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

Lussier CR, Brial F, Roy SA, Langlois MJ, Verdu EF, Rivard N, Perreault N, Boudreau F - PLoS ONE (2010)

Bottom Line: Although Hnf1alpha is crucial for pancreas and liver functions, it is believed to play a limited functional role for intestinal epithelial functions.Changes in global gene expression were also analyzed.This phenotype was associated with a decrease of the mucosal barrier function and lumen-to-blood glucose delivery.

View Article: PubMed Central - PubMed

Affiliation: Département d'Anatomie et Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Quebec, Canada.

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 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.

Show MeSH

Related in: MedlinePlus

Hnf1α  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.
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pone-0012378-g004: Hnf1α 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.

Mentions: Adult Hnf1α mutant mice displayed a dramatic increase in intestinal crypt proliferation resulting in organomegaly. Because the Wnt/β-catenin signaling pathway is crucial in the regulation of gut epithelial cell proliferation [1], the level of β-catenin activity was next compared between Hnf1α mutant and control mice. Western blot analysis on total extracts obtained from small intestinal mucosa scrapping showed no significant modulation of total β-catenin between Hnf1α mutant and control mice (Figure 4A). No change in the level of unphosphorylated active form of β-catenin on serine 37 was observed. We have also verified the phosphorylation status of β-catenin on serine 552, a process regulated by AKT and reported to promote β-catenin stability and nuclear localization [18]. The phosphorylation status of β-catenin at this specific amino acid residue remained unchanged between Hnf1α mutant and controls (Figure 4A). This finding was also confirmed by immunofluorescence for which no difference in nuclear β-catenin labelling was observed (data not shown). The expression of the Axin2 and stem cells expressing leucine rich repeat-containing G protein-coupled receptor 5 (Lgr5) gene transcripts, two well established targets of Wnt signalling in intestinal epithelial cells [19], [20], was not affected by the loss of Hnf1α (Figure 4B). One specific signalling pathway that regulates cell growth and metabolism is mTOR [21]. To investigate the activation status of the mTOR signalling pathway, the phosphorylation of S6, which is catalyzed by S6 kinase in an mTOR-dependent manner [22], was monitored in the small intestinal epithelium of Hnf1α mutant and controls mice. Western blot analysis showed that the S6 phosphorylation at Ser235/236 was elevated more than 5-fold in the small intestinal epithelium of 4 month-old Hnf1α mutant mice as compared with control mice (Figure 4C–D). S6 phosphorylation levels was not yet significantly modulated in the small intestine of 1 month-old Hnf1α mutant mice (Figure 4D) which was consistent with the observed delay in the amplification of crypt unit proliferation during the adult life of these mutant mice (Figure 3). One crucial negative regulator of mTOR signalling is the tuberous sclerosis protein TSC2 [21]. Although TSC2 gene transcript remained stable during post-natal development and adult life (data not shown), TSC2 protein was down modulated by 50% in the small intestinal epithelium of adult Hnf1α mutant mice as compared with control mice (Figure 4C–D). The PI3K/Akt and AMP-activated protein kinase (AMPK) pathways are important regulators of TSC2 protein function and stability. AMPK and AKT phosphorylation-activated status was not significantly modulated in the small intestinal epithelium of 4-month-old Hnf1α mutant mice as compared with control mice under our experimental conditions (Figure 4E). Another important molecular entity that inhibits mTOR signalling is the Ddit4l/REDD2 that acts downstream of Akt and upstream of TSC2 preventing the inhibitory binding of 14-3-3 to TSC2 [23]. Since no commercial antibodies were available for Ddit4l/REDD2, total RNA was isolated from the small intestine of Hnf1α mutant and control mice at different time points during post-natal development. qRT-PCR analyses showed that the Ddit4l/REDD2 gene transcript was significantly increased during the suckling-weaning transition in control mice, a tendency that was abolished in the small intestine of Hnf1α mice (Figure 4F).


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

Lussier CR, Brial F, Roy SA, Langlois MJ, Verdu EF, Rivard N, Perreault N, Boudreau F - PLoS ONE (2010)

Hnf1α  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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2927538&req=5

pone-0012378-g004: Hnf1α 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.
Mentions: Adult Hnf1α mutant mice displayed a dramatic increase in intestinal crypt proliferation resulting in organomegaly. Because the Wnt/β-catenin signaling pathway is crucial in the regulation of gut epithelial cell proliferation [1], the level of β-catenin activity was next compared between Hnf1α mutant and control mice. Western blot analysis on total extracts obtained from small intestinal mucosa scrapping showed no significant modulation of total β-catenin between Hnf1α mutant and control mice (Figure 4A). No change in the level of unphosphorylated active form of β-catenin on serine 37 was observed. We have also verified the phosphorylation status of β-catenin on serine 552, a process regulated by AKT and reported to promote β-catenin stability and nuclear localization [18]. The phosphorylation status of β-catenin at this specific amino acid residue remained unchanged between Hnf1α mutant and controls (Figure 4A). This finding was also confirmed by immunofluorescence for which no difference in nuclear β-catenin labelling was observed (data not shown). The expression of the Axin2 and stem cells expressing leucine rich repeat-containing G protein-coupled receptor 5 (Lgr5) gene transcripts, two well established targets of Wnt signalling in intestinal epithelial cells [19], [20], was not affected by the loss of Hnf1α (Figure 4B). One specific signalling pathway that regulates cell growth and metabolism is mTOR [21]. To investigate the activation status of the mTOR signalling pathway, the phosphorylation of S6, which is catalyzed by S6 kinase in an mTOR-dependent manner [22], was monitored in the small intestinal epithelium of Hnf1α mutant and controls mice. Western blot analysis showed that the S6 phosphorylation at Ser235/236 was elevated more than 5-fold in the small intestinal epithelium of 4 month-old Hnf1α mutant mice as compared with control mice (Figure 4C–D). S6 phosphorylation levels was not yet significantly modulated in the small intestine of 1 month-old Hnf1α mutant mice (Figure 4D) which was consistent with the observed delay in the amplification of crypt unit proliferation during the adult life of these mutant mice (Figure 3). One crucial negative regulator of mTOR signalling is the tuberous sclerosis protein TSC2 [21]. Although TSC2 gene transcript remained stable during post-natal development and adult life (data not shown), TSC2 protein was down modulated by 50% in the small intestinal epithelium of adult Hnf1α mutant mice as compared with control mice (Figure 4C–D). The PI3K/Akt and AMP-activated protein kinase (AMPK) pathways are important regulators of TSC2 protein function and stability. AMPK and AKT phosphorylation-activated status was not significantly modulated in the small intestinal epithelium of 4-month-old Hnf1α mutant mice as compared with control mice under our experimental conditions (Figure 4E). Another important molecular entity that inhibits mTOR signalling is the Ddit4l/REDD2 that acts downstream of Akt and upstream of TSC2 preventing the inhibitory binding of 14-3-3 to TSC2 [23]. Since no commercial antibodies were available for Ddit4l/REDD2, total RNA was isolated from the small intestine of Hnf1α mutant and control mice at different time points during post-natal development. qRT-PCR analyses showed that the Ddit4l/REDD2 gene transcript was significantly increased during the suckling-weaning transition in control mice, a tendency that was abolished in the small intestine of Hnf1α mice (Figure 4F).

Bottom Line: Although Hnf1alpha is crucial for pancreas and liver functions, it is believed to play a limited functional role for intestinal epithelial functions.Changes in global gene expression were also analyzed.This phenotype was associated with a decrease of the mucosal barrier function and lumen-to-blood glucose delivery.

View Article: PubMed Central - PubMed

Affiliation: Département d'Anatomie et Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Quebec, Canada.

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 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.

Show MeSH
Related in: MedlinePlus