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Postnatal expansion of the pancreatic beta-cell mass is dependent on survivin.

Jiang Y, Nishimura W, Devor-Henneman D, Kusewitt D, Wang H, Holloway MP, Dohi T, Sabo E, Robinson ML, Altieri DC, Sharma A, Altura RA - Diabetes (2008)

Bottom Line: Selective deletion of survivin in pancreatic endocrine cells in the mouse had no discernible effects during embryogenesis but was associated with striking decreases in beta-cell number after birth, leading to hyperglycemia and early-onset diabetes by 4 weeks of age.Serum insulin levels were significantly decreased in animals lacking endocrine cell survivin, with relative stability of other hormones.Exogenous expression of survivin in mature beta-cells lacking endogenous survivin completely rescued the hyperglycemic phenotype and the decrease in beta-cell mass, confirming the specificity of the survivin effect in these cells.

View Article: PubMed Central - PubMed

Affiliation: The Research Institute at Nationwide Children's Hospital, Columbus, Ohio, USA.

ABSTRACT

Objective: Diabetes results from a deficiency of functional beta-cells due to both an increase in beta-cell death and an inhibition of beta-cell replication. The molecular mechanisms responsible for these effects in susceptible individuals are mostly unknown. The objective of this study was to determine whether a gene critical for cell division and cell survival in cancer cells, survivin, might also be important for beta-cells.

Research design and methods: We generated mice harboring a conditional deletion of survivin in pancreatic endocrine cells using mice with a Pax-6-Cre transgene promoter construct driving tissue-specific expression of Cre-recombinase in these cells. We performed metabolic studies and immunohistochemical analyses to determine the effects of a mono- and biallelic deletion of survivin.

Results: Selective deletion of survivin in pancreatic endocrine cells in the mouse had no discernible effects during embryogenesis but was associated with striking decreases in beta-cell number after birth, leading to hyperglycemia and early-onset diabetes by 4 weeks of age. Serum insulin levels were significantly decreased in animals lacking endocrine cell survivin, with relative stability of other hormones. Exogenous expression of survivin in mature beta-cells lacking endogenous survivin completely rescued the hyperglycemic phenotype and the decrease in beta-cell mass, confirming the specificity of the survivin effect in these cells.

Conclusions: Our findings implicate survivin in the maintenance of beta-cell mass through both replication and antiapoptotic mechanisms. Given the widespread involvement of survivin in cancer, a novel role for survivin may well be exploited in beta-cell regulation in diseased states, such as diabetes.

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Defects in replication and apoptosis pathways. A: PCNA staining of pancreatic tissues indicates a 50% decrease in the proliferation of islet cells isolated from survivin-deficient animals (□, n = 5) compared with their littermate controls (▪, n = 5). Data are mean (±SD) percentages of positive cells. *Significance at P < 0.05. B: Caspase 3 activity assays performed on islets isolated from 2-week-old mice indicate a two- to threefold increase in caspase 3 activation in islet cells isolated from survivin-deficient animals (□) compared with their littermate controls (▪). Data are mean (±SD) percentages of positive cells. *Significance at P < 0.05. C: Aberrant cell cycle progression in islet cells lacking survivin. Islet cells isolated from 2-week-old Pax6-cre;survivinlox/lox mice and their littermate controls were fixed, stained with propidium iodide, and analyzed by flow cytometry. Comparisons between survivin-deficient animals and their controls are shown. D: Fold changes in the expression of the indicated genes as determined by quantitative PCR of isolated islets at 1–2 weeks of age. ▪, Pax6-cre;survivinlox/lox cells in comparison with controls. Data are mean (±SD).
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f5: Defects in replication and apoptosis pathways. A: PCNA staining of pancreatic tissues indicates a 50% decrease in the proliferation of islet cells isolated from survivin-deficient animals (□, n = 5) compared with their littermate controls (▪, n = 5). Data are mean (±SD) percentages of positive cells. *Significance at P < 0.05. B: Caspase 3 activity assays performed on islets isolated from 2-week-old mice indicate a two- to threefold increase in caspase 3 activation in islet cells isolated from survivin-deficient animals (□) compared with their littermate controls (▪). Data are mean (±SD) percentages of positive cells. *Significance at P < 0.05. C: Aberrant cell cycle progression in islet cells lacking survivin. Islet cells isolated from 2-week-old Pax6-cre;survivinlox/lox mice and their littermate controls were fixed, stained with propidium iodide, and analyzed by flow cytometry. Comparisons between survivin-deficient animals and their controls are shown. D: Fold changes in the expression of the indicated genes as determined by quantitative PCR of isolated islets at 1–2 weeks of age. ▪, Pax6-cre;survivinlox/lox cells in comparison with controls. Data are mean (±SD).

Mentions: To test the hypothesis that survivin regulates cell division and/or cell death in pancreatic β-cells during a time period shortly after birth, we examined tissue sections isolated from 2-week-old animals for the expression of proliferating cell nuclear antigen (PCNA), a marker of cell cycle proliferation, and for transferase-mediated dUTP nick-end labeling (TUNEL), a marker for apoptosis. We observed a 50% decrease in PCNA staining in the survivin-deficient β-cells (Fig. 5A) but no significant change in the number of TUNEL+ cells (not shown). To increase the sensitivity for the detection of cell death, we isolated islet cells from the pancreata of 1- to 2-week-old animals and subjected them to functional caspase 3 activity assays. With this methodology, we did observe a twofold increase in caspase 3 activity in the survivin-deficient cells (Fig. 5B), suggesting an effect on a caspase 3–dependent cell death pathway. To further characterize the cell cycle abnormalities, we performed flow cytometry analyses on the same isolated islets. This revealed an excess of cells with >4N modal chromosome numbers in the survivin-deficient islets (14 vs. 9%; Fig. 5C) and an accumulation of survivin-deficient islets in late S/G2 (36 vs. 23%; Fig. 5C), suggesting a delay in cell cycle progression. To attempt to gain further insight into potential cell cycle proteins regulated by survivin in β-cells, we performed quantitative PCR on RNA from the isolated islets for genes involved in cell cycle progression. These analyses revealed a significant (average threefold) increase in expression of the cell cycle inhibitor p21WAF1 and a twofold decrease in expression in cyclin E in the survivin-deficient cells (Fig. 5D). No significant changes in the expression levels of Cyclin A, B1, B2, C, D1, F, p27, Cdk2, or Cdk4 were seen (Fig. 5D). Taken together, our findings support the hypothesis that survivin regulates cell cycle progression in pancreatic β-cells. These effects could be mediated through repression of p21WAF1, which might occur as a consequence of survivin-dependent repression of p53 protein (37,38).


Postnatal expansion of the pancreatic beta-cell mass is dependent on survivin.

Jiang Y, Nishimura W, Devor-Henneman D, Kusewitt D, Wang H, Holloway MP, Dohi T, Sabo E, Robinson ML, Altieri DC, Sharma A, Altura RA - Diabetes (2008)

Defects in replication and apoptosis pathways. A: PCNA staining of pancreatic tissues indicates a 50% decrease in the proliferation of islet cells isolated from survivin-deficient animals (□, n = 5) compared with their littermate controls (▪, n = 5). Data are mean (±SD) percentages of positive cells. *Significance at P < 0.05. B: Caspase 3 activity assays performed on islets isolated from 2-week-old mice indicate a two- to threefold increase in caspase 3 activation in islet cells isolated from survivin-deficient animals (□) compared with their littermate controls (▪). Data are mean (±SD) percentages of positive cells. *Significance at P < 0.05. C: Aberrant cell cycle progression in islet cells lacking survivin. Islet cells isolated from 2-week-old Pax6-cre;survivinlox/lox mice and their littermate controls were fixed, stained with propidium iodide, and analyzed by flow cytometry. Comparisons between survivin-deficient animals and their controls are shown. D: Fold changes in the expression of the indicated genes as determined by quantitative PCR of isolated islets at 1–2 weeks of age. ▪, Pax6-cre;survivinlox/lox cells in comparison with controls. Data are mean (±SD).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2551682&req=5

f5: Defects in replication and apoptosis pathways. A: PCNA staining of pancreatic tissues indicates a 50% decrease in the proliferation of islet cells isolated from survivin-deficient animals (□, n = 5) compared with their littermate controls (▪, n = 5). Data are mean (±SD) percentages of positive cells. *Significance at P < 0.05. B: Caspase 3 activity assays performed on islets isolated from 2-week-old mice indicate a two- to threefold increase in caspase 3 activation in islet cells isolated from survivin-deficient animals (□) compared with their littermate controls (▪). Data are mean (±SD) percentages of positive cells. *Significance at P < 0.05. C: Aberrant cell cycle progression in islet cells lacking survivin. Islet cells isolated from 2-week-old Pax6-cre;survivinlox/lox mice and their littermate controls were fixed, stained with propidium iodide, and analyzed by flow cytometry. Comparisons between survivin-deficient animals and their controls are shown. D: Fold changes in the expression of the indicated genes as determined by quantitative PCR of isolated islets at 1–2 weeks of age. ▪, Pax6-cre;survivinlox/lox cells in comparison with controls. Data are mean (±SD).
Mentions: To test the hypothesis that survivin regulates cell division and/or cell death in pancreatic β-cells during a time period shortly after birth, we examined tissue sections isolated from 2-week-old animals for the expression of proliferating cell nuclear antigen (PCNA), a marker of cell cycle proliferation, and for transferase-mediated dUTP nick-end labeling (TUNEL), a marker for apoptosis. We observed a 50% decrease in PCNA staining in the survivin-deficient β-cells (Fig. 5A) but no significant change in the number of TUNEL+ cells (not shown). To increase the sensitivity for the detection of cell death, we isolated islet cells from the pancreata of 1- to 2-week-old animals and subjected them to functional caspase 3 activity assays. With this methodology, we did observe a twofold increase in caspase 3 activity in the survivin-deficient cells (Fig. 5B), suggesting an effect on a caspase 3–dependent cell death pathway. To further characterize the cell cycle abnormalities, we performed flow cytometry analyses on the same isolated islets. This revealed an excess of cells with >4N modal chromosome numbers in the survivin-deficient islets (14 vs. 9%; Fig. 5C) and an accumulation of survivin-deficient islets in late S/G2 (36 vs. 23%; Fig. 5C), suggesting a delay in cell cycle progression. To attempt to gain further insight into potential cell cycle proteins regulated by survivin in β-cells, we performed quantitative PCR on RNA from the isolated islets for genes involved in cell cycle progression. These analyses revealed a significant (average threefold) increase in expression of the cell cycle inhibitor p21WAF1 and a twofold decrease in expression in cyclin E in the survivin-deficient cells (Fig. 5D). No significant changes in the expression levels of Cyclin A, B1, B2, C, D1, F, p27, Cdk2, or Cdk4 were seen (Fig. 5D). Taken together, our findings support the hypothesis that survivin regulates cell cycle progression in pancreatic β-cells. These effects could be mediated through repression of p21WAF1, which might occur as a consequence of survivin-dependent repression of p53 protein (37,38).

Bottom Line: Selective deletion of survivin in pancreatic endocrine cells in the mouse had no discernible effects during embryogenesis but was associated with striking decreases in beta-cell number after birth, leading to hyperglycemia and early-onset diabetes by 4 weeks of age.Serum insulin levels were significantly decreased in animals lacking endocrine cell survivin, with relative stability of other hormones.Exogenous expression of survivin in mature beta-cells lacking endogenous survivin completely rescued the hyperglycemic phenotype and the decrease in beta-cell mass, confirming the specificity of the survivin effect in these cells.

View Article: PubMed Central - PubMed

Affiliation: The Research Institute at Nationwide Children's Hospital, Columbus, Ohio, USA.

ABSTRACT

Objective: Diabetes results from a deficiency of functional beta-cells due to both an increase in beta-cell death and an inhibition of beta-cell replication. The molecular mechanisms responsible for these effects in susceptible individuals are mostly unknown. The objective of this study was to determine whether a gene critical for cell division and cell survival in cancer cells, survivin, might also be important for beta-cells.

Research design and methods: We generated mice harboring a conditional deletion of survivin in pancreatic endocrine cells using mice with a Pax-6-Cre transgene promoter construct driving tissue-specific expression of Cre-recombinase in these cells. We performed metabolic studies and immunohistochemical analyses to determine the effects of a mono- and biallelic deletion of survivin.

Results: Selective deletion of survivin in pancreatic endocrine cells in the mouse had no discernible effects during embryogenesis but was associated with striking decreases in beta-cell number after birth, leading to hyperglycemia and early-onset diabetes by 4 weeks of age. Serum insulin levels were significantly decreased in animals lacking endocrine cell survivin, with relative stability of other hormones. Exogenous expression of survivin in mature beta-cells lacking endogenous survivin completely rescued the hyperglycemic phenotype and the decrease in beta-cell mass, confirming the specificity of the survivin effect in these cells.

Conclusions: Our findings implicate survivin in the maintenance of beta-cell mass through both replication and antiapoptotic mechanisms. Given the widespread involvement of survivin in cancer, a novel role for survivin may well be exploited in beta-cell regulation in diseased states, such as diabetes.

Show MeSH
Related in: MedlinePlus