PKD1 Signaling and Crosstalk Mechanisms in Intestinal Epithelial Cell Regulation

肠上皮细胞调节中的 PKD1 信号传导和串扰机制

• 批准号: 8915687
• 负责人: JUAN ENRIQUE ROZENGURT
• 金额: $ 33.5万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8915687
• 关键词: Agonist; Biology; Cell Nucleus; Cell Proliferation; Cell Proliferation Regulation; Cells; Cessation of life; Characteristics; Chemicals; Chronic; Complex; DNA biosynthesis; Digestive System Disorders; Disease; Employee Strikes; Epithelial; Epithelial Cell Proliferation; Epithelial Cells; Epithelium; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; Gastrointestinal tract structure; Growth Factor; Health; In Vitro; Inflammatory; Intestinal Mucosa; Intestines; Knowledge; Laboratories; Link; Lipids; Malignant Neoplasms; Mediating; Membrane; N-terminal; Neurotransmitters; Nuclear; Nuclear Import; Organelles; Organoids; Outcome; Pathogenesis; Pathway interactions; Peptides; Phosphorylation; Play; Process; Protein Kinase; Proteins; Regulation; Role; Signal Transduction; Signal Transduction Pathway; Stem cells; Stimulus; Stratum Basale; System; Testing; Therapeutic Intervention; Transcriptional Activation; Transgenic Mice; Wound Healing; base; cell type; gastrointestinal; human disease; in vivo; innovation; interest; intestinal crypt; intestinal homeostasis; migration; mouse model; novel; p21 activated kinase; protein kinase D; receptor; repaired; response; response to injury; stem

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.

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。尽管它对于理解肠道稳态、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。许多胃肠道 (GI) 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) 在靶细胞中启动其特有的作用。蛋白激酶 D (PKD1) 正在成为 GPCR 信号转导中的关键节点，因此对 PKD1 调控和功能的理解引起了人们的强烈兴趣和潜在影响。这里提出的研究将确定 GPCR/PKD1 信号传导与控制肠上皮细胞迁移和增殖的关键途径之间的新联系。待探索的首要假设是 PKD1 在导致肠上皮细胞增殖的信号转导途径中发挥着关键作用。提出了三个具体目标： 具体目标 1：表征 PKD1 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。该目标将重点关注体内 PKD1 信号传导，使用转基因小鼠模型（包括 PKD1 转基因小鼠和干细胞衍生的肠道类器官），通过干/祖细胞增殖和修复响应肠粘膜损伤，检查其在稳态更新中的功能。具体目标 2) 表征肠上皮细胞中 PKD1 和 β-catenin 信号系统之间的串扰机制。该目标中提出的研究将表征 GPCR/PKD1 和β-连环蛋白信号传导之间的新型串扰，导致 PKD1/β-连环蛋白复合物形成、β-连环蛋白易位至细胞核、Ser552 处的刺激性磷酸化和转录激活；具体目标 3) 通过 PAK 介导的 PKD1 N 端残基 Ser203 磷酸化来确定 PKD1 调节的新机制。基于新的初步结果，我们将鉴定 PKD1 N 端结构域中的一种新型磷酸化，以响应肠上皮细胞中由 p21 激活激酶 (PAK)、Rac 和 Cdc42 效应子介导的 GPCR 激动剂。 PAK/PKD1 级联的发现揭示了 GPCR 激动剂启动的信号转导途径中的一个新整合点。我们预计本申请中提出的机制研究将确定 PKD1 调节肠上皮细胞增殖和迁移的新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依据。