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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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The MEF2 transcription factors are expressed in the pancreas and the MC1568 inhibitor increases β- and δ-cell mass. A: qPCR analysis of Mef2A expression in E15.5 and E18.5 mouse pancreas, and E18.5 heart and muscle. B: Immunohistological analysis of MEF2A (red) in E18.5 mouse pancreas. β-Cells were detected with insulin (INS) staining (green). The arrow shows one cell coexpressing MEFA2 and insulin. C: qPCR analysis of Pax4 mRNA expression between 3 and 14 days in culture (D3 to D14), in E13.5 pancreatic explants that were treated or not with MC1568 during 14 days. D: qPCR analysis of insulin mRNA expression from D3 to D14 in cultured pancreatic explants that were treated or not with MC1568. E: Immunohistological analyses of pancreata after 7 days in culture, with and without MC1568 treatment. β-Cell development was evaluated with insulin staining (red). Absolute areas that were occupied by the insulin-positive cells were quantified. F: qPCR analysis of MafA mRNA expression from D3 to D14 in pancreatic explants treated or not with MC1568. G: qPCR analysis of Znt8 mRNA expression from D3 to D14 in pancreatic explants treated or not with MC1568. H: qPCR analysis of somatostatin mRNA expression from D3 to D14 in pancreatic explants treated or not with MC1568. I: Immunohistological analyses of pancreata after 7 days in culture, with and without MC1568 treatment. δ-Cell development was evaluated with somatostatin (SST) staining (green). Absolute areas that were occupied by the somatostatin-positive cells were quantified. In E and I, nuclei were stained with Hoechst stain (blue). qPCR data and immunohistological analyses are the means ± SEM of four and six 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 8: The MEF2 transcription factors are expressed in the pancreas and the MC1568 inhibitor increases β- and δ-cell mass. A: qPCR analysis of Mef2A expression in E15.5 and E18.5 mouse pancreas, and E18.5 heart and muscle. B: Immunohistological analysis of MEF2A (red) in E18.5 mouse pancreas. β-Cells were detected with insulin (INS) staining (green). The arrow shows one cell coexpressing MEFA2 and insulin. C: qPCR analysis of Pax4 mRNA expression between 3 and 14 days in culture (D3 to D14), in E13.5 pancreatic explants that were treated or not with MC1568 during 14 days. D: qPCR analysis of insulin mRNA expression from D3 to D14 in cultured pancreatic explants that were treated or not with MC1568. E: Immunohistological analyses of pancreata after 7 days in culture, with and without MC1568 treatment. β-Cell development was evaluated with insulin staining (red). Absolute areas that were occupied by the insulin-positive cells were quantified. F: qPCR analysis of MafA mRNA expression from D3 to D14 in pancreatic explants treated or not with MC1568. G: qPCR analysis of Znt8 mRNA expression from D3 to D14 in pancreatic explants treated or not with MC1568. H: qPCR analysis of somatostatin mRNA expression from D3 to D14 in pancreatic explants treated or not with MC1568. I: Immunohistological analyses of pancreata after 7 days in culture, with and without MC1568 treatment. δ-Cell development was evaluated with somatostatin (SST) staining (green). Absolute areas that were occupied by the somatostatin-positive cells were quantified. In E and I, nuclei were stained with Hoechst stain (blue). qPCR data and immunohistological analyses are the means ± SEM of four and six 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 how the class IIa HDACs control β- and δ-cell development, we assessed expression of MEF2 transcription factors, which associate with class IIa HDACs in cardiac muscle development, skeletal muscle differentiation, and T-cell apoptosis (7). We analyzed Mef2A, Mef2C, and Mef2D expression in the embryonic pancreas. Whereas Mef2C mRNA was detected at a low level (data not shown), Mef2A and Mef2D mRNAs, which are expressed at high levels in embryonic heart and skeletal muscle (32), were detected in E15.5 and E18.5 pancreata at levels similar to those observed in E18.5 heart and skeletal muscle (Fig. 8A and Supplementary Fig. 7A). Immunohistochemistry showed the expression of MEF2A in the pancreas at E18.5. MEF2A was expressed in endocrine cells and smooth muscle cells and not in acinar cells (Fig. 8B and Supplementary Fig. 7B–E).


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)

The MEF2 transcription factors are expressed in the pancreas and the MC1568 inhibitor increases β- and δ-cell mass. A: qPCR analysis of Mef2A expression in E15.5 and E18.5 mouse pancreas, and E18.5 heart and muscle. B: Immunohistological analysis of MEF2A (red) in E18.5 mouse pancreas. β-Cells were detected with insulin (INS) staining (green). The arrow shows one cell coexpressing MEFA2 and insulin. C: qPCR analysis of Pax4 mRNA expression between 3 and 14 days in culture (D3 to D14), in E13.5 pancreatic explants that were treated or not with MC1568 during 14 days. D: qPCR analysis of insulin mRNA expression from D3 to D14 in cultured pancreatic explants that were treated or not with MC1568. E: Immunohistological analyses of pancreata after 7 days in culture, with and without MC1568 treatment. β-Cell development was evaluated with insulin staining (red). Absolute areas that were occupied by the insulin-positive cells were quantified. F: qPCR analysis of MafA mRNA expression from D3 to D14 in pancreatic explants treated or not with MC1568. G: qPCR analysis of Znt8 mRNA expression from D3 to D14 in pancreatic explants treated or not with MC1568. H: qPCR analysis of somatostatin mRNA expression from D3 to D14 in pancreatic explants treated or not with MC1568. I: Immunohistological analyses of pancreata after 7 days in culture, with and without MC1568 treatment. δ-Cell development was evaluated with somatostatin (SST) staining (green). Absolute areas that were occupied by the somatostatin-positive cells were quantified. In E and I, nuclei were stained with Hoechst stain (blue). qPCR data and immunohistological analyses are the means ± SEM of four and six 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 8: The MEF2 transcription factors are expressed in the pancreas and the MC1568 inhibitor increases β- and δ-cell mass. A: qPCR analysis of Mef2A expression in E15.5 and E18.5 mouse pancreas, and E18.5 heart and muscle. B: Immunohistological analysis of MEF2A (red) in E18.5 mouse pancreas. β-Cells were detected with insulin (INS) staining (green). The arrow shows one cell coexpressing MEFA2 and insulin. C: qPCR analysis of Pax4 mRNA expression between 3 and 14 days in culture (D3 to D14), in E13.5 pancreatic explants that were treated or not with MC1568 during 14 days. D: qPCR analysis of insulin mRNA expression from D3 to D14 in cultured pancreatic explants that were treated or not with MC1568. E: Immunohistological analyses of pancreata after 7 days in culture, with and without MC1568 treatment. β-Cell development was evaluated with insulin staining (red). Absolute areas that were occupied by the insulin-positive cells were quantified. F: qPCR analysis of MafA mRNA expression from D3 to D14 in pancreatic explants treated or not with MC1568. G: qPCR analysis of Znt8 mRNA expression from D3 to D14 in pancreatic explants treated or not with MC1568. H: qPCR analysis of somatostatin mRNA expression from D3 to D14 in pancreatic explants treated or not with MC1568. I: Immunohistological analyses of pancreata after 7 days in culture, with and without MC1568 treatment. δ-Cell development was evaluated with somatostatin (SST) staining (green). Absolute areas that were occupied by the somatostatin-positive cells were quantified. In E and I, nuclei were stained with Hoechst stain (blue). qPCR data and immunohistological analyses are the means ± SEM of four and six 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 how the class IIa HDACs control β- and δ-cell development, we assessed expression of MEF2 transcription factors, which associate with class IIa HDACs in cardiac muscle development, skeletal muscle differentiation, and T-cell apoptosis (7). We analyzed Mef2A, Mef2C, and Mef2D expression in the embryonic pancreas. Whereas Mef2C mRNA was detected at a low level (data not shown), Mef2A and Mef2D mRNAs, which are expressed at high levels in embryonic heart and skeletal muscle (32), were detected in E15.5 and E18.5 pancreata at levels similar to those observed in E18.5 heart and skeletal muscle (Fig. 8A and Supplementary Fig. 7A). Immunohistochemistry showed the expression of MEF2A in the pancreas at E18.5. MEF2A was expressed in endocrine cells and smooth muscle cells and not in acinar cells (Fig. 8B and Supplementary Fig. 7B–E).

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