Cell adhesion-dependent mechanisms of beta cell growth and homeostasis

总结 我们的实验室最近发现，α E-连环蛋白，一种细胞粘附过程的调节剂，在细胞粘附过程中起着关键作用。 由于其作为Sonic Hedgehog抑制因子的功能，在胰岛细胞谱系发育中的作用 (SHH)通路我们发现，在Pdx 1+多能胰腺祖细胞中α E-连环蛋白的缺失导致胰腺癌的发生。 未成熟的双能Sox 9+祖细胞的积累。这些αE-cateninnull/Sox 9+祖细胞不能 由于SHH途径的组成性激活而采用内分泌细胞表型。有趣的是， 药物阻断这些α E-cateninnull/Sox 9+祖细胞中的SHH通路， 分化为表达激素的胰岛细胞。最近，我们发现， 在人成年胰岛中通过siRNA的α E-连环蛋白可以引起显著的β细胞复制。因此，根据这些结果， 基于α E-连环蛋白也能阻断Wnt信号的观点，我们假设在分化的胰岛细胞中， α E-连环蛋白可能是另一个细胞周期进入的“刹车”，因为它在信号传导上具有相反的功能 SHH和Wnt途径，通常会促进细胞增殖。 为了验证这一假设，我们将把研究重点放在以下具体目标上： 目的1：确定α E-连环蛋白在胚胎发育过程中作为β细胞生长调节剂的作用， 在出生后的生活中，在生理条件下和在损伤环境中。在这些实验中，还将测试 在胚胎和出生后的β细胞中，α E-连环蛋白的条件性缺失将解除SHH和Wnt的抑制， 转预期引起细胞周期进入。在平行研究中，我们还将测试αE- 在β-细胞损伤的链脲佐菌素模型中，和/或在 对代谢应激源的反应，如暴露于高脂肪饮食。 目的2：靶向α E-连环蛋白依赖的信号传导轴，用于人胰岛细胞的体外扩增， 导管细胞群的重编程，在体外和体内细胞移植模型中。基于 人类胰岛细胞对促生长刺激物表现出适度的反应倾向，这些研究将 测试敲低成人β细胞中的α E-连环蛋白是否会解除SHH和Wnt的抑制，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，通常从胰岛分离程序中丢弃， 为了测试它们是否能在体外和体内重新获得胚胎样分化为内分泌细胞的能力， 体内细胞移植模型。 总的来说，我们暂时下调α E-连环蛋白表达的策略， SHH通路的激活可能被证明是促进β-细胞体外扩增和/或再增殖的有力策略。 指导成体导管细胞向β细胞表型的分化能力。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转化价值。

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.