Cell adhesion-dependent mechanisms of beta cell growth and homeostasis

β细胞生长和稳态的细胞粘附依赖性机制

• 批准号: 10713361
• 负责人: VINCENZINO CIRULLI
• 金额: $ 9.8万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10713361
• 关键词: Ablation; Adopted; Adult; Alpha Cell; Animal Model; Beta Cell; Cell Adhesion; Cell Adhesion Process; Cell Cycle; Cell Lineage; Cell Proliferation; Cell Transplantation; Cells; Competence; D Cells; Development; Diabetes Mellitus; Down-Regulation; Duct (organ) structure; Ductal Epithelial Cell; Embryo; Embryonic Development; Endocrine; Exhibits; Exposure to; Fostering; Future; Gene Deletion; Growth; Growth Factor; High Fat Diet; Homeostasis; Hormones; Human; In Vitro; Injury; Intervention; Islet Cell; Islets of Langerhans; Laboratories; Life; Metabolic; Modeling; Molecular; Natural regeneration; Organogenesis; Pancreas; Pathway interactions; Phenotype; Physiological; Play; Population; Procedures; Proliferating; Repression; Resistance; Rodent; Role; SHH gene; Signal Transduction; Small Interfering RNA; Sonic Hedgehog Pathway; Stimulus; Streptozocin; Structure of beta Cell of islet; Testing; Tissues; Type 2 diabetic; WNT Signaling Pathway; Work; alpha catenin; cell growth; cell injury; cell regeneration; cell replacement therapy; cell type; derepression; directed differentiation; drug development; endocrine pancreas development; experimental study; in vivo; islet; islet stem cells; knock-down; loss of function; patient retention; pharmacologic; postnatal; postnatal human; progenitor; programs; response; smoothened signaling pathway; stressor; transdifferentiation; transplant model

Our laboratory has recently discovered that αE-catenin, a regulator of cell adhesion processes, plays a critical role in the development of the islet cell lineage by virtue of its function as a repressor of the Sonic Hedgehog (SHH) pathway. We found that deletion of αE-catenin in Pdx1+ multipotent pancreatic progenitors results in the accumulation of immature bipotent Sox9+ progenitors. These αE-cateninnull/Sox9+ progenitors are unable to adopt an endocrine cell phenotype due to a constitutive activation of the SHH pathway. Interestingly, pharmacological blockade of the SHH pathway in these αE-cateninnull/Sox9+ progenitors restored their ability to differentiate into hormone expressing islet cells. More recently, we found that the temporal downregulation of αE-catenin by siRNA in human adult islets can elicit significant β-cell replication. Hence, based on these results, and on the notion that αE-catenin can also block Wnt signaling, we hypothesize that in differentiated islet cells αE-catenin may represent yet another “brake” on cell cycle entry by virtue of its opposing functions on signaling SHH and Wnt pathways that would normally promote cell proliferation. To test this hypothesis, we will focus our studies on the following Specific Aims: Aim 1: Determine the role of αE-catenin as a modulator of β-cell growth during embryonic development and in postnatal life, under physiologic conditions and in injury settings. In these experiments will also test if the conditional deletion of αE-catenin in embryonic and in postnatal β-cells will de-repress SHH and Wnt, which in turn are expected to elicit cell cycle entry. In parallel studies we will also test if the conditional ablation of αE-catenin in postnatal life will enhance β-cell regeneration in the streptozotocin model of β-cell injury, and/or in response to metabolic stressors such as exposure to high fat diet. Aim 2: Targeting αE-catenin-dependent signaling axis for the ex vivo expansion of human islet cells and for the reprogramming of ductal cell populations, both in vitro and in vivo in cell transplantation models. Based on the notion that human islet cells exhibit a modest propensity to respond to pro-growth stimuli, these studies will test if knocking down αE-catenin in human adult β-cells will de-repress SHH and Wnt, and foster cell proliferation. A similar strategy will be tested on human adult ductal tissue, usually discarded from islet isolation procedures, to test if they can regain an embryonic-like competency to differentiate into endocrine cells, both in vitro and in vivo in cell transplantation models. Collectively, our strategy to transiently down-regulate αE-catenin expression, allowing for the de-repression of the SHH pathway may prove as a powerful strategy to promote the ex vivo expansion of β-cells, and/or re-direct the differentiation competency of adult ductal cells toward a β-cell phenotype. Hence, we anticipate that these studies harbor significant translational value for cell replacement therapies in diabetes.

我们的实验室最近发现，αE-连环蛋白是细胞黏附过程的调节者，通过其作为Sonic Hedgehog(SHH)途径的抑制因子在胰岛细胞谱系的发育中发挥关键作用。我们发现在Pdx1+多能胰腺祖细胞中αE-catenin的缺失导致未成熟的双能Sox9+祖细胞的聚集。由于α途径的结构性激活，这些SHHE-cateninull/Sox9+前体细胞不能采用内分泌细胞表型。有趣的是，药物阻断了这些αE-连环蛋白缺失/Sox9+前体细胞中的SHH通路，恢复了它们分化为激素表达的胰岛细胞的能力。最近，我们发现，在人类成年胰岛中，小干扰RNA对αE-连环蛋白的瞬时下调可以诱导显著的β细胞复制。因此，基于这些结果，以及αE-连环蛋白也可以阻断WNT信号通路的概念，我们推测在分化的胰岛细胞中，αE-连环蛋白可能代表着另一种细胞周期进入的“刹车”，因为它在正常情况下促进细胞增殖的信号通路SHH和WNT上具有相反的功能。为了验证这一假设，我们的研究将集中在以下具体目标上：目的1：确定αE-连环蛋白在胚胎发育和出生后生命、生理条件和损伤环境中作为β细胞生长调节器的作用。在这些实验中，还将测试在胚胎和出生后的α细胞中有条件地删除βE-连环蛋白是否会抑制SHH和WNT，这反过来预计会引发细胞周期进入。在平行研究中，我们还将测试在出生后生命中有条件地切除αE-连环蛋白是否会在链脲佐菌素的β细胞损伤模型中增强β细胞再生，和/或对代谢应激因素(如暴露于高脂肪饮食)的反应。目的2：靶向αE-连环蛋白依赖的信号轴，用于体外扩增人胰岛细胞，并在细胞移植模型中对体内和体外的导管细胞群体进行重新编程。基于人类胰岛细胞对促生长刺激表现出适度倾向的概念，这些研究将测试敲除人类成年α细胞中的βE-连环蛋白是否会抑制SHH和WNT，并促进细胞增殖。类似的策略将在成人导管组织上进行测试，这些组织通常是从胰岛分离程序中丢弃的，以测试它们能否在体外和体内细胞移植模型中重新获得类似胚胎的能力，分化为内分泌细胞。总之，我们暂时下调αE-连环蛋白表达，允许抑制SHH途径的策略可能被证明是促进β细胞体外扩增和/或将成年导管细胞的分化能力重定向为β细胞表型的有效策略。因此，我们预计这些研究对糖尿病的细胞替代疗法具有重要的翻译价值。