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The retinoblastoma protein and its homolog p130 regulate the G1/S transition in pancreatic beta-cells.

Harb G, Vasavada RC, Cobrinik D, Stewart AF - Diabetes (2009)

Bottom Line: In vivo loss of either p107 or p130 did not affect beta-cell replication or function.Combined pRb/p130 loss, however, resulted in dramatically accelerated proliferation as well as apoptotic cell death.Pancreas and beta-cell mass were significantly reduced in double mutants.

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology and Metabolism, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

ABSTRACT

Objective: The retinoblastoma protein family (pRb, p130, p107) plays a central role in the regulation of cell cycle progression. Surprisingly, loss of pRb in the beta-cell has no discernible effect on cell cycle control. Therefore, we explored the effects of individual loss of either p130 or p107 in addition to the simultaneous loss of both pRb/p130 on the beta-cell.

Research design and methods: Adult mice deficient in either p130 or p107 or both pRb/p130 were examined for effects on beta-cell replication, function, and survival. The Cre-Lox system was also used to inactivate pRb in wild-type and p130-deficient beta-cells in vitro.

Results: In vivo loss of either p107 or p130 did not affect beta-cell replication or function. Combined pRb/p130 loss, however, resulted in dramatically accelerated proliferation as well as apoptotic cell death. Pancreas and beta-cell mass were significantly reduced in double mutants. Despite this, overall glucose tolerance was normal, except for mild postprandial hyperglycemia. Ex vivo, acute deletion of pRb in p130-deficient beta-cells also caused a striking increase in proliferation. The combined deletion of pRb/p130 upregulated islet expression of E2F2 but not E2F1.

Conclusions: These studies define an essential role for the pocket proteins in controlling the G(1)/S transition in beta-cells. When deficient in both pRb and p130, beta-cells undergo unrestrained cell cycle reentry and activation of apoptosis. These studies underscore the central role of the pRb pathway in controlling beta-cell turnover and provide new cellular targets for beta-cell regeneration.

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Acute in vitro deletion of Rb in wild-type or p130−/− β-cells. A: Immunofluorescent detection of Cre (red) expression in the nuclei of β-cells stained for insulin (green) 24 h after transduction with Ad.Cre. B: PCR detection of Cre-mediated recombination of the Rb gene in Rblox/lox;p130+/+ or Rblox/lox;p130−/− islets. PCR analysis of the floxed (670-bp) and recombined (260-bp) Rb alleles was performed 72 h after Ad.Cre transduction. The primers used to detect Rb and Gapdh alleles were previously reported by Vasavada et al. (14). C: Representative immunoblots for Cre recombinase, the pocket protein family, E2F1 and E2F2 from Ad.GFP, or Ad.Cre-transduced Rblox/lox;p130+/+ or Rblox/lox;p130−/− islets (n = 3–4). D: Immunofluorescent staining of insulin (green) and BrdU (red) in Rb+/+;p130+/+, Rblox/lox;p130+/+, or Rblox/lox;p130−/− β-cells transduced with either Ad.GFP or Ad.Cre followed by 72 h of culture (×400 magnification). E: Quantification of BrdU incorporation into Rb+/+;p130+/+ (n = 6), Rblox/lox;p130+/+ (n = 7), or Rblox/lox;p130−/− (n = 6) β-cells transduced with either Ad.GFP (white bars) or Ad.Cre (black bars). *P < 0.05 versus Ad.GFP transduced β-cells from both groups. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 8: Acute in vitro deletion of Rb in wild-type or p130−/− β-cells. A: Immunofluorescent detection of Cre (red) expression in the nuclei of β-cells stained for insulin (green) 24 h after transduction with Ad.Cre. B: PCR detection of Cre-mediated recombination of the Rb gene in Rblox/lox;p130+/+ or Rblox/lox;p130−/− islets. PCR analysis of the floxed (670-bp) and recombined (260-bp) Rb alleles was performed 72 h after Ad.Cre transduction. The primers used to detect Rb and Gapdh alleles were previously reported by Vasavada et al. (14). C: Representative immunoblots for Cre recombinase, the pocket protein family, E2F1 and E2F2 from Ad.GFP, or Ad.Cre-transduced Rblox/lox;p130+/+ or Rblox/lox;p130−/− islets (n = 3–4). D: Immunofluorescent staining of insulin (green) and BrdU (red) in Rb+/+;p130+/+, Rblox/lox;p130+/+, or Rblox/lox;p130−/− β-cells transduced with either Ad.GFP or Ad.Cre followed by 72 h of culture (×400 magnification). E: Quantification of BrdU incorporation into Rb+/+;p130+/+ (n = 6), Rblox/lox;p130+/+ (n = 7), or Rblox/lox;p130−/− (n = 6) β-cells transduced with either Ad.GFP (white bars) or Ad.Cre (black bars). *P < 0.05 versus Ad.GFP transduced β-cells from both groups. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: Off-target expression of the RIP promoter in the CNS is well described and conceivably might account for an islet phenotype. Thus, to determine whether the increase in β-cell proliferation in the double-mutant mice resulted from direct (β-cell) or indirect (CNS) effects of pRb/p130 loss, we disrupted the Rb gene ex vivo in both wild-type and p130−/− β-cells using the Cre-LoxP method of gene recombination. Isolated β-cells from Rblox/lox;p130+/+ or Rblox/lox;p130−/− animals were transduced with an adenovirus expressing Cre recombinase (Ad.Cre). Expression of Cre in β-cell nuclei could be detected within 24 h after infection (Fig. 8A). This resulted in efficient deletion of exon 19 at 72 h after transduction (Fig. 8B). Cre-mediated deletion of pRb resulted in a reduction in pRb protein levels, associated with an increase in both p130 and p107 in Rblox/lox;p130+/+ islets (Fig. 8C). Rblox/lox;p130−/− islets displayed upregulated p107 expression. This was further increased after deletion of pRb. Loss of both pRb and p130 resulted in elevated E2F2, but not E2F1, as assessed by immunoblot.


The retinoblastoma protein and its homolog p130 regulate the G1/S transition in pancreatic beta-cells.

Harb G, Vasavada RC, Cobrinik D, Stewart AF - Diabetes (2009)

Acute in vitro deletion of Rb in wild-type or p130−/− β-cells. A: Immunofluorescent detection of Cre (red) expression in the nuclei of β-cells stained for insulin (green) 24 h after transduction with Ad.Cre. B: PCR detection of Cre-mediated recombination of the Rb gene in Rblox/lox;p130+/+ or Rblox/lox;p130−/− islets. PCR analysis of the floxed (670-bp) and recombined (260-bp) Rb alleles was performed 72 h after Ad.Cre transduction. The primers used to detect Rb and Gapdh alleles were previously reported by Vasavada et al. (14). C: Representative immunoblots for Cre recombinase, the pocket protein family, E2F1 and E2F2 from Ad.GFP, or Ad.Cre-transduced Rblox/lox;p130+/+ or Rblox/lox;p130−/− islets (n = 3–4). D: Immunofluorescent staining of insulin (green) and BrdU (red) in Rb+/+;p130+/+, Rblox/lox;p130+/+, or Rblox/lox;p130−/− β-cells transduced with either Ad.GFP or Ad.Cre followed by 72 h of culture (×400 magnification). E: Quantification of BrdU incorporation into Rb+/+;p130+/+ (n = 6), Rblox/lox;p130+/+ (n = 7), or Rblox/lox;p130−/− (n = 6) β-cells transduced with either Ad.GFP (white bars) or Ad.Cre (black bars). *P < 0.05 versus Ad.GFP transduced β-cells from both groups. (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 8: Acute in vitro deletion of Rb in wild-type or p130−/− β-cells. A: Immunofluorescent detection of Cre (red) expression in the nuclei of β-cells stained for insulin (green) 24 h after transduction with Ad.Cre. B: PCR detection of Cre-mediated recombination of the Rb gene in Rblox/lox;p130+/+ or Rblox/lox;p130−/− islets. PCR analysis of the floxed (670-bp) and recombined (260-bp) Rb alleles was performed 72 h after Ad.Cre transduction. The primers used to detect Rb and Gapdh alleles were previously reported by Vasavada et al. (14). C: Representative immunoblots for Cre recombinase, the pocket protein family, E2F1 and E2F2 from Ad.GFP, or Ad.Cre-transduced Rblox/lox;p130+/+ or Rblox/lox;p130−/− islets (n = 3–4). D: Immunofluorescent staining of insulin (green) and BrdU (red) in Rb+/+;p130+/+, Rblox/lox;p130+/+, or Rblox/lox;p130−/− β-cells transduced with either Ad.GFP or Ad.Cre followed by 72 h of culture (×400 magnification). E: Quantification of BrdU incorporation into Rb+/+;p130+/+ (n = 6), Rblox/lox;p130+/+ (n = 7), or Rblox/lox;p130−/− (n = 6) β-cells transduced with either Ad.GFP (white bars) or Ad.Cre (black bars). *P < 0.05 versus Ad.GFP transduced β-cells from both groups. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: Off-target expression of the RIP promoter in the CNS is well described and conceivably might account for an islet phenotype. Thus, to determine whether the increase in β-cell proliferation in the double-mutant mice resulted from direct (β-cell) or indirect (CNS) effects of pRb/p130 loss, we disrupted the Rb gene ex vivo in both wild-type and p130−/− β-cells using the Cre-LoxP method of gene recombination. Isolated β-cells from Rblox/lox;p130+/+ or Rblox/lox;p130−/− animals were transduced with an adenovirus expressing Cre recombinase (Ad.Cre). Expression of Cre in β-cell nuclei could be detected within 24 h after infection (Fig. 8A). This resulted in efficient deletion of exon 19 at 72 h after transduction (Fig. 8B). Cre-mediated deletion of pRb resulted in a reduction in pRb protein levels, associated with an increase in both p130 and p107 in Rblox/lox;p130+/+ islets (Fig. 8C). Rblox/lox;p130−/− islets displayed upregulated p107 expression. This was further increased after deletion of pRb. Loss of both pRb and p130 resulted in elevated E2F2, but not E2F1, as assessed by immunoblot.

Bottom Line: In vivo loss of either p107 or p130 did not affect beta-cell replication or function.Combined pRb/p130 loss, however, resulted in dramatically accelerated proliferation as well as apoptotic cell death.Pancreas and beta-cell mass were significantly reduced in double mutants.

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology and Metabolism, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

ABSTRACT

Objective: The retinoblastoma protein family (pRb, p130, p107) plays a central role in the regulation of cell cycle progression. Surprisingly, loss of pRb in the beta-cell has no discernible effect on cell cycle control. Therefore, we explored the effects of individual loss of either p130 or p107 in addition to the simultaneous loss of both pRb/p130 on the beta-cell.

Research design and methods: Adult mice deficient in either p130 or p107 or both pRb/p130 were examined for effects on beta-cell replication, function, and survival. The Cre-Lox system was also used to inactivate pRb in wild-type and p130-deficient beta-cells in vitro.

Results: In vivo loss of either p107 or p130 did not affect beta-cell replication or function. Combined pRb/p130 loss, however, resulted in dramatically accelerated proliferation as well as apoptotic cell death. Pancreas and beta-cell mass were significantly reduced in double mutants. Despite this, overall glucose tolerance was normal, except for mild postprandial hyperglycemia. Ex vivo, acute deletion of pRb in p130-deficient beta-cells also caused a striking increase in proliferation. The combined deletion of pRb/p130 upregulated islet expression of E2F2 but not E2F1.

Conclusions: These studies define an essential role for the pocket proteins in controlling the G(1)/S transition in beta-cells. When deficient in both pRb and p130, beta-cells undergo unrestrained cell cycle reentry and activation of apoptosis. These studies underscore the central role of the pRb pathway in controlling beta-cell turnover and provide new cellular targets for beta-cell regeneration.

Show MeSH
Related in: MedlinePlus