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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 is highly enriched in β- and δ-cells. A: qPCR analysis of Hdac5 mRNA expression in embryonic pancreas, adult islets, and adult exocrine tissue. B–D: Immunohistological analysis of HDAC5 (brown) in E15.5 and adult pancreas. B: β-Cells were detected with insulin (INS) staining (red) and PDX1 staining (green) was used to detect pancreatic epithelium (PDX1low) and β-cells (PDX1high). Some β-cell PDX1high+/insulin+ expressing HDAC5 are framed. C: β- and α-cells were detected using insulin (red) and glucagon (GLU) (green) staining, respectively. Some β-cell insulin+/HDAC5+ and some α-cell glucagon+/HDAC5– are framed. D: δ-Cells were detected with somatostatin (SST) staining (green), and the arrow shows a δ-cell somatostatin+/HDAC5+. Nuclei were stained with Hoechst stain (blue). Scale bar, 50 μm (B and C) and 10 μm (D). (A high-quality digital representation of this figure is available in the online issue.)
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Figure 2: HDAC5 is highly enriched in β- and δ-cells. A: qPCR analysis of Hdac5 mRNA expression in embryonic pancreas, adult islets, and adult exocrine tissue. B–D: Immunohistological analysis of HDAC5 (brown) in E15.5 and adult pancreas. B: β-Cells were detected with insulin (INS) staining (red) and PDX1 staining (green) was used to detect pancreatic epithelium (PDX1low) and β-cells (PDX1high). Some β-cell PDX1high+/insulin+ expressing HDAC5 are framed. C: β- and α-cells were detected using insulin (red) and glucagon (GLU) (green) staining, respectively. Some β-cell insulin+/HDAC5+ and some α-cell glucagon+/HDAC5– are framed. D: δ-Cells were detected with somatostatin (SST) staining (green), and the arrow shows a δ-cell somatostatin+/HDAC5+. Nuclei were stained with Hoechst stain (blue). Scale bar, 50 μm (B and C) and 10 μm (D). (A high-quality digital representation of this figure is available in the online issue.)

Mentions: During mouse pancreas development at E15.5 and in the adult pancreas, immunohistochemistry showed that nuclear HDAC1 (Supplementary Fig. 1) and HDAC2 (data not shown) were detected in all pancreatic cell types, consistent with reports of ubiquitous expression of class I HDACs in many tissues (24). By contrast, class IIa HDAC expression was cell type specific. At E15.5, Hdac4, Hdac5, and Hdac9 were expressed in the developing pancreas, as determined by quantitative PCR (qPCR) (Figs. 1A, 2A, and 3A). In adult pancreas, Hdac4, Hdac5, and Hdac9 expression was restricted to endocrine islets and was not detected in exocrine tissue (Figs. 1A, 2A, and 3A). Purity of endocrine versus exocrine fractions was validated by insulin and amylase mRNA expression, respectively (Supplementary Fig. 2). To determine which endocrine cell type expresses class IIa HDACs, we performed immunohistochemistry and showed that HDAC4 was detected at E15.5 and E18.5 in insulin-positive cells (Fig. 1B and data not shown). At P7 and in the adult pancreas, we observed two different expression levels of HDAC4. Low expression of HDAC4 was seen in cells stained positive for insulin (Fig. 1B and data not shown), whereas greater expression of HDAC4 was observed in cells expressing somatostatin (Fig. 1C). HDAC5 was specifically detected in β-cells at E15.5 and E18.5 (Fig. 2B and data not shown). At P7 and in the adult pancreas, HDAC5 was detected in both insulin-expressing cells and in somatostatin-expressing cells (Fig. 2C and D and data not shown). As was seen with HDAC5 expression, HDAC9 was selectively detected in insulin-positive cells at E15.5, E18.5, and P7 and in the adult pancreas (Fig. 3B and data not shown). In contrast, HDAC9 was not detected in somatostatin-expressing cells (Fig. 3C). Strikingly, we found no expression of HDAC4, -5, and -9 in glucagon-expressing cells or in the acinar pancreatic tissue (Figs. 1D, 2B and C, and 3D). Thus, HDAC4 is highly enriched in δ-cells and at a low level in β-cells, HDAC5 expression is highly enriched in β- and δ-cells, and HDAC9 is highly enriched in β-cells. Finally, immunohistochemical analysis showed that the fourth member of the class IIa HDACs (HDAC7) was expressed in vascular endothelial cells and absent from endocrine cells (Supplementary Fig. 3). Altogether, these results reveal specific expression of three class IIa HDACs (HDAC4, -5, and -9) in pancreatic endocrine β- and δ-cells.


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 is highly enriched in β- and δ-cells. A: qPCR analysis of Hdac5 mRNA expression in embryonic pancreas, adult islets, and adult exocrine tissue. B–D: Immunohistological analysis of HDAC5 (brown) in E15.5 and adult pancreas. B: β-Cells were detected with insulin (INS) staining (red) and PDX1 staining (green) was used to detect pancreatic epithelium (PDX1low) and β-cells (PDX1high). Some β-cell PDX1high+/insulin+ expressing HDAC5 are framed. C: β- and α-cells were detected using insulin (red) and glucagon (GLU) (green) staining, respectively. Some β-cell insulin+/HDAC5+ and some α-cell glucagon+/HDAC5– are framed. D: δ-Cells were detected with somatostatin (SST) staining (green), and the arrow shows a δ-cell somatostatin+/HDAC5+. Nuclei were stained with Hoechst stain (blue). Scale bar, 50 μm (B and C) and 10 μm (D). (A high-quality digital representation of this figure is available in the online issue.)
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Figure 2: HDAC5 is highly enriched in β- and δ-cells. A: qPCR analysis of Hdac5 mRNA expression in embryonic pancreas, adult islets, and adult exocrine tissue. B–D: Immunohistological analysis of HDAC5 (brown) in E15.5 and adult pancreas. B: β-Cells were detected with insulin (INS) staining (red) and PDX1 staining (green) was used to detect pancreatic epithelium (PDX1low) and β-cells (PDX1high). Some β-cell PDX1high+/insulin+ expressing HDAC5 are framed. C: β- and α-cells were detected using insulin (red) and glucagon (GLU) (green) staining, respectively. Some β-cell insulin+/HDAC5+ and some α-cell glucagon+/HDAC5– are framed. D: δ-Cells were detected with somatostatin (SST) staining (green), and the arrow shows a δ-cell somatostatin+/HDAC5+. Nuclei were stained with Hoechst stain (blue). Scale bar, 50 μm (B and C) and 10 μm (D). (A high-quality digital representation of this figure is available in the online issue.)
Mentions: During mouse pancreas development at E15.5 and in the adult pancreas, immunohistochemistry showed that nuclear HDAC1 (Supplementary Fig. 1) and HDAC2 (data not shown) were detected in all pancreatic cell types, consistent with reports of ubiquitous expression of class I HDACs in many tissues (24). By contrast, class IIa HDAC expression was cell type specific. At E15.5, Hdac4, Hdac5, and Hdac9 were expressed in the developing pancreas, as determined by quantitative PCR (qPCR) (Figs. 1A, 2A, and 3A). In adult pancreas, Hdac4, Hdac5, and Hdac9 expression was restricted to endocrine islets and was not detected in exocrine tissue (Figs. 1A, 2A, and 3A). Purity of endocrine versus exocrine fractions was validated by insulin and amylase mRNA expression, respectively (Supplementary Fig. 2). To determine which endocrine cell type expresses class IIa HDACs, we performed immunohistochemistry and showed that HDAC4 was detected at E15.5 and E18.5 in insulin-positive cells (Fig. 1B and data not shown). At P7 and in the adult pancreas, we observed two different expression levels of HDAC4. Low expression of HDAC4 was seen in cells stained positive for insulin (Fig. 1B and data not shown), whereas greater expression of HDAC4 was observed in cells expressing somatostatin (Fig. 1C). HDAC5 was specifically detected in β-cells at E15.5 and E18.5 (Fig. 2B and data not shown). At P7 and in the adult pancreas, HDAC5 was detected in both insulin-expressing cells and in somatostatin-expressing cells (Fig. 2C and D and data not shown). As was seen with HDAC5 expression, HDAC9 was selectively detected in insulin-positive cells at E15.5, E18.5, and P7 and in the adult pancreas (Fig. 3B and data not shown). In contrast, HDAC9 was not detected in somatostatin-expressing cells (Fig. 3C). Strikingly, we found no expression of HDAC4, -5, and -9 in glucagon-expressing cells or in the acinar pancreatic tissue (Figs. 1D, 2B and C, and 3D). Thus, HDAC4 is highly enriched in δ-cells and at a low level in β-cells, HDAC5 expression is highly enriched in β- and δ-cells, and HDAC9 is highly enriched in β-cells. Finally, immunohistochemical analysis showed that the fourth member of the class IIa HDACs (HDAC7) was expressed in vascular endothelial cells and absent from endocrine cells (Supplementary Fig. 3). Altogether, these results reveal specific expression of three class IIa HDACs (HDAC4, -5, and -9) in pancreatic endocrine β- and δ-cells.

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