Cell adhesion-dependent mechanisms of beta cell growth and homeostasis

摘要 我们的实验室最近发现，αE-连环蛋白，一个细胞黏附过程的调节者，在细胞黏附过程中发挥着关键的作用 通过其作为Sonic Hedgehog的抑制物在胰岛细胞谱系发育中的作用 (Shh)途径。我们发现在Pdx1多能胰腺祖细胞中αE-连环蛋白的缺失导致 未成熟的双能Sox9祖细胞的积累。这些αE-cateninNull/SOX9前身无法 由于SHH途径的结构性激活，采用内分泌细胞表型。有趣的是， 药物阻断这些αE-连环蛋白缺失/SOX9前体细胞中的SHH通路，恢复了它们的能力 分化为分泌激素的胰岛细胞。最近，我们发现，时间上的下调 人成人胰岛中的αE-连环蛋白可诱导显著的β细胞复制。因此，根据这些结果， 基于αE-连环蛋白也可以阻断Wnt信号的概念，我们假设在分化的胰岛细胞中 αE-连环蛋白可能通过其相反的信号传递功能而代表着细胞周期进入的另一种“刹车” Shh和Wnt途径通常会促进细胞增殖。 为了验证这一假设，我们将重点研究以下具体目标： 目的1：确定αE-连环蛋白在胚胎发育和β-细胞生长调节中的作用 在出生后的生活中，在生理条件下和在受伤的环境中。在这些实验中还将测试是否 αE-连环蛋白在胚胎和出生后的β细胞中的条件性缺失将降低SHH和WNT，这在 TURN有望诱导细胞周期进入。在平行研究中，我们还将测试条件消融αE- 在链脲佐菌素的β细胞损伤模型中，出生后生命中的连接素将促进β细胞的再生，和/或在 对代谢应激源的反应，如暴露在高脂肪饮食中。 目的2：靶向αE-连环蛋白依赖的信号轴体外扩增人胰岛细胞 在细胞移植模型中，体内和体外的导管细胞群体的重新编程。基于 人类胰岛细胞对促生长刺激表现出适度的反应倾向，这些研究将 测试敲除成人α细胞中的βE-连环蛋白是否会降低SHH和WNT的表达，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， 在体外和体外，测试它们是否能够重新获得类似胚胎的能力，分化为内分泌细胞 活体细胞移植模型。 总而言之，我们暂时下调αE-连环蛋白表达的策略，允许去抑制 SHH通路的激活可能被证明是促进β-细胞体外扩增和/或再扩增的有效策略。 成年导管细胞具有向β细胞表型分化的能力。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。

SUMMARY Our laboratory has recently discovered that αE-catenin, a regulator of cell adhesion processes, play 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.