Calcium-sensing receptor signaling in the regulation of colonic epithelial cells

钙敏感受体信号传导在结肠上皮细胞的调节中

• 批准号: 8246331
• 负责人: JUAN ENRIQUE ROZENGURT
• 金额: --
• 依托单位: VA GREATER LOS ANGELES HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8246331
• 关键词: Ablation; Adult; Amino Acids; Architecture; Aromatic Amino Acids; Calcium-Sensing Receptors; Cell Nucleus; Cell Proliferation; Cell membrane; Cell physiology; Cells; Cessation of life; Chemoprevention; Chief Cell; Colon; Colorectal Cancer; Diet; Down-Regulation; Employee Strikes; Epithelial Cell Proliferation; Epithelial Cells; Family; G-Protein-Coupled Receptors; GTP Binding; Gastrointestinal tract structure; General Population; Genetic; Genetic Transcription; Homeostasis; Hormones; Human; Inflammation; Inflammatory Bowel Diseases; Intestines; Ions; Knockout Mice; Life; Maintenance; Malignant Neoplasms; Mediating; Mucous Membrane; Mus; Nutrient; Organ; Organism; Parathyroid gland; Pathogenesis; Peptides; Phosphorylation; Play; Population; Process; Protein Dephosphorylation; Publishing; Receptor Activation; Receptor Signaling; Regulation; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Stimulus; Stratum Basale; Time; Tissues; Veterans; Wound Healing; base; beta catenin; cell type; colonic crypt; crypt cell; extracellular; gastrointestinal system; human disease; in vivo; member; migration; mouse model; novel; phospholipase C beta; prevent; protective effect; response; restoration

DESCRIPTION (provided by applicant): The sequential proliferation, lineage-specific differentiation, migration, and death of the epithelial cells of the colonic mucosa is a tightly regulated process modulated by a broad range of regulatory peptides, differentiation signals and luminal stimuli. Despite its fundamental importance for understanding the pathogenesis of human diseases, including inflammatory bowel diseases (IBD) and colorectal cancer (CRC), the signaling mechanisms involved remain incompletely understood. In this context, GTP-binding (G) protein-coupled receptors (GPCRs) and their intracellular signal transduction pathways play a critical role in the regulation of multiple functions of the digestive system, including cell proliferation, inflammation and promotion of CRC. The extracellular calcium-sensing receptor (CaSR), a member of the GPCR family, is prominently expressed by epithelial cells of the gastrointestinal (GI) tract but its function and mechanism in the regulation of normal and/or abnormal intestinal epithelial cell proliferation in vivo remains unknown. We demonstrated that CaSR stimulation by extracellular Ca2+ inhibits the proliferation of colon-derived epithelial cells. Our new preliminary studies obtained with a novel CaSR intestinal-specific knock out mouse show that the genetic ablation of the CaSR strikingly increases colonic crypt proliferation. Further preliminary results with colon-derived cells in culture demonstrate that CaSR stimulation strikingly decreases 2-catenin phosphorylation at Ser-552 and Ser-675, two amino acid residues that regulate 2-catenin localization and transcriptional activity. CaSR-mediated 2-catenin dephosphorylation at Ser-552 and Ser-675 coincided with its translocation from the nucleus to the plasma membrane and with 2-catenin-mediated transcription inhibition. Based on our preliminary results, the overarching hypothesis of this proposal is that CaSR signaling inhibits colon epithelial cell proliferation via down-regulation of b-catenin signaling. We plan to explore this hypothesis by pursuing the following Specific Aims: 1). Characterize the role of the CaSR in colon epithelial cell proliferation, differentiation, and cancer using a novel CaSR GI tract-specific knock-out mouse model; 2) Identify the signal transduction pathways that mediate the decrease in b-catenin phosphorylation at Ser- 552 and Ser-675 in response to CaSR stimulation in colon-derived epithelial cells. 3). Characterize b- catenin sub-cellular distribution and transcriptional activity in response to CaSR stimulation in colon- derived epithelial cells. We anticipate that the mechanistic studies proposed with genetically modified mice and human epithelial cells in culture will demonstrate that the CaSR inhibits colonic epithelial cell proliferation via negative crosstalk with the b-catenin signaling. These studies will also establish a robust rationale to explore protective effects of CaSR activation (e.g. via dietary Ca2+) in preventing CRC, one of the most common malignancies in the general population as well as in the US veteran population. PUBLIC HEALTH RELEVANCE: The mechanistic studies proposed in this application on the function of the extracellular calcium-sensing receptor (CaSR) using genetically modified mice and human colon- derived cells in culture are of importance to define the role of the CaSR in the regulation of intestinal epithelial cell proliferation and consequently, are of high significance for Colorectal Cancer (CRC) and Inflammatory Bowel Disease (IBD). The completion of the proposed studies will provide a robust intellectual framework for targeting the colonic CaSR in the chemoprevention of CRC in US veterans as well as in the US population at large.

描述（由申请人提供）： 结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、分化信号和管腔刺激调节的严格调节过程。尽管它对于理解人类疾病的发病机制具有根本重要性，包括炎症性肠病（IBD）和结直肠癌（CRC），但所涉及的信号传导机制仍不完全清楚。在这种情况下，GTP结合（G）蛋白偶联受体（GPCR）及其细胞内信号转导途径在消化系统的多种功能的调节中起关键作用，包括细胞增殖、炎症和CRC的促进。细胞外钙敏感受体（CaSR）是GPCR家族的成员之一，主要表达于胃肠道上皮细胞，但其在体内调节正常和/或异常肠上皮细胞增殖的功能和机制尚不清楚。我们证明，细胞外Ca 2+的CaSR刺激抑制结肠源性上皮细胞的增殖。我们的新的初步研究获得了一种新的CaSR膀胱特异性敲除小鼠表明，基因消融的CaSR显着增加结肠隐窝增殖。进一步的初步结果与结肠来源的细胞培养表明，CaSR刺激显着降低2-连环蛋白磷酸化的Ser-552和Ser-675，两个氨基酸残基，调节2-连环蛋白的定位和转录活性。CaSR介导的2-catenin在Ser-552和Ser-675处的去磷酸化与其从细胞核到质膜的易位以及2-catenin介导的转录抑制一致。基于我们的初步结果，该提议的总体假设是CaSR信号通过下调β-连环蛋白信号抑制结肠上皮细胞增殖。我们计划通过追求以下具体目标来探索这一假设：1）。使用新型CaSR GI道特异性敲除小鼠模型表征CaSR在结肠上皮细胞增殖、分化和癌症中的作用; 2）鉴定在结肠来源的上皮细胞中响应于CaSR刺激而介导Ser- 552和Ser-675处的b-连环蛋白磷酸化降低的信号转导途径。3）。表征结肠来源的上皮细胞中响应于CaSR刺激的B-连环蛋白亚细胞分布和转录活性。我们预计，提出的机制研究与遗传修饰的小鼠和人类上皮细胞培养将证明，CaSR抑制结肠上皮细胞增殖通过负串扰与β-连环蛋白信号。这些研究还将建立一个强大的理论基础，以探索CaSR激活（例如，通过饮食Ca 2+）在预防CRC（一般人群以及美国退伍军人人群中最常见的恶性肿瘤之一）中的保护作用。 公共卫生关系： 本申请中提出的使用遗传修饰的小鼠和培养的人结肠衍生细胞对细胞外钙敏感受体（CaSR）的功能进行的机制研究对于确定CaSR在肠上皮细胞增殖调节中的作用是重要的，因此对于结肠直肠癌（CRC）和炎性肠病（IBD）具有高度意义。拟议研究的完成将为靶向结肠CaSR在美国退伍军人以及整个美国人群中化学预防CRC提供一个强大的知识框架。