Loss of hepatocyte-nuclear-factor-1alpha impacts on adult mouse intestinal epithelial cell growth and cell lineages differentiation

Abstract

Background and aims: Although Hnf1alpha is crucial for pancreas and liver functions, it is believed to play a limited functional role for intestinal epithelial functions. The aim of this study was to assess the consequences of abrogating Hnf1alpha on the maintenance of adult small intestinal epithelial functions.

Methodology/principal findings: An Hnf1alpha knockout mouse model was used. Assessment of histological abnormalities, crypt epithelial cell proliferation, epithelial barrier, glucose transport and signalling pathways were measured in these animals. Changes in global gene expression were also analyzed. Mice lacking Hnf1alpha displayed increased crypt proliferation and intestinalomegaly as well as a disturbance of intestinal epithelial cell lineages production during adult life. This phenotype was associated with a decrease of the mucosal barrier function and lumen-to-blood glucose delivery. The mammalian target of rapamycin (mTOR) signalling pathway was found to be overly activated in the small intestine of adult Hnf1alpha mutant mice. The intestinal epithelium of Hnf1alpha null mice displayed a reduction of the enteroendocrine cell population. An impact was also observed on proper Paneth cell differentiation with abnormalities in the granule exocytosis pathway.

Conclusions/significance: Together, these results unravel a functional role for Hnf1alpha in regulating adult intestinal growth and sustaining the functions of intestinal epithelial cell lineages.

Figure 1. Hnf1α null mice display intestinalomegaly.

(A) RT-PCR detection of Hnf1α was performed on total small intestinal RNA extracts. β2-microglobulin (β2mic) mRNA level was monitored as a housekeeping gene control. (B) Western blot analysis was performed on total small intestinal mucosa lysates with an antibody specific for the detection of Hnf1α. Specific detection of actin was done to control for protein integrity. (C) Photograph of representative 5-month-old control and Hnf1α null small intestines. Statistical analysis of 4-month-old mice small intestine (SI) relative length (D), relative weight per mm (E) and weight relative to mouse total body weight (F). n = 5-10; *P<0.05; **P<0.01;***P<0.001.

Figure 2. Loss of Hnf1α results in disturbed crypt-villus architecture.

Haematoxylin and eosin stained micrographs of the jejunum (A) and the ileum (B) of 4-month-old mice. Bar = 50 µm. Statistical analysis of villus (C) and crypt length (D) of jejunum and ileum of 1-month and 4-month-old mice. n = 3; total of 99-120 villi and 120 crypts. *P<0.05; ***P<0.001.

Figure 3. Loss of Hnf1α results in increased crypt proliferation index.

(A) Haematoxylin and eosin stained micrograph of a representative dividing crypt event. Bar = 30 µm. (B) Statistical analysis of the percentage of dividing crypt unit (crypt proliferation index) in the jejunum and ileum of 1-month and 4-month-old mice. n = 3-4. (C) Representative immunofluorescence for BrdU performed on sections of jejunum of both control (Ctl) and Hnf1α null mice (KO) (left panels). A statistical analysis of the number of BrdU labeled cells in the jejunum of 1-month and 4-month-old mice. n = 3; total of 85 and 105 crypts, respectively (right panel). **P<0.01; ***P<0.001.

Figure 4. Hnf1α null mice display an increase in mTOR signalling.

(A) Total protein extracts of 4-month-old mice were analyzed by Western blot for the detection of the active form of β-catenin dephosphorylated on Ser37 or Thr41, the active form of β-catenin phosphorylated on Ser552 and the total β-catenin. Band densities relative to the actin level were calculated for each sample and expression variation was statistically validated. n = 4. (B) qRT-PCR analysis of axin2 and lgr5 was performed on total RNA extracts from 4-month-old mice. n = 5-10. (C) Total protein extracts of 4-month-old mice were analyzed by Western blot for the detection of S6 ribosomal protein phosphorylated on serine 235/236, total S6 and total tuberous sclerosis 2 (TSC2). Specific detection of actin was done to control for protein integrity. (D) Total protein extracts were analyzed by Western blot for the detection of S6 ribosomal protein phosphorylated on serine 235/236 and total TSC2. Band densities relative to the actin level were calculated for each sample and expression variation was statistically validated. n = 3-6. (E) Total protein extracts of 4-month-old mice were analyzed by Western blot for the detection of total and activated forms of AMPKα and AKT. Band densities relative to the actin level were calculated for each sample and expression variation was statistically validated. n = 4-7. (F) qRT-PCR analysis of Ddit4L/Redd2 was performed on total RNA extracts at different time points during post-natal development. n = 5-10 per time point. *P<0.05; **P<0.01.

Figure 5. Barrier functions and glucose transport are deregulated in the Hnf1α mutant intestine.

(A) Statistical analysis of small intestinal permeability for ⁵¹CR-EDTA and ion transport as measured by short circuit current (I _sc). n = 5-6. (B) Glucose plasma concentration at 0 (before) and 5, 10, 15, 30, 60, and 120 min after administration of glucose through gastric gavage. n = 6-8. (C) Glucose plasma concentration net increase after calibration of starting glucose concentration for each group at 0. (D) Linear regression T test analysis of glucose plasma concentration between 0 and 15 min. *P<0.05; ***P<0.001.

Figure 6. Loss of Hnf1α leads to deregulation of Paneth cells homeostasis.

(A) Jejunum and ileum tissue sections were used to perform alcian blue staining. Representative micrographs and statistical analyses of the number of positive labeled-cells per length units of crypts and villi. n = 3; total of 75 villi and 60 crypts. Bar = 50 µm (B) Quantitative RT-PCR (qRT-PCR) analysis of trefoil factor 3 (Tff3) and mucin 2 (Muc2) was performed on total RNA extracts from 4-month-old mice. n = 5-10. (C) Lysozyme immunodetection was performed on ileum tissue sections of 4-month-old mice. Nuclei were stained in blue with Dapi. Representative micrographs and statistical analyses of the number of positive cells per crypt. n = 3-4; average of 35 crypts per animal. Bar = 20 µm (D) qRT-PCR analysis of lysozyme was performed on total RNA extracts from 4-month-old mice. n = 5-10. (E) Electron microscopic analysis of 4-month-old mice ileum sections. Bar = 10 µm. *P<0.05; ***P<0.001.

Figure 7. Loss of Hnf1α leads to deregulation of enteroendocrine cell production.

(A) Chromogranin A immunodetection was performed on ileum tissue sections. Representative micrographs and statistical analyses of the number of positive labeled-cells per crypt-villus axis. n = 3-4; average of 40 crypt-villus axis per mouse. (B) qRT-PCR analysis performed on total RNA extracts from 4-month-old mice (means +/- SEM). n = 5-10. *P<0.05; **P<0.01; ***P<0.001.