Cell Cycle Regulators in Pancreatic Development and Disease

胰腺发育和疾病中的细胞周期调节因子

• 批准号: 8741504
• 负责人: Sushil Rane
• 金额: $ 33.97万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8741504
• 关键词: Acinar Cell; Address; Adult; Apoptosis; Area; Autoimmune Process; Beta Cell; Binding; Biological Process; Bone Marrow; CDKN2A gene; Cell Cycle; Cell Differentiation process; Cell Lineage; Cell Proliferation; Cell division; Cell physiology; Cells; Cellular biology; Chemical Warfare; Clinical; Complex; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; DNA; Development; Diabetes Mellitus; Diagnostic; Disease; Ductal; Ductal Epithelial Cell; Ductal Epithelium; E2F Transcription Factor 1; E2F1 gene; Embryo; Endocrine; Epithelial; Epithelium; Exhibits; Gene Targeting; Generations; Genes; Glucose; Goals; Growth; Growth and Development function; Hour; Hyperplasia; Inherited; Injury; Insulin; Investigation; Islets of Langerhans; Kinetics; Knockout Mice; Lead; Location; Maintenance; Mammalian Cell; Mediating; Mesenchymal; Mesenchyme; Modeling; Morphogenesis; Mus; Mutant Strains Mice; Natural regeneration; Obesity; Organ; Organogenesis; Pancreas; Pancreatectomy; Pancreatic duct; Pathogenesis; Pathway interactions; Phosphotransferases; Physiological; Play; Pluripotent Stem Cells; Point Mutation; Primary Cell Cultures; Process; Property; Recruitment Activity; Regulation; Relative (related person); Reporting; Research; Retinoblastoma Protein; Role; Source; Specific qualifier value; Splenocyte; Stem cells; Sum; Therapeutic; Tissues; Toxin; Translating; analog; base; cell growth; clinical application; cyclin D2; diabetes management; diabetes mellitus therapy; embryonic stem cell; established cell line; human CDK2 protein; in vivo; inhibitor/antagonist; islet; mathematical model; mortality; mouse model; novel; pancreas development; precursor cell; progenitor; programs; promoter; reconstitution; regenerative; response; transcription factor; transdifferentiation; treatment strategy

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.

答：CDK4调节静止期的β细胞和导管上皮祖细胞的募集，以重建β细胞团。 在糖尿病中，产生胰岛素的胰岛β细胞被破坏、严重耗尽或功能受损。因此，更换功能性的β细胞团将促进临床糖尿病的治疗。我们以前已经证明了CDK4在调节β细胞质量中的重要性。CDK4基因缺陷的小鼠表现出β细胞发育不良并发展为糖尿病，而表达活性CDK4R24C激酶的小鼠则观察到β细胞增殖。虽然β细胞复制似乎是导致β细胞数量增加的主要机制，但相当多的证据也支持胰腺导管上皮在产生新的β细胞方面的贡献。此外，虽然人们认为大多数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使静止的细胞参与β细胞的再生反应。我们使用CDK4突变小鼠的部分胰腺切除(PX)模型来解决这些问题。为了准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大量增殖。有趣的是，数学模型显示，静止细胞重新进入活跃的细胞周期促进了CDK4R24C胰腺中的β细胞质量重建。此外，在PX术后24-48小时内，表达转录因子PDX-1的导管上皮细胞显著增加。我们还在导管上皮中检测到胰岛素阳性细胞，并在CDK4R24C胰腺中检测到显著增加的胰岛样细胞团。我们的结论是，CDK4不仅促进了β细胞的复制，还促进了导管上皮中的β细胞前体细胞的激活。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团簇对胰岛素阳性细胞总数增加的贡献，表明迄今尚未确定的大的非增殖性贡献。 B：CDK4-E2F1通路通过靶向Pdx1+祖细胞和Ngn3+内分泌前体调控早期胰腺发育。 细胞分裂和细胞分化是错综复杂的生物过程，对器官发育至关重要。细胞周期蛋白依赖性蛋白激酶(CDK)是细胞周期的主要调节者，协调细胞分裂和分化程序。CDK1是推动细胞分裂所必需的，也是第一次胚胎分裂所必需的。相反，其他CDK(2、4和6)虽然对器官发生来说是不必要的，但被认为对组织特异性细胞的发育至关重要。在这里，我们说明了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细胞的广泛形态发生、增殖和分化，从而形成不同的成人胰腺细胞系。然而，在早期胰腺中指定谱系承诺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在二次过渡之前和期间调节胰腺的发育。CDK4缺乏会导致胚胎胰腺体积缩小，这是由于间充质发育受损和Pdx1+胰祖细胞数量受限所致。有趣的是，活化的CDK4R24C激酶的表达导致Nkx2.2+和Nkx6.1+细胞的增加，以及Ngn3+内分泌前体细胞的数量和增殖增加，从而导致细胞谱系的扩大。此外，我们发现E2F1结合并激活了Ngn3启动子，从而以一种依赖于CDK4的方式调节胚胎胰腺中Ngn3的表达水平。这些结果表明，CDK4通过引导E2F1介导的Ngn3的激活和增加内分泌前体的池来促进细胞的发育。这些结果证实CDK4是早期胰腺发育的重要调节因子，因为它能够调节胰腺前体细胞和内分泌前体细胞的增殖潜能。 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对于胚胎胰腺发育和成年细胞功能是必不可少的。有趣的是，PDX-1利用了一个保守的RB相互作用基序(RIM)，该基序也存在于E2F中。RIM内的点突变减少了RB-PDX-1复合体的形成，破坏了PDX-1的稳定性，并促进了其蛋白酶体的降解。葡萄糖调节Rb和PDX-1水平、Rb/PDX-1复合体的形成和PDX-1的降解。Rb基因是细胞特异性基因的启动子，Rb基因的敲除会导致PDX-1及其靶基因表达的降低。此外，体内Rb缺乏导致胰腺体积缩小，原因是PDX-1+胰祖细胞增殖减少，细胞凋亡增加，胰腺发育调节因子异常表达。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，其中包括RB介导的胰腺特异性转录因子PDX-1的稳定。