Islet transcription factor activation: FoxPs are required for postnatal endocrine cell proliferation while Pdx1 recruited chromatin remodeling enzymes impact pancreas size

 描述(申请人提供)：糖尿病是一种代谢状态，其特征是胰腺β细胞群的功能和/或质量受损，导致 严重的高血糖。国家卫生研究院支持的努力旨在将β细胞替换或将其他类型的胰岛细胞重新编程为具有功能的β细胞(例如，胰岛α细胞)。因此，控制α-和β-细胞增殖的机制的发现将为开发治疗糖尿病的药物提供重要的启示。斯坦实验室的直接兴趣在于确定控制胰腺大小和胰岛发育的转录因子及其强制性共同调节因子的特征。在这方面，我们发现FoxP1、FoxP2和FoxP4对α细胞的增殖是必不可少的，并将研究Pdx1招募的分子影响 SWI/SNF染色质重塑复合体对胰腺大小的调节作用FoxP类转录因子在胰岛中的作用尚不清楚。在这项建议的目标1中，我和查德·亨特博士一起培育了内分泌特异性FoxP1/2/4基因敲除的转基因小鼠。我们的数据显示，这组小鼠在4周大的禁食条件下正常清除葡萄糖，但有低血糖和血清胰高血糖素水平下降。所有内分泌细胞类型(α-，-，β-)的增殖能力都降低；然而，α细胞群体受到的影响最大(细胞数量减少约90%)。以往的报道表明，正常血糖和正常血糖只需要3%的α细胞群，这表明在FoxP1/2/4条件性基因敲除小鼠中，其余10%的α细胞功能障碍。这个问题将通过分离胰岛的胰升糖素分泌分析和4周龄实验组胰腺的免疫组织化学技术来解决。PDX1是最早在胰腺发生过程中表达的转录因子之一，其转录活性是胰腺发育和出生后β细胞功能所必需的。为了研究Pdx1的活性是如何被调节的，我们的实验室已经证明Swi/SNF染色质重塑复合体与(体内和体外)相互作用并调节Pdx1的转录活性。初步结果表明，RNAi介导的BRG1基因敲除对糖稳态基因的表达产生负面影响。BRG1是一种对Swi/SNF染色质重塑活动至关重要的ATPase。在胰腺发生的早期阶段有条件地敲除BRG1会导致发育不良的小鼠胰腺发育不良，大约是对照小鼠的50%。该条件性基因敲除模型将在AIM 2中用于研究BRG1如何影响胰腺大小。我将检查BRG1突变体中被指定为所有胰腺细胞类型的细胞群，即多能祖细胞(MPC)，是否在数量上减少，表现出增殖减少或凋亡增加，这可以解释胰腺发育不良。总而言之，这项建议将对糖尿病领域产生重大影响，因为它将阐明SWI/SnF染色质重塑复合体如何影响胰腺质量，以及FoxP1/2/4调节因子如何影响正确的α细胞发育和功能。

 DESCRIPTION (provided by applicant): Diabetes mellitus is metabolic condition that is characterized by impaired function and/or mass of the pancreatic β-cell population that results in severe hyperglycemia. Efforts supported by the NIH are aimed at β-cell replacement or reprogramming other islet cell types into functional β-cells (e.g. islet α-cells). Thus, the discovery of mechanisms which control α- and β-cell proliferation will provide significant insigh into developing therapeutics to treat diabetes. The Stein lab has a direct interest in characterizing the transcription factors and their obligatory coregulators that govern pancreas size and islet development. In this regard, we have discovered that FoxP1, FoxP2 and FoxP4 are essential for α-cell proliferation and will investigate the molecular impact of Pdx1 recruited Swi/Snf chromatin remodeling complex on regulating pancreas size. The role of the FoxP class of transcription factors have yet to be characterized in the islet. In aim 1 of this proposal I hav generated, along with Dr. Chad Hunter, transgenic mice with an endocrine specific knockout of FoxP1/2/4. Our data reveals that this cohort of mice clear glucose normally, but have hypoglycemia and decreased serum glucagon levels under fasting conditions at 4 weeks of age. All endocrine cell types (α-, -, β-) have reduced proliferative capability; however, the α-cel population is the most profoundly impacted (~90% reduction in cell number). Previous reports show that only 3% of the α-cell population is required for euglycemia and normoglucagonemia, which suggests the remaining 10% of α-cells in the FoxP1/2/4 conditional knockout mice are dysfunctional. This question will be addressed using glucagon secretion assays from isolated islets and immunohistochemical techniques in pancreata from 4 week old experimental groups. Pdx1 is one of the first transcription factors expressed during pancreatogenesis and its transcriptional activity is required in both pancreas development and postnatal β-cell functions. To address how the activity of Pdx1 is modulated, our lab has shown that the Swi/Snf chromatin remodeling complex interacts with (both in vivo and in vitro) and regulates Pdx1 transcriptional activity. Preliminary results have shown that RNAi- mediated knockdown of Brg1, an ATPase essential for Swi/Snf chromatin remodeling activity, negatively impacts the expression of glucose homeostasis genes. Conditional knockout of Brg1 during the early stages of pancreatogenesis results in mice with a hypoplastic pancreas, approximately 50% the size of control littermates. This conditional knockout model will be used in Aim 2 to examine how Brg1 impacts pancreas size. I will examine whether the population of cells in the Brg1 mutant that are designated to become all pancreatic cell types, known as the multipotent progenitor cells (MPCs), are decreased in number, exhibit decreased proliferation or increased apoptosis that could explain the hypoplastic pancreas. Collectively, this proposal will be of major interest to th diabetes field as it will shed light on how the Swi/Snf chromatin remodeling complex impacts pancreas mass and the FoxP1/2/4 regulators influence proper α-cell development and function.