ES cells.

Fig. 9

A model for functional interaction between Pnn, Wnt/β-catenin signaling, and Cdx proteins. Pnn may modulate Wnt/β-catenin activity through its interaction with β-catenin and, in turn, influence Cdx2 expression. Besides, Pnn might be directly involved in Cdx2 regulation at its promoter. It is very tempting to contemplate that Pnn may coordinate these two possible roles in an interdependent manner to ensure tight regulation of Cdx2 expression during mouse small intestinal development. Autoregulatory mechanism and inhibitory role of Cdx proteins on Wnt/β-catenin activity are shown in blue. The impact of Wnt/β-catenin activity on Cdx genes is shown in green.

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Pinin modulates expression of an intestinal homeobox gene, Cdx2, and plays an essential role for small intestinal morphogenesis - PubMed (original) (raw)