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: 6576109
• 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 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6576109
• 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研究的科学家的专业知识。所提出的实验计划将使用人胎儿和成人胰腺细胞群（PI实验室）以及用于连接蛋白作为胰岛细胞生长和分化的假定调节因子功能的体内动态研究的小鼠模型（梅达博士实验室）。这两项研究计划的综合互动提供了一个独特的机会，可以交换数据和试剂，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。 我们实验室和梅达博士的初步研究表明，特定的“连接蛋白”，建立间隙连接通道的单个单体蛋白，表征了人类和小鼠胰腺中不同的胰腺上皮细胞群体。因此，虽然Cx 32似乎在大多数胰腺未分化上皮细胞中表达，但Cx 36主要限于发育中的β细胞。相反，Cx43突出了与发育中的胰岛簇和/或从导管上皮出现相关的离散细胞群。我们的数据还表明，Cx 36的表达在β细胞个体发育过程中受到发育调节，在成熟的（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性基因转移研究显示，在胎儿胰腺细胞中转导Cx 32导致胰岛素和胰高血糖素基因表达的显著下调，而转导Cx 36导致胰岛素基因表达的增加。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。 该建议的中心假设是，特定的连接蛋白同种型，每个组装成具有不同门控能力的特定间隙连接通道，可能对胰岛细胞个体发育的不同发育阶段和成人β细胞稳态贡献独特的信号传导功能。因此，我们的目的是测试Cx 32、Cx 36和/或Cx43是否可以用作分子工具来促进从未分化的胰腺祖细胞群体发育的β细胞，和/或触发完全成熟的成体β细胞中的生长途径。为了进行这些研究，我们已经产生了一组慢病毒载体，其携带由CMV启动子或组织特异性启动子如PDX-1、ngn 3和胰岛素启动子控制的选择连接蛋白的编码序列。在平行实验中，我们将产生Cre/7 ox小鼠模型来剖析Cx 36和Cx43在胰岛细胞谱系的不同发育阶段的体内功能作用。我们的具体目标是：1）研究Cx 32、Cx 36和Cx43在人胎胰腺细胞中的功能，2）研究Cx 32、Cx 36和Cx43基因的瞬时和稳定转染对人胎胰腺细胞的发育影响; 3）测试Cx 32、Cx 36和Cx43刺激成熟胰岛细胞的细胞生长或来自人成人胰腺的导管细胞分化的能力;（4）用Cre/lox小鼠模型研究Cx 36和Cx43在体内失活对发育的影响。