Cell Cycle Regulators in Pancreatic Development and Disease

A：Cdk 4调节静止β细胞和导管上皮祖细胞的募集以重建β细胞团。 产生胰岛素的胰岛β细胞在糖尿病中被破坏、严重耗尽或功能受损。因此，替换功能性β细胞群将推进临床糖尿病管理。我们以前已经证明了Cdk 4在调节β细胞群中的重要性。Cdk 4缺陷小鼠显示β细胞发育不全并发展为糖尿病，而在表达活性Cdk 4 R24 C激酶的小鼠中观察到β细胞增生。虽然β细胞复制似乎是导致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产生新β细胞中的贡献。此外，虽然据信大多数辟田胞处于休眠状态，但尚不清楚静止细胞是否以及在何种程度上可以被诱导参与辟田胞再生反应。我们使用Cdk 4突变小鼠的部分胰腺切除术（PX）模型解决了这些问题。为了精确地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。在PX后一周内，我们观察到胰岛β细胞和导管上皮细胞的大量增殖。有趣的是，数学模型显示，静止细胞进入活跃细胞周期的募集促进Cdk 4 R24 C胰腺中的β细胞团重建。此外，在PX后24-48小时内，表达转录因子Pdx-1的导管上皮细胞显著增加。我们还在导管上皮沿着检测到胰岛素阳性细胞，在Cdk 4 R24 C胰腺中胰岛样细胞簇显著增加。我们得出结论，Cdk 4不仅促进β细胞复制，而且还促进导管上皮中β细胞祖细胞的激活。此外，我们发现Cdk 4通过招募静止细胞进入细胞周期来控制β细胞群。比较细胞增殖和胰岛样簇对胰岛素阳性细胞总增加的贡献表明迄今为止未表征的大的非增殖性贡献。 B：Cdk 4-E2 F1途径通过靶向Pdx 1+祖细胞和Ngn 3+内分泌前体来调节早期胰腺发育。 细胞分裂和细胞分化是对器官发育至关重要的复杂调节的生物过程。细胞周期蛋白依赖性激酶（Cdks）是细胞周期的主要调节因子，其协调细胞分裂和分化程序。cdk 1是驱动细胞分裂所必需的，也是第一次胚胎分裂所必需的。相比之下，其他Cdks（2，4和6），虽然对器官发生，被认为是至关重要的组织特异性细胞的发展。在这里，我们说明了Cdk 4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，以产生成人胰腺的不同细胞谱系。然而，在早期胰腺中指定谱系定型的细胞周期分子的身份是未知的。我们发现，Cdk 4和它的下游转录因子E2 F1调节胰腺发育之前和期间的次级转换。由于间充质发育受损和Pdx 1+胰腺祖细胞数量的限制，Cdk 4的缺乏导致胚胎胰腺大小减小。有趣的是，活化的Cdk 4 R24 C激酶的表达导致Nkx2.2+和Nkx6.1+细胞的增加以及Ngn 3+内分泌前体细胞的数量和增殖的增加，从而导致细胞谱系的扩增。此外，我们表明，E2 F1结合并激活Ngn 3启动子，从而调节Ngn 3在胚胎胰腺中的表达水平在Cdk 4依赖的方式。这些结果表明，Cdk 4通过指导E2 F1介导的Ngn 3激活和增加内分泌前体的库来促进细胞发育。这些结果确定Cdk 4作为早期胰腺发育的重要调节因子，其能够调节胰腺祖细胞和内分泌前体细胞的增殖潜力。 C：RB通过稳定Pdx-1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抑制促进细胞增殖的E2 Fs或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相关并稳定Pdx-1，Pdx-1对胚胎胰腺发育和成体细胞功能至关重要。有趣的是，Pdx-1利用了一个保守的RB相互作用基序（RIM），也存在于E2 F中。RIM内的点突变减少RB-Pdx-1复合物的形成，使Pdx-1不稳定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，并且RB的敲低导致Pdx-1及其靶基因的表达降低。此外，由于Pdx-1+胰腺祖细胞的增殖减少、细胞凋亡增加和胰腺发育调节因子的异常表达，体内RB缺乏导致胰腺尺寸减小。这些结果表明，胰腺发育和细胞功能，其中涉及RB介导的胰腺特异性转录因子Pdx-1的稳定一个意想不到的监管机制。 我们已经开始系统分析胰腺发育和功能中的其他细胞周期调节因子

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