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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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Simultaneous inactivation of Rb and p130 genes in double-mutant mice. A: Generation of β-cell–specific pRb conditional knockout mice (RbCKO) has been previously described by our group (14). Mice that contain loxP sequences surrounding exon 19 of the RB gene were crossed with RIP-Cre mice resulting in excision of the flanked region (18) B: The RbCKO mice were crossed with p130−/− mice to obtain RbCKO;p130−/− double-mutant mice. C: PCR analysis of the Rb 283-bp product represents the floxed allele whereas the 235-bp allele represents the wild-type Rb gene. The 320-bp product represents mutant p130. The lower panel indicates uniform amplification of the Gapdh housekeeping standard and the Cre transgene. D: Immunoblot analysis of pRb and p130 protein in isolated pancreatic islets reveals deficiency of the two proteins in RbCKO;p130−/− double-mutant animals.
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Figure 4: Simultaneous inactivation of Rb and p130 genes in double-mutant mice. A: Generation of β-cell–specific pRb conditional knockout mice (RbCKO) has been previously described by our group (14). Mice that contain loxP sequences surrounding exon 19 of the RB gene were crossed with RIP-Cre mice resulting in excision of the flanked region (18) B: The RbCKO mice were crossed with p130−/− mice to obtain RbCKO;p130−/− double-mutant mice. C: PCR analysis of the Rb 283-bp product represents the floxed allele whereas the 235-bp allele represents the wild-type Rb gene. The 320-bp product represents mutant p130. The lower panel indicates uniform amplification of the Gapdh housekeeping standard and the Cre transgene. D: Immunoblot analysis of pRb and p130 protein in isolated pancreatic islets reveals deficiency of the two proteins in RbCKO;p130−/− double-mutant animals.

Mentions: The preceding data suggest that in the absence of an individual pocket protein, functional redundancy among the remaining members may be sufficient to maintain cell cycle arrest in β-cells. Therefore, we generated mutant mice in which β-cells were deficient for both pRb as well as p130. Inactivation of both the Rb and p130 genes was selected because disruption of both p130 and p107 is embryonic lethal (17) and because a previous study reported that one out of 15 Rb+/−p130−/− mutant mice developed an insulinoma, an effect not observed in Rb+/−p107−/− mutant mice (21). To disrupt both Rb and p130 simultaneously, β-cell–specific pRb CKO (RbCKO) mice (Fig. 4A) (14) were crossed with p130- mice to generate compound RbCKO;p130−/− double-knockout mutant mice (Fig. 4B). In addition, mice that were wild type or heterozygous for Rb on a p130−/− background (RbWT;p130−/− and RbHT;p130−/−, respectively) were also analyzed for comparison. PCR analysis of genomic DNA was used for identification of double-mutant animals based on the presence of floxed Rb alleles (283-bp), the Cre transgene, and the 320-bp mutant p130 gene (Fig. 4C). Immunoblot analysis of pRb and p130 revealed only faint residual levels of pRb, likely reflecting expression of pRb in the few non–β-cells of isolated islets (fibroblasts, α-cells, endothelial cells, etc.) and absent p130 protein in extracts of isolated RbCKO;p130−/− islets (Fig. 4D).


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)

Simultaneous inactivation of Rb and p130 genes in double-mutant mice. A: Generation of β-cell–specific pRb conditional knockout mice (RbCKO) has been previously described by our group (14). Mice that contain loxP sequences surrounding exon 19 of the RB gene were crossed with RIP-Cre mice resulting in excision of the flanked region (18) B: The RbCKO mice were crossed with p130−/− mice to obtain RbCKO;p130−/− double-mutant mice. C: PCR analysis of the Rb 283-bp product represents the floxed allele whereas the 235-bp allele represents the wild-type Rb gene. The 320-bp product represents mutant p130. The lower panel indicates uniform amplification of the Gapdh housekeeping standard and the Cre transgene. D: Immunoblot analysis of pRb and p130 protein in isolated pancreatic islets reveals deficiency of the two proteins in RbCKO;p130−/− double-mutant animals.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2712776&req=5

Figure 4: Simultaneous inactivation of Rb and p130 genes in double-mutant mice. A: Generation of β-cell–specific pRb conditional knockout mice (RbCKO) has been previously described by our group (14). Mice that contain loxP sequences surrounding exon 19 of the RB gene were crossed with RIP-Cre mice resulting in excision of the flanked region (18) B: The RbCKO mice were crossed with p130−/− mice to obtain RbCKO;p130−/− double-mutant mice. C: PCR analysis of the Rb 283-bp product represents the floxed allele whereas the 235-bp allele represents the wild-type Rb gene. The 320-bp product represents mutant p130. The lower panel indicates uniform amplification of the Gapdh housekeeping standard and the Cre transgene. D: Immunoblot analysis of pRb and p130 protein in isolated pancreatic islets reveals deficiency of the two proteins in RbCKO;p130−/− double-mutant animals.
Mentions: The preceding data suggest that in the absence of an individual pocket protein, functional redundancy among the remaining members may be sufficient to maintain cell cycle arrest in β-cells. Therefore, we generated mutant mice in which β-cells were deficient for both pRb as well as p130. Inactivation of both the Rb and p130 genes was selected because disruption of both p130 and p107 is embryonic lethal (17) and because a previous study reported that one out of 15 Rb+/−p130−/− mutant mice developed an insulinoma, an effect not observed in Rb+/−p107−/− mutant mice (21). To disrupt both Rb and p130 simultaneously, β-cell–specific pRb CKO (RbCKO) mice (Fig. 4A) (14) were crossed with p130- mice to generate compound RbCKO;p130−/− double-knockout mutant mice (Fig. 4B). In addition, mice that were wild type or heterozygous for Rb on a p130−/− background (RbWT;p130−/− and RbHT;p130−/−, respectively) were also analyzed for comparison. PCR analysis of genomic DNA was used for identification of double-mutant animals based on the presence of floxed Rb alleles (283-bp), the Cre transgene, and the 320-bp mutant p130 gene (Fig. 4C). Immunoblot analysis of pRb and p130 revealed only faint residual levels of pRb, likely reflecting expression of pRb in the few non–β-cells of isolated islets (fibroblasts, α-cells, endothelial cells, etc.) and absent p130 protein in extracts of isolated RbCKO;p130−/− islets (Fig. 4D).

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