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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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HDAC5 gain-of-function represses β- and δ-cell mass. A: qPCR analysis of Hdac5,insulin, somatostatin, glucagon, and amylase mRNAs expression in pancreatic spheres transduced with CMV-GFP or CMV-HDAC4 lentivirus, followed by a 7-day culture period. B: qPCR analysis of NeuroD1, Pdx1, MafA, Nkx2.2, Znt8, and Ia1 mRNA expression in pancreatic spheres transduced with CMV-GFP or CMV-HDAC5 lentivirus, followed by a 7-day culture period. C: Immunohistological analyses of pancreatic spheres transduced with a lentivirus expressing eGFP or HDAC5 followed by a 7-day culture period. β-Cells and δ-cells were detected with insulin (INS) (red) and antibody somatostatin (SST) (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-Cell and δ-cell areas are presented as a percentage of the total tissue area. qPCR data and immunohistological analyses are the means ± SEM of three and four independent experiments, respectively. *P < 0.05; **P < 0.005; ***P < 0.001. Scale bar, 50 μm. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 6: HDAC5 gain-of-function represses β- and δ-cell mass. A: qPCR analysis of Hdac5,insulin, somatostatin, glucagon, and amylase mRNAs expression in pancreatic spheres transduced with CMV-GFP or CMV-HDAC4 lentivirus, followed by a 7-day culture period. B: qPCR analysis of NeuroD1, Pdx1, MafA, Nkx2.2, Znt8, and Ia1 mRNA expression in pancreatic spheres transduced with CMV-GFP or CMV-HDAC5 lentivirus, followed by a 7-day culture period. C: Immunohistological analyses of pancreatic spheres transduced with a lentivirus expressing eGFP or HDAC5 followed by a 7-day culture period. β-Cells and δ-cells were detected with insulin (INS) (red) and antibody somatostatin (SST) (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-Cell and δ-cell areas are presented as a percentage of the total tissue area. qPCR data and immunohistological analyses are the means ± SEM of three and four independent experiments, respectively. *P < 0.05; **P < 0.005; ***P < 0.001. Scale bar, 50 μm. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: To further investigate the role of HDAC5 in β- and δ-cell development, we used lentivirus-mediated gene transfer to overexpress HDAC5 in E13.5 pancreas. In spheres infected with a lentiviral vector expressing HDAC5 (CMV-Hdac5), we observed by qPCR a 10-fold increase in Hdac5 expression (Fig. 6A). This result was associated with a 21.2 ± 1.9% decrease in insulin expression and a 42.5 ± 12.9% decrease in somatostatin expression (Fig. 6A). Interestingly, the expression of NeuroD1, Pdx1, Nkx2.2, MafA, Znt8, and Ia1 was significantly lower in spheres overexpressing HDAC5 (Fig. 6B). In contrast, we did not observe any change in glucagon or amylase expression (Fig. 6A). Finally, immunohistochemical analysis confirmed a decrease in both β-cells (a 28.9 ± 11% decrease) and δ-cells (a 61.3 ± 6.9% decrease) in spheres overexpressing HDAC5 (Fig. 6C), whereas no change in α-cells was observed (data not shown). Taken together, these results demonstrate that HDAC5 is involved in the control of β- and δ-cell development.


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)

HDAC5 gain-of-function represses β- and δ-cell mass. A: qPCR analysis of Hdac5,insulin, somatostatin, glucagon, and amylase mRNAs expression in pancreatic spheres transduced with CMV-GFP or CMV-HDAC4 lentivirus, followed by a 7-day culture period. B: qPCR analysis of NeuroD1, Pdx1, MafA, Nkx2.2, Znt8, and Ia1 mRNA expression in pancreatic spheres transduced with CMV-GFP or CMV-HDAC5 lentivirus, followed by a 7-day culture period. C: Immunohistological analyses of pancreatic spheres transduced with a lentivirus expressing eGFP or HDAC5 followed by a 7-day culture period. β-Cells and δ-cells were detected with insulin (INS) (red) and antibody somatostatin (SST) (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-Cell and δ-cell areas are presented as a percentage of the total tissue area. qPCR data and immunohistological analyses are the means ± SEM of three and four independent experiments, respectively. *P < 0.05; **P < 0.005; ***P < 0.001. Scale bar, 50 μm. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 6: HDAC5 gain-of-function represses β- and δ-cell mass. A: qPCR analysis of Hdac5,insulin, somatostatin, glucagon, and amylase mRNAs expression in pancreatic spheres transduced with CMV-GFP or CMV-HDAC4 lentivirus, followed by a 7-day culture period. B: qPCR analysis of NeuroD1, Pdx1, MafA, Nkx2.2, Znt8, and Ia1 mRNA expression in pancreatic spheres transduced with CMV-GFP or CMV-HDAC5 lentivirus, followed by a 7-day culture period. C: Immunohistological analyses of pancreatic spheres transduced with a lentivirus expressing eGFP or HDAC5 followed by a 7-day culture period. β-Cells and δ-cells were detected with insulin (INS) (red) and antibody somatostatin (SST) (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-Cell and δ-cell areas are presented as a percentage of the total tissue area. qPCR data and immunohistological analyses are the means ± SEM of three and four independent experiments, respectively. *P < 0.05; **P < 0.005; ***P < 0.001. Scale bar, 50 μm. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: To further investigate the role of HDAC5 in β- and δ-cell development, we used lentivirus-mediated gene transfer to overexpress HDAC5 in E13.5 pancreas. In spheres infected with a lentiviral vector expressing HDAC5 (CMV-Hdac5), we observed by qPCR a 10-fold increase in Hdac5 expression (Fig. 6A). This result was associated with a 21.2 ± 1.9% decrease in insulin expression and a 42.5 ± 12.9% decrease in somatostatin expression (Fig. 6A). Interestingly, the expression of NeuroD1, Pdx1, Nkx2.2, MafA, Znt8, and Ia1 was significantly lower in spheres overexpressing HDAC5 (Fig. 6B). In contrast, we did not observe any change in glucagon or amylase expression (Fig. 6A). Finally, immunohistochemical analysis confirmed a decrease in both β-cells (a 28.9 ± 11% decrease) and δ-cells (a 61.3 ± 6.9% decrease) in spheres overexpressing HDAC5 (Fig. 6C), whereas no change in α-cells was observed (data not shown). Taken together, these results demonstrate that HDAC5 is involved in the control of β- and δ-cell development.

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