Cell Cycle Regulators in Pancreatic Development and Disease

答：Cdk4 调节静止 β 细胞和导管上皮祖细胞的募集以重建 β 细胞群。 在糖尿病中，产生胰岛素的胰岛β细胞被破坏、严重耗竭或功能受损。因此，替代功能性β细胞群将促进临床糖尿病管理。我们之前已经证明了 Cdk4 在调节 β 细胞质量中的重要性。 Cdk4 缺陷小鼠表现出 β 细胞发育不全并发展为糖尿病，而在表达活性 Cdk4R24C 激酶的小鼠中观察到 β 细胞增生。虽然β细胞复制似乎是β细胞质量增加的主要机制，但大量证据也支持胰腺导管上皮在新β细胞生成中的贡献。此外，虽然据信大多数β细胞处于休眠状态，但尚不清楚是否以及在多大程度上可以诱导休眠细胞参与β细胞再生反应。我们使用 Cdk4 突变小鼠的部分胰腺切除术 (PX) 模型解决了这些问题。为了精确研究再生过程的动力学，我们进行了基于 DNA 类似物的谱系追踪研究，然后进行了数学建模。 PX 后一周内，我们观察到胰岛β细胞和导管上皮细胞大量增殖。有趣的是，数学模型表明，将静止细胞招募到活跃细胞周期中可以促进 Cdk4R24C 胰腺中 β 细胞团的重建。此外，在 PX 后 24-48 小时内，表达转录因子 Pdx-1 的导管上皮细胞急剧增加。我们还在导管上皮中检测到胰岛素阳性细胞，并且 Cdk4R24C 胰腺中的胰岛样细胞簇显着增加。我们得出结论，Cdk4不仅促进β细胞复制，而且促进导管上皮中β细胞祖细胞的激活。此外，我们还发现 Cdk4 通过招募静止细胞进入细胞周期来控制 β 细胞质量。比较细胞增殖和胰岛样簇对胰岛素阳性细胞总数增加的贡献，表明迄今为止未表征的巨大非增殖贡献。 B：Cdk4-E2F1 通路通过靶向 Pdx1 祖细胞和 Ngn3 内分泌前体来调节早期胰腺发育。 细胞分裂和细胞分化是复杂调控的生物过程，对器官发育至关重要。细胞周期蛋白依赖性激酶 (Cdks) 是细胞周期的主要调节因子，负责协调细胞分裂和分化程序。 Cdk1 对于驱动细胞分裂至关重要，并且是第一次胚胎分裂所必需的。相比之下，其他 Cdks（2、4 和 6）虽然对于器官发生来说是可有可无的，但被认为对于组织特异性细胞的发育至关重要。在这里，我们阐述了 Cdk4 在调节早期胰腺发育中的重要作用。胰腺发育涉及胰腺上皮的广泛形态发生、增殖和分化，以产生成体胰腺的独特细胞谱系。然而，在早期胰腺内指定谱系定型的细胞周期分子的身份尚不清楚。我们发现 Cdk4 及其下游转录因子 E2F1 在二次转化之前和期间调节胰腺发育。 Cdk4 缺乏会由于间充质发育受损和 Pdx1 胰腺祖细胞数量限制而导致胚胎胰腺尺寸减小。有趣的是，激活的 Cdk4R24C 激酶的表达导致 Nkx2.2 和 Nkx6.1 细胞增加，以及 Ngn3 内分泌前体细胞数量和增殖的增加，从而导致细胞谱系扩张。此外，我们发现E2F1结合并激活Ngn3启动子，从而以Cdk4依赖性方式调节胚胎胰腺中Ngn3的表达水平。这些结果表明，Cdk4 通过指导 E2F1 介导的 Ngn3 激活和增加内分泌前体库来促进细胞发育。这些结果表明，Cdk4 具有调节胰腺祖细胞和内分泌前体细胞增殖潜力的能力，是早期胰腺发育的重要调节因子。 C：RB 通过稳定 Pdx-1 来调节胰腺发育。 RB 是 Cdks 的关键底物，也是哺乳动物细胞周期的重要调节因子。 RB 要么抑制促进细胞增殖的 E2F，要么增强促进分化的细胞特异性因子的活性，尽管促进这种双重相互作用的机制尚不清楚。在这里，我们证明 RB 与 Pdx-1 结合并稳定 Pdx-1，这对于胚胎胰腺发育和成体细胞功能至关重要。有趣的是，Pdx-1 利用了 E2F 中也存在的保守 RB 相互作用基序 (RIM)。 RIM 内的点突变会减少 RB-Pdx-1 复合物的形成，破坏 Pdx-1 的稳定性并促进其蛋白酶体降解。葡萄糖调节 RB 和 Pdx-1 水平、RB/Pdx-1 复合物形成和 Pdx-1 降解。 RB占据细胞特异性基因的启动子，敲低RB会导致Pdx-1及其靶基因的表达降低。此外，由于 Pdx-1 胰腺祖细胞增殖减少、细胞凋亡增加和胰腺发育调节因子的异常表达，体内 RB 缺乏会导致胰腺大小减小。这些结果证明了胰腺发育和细胞功能的一种意想不到的调节机制，其中涉及 RB 介导的胰腺特异性转录因子 Pdx-1 的稳定性。 我们已经开始对胰腺发育和功能中的其他细胞周期调节因子进行系统分析

A: Cdk4 regulates recruitment of quiescent beta cells and ductal epithelial progenitors to reconstitute beta cell Mass. Insulin-producing pancreatic islet beta cells are destroyed, severely depleted or functionally impaired in diabetes. Therefore, replacing functional beta cell mass would advance clinical diabetes management. We have previously demonstrated the importance of Cdk4 in regulating beta cell mass. Cdk4-deficient mice display beta cell hypoplasia and develop diabetes, whereas beta cell hyperplasia is observed in mice expressing an active Cdk4R24C kinase. While beta cell replication appears to be the primary mechanism responsible for beta cell mass increase, considerable evidence also supports a contribution from the pancreatic ductal epithelium in generation of new beta cells. Further, while it is believed that majority of beta 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 beta cell regenerative response. We addressed 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 beta cells and ductal epithelial cells. Interestingly, mathematical models showed that recruitment of quiescent cells into the active cell cycle promotes beta 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 beta cell replication, but also facilitates the activation of beta cell progenitors in the ductal epithelium. In addition, we show that Cdk4 controls beta 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 thatCdk4 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. We have begun a systematic analyses of other cell cycle regulators in pancreas development and function