A: Cdk4 regulates recruitment of quiescent cells and ductal epithelial progenitors to reconstitute beta cell Mass. Insulin-producing pancreatic islet cells are destroyed, severely depleted or functionally impaired in diabetes. Therefore, replacing functional -cell mass would advance clinical diabetes management. We have previously demonstrated the importance of Cdk4 in regulating -cell mass. Cdk4-deficient mice display -cell hypoplasia and develop diabetes, whereas -cell hyperplasia is observed in mice expressing an active Cdk4R24C kinase. While -cell replication appears to be the primary mechanism responsible for -cell mass increase, considerable evidence also supports a contribution from the pancreatic ductal epithelium in generation of new -cells. Further, while it is believed that majority of -cells are in a state of dormancy, it is unclear if and to what extent the quiescent cells can be coaxed to participate in the -cell regenerative response. Here, we address these queries using a model of partial pancreatectomy (PX) in Cdk4 mutant mice. To investigate the kinetics of the regeneration process precisely, we performed DNA analog-based lineage-tracing studies followed by mathematical modeling. Within a week after PX, we observed considerable proliferation of islet -cells and ductal epithelial cells. Interestingly, the mathematical model showed that recruitment of quiescent cells into the active cell cycle promotes -cell mass reconstitution in the Cdk4R24C pancreas. Moreover, within 24-48 hours post-PX, ductal epithelial cells expressing the transcription factor Pdx-1 dramatically increased. We also detected insulin-positive cells in the ductal epithelium along with a significant increase of islet-like cell clusters in the Cdk4R24C pancreas. We conclude that Cdk4 not only promotes -cell replication, but also facilitates the activation of -cell progenitors in the ductal epithelium. In addition, we show that Cdk4 controls -cell mass by recruiting quiescent cells to enter the cell cycle. Comparing the contribution of cell proliferation and islet-like clusters to the total increase in insulin-positive cells suggests a hitherto uncharacterized large non-proliferative contribution. B: Cdk4-E2F1 pathway regulates early pancreas development by targeting Pdx1+ progenitors and Ngn3+ endocrine precursors. Cell division and cell differentiation are intricately regulated biological processes that are vital to organ development. Cyclin-dependent kinases (Cdks) are master regulators of the cell cycle that orchestrates the cell division and differentiation programs. Cdk1 is essential to drive cell division and is required for the first embryonic divisions. In contrast, the other Cdks (2, 4 and 6), while dispensable for organogenesis, are considered vital for development of tissue-specific cells. Here, we illustrate an important role for Cdk4 in regulating early pancreas development. Pancreatic development involves extensive morphogenesis, proliferation and differentiation of the pancreatic epithelium to give rise to the distinct cell lineages of the adult pancreas. However, the identity of cell cycle molecules that specify lineage commitment within the early pancreas is unknown. We show that Cdk4 and its downstream transcription factor E2F1 regulate pancreas development prior to and during the secondary transition. Deficiency of Cdk4 results in reduced embryonic pancreas size due to impaired mesenchyme development and limitation of the number of Pdx1+ pancreatic progenitor cells. Interestingly, expression of activated Cdk4R24C kinase leads to increased Nkx2.2+ and Nkx6.1+ cells and a rise in the number and proliferation of Ngn3+ endocrine precursor cells resulting in expansion of the cell lineage. Further, we show that E2F1 binds and activates the Ngn3 promoter thereby modulating Ngn3 expression levels in the embryonic pancreas in a Cdk4-dependent manner. These results suggest that Cdk4 promotes cell development by directing E2F1-mediated activation of Ngn3 and increasing the pool of endocrine precursors. These results identify Cdk4 as an important regulator of early pancreas development by virtue of its ability to modulate the proliferation potential of pancreatic progenitors and endocrine precursors. C: RB regulates pancreas development by stabilizing Pdx-1. RB is a key substrate of Cdks and an important regulator of the mammalian cell cycle. RB either represses E2Fs that promote cell proliferation or enhances the activity of cell-specific factors that promote differentiation, although the mechanism that facilitates this dual interaction is unclear. Here, we demonstrate that RB associates with and stabilizes Pdx-1 that is essential for embryonic pancreas development and adult -cell function. Interestingly, Pdx-1 utilizes a conserved RB-interaction motif (RIM) that is also present in E2Fs. Point mutations within the RIM reduce RB-Pdx-1 complex formation, destabilize Pdx-1 and promote its proteasomal degradation. Glucose regulates RB and Pdx-1 levels, RB/Pdx-1 complex formation and Pdx-1 degradation. RB occupies the promoters of -cell specific genes, and knockdown of RB results in reduced expression of Pdx-1 and its target genes. Further, RB-deficiency in vivo results in reduced pancreas size due to decreased proliferation of Pdx-1+ pancreatic progenitors, increased apoptosis and aberrant expression of regulators of pancreatic development. These results demonstrate an unanticipated regulatory mechanism for pancreatic development and -cell function, which involves RB-mediated stabilization of the pancreas-specific transcription factor Pdx-1.

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Kim, So Yoon; Lee, Ji-Hyeon; Merrins, Matthew J et al. (2017) Loss of Cyclin-dependent Kinase 2 in the Pancreas Links Primary ?-Cell Dysfunction to Progressive Depletion of ?-Cell Mass and Diabetes. J Biol Chem 292:3841-3853
Lee, Ji-Hyeon; Anver, Miriam; Kost-Alimova, Maria et al. (2014) Telomere dysfunction suppresses multiple endocrine neoplasia in mice. Genes Cancer 5:306-19
Kim, So Yoon; Rane, Sushil G (2011) The Cdk4-E2f1 pathway regulates early pancreas development by targeting Pdx1+ progenitors and Ngn3+ endocrine precursors. Development 138:1903-12
Kim, Yong-Chul; Kim, So Yoon; Mellado-Gil, Jose Manuel et al. (2011) RB regulates pancreas development by stabilizing Pdx1. EMBO J 30:1563-76
Lee, Ji-Hyeon; Jo, Junghyo; Hardikar, Anandwardhan A et al. (2010) Cdk4 regulates recruitment of quiescent beta-cells and ductal epithelial progenitors to reconstitute beta-cell mass. PLoS One 5:e8653
Lin, Huei-Min; Lee, Ji-Hyeon; Yadav, Hariom et al. (2009) Transforming growth factor-beta/Smad3 signaling regulates insulin gene transcription and pancreatic islet beta-cell function. J Biol Chem 284:12246-57