Role of Connexins in Beta-Cell Development and Function

连接蛋白在 β 细胞发育和功能中的作用

• 批准号: 6667126
• 负责人: VINCENZINO CIRULLI
• 金额: $ 31.5万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-30 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6667126
• 关键词: Lentivirus; athymic mouse; biological signal transduction; cell cell interaction; cell component structure /function; cell differentiation; cell proliferation; confocal scanning microscopy; diabetes mellitus genetics; electron microscopy; flow cytometry; gap junctions; gene expression; genetic transduction; glucagon; human tissue; insulin; insulin dependent diabetes mellitus; pancreatic islet transplantation; pancreatic islets; stem cells; terminal nick end labeling; transfection /expression vector

DESCRIPTION (provided by applicant): This proposal integrates the expertise of scientists working in complementary model systems of pancreatic developmental biology. The experimental plan presented will use both human fetal and adult pancreatic cell populations (PI's laboratory), as well as murine models for the in vivo dynamic study of connexins function as putative regulators of islet cell growth and differentiation (Dr. Meda's laboratory). The integrated interaction of these two research initiatives provides a unique opportunity to exchange data and reagents for the translation of knowledge gathered from animal studies into the biology of human islet cells. Preliminary studies from both our laboratory and Dr. Meda's demonstrate that specific "connexins", the individual monomeric proteins building up gap junction channels, characterize distinct populations of pancreatic epithelial cells in the human and murine pancreas. Thus, while Cx32 appears expressed in most pancreatic undifferentiated epithelial cells, Cx36 is mainly restricted to developing beta-cells. Conversely, Cx43 highlights a discrete population of cells associated with developing islet clusters and/or emerging from the ductal epithelium. Our data also demonstrate that expression of Cx36 is developmentally regulated during (beta-cell ontogeny, with the highest levels detected in mature (glucose-responsive) islet beta-cells. Functional gene transfer studies using Lentiviral vectors show that transduction of Cx32 in fetal pancreatic cells causes a significant down-regulation of insulin and glucagon gene expression, whereas transduction of Cx36 causes an increased insulin gene expression. These data provide direct proof of principle for a role of these connexins in pancreatic cell growth and differentiation. The central hypothesis of this proposal is that specific connexin isoforms, each assembling into specific gap junction channels with distinct gating capabilities, may contribute unique signaling functions to distinct developmental stages of islet cell ontogeny and in adult beta -cell homeostasis. Hence, our objective is to test whether Cx32, Cx36, and/or Cx43 can be used as molecular tools to promote beta-cell development from populations of undifferentiated pancreatic progenitor cells, and/or trigger a growth pathway in fully mature adult beta -cells. To perform these studies, we have generated a panel of Lentiviral vectors carrying the coding sequence of select connexins controlled by either a CMV promoter, or by tissue specific promoters such as the PDX-1, ngn3, and the Insulin promoter. In parallel experiments, we will generate Cre/7ox murine models to dissect the functional roles of Cx36 and Cx43 in vivo at distinct developmental stages of the islet cell lineage. Our Specific Aims are: 1) To study the function of Cx32, Cx36, and Cx43 in human fetal pancreatic cell populations, by inhibitory peptides; 2) To study the developmental effects of transient and stable gene transfer of Cx32, Cx36, and Cx43 in human fetal pancreatic cell populations; 3) To test the ability of Cx32, Cx36, and Cx43 to stimulate either cell growth of mature islet cells, or differentiation of ductal cells from the human adult pancreas; and 4) To investigate the developmental effects of in vivo inactivation of Cx36 and Cx43 using Cre/lox murine models.

描述（由申请人提供）： 该提案整合了从事胰腺发育生物学互补模型系统工作的科学家的专业知识。提出的实验计划将使用人类胎儿和成人胰腺细胞群（PI 的实验室）以及小鼠模型，对连接蛋白作为胰岛细胞生长和分化的假定调节剂的功能进行体内动态研究（Meda 博士的实验室）。这两项研究计划的综合相互作用提供了一个独特的机会来交换数据和试剂，以便将从动物研究中收集的知识转化为人类胰岛细胞的生物学。 我们实验室和 Meda 博士实验室的初步研究表明，特定的“连接蛋白”（建立间隙连接通道的单个单体蛋白）表征了人和小鼠胰腺中不同的胰腺上皮细胞群体。因此，虽然 Cx32 在大多数胰腺未分化上皮细胞中表达，但 Cx36 主要限于发育中的 β 细胞。相反，Cx43 突出显示与发育中的胰岛簇和/或从导管上皮中出现相关的离散细胞群。我们的数据还表明，Cx36 的表达在β细胞个体发育过程中受到发育调节，在成熟（葡萄糖反应性）胰岛β细胞中检测到最高水平。使用慢病毒载体的功能基因转移研究表明，胎儿胰腺细胞中转导Cx32会导致胰岛素和胰高血糖素基因表达显着下调，而转导 Cx36 导致胰岛素基因表达增加。这些数据为这些连接蛋白在胰腺细胞生长和分化中的作用提供了直接的原理证明。 该提议的中心假设是，特定的连接蛋白亚型，每种亚型组装成具有不同门控能力的特定间隙连接通道，可能为胰岛细胞个体发育和成体β细胞稳态的不同发育阶段贡献独特的信号传导功能。因此，我们的目标是测试 Cx32、Cx36 和/或 Cx43 是否可以用作分子工具来促进未分化胰腺祖细胞群的 β 细胞发育，和/或触发完全成熟的成体 β 细胞的生长途径。为了进行这些研究，我们生成了一组慢病毒载体，其携带由 CMV 启动子或组织特异性启动子（例如 PDX-1、ngn3 和胰岛素启动子）控制的精选连接蛋白的编码序列。在平行实验中，我们将生成 Cre/7ox 小鼠模型，以剖析 Cx36 和 Cx43 在胰岛细胞谱系不同发育阶段的体内功能作用。我们的具体目标是： 1) 通过抑制肽研究 Cx32、Cx36 和 Cx43 在人胎儿胰腺细胞群中的功能； 2) 研究Cx32、Cx36和Cx43瞬时和稳定基因转移对人胎儿胰腺细胞群的发育影响； 3) 测试Cx32、Cx36和Cx43刺激成熟胰岛细胞的细胞生长或成人胰腺导管细胞分化的能力； 4) 使用 Cre/lox 小鼠模型研究体内 Cx36 和 Cx43 失活对发育的影响。

项目成果

[Optical diagnosis of cervical dysplasia]

【宫颈发育不良的光学诊断】

• 发表时间: 2004
• 期刊: Bulletin du cancer
• 影响因子: 1.2
• 作者: Charvet,Igor;Meda,Paolo;Genet,Magalie;Pelte,Marie-Francoise;Vlastos,Anne-Therese
• 通讯作者: Vlastos,Anne-Therese

On-line analysis of gap junctions reveals more efficient electrical than dye coupling between islet cells.

间隙连接的在线分析揭示了胰岛细胞之间的电耦合比染料耦合更有效。

• DOI: 10.1152/ajpendo.00473.2002
• 发表时间: 2003
• 期刊: American journal of physiology. Endocrinology and metabolism.
• 作者: Quesada,Ivan;Fuentes,Esther;Andreu,Etelvina;Meda,Paolo;Nadal,Angel;Soria,Bernat
• 通讯作者: Soria,Bernat

Association of the connexin36 gene with juvenile myoclonic epilepsy.

connexin36 基因与青少年肌阵挛性癫痫的关联。

• DOI: 10.1136/jmg.2003.017954
• 发表时间: 2004
• 期刊: Journal of medical genetics
• 影响因子: 4
• 作者: Mas,C;Taske,N;Deutsch,S;Guipponi,M;Thomas,P;Covanis,A;Friis,M;Kjeldsen,MJ;Pizzolato,GP;Villemure,J-G;Buresi,C;Rees,M;Malafosse,A;Gardiner,M;Antonarakis,SE;Meda,P
• 通讯作者: Meda,P

Cx36 involvement in insulin secretion: characteristics and mechanism.

Cx36 参与胰岛素分泌：特征和机制。

• DOI: 10.1080/cac.10.4-6.431.435
• 发表时间: 2003
• 期刊: Cell communication & adhesion
• 作者: Meda,Paolo

Ectopic expression of syncollin in INS-1 beta-cells sorts it into granules and impairs regulated secretion.

INS-1 β 细胞中 Syncollin 的异位表达将其分类为颗粒并损害调节的分泌。

• DOI: 10.1021/bi048894d
• 发表时间: 2005
• 期刊: Biochemistry.
• 作者: Li,Jingsong;Luo,Ruihua;Hooi,ShingChuan;Ruga,Pilar;Zhang,Jiping;Meda,Paolo;Li,GuoDong
• 通讯作者: Li,GuoDong