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Specific control of pancreatic endocrine β- and δ-cell mass by class IIa histone deacetylases HDAC4, HDAC5, and HDAC9.

Lenoir O, Flosseau K, Ma FX, Blondeau B, Mai A, Bassel-Duby R, Ravassard P, Olson EN, Haumaitre C, Scharfmann R - Diabetes (2011)

Bottom Line: Conversely, HDAC4 and HDAC5 overexpression showed a decreased pool of insulin-producing β-cells and somatostatin-producing δ-cells.We conclude that HDAC4, -5, and -9 are key regulators to control the pancreatic β/δ-cell lineage.These results highlight the epigenetic mechanisms underlying the regulation of endocrine cell development and suggest new strategies for β-cell differentiation-based therapies.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale, U845, Research Center Growth and Signalling, Paris Descartes University, Sorbonne Paris Cité, Necker Hospital, Paris, France.

ABSTRACT

Objective: Class IIa histone deacetylases (HDACs) belong to a large family of enzymes involved in protein deacetylation and play a role in regulating gene expression and cell differentiation. Previously, we showed that HDAC inhibitors modify the timing and determination of pancreatic cell fate. The aim of this study was to determine the role of class IIa HDACs in pancreas development.

Research design and methods: We took a genetic approach and analyzed the pancreatic phenotype of mice lacking HDAC4, -5, and -9. We also developed a novel method of lentiviral infection of pancreatic explants and performed gain-of-function experiments.

Results: We show that class IIa HDAC4, -5, and -9 have an unexpected restricted expression in the endocrine β- and δ-cells of the pancreas. Analyses of the pancreas of class IIa HDAC mutant mice revealed an increased pool of insulin-producing β-cells in Hdac5(-/-) and Hdac9(-/-) mice and an increased pool of somatostatin-producing δ-cells in Hdac4(-/-) and Hdac5(-/-) mice. Conversely, HDAC4 and HDAC5 overexpression showed a decreased pool of insulin-producing β-cells and somatostatin-producing δ-cells. Finally, treatment of pancreatic explants with the selective class IIa HDAC inhibitor MC1568 enhances expression of Pax4, a key factor required for proper β-and δ-cell differentiation and amplifies endocrine β- and δ-cells.

Conclusions: We conclude that HDAC4, -5, and -9 are key regulators to control the pancreatic β/δ-cell lineage. These results highlight the epigenetic mechanisms underlying the regulation of endocrine cell development and suggest new strategies for β-cell differentiation-based therapies.

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HDAC4 loss-of-function enhances δ-cell mass, whereas HDAC4 gain-of-function represses β- and δ-cell mass. A: Immunohistological analyses of wild-type and Hdac4−/− pancreas at P1. β-Cells and δ-cells were detected with insulin (INS) (brown, left panels) and somatostatin (SST) (brown, right panels) stainings. B: Morphometric analysis of β- and δ-cell surfaces by quantification of areas occupied by insulin- and somatostatin-positive cells. β-Cell and δ-cell surfaces were normalized to wild-type (WT) values (100%). Data are shown as means ± SEM. Four pancreata were analyzed for each genotype. C: qPCR analysis of Hdac4,insulin, somatostatin, glucagon, and amylase mRNA expression in pancreatic spheres transduced with CMV-GFP or CMV-HDAC4 lentivirus, followed by a 7-day culture period. D: qPCR analysis of NeuroD1, Pdx1, MafA, Nkx2.2, Znt8, and Ia1 mRNA expression in pancreatic spheres transduced with CMV-GFP or CMV-HDAC4 lentivirus, followed by a 7-day culture period. E: Immunohistological analyses of pancreatic spheres transduced with a lentivirus expressing enhanced GFP or HDAC4 followed by a 7-day culture period. β-Cells and δ-cells were detected with insulin (red) and antibody somatostatin (green) stainings. Nuclei were stained with Hoechst stain (blue). The absolute areas that were occupied by the insulin- and somatostatin-positive cells were quantified. β- and δ-Cell areas are presented as a percentage of the total tissue area. qPCR data and immunohistological analyses are the means ± SEM of five and six independent experiments, respectively. *P < 0.05; **P < 0.005; ***P < 0.001. Scale bar, 100 μm (A) and 50 μm (E). (A high-quality digital representation of this figure is available in the online issue.)
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Figure 4: HDAC4 loss-of-function enhances δ-cell mass, whereas HDAC4 gain-of-function represses β- and δ-cell mass. A: Immunohistological analyses of wild-type and Hdac4−/− pancreas at P1. β-Cells and δ-cells were detected with insulin (INS) (brown, left panels) and somatostatin (SST) (brown, right panels) stainings. B: Morphometric analysis of β- and δ-cell surfaces by quantification of areas occupied by insulin- and somatostatin-positive cells. β-Cell and δ-cell surfaces were normalized to wild-type (WT) values (100%). Data are shown as means ± SEM. Four pancreata were analyzed for each genotype. C: qPCR analysis of Hdac4,insulin, somatostatin, glucagon, and amylase mRNA expression in pancreatic spheres transduced with CMV-GFP or CMV-HDAC4 lentivirus, followed by a 7-day culture period. D: qPCR analysis of NeuroD1, Pdx1, MafA, Nkx2.2, Znt8, and Ia1 mRNA expression in pancreatic spheres transduced with CMV-GFP or CMV-HDAC4 lentivirus, followed by a 7-day culture period. E: Immunohistological analyses of pancreatic spheres transduced with a lentivirus expressing enhanced GFP or HDAC4 followed by a 7-day culture period. β-Cells and δ-cells were detected with insulin (red) and antibody somatostatin (green) stainings. Nuclei were stained with Hoechst stain (blue). The absolute areas that were occupied by the insulin- and somatostatin-positive cells were quantified. β- and δ-Cell areas are presented as a percentage of the total tissue area. qPCR data and immunohistological analyses are the means ± SEM of five and six independent experiments, respectively. *P < 0.05; **P < 0.005; ***P < 0.001. Scale bar, 100 μm (A) and 50 μm (E). (A high-quality digital representation of this figure is available in the online issue.)

Mentions: HDAC4 is specifically expressed in chondrocytes, skeletal muscle, heart, brain, and retina (25–27) and plays a central role in the formation of the skeleton (16). As Hdac4−/− mice die by P7 because of bone abnormalities (16), we assessed the pancreas of Hdac4−/− mice at P1. No difference was detected in pancreatic weight between Hdac4−/− and wild-type mice (data not shown). Quantification of insulin staining indicated that β-cell mass of Hdac4−/− and wild-type mice was similar (Fig. 4A and B), as was the case for the α-cell mass quantified by glucagon staining (data not shown). Quantification of somatostatin staining indicated that δ-cell mass was 1.46 ± 0.09 higher in Hdac4−/− mice than in wild-type mice (Fig. 4A and B).


Specific control of pancreatic endocrine β- and δ-cell mass by class IIa histone deacetylases HDAC4, HDAC5, and HDAC9.

Lenoir O, Flosseau K, Ma FX, Blondeau B, Mai A, Bassel-Duby R, Ravassard P, Olson EN, Haumaitre C, Scharfmann R - Diabetes (2011)

HDAC4 loss-of-function enhances δ-cell mass, whereas HDAC4 gain-of-function represses β- and δ-cell mass. A: Immunohistological analyses of wild-type and Hdac4−/− pancreas at P1. β-Cells and δ-cells were detected with insulin (INS) (brown, left panels) and somatostatin (SST) (brown, right panels) stainings. B: Morphometric analysis of β- and δ-cell surfaces by quantification of areas occupied by insulin- and somatostatin-positive cells. β-Cell and δ-cell surfaces were normalized to wild-type (WT) values (100%). Data are shown as means ± SEM. Four pancreata were analyzed for each genotype. C: qPCR analysis of Hdac4,insulin, somatostatin, glucagon, and amylase mRNA expression in pancreatic spheres transduced with CMV-GFP or CMV-HDAC4 lentivirus, followed by a 7-day culture period. D: qPCR analysis of NeuroD1, Pdx1, MafA, Nkx2.2, Znt8, and Ia1 mRNA expression in pancreatic spheres transduced with CMV-GFP or CMV-HDAC4 lentivirus, followed by a 7-day culture period. E: Immunohistological analyses of pancreatic spheres transduced with a lentivirus expressing enhanced GFP or HDAC4 followed by a 7-day culture period. β-Cells and δ-cells were detected with insulin (red) and antibody somatostatin (green) stainings. Nuclei were stained with Hoechst stain (blue). The absolute areas that were occupied by the insulin- and somatostatin-positive cells were quantified. β- and δ-Cell areas are presented as a percentage of the total tissue area. qPCR data and immunohistological analyses are the means ± SEM of five and six independent experiments, respectively. *P < 0.05; **P < 0.005; ***P < 0.001. Scale bar, 100 μm (A) and 50 μm (E). (A high-quality digital representation of this figure is available in the online issue.)
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Related In: Results  -  Collection

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Figure 4: HDAC4 loss-of-function enhances δ-cell mass, whereas HDAC4 gain-of-function represses β- and δ-cell mass. A: Immunohistological analyses of wild-type and Hdac4−/− pancreas at P1. β-Cells and δ-cells were detected with insulin (INS) (brown, left panels) and somatostatin (SST) (brown, right panels) stainings. B: Morphometric analysis of β- and δ-cell surfaces by quantification of areas occupied by insulin- and somatostatin-positive cells. β-Cell and δ-cell surfaces were normalized to wild-type (WT) values (100%). Data are shown as means ± SEM. Four pancreata were analyzed for each genotype. C: qPCR analysis of Hdac4,insulin, somatostatin, glucagon, and amylase mRNA expression in pancreatic spheres transduced with CMV-GFP or CMV-HDAC4 lentivirus, followed by a 7-day culture period. D: qPCR analysis of NeuroD1, Pdx1, MafA, Nkx2.2, Znt8, and Ia1 mRNA expression in pancreatic spheres transduced with CMV-GFP or CMV-HDAC4 lentivirus, followed by a 7-day culture period. E: Immunohistological analyses of pancreatic spheres transduced with a lentivirus expressing enhanced GFP or HDAC4 followed by a 7-day culture period. β-Cells and δ-cells were detected with insulin (red) and antibody somatostatin (green) stainings. Nuclei were stained with Hoechst stain (blue). The absolute areas that were occupied by the insulin- and somatostatin-positive cells were quantified. β- and δ-Cell areas are presented as a percentage of the total tissue area. qPCR data and immunohistological analyses are the means ± SEM of five and six independent experiments, respectively. *P < 0.05; **P < 0.005; ***P < 0.001. Scale bar, 100 μm (A) and 50 μm (E). (A high-quality digital representation of this figure is available in the online issue.)
Mentions: HDAC4 is specifically expressed in chondrocytes, skeletal muscle, heart, brain, and retina (25–27) and plays a central role in the formation of the skeleton (16). As Hdac4−/− mice die by P7 because of bone abnormalities (16), we assessed the pancreas of Hdac4−/− mice at P1. No difference was detected in pancreatic weight between Hdac4−/− and wild-type mice (data not shown). Quantification of insulin staining indicated that β-cell mass of Hdac4−/− and wild-type mice was similar (Fig. 4A and B), as was the case for the α-cell mass quantified by glucagon staining (data not shown). Quantification of somatostatin staining indicated that δ-cell mass was 1.46 ± 0.09 higher in Hdac4−/− mice than in wild-type mice (Fig. 4A and B).

Bottom Line: Conversely, HDAC4 and HDAC5 overexpression showed a decreased pool of insulin-producing β-cells and somatostatin-producing δ-cells.We conclude that HDAC4, -5, and -9 are key regulators to control the pancreatic β/δ-cell lineage.These results highlight the epigenetic mechanisms underlying the regulation of endocrine cell development and suggest new strategies for β-cell differentiation-based therapies.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale, U845, Research Center Growth and Signalling, Paris Descartes University, Sorbonne Paris Cité, Necker Hospital, Paris, France.

ABSTRACT

Objective: Class IIa histone deacetylases (HDACs) belong to a large family of enzymes involved in protein deacetylation and play a role in regulating gene expression and cell differentiation. Previously, we showed that HDAC inhibitors modify the timing and determination of pancreatic cell fate. The aim of this study was to determine the role of class IIa HDACs in pancreas development.

Research design and methods: We took a genetic approach and analyzed the pancreatic phenotype of mice lacking HDAC4, -5, and -9. We also developed a novel method of lentiviral infection of pancreatic explants and performed gain-of-function experiments.

Results: We show that class IIa HDAC4, -5, and -9 have an unexpected restricted expression in the endocrine β- and δ-cells of the pancreas. Analyses of the pancreas of class IIa HDAC mutant mice revealed an increased pool of insulin-producing β-cells in Hdac5(-/-) and Hdac9(-/-) mice and an increased pool of somatostatin-producing δ-cells in Hdac4(-/-) and Hdac5(-/-) mice. Conversely, HDAC4 and HDAC5 overexpression showed a decreased pool of insulin-producing β-cells and somatostatin-producing δ-cells. Finally, treatment of pancreatic explants with the selective class IIa HDAC inhibitor MC1568 enhances expression of Pax4, a key factor required for proper β-and δ-cell differentiation and amplifies endocrine β- and δ-cells.

Conclusions: We conclude that HDAC4, -5, and -9 are key regulators to control the pancreatic β/δ-cell lineage. These results highlight the epigenetic mechanisms underlying the regulation of endocrine cell development and suggest new strategies for β-cell differentiation-based therapies.

Show MeSH
Related in: MedlinePlus