Role of Connexins in Beta-Cell Development and Function

描述(由申请人提供)： 这项建议整合了胰腺发育生物学互补模型系统中科学家的专业知识。提出的实验计划将使用人类胎儿和成人胰腺细胞群体(Pi的实验室)，以及用于体内动态研究连接蛋白作为胰岛细胞生长和分化假定调节因子的小鼠模型(Meda博士的实验室)。这两项研究举措的综合互动为将动物研究收集的知识转化为人类胰岛细胞生物学提供了独特的交换数据和试剂的机会。 来自我们实验室和Meda博士的初步研究表明，特定的“连接蛋白”，即建立缝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群体的胰腺上皮细胞。因此，虽然Cx32似乎在大多数胰腺未分化上皮细胞中表达，但Cx36主要局限于发育中的β细胞。相反，Cx43突出显示了与发育中的胰岛簇和/或从导管上皮中冒出的离散细胞群。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，在成熟的(葡萄糖反应的)胰岛β细胞中检测到最高水平。利用慢病毒载体进行的功能性基因转移研究表明，在胚胎胰腺细胞中转导Cx32导致胰岛素和胰升糖素基因表达显著下调，而转导Cx36导致胰岛素基因表达上调。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据。 这一建议的中心假设是，特定的连接蛋白亚型，每个亚型组装成具有不同门控能力的特定缝隙连接通道，可能在胰岛细胞个体发育的不同阶段和成年β细胞的动态平衡中贡献独特的信号功能。因此，我们的目标是测试Cx32、Cx36和/或Cx43是否可以作为分子工具来促进未分化的胰岛前体细胞群体中的β细胞发育，和/或在完全成熟的成年β细胞中触发生长途径。为了进行这些研究，我们构建了一组慢病毒载体，携带由CMV启动子或组织特异性启动子(如PDX-1、NGN3和Insulin启动子)控制的选择性连接蛋白的编码序列。在平行实验中，我们将建立Cre/7ox小鼠模型，以剖析Cx36和Cx43在体内胰岛细胞谱系不同发育阶段的功能作用。我们的具体目标是：1)通过抑制肽研究Cx32、Cx36和Cx43在人胚胎胰腺细胞群体中的功能；2)研究瞬时和稳定的Cx32、Cx36和Cx43基因转移对人胚胎胰腺细胞群体的发育影响；3)检测Cx32、Cx36和Cx43刺激成熟胰岛细胞生长或成人胰腺导管细胞分化的能力；4)利用CRE/LOX小鼠模型研究Cx36和Cx43体内灭活对发育的影响。

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.