PKD1 Signaling and Crosstalk Mechanisms in Intestinal Epithelial Cell Regulation

描述（由申请人提供）：肠粘膜上皮细胞的连续增殖、谱系特异性分化、迁移和死亡是一个受到广泛调节肽、神经递质、生物活性脂质和分化信号调节的严格调节过程。尽管其对于理解肠道内环境稳定、伤口愈合和人类疾病的发病机制具有根本重要性，但所涉及的信号传导机制仍不完全清楚。许多胃肠肽、神经递质和生物活性脂质通过七螺旋G蛋白偶联受体（GPCR）在其靶细胞中启动其特征性作用。蛋白激酶D（PKD 1）正在成为GPCR信号转导的关键节点，因此对PKD 1调控和功能的理解具有强烈的兴趣和潜在的影响。本文提出的研究将确定GPCR/PKD 1信号传导与控制肠上皮细胞迁移和增殖的关键通路之间的新联系。要探索的首要假设是PKD 1在导致肠上皮细胞增殖的信号转导途径中起关键作用。提出了三个具体目标：具体目标1：表征PKD 1信号传导在体内肠上皮细胞增殖和干细胞衍生的肠类器官中的作用。该目标将集中在PKD 1信号在体内，检查其功能的稳态周转通过干/祖细胞增殖和修复响应肠粘膜损伤，使用基因修饰的小鼠模型，包括PKD 1转基因小鼠和干细胞衍生的肠类器官。具体目标2）表征肠上皮细胞中PKD 1和β-catenin信号系统之间的串扰机制。本研究的主要目的是研究GPCR/PKD 1与β-catenin信号通路之间的一种新的相互作用，导致PKD 1/β- catenin复合物的形成，β-catenin易位到细胞核，Ser 552的刺激性磷酸化和转录激活;具体目的3）通过PAK介导的PKD 1在N端残基Ser 203的磷酸化来鉴定PKD 1调控的一种新机制。基于新的初步结果，我们将确定一种新的磷酸化的N-末端结构域的PKD 1在响应GPCR激动剂在肠上皮细胞介导的p21激活的激酶（PAKs），效应Rac和Cdc 42。PAK/PKD 1级联的发现揭示了GPCR激动剂引发的信号转导途径中的新整合点。我们预期，本申请中提出的机制研究将鉴定PKD 1调节肠上皮细胞增殖和迁移的新型上游途径和下游串扰机制，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.