Cell adhesion-dependent mechanisms of beta cell growth and homeostasis

我们的实验室最近发现αE-catenin是一种细胞粘附过程的调节剂，由于其作为Sonic Hedgehog （SHH）通路的抑制因子的功能，在胰岛细胞谱系的发育中起着关键作用。我们发现αE-catenin在Pdx1+多能胰腺祖细胞中的缺失导致未成熟的双能Sox9+祖细胞的积累。这些αE-cateninnull/Sox9+祖细胞由于SHH通路的组成性激活而无法采用内分泌细胞表型。有趣的是，通过药物阻断这些αE-cateninnull/Sox9+祖细胞的SHH通路可以恢复它们向表达激素的胰岛细胞分化的能力。最近，我们发现成人胰岛中siRNA对αE-catenin的时间下调可以引起显著的β细胞复制。因此，基于这些结果，并基于αE-catenin也可以阻断Wnt信号传导的概念，我们假设在分化的胰岛细胞中，αE-catenin可能代表了细胞周期进入的另一个“制动”，因为它在SHH和Wnt信号通路上具有相反的功能，而SHH和Wnt信号通路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：目标1：确定αE-catenin在胚胎发育和出生后生命、生理条件和损伤环境下作为β细胞生长调节剂的作用。在这些实验中，我们还将测试胚胎和出生后β细胞中αE-catenin的条件缺失是否会抑制SHH和Wnt，进而引发细胞周期进入。在平行研究中，我们还将测试αE-catenin在出生后的条件消融是否会增强β细胞损伤链脲佐菌素模型中的β细胞再生，和/或对代谢应激源（如暴露于高脂肪饮食）的反应。目的2：在体外和体内细胞移植模型中，靶向α e -catenin依赖性信号轴用于人胰岛细胞的体外扩增和导管细胞群的重编程。基于人类胰岛细胞对促生长刺激表现出适度的反应倾向的观点，这些研究将测试在成人β细胞中敲除αE-catenin是否会抑制SHH和Wnt，并促进细胞增殖。类似的策略将在人类成人导管组织上进行测试，通常在胰岛分离过程中被丢弃，以测试它们是否能在体外和体内细胞移植模型中重新获得胚胎样的能力，分化为内分泌细胞。总的来说，我们暂时下调αE-catenin表达的策略，允许SHH通路的去抑制，可能被证明是促进β细胞体外扩增的有力策略，和/或将成人导管细胞的分化能力重新定向到β细胞表型。因此，我们预计这些研究对糖尿病细胞替代疗法具有重要的转化价值。

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