PKD1 Signaling and Crosstalk Mechanisms in Intestinal Epithelial Cell Regulation

描述(申请人提供)：肠粘膜上皮细胞的连续增殖、谱系特异性分化、迁移和死亡是一个严格调控的过程，受广泛的调节肽、神经递质、生物活性脂和分化信号的调控。尽管它对了解肠道内环境平衡、伤口愈合和人类疾病的发病机制具有重要意义，但其涉及的信号机制仍不完全清楚。许多胃肠道(GI)多肽、神经递质和生物活性脂类通过七螺旋G蛋白偶联受体(GPCRs)在靶细胞中启动其特有的作用。蛋白激酶D(PKD1)是GPCR信号通路中的一个关键节点，因此对其调控和功能的了解具有重要意义和潜在意义。本文提出的研究将确定GPCR/PKD1信号与控制肠上皮细胞迁移和增殖的关键通路之间的新联系。有待探索的最重要的假设是，PKD1在导致肠上皮细胞增殖的信号转导通路中发挥关键作用。提出了三个特定的目标：特定的目标1：研究PKD1信号在体内和干细胞来源的肠道器官中对肠上皮细胞增殖的作用。这一目标将集中在体内的PKD1信号转导，利用转基因小鼠模型，包括PKD1转基因小鼠和干细胞衍生的肠道器官，研究其在干细胞/祖细胞增殖和修复肠粘膜损伤的动态平衡更新中的作用。具体目的2)研究肠上皮细胞中PKD 1和�-连环蛋白信号系统之间的串扰机制。本研究的目的是研究gpr/pkd1与�-catenin信号之间的一种新的串扰，导致pkd1/�-catenin复合体的形成、�-catenin转位到细胞核、Ser552处的刺激性磷酸化和转录激活；基于新的初步结果，我们将在肠上皮细胞中发现一种新的PKD1 N末端结构域的磷酸化，以响应肠上皮细胞中由p21激活的蛋白激酶(PAKs)介导的GPCR激动剂，p21激活的激酶是RAC和CDC42的效应者。PAK/PKD1级联信号通路的发现揭示了GPCR激动剂启动的信号转导通路中的一个新的整合点。我们期待在本申请中提出的机制研究将发现新的上游途径(S)和下游串扰机制，通过它调控肠上皮细胞的增殖和迁移，从而为消化系统疾病的创新治疗干预提供理论基础。

DESCRIPTION (provided by applicant): The sequential proliferation, lineage-specific differentiation, migration and death of the epithelial cells of the intestinal mucosa is a tightly regulated process modulated by a broad range of regulatory peptides, neurotransmitters, bioactive lipids and differentiation signals. Despite its fundamental importance for understanding intestinal homeostasis, wound healing and pathogenesis of human diseases, the signaling mechanisms involved remain incompletely understood. Many gastrointestinal (GI) peptides, neurotransmitters and bioactive lipids initiate their characteristic effects in their target cells through heptahelical G protein-coupled receptors (GPCRs). Protein kinase D (PKD1) is emerging as a key node in GPCR signaling and consequently the understanding of PKD1 regulation and function is of intense interest and potential impact. The studies proposed here will identify new links between GPCR/PKD1 signaling and pivotal pathways that control migration and proliferation of intestinal epithelial cells. The overarching hypothesis to be explored is that PKD1 plays a critical role in the signal transduction pathways leading to intestinal epithelial cell proliferation. Three Specific Aims are proposed: Specific Aim 1: Characterize the role of PKD1 signaling in intestinal epithelial cell proliferation in vivo and in stem cell-derived intestinal organoids. This Aim will focus on PKD1 signaling in vivo, examining its function in homeostatic turnover via stem/progenitor cell proliferation and repair in response to injury of the intestinal mucosa, using genetically modified mouse models, including PKD1 transgenic mice and stem-cell derived intestinal organoids. Specific Aim 2) Characterize crosstalk mechanisms between PKD1 and �-catenin signaling systems in intestinal epithelial cells. The studies proposed in this Aim will characterize an novel crosstalk between GPCR/PKD1 and �-catenin signaling, leading to PKD1/�- catenin complex formation, �-catenin translocation to the nucleus, stimulatory phosphorylation at Ser552 and transcriptional activation; Specific Aim 3) Identify a novel mechanism of PKD1 regulation through PAK-mediated PKD1 phosphorylation at the N-terminal residue Ser203. Based on new preliminary results, we will identify a novel phosphorylation in the N-terminal domain of PKD1 in response to GPCR agonists in intestinal epithelial cells mediated by p21-activated kinases (PAKs), effectors for Rac and Cdc42. The discovery of a PAK/PKD1 cascade uncovers a new point of integration in the signal transduction pathways initiated by GPCR agonists. We anticipate that the mechanistic studies proposed in this application will identify novel upstream pathway(s) and downstream crosstalk mechanisms by which PKD1 regulates the proliferation and migration of intestinal epithelial cells thereby providing the rationale for innovative therapeutic intervention in diseases of the digestive system.