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: 8970680
• 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/8970680
• 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; intestinal homeostasis; 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.

描述（由申请人提供）： 结肠粘膜上皮细胞的顺序增殖，谱系特异性分化，迁移和死亡是由广泛调节肽，分化信号和管腔刺激的严格调节过程。尽管它对于理解人类疾病的发病机理的基本重要性，包括炎症性肠病（IBD）和结直肠癌（CRC），但所涉及的信号机制仍未完全理解。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调节消化系统的多种功能中起关键作用，包括细胞增殖，炎症和CRC的促进。 GPCR家族的一个成员的细胞外钙 - 感应受体（CASR）由胃肠道（GI）的上皮细胞显着表达，但其在调节正常和/或异常肠道肠上皮上皮细胞增殖中的功能和机制仍然是未知的。我们证明，细胞外Ca2+的CASR刺激抑制了结肠衍生的上皮细胞的增殖。我们通过新的CASR肠道特异性敲除小鼠获得的新初步研究表明，CASR的遗传消融显着增加了结肠隐窝的增殖。培养中结肠衍生细胞的进一步初步结果表明，CASR刺激在SER-552和SER-675（两个调节2-catenin局部化和转录活性的氨基酸残基）下显着降低了2-catenin磷酸化。 CASR介导的Ser-552和Ser-675在Ser-552和Ser-675处介导的2-catenin去磷酸化伴随着其从细胞核到质膜的易位，并伴随着2-catenin介导的转录抑制。基于我们的初步结果，该提案的总体假设是CASR信号传导通过下调B-catenin信号传导抑制结肠上皮细胞的增殖。我们计划通过追求以下特定目的来探讨这一假设：1）。使用新型CASR GI特异性敲除小鼠模型来表征CASR在结肠上皮细胞增殖，分化和癌症中的作用； 2）确定介导Ser-552和Ser-675介导的B-catenin磷酸化降低的信号转导途径，以响应于结肠衍生的上皮细胞中的CASR刺激。 3）。表征B- catenin亚细胞分布和转录活性，响应于结肠衍生的上皮细胞中的CASR刺激。我们预计，在培养物中使用转基因小鼠和人类上皮细胞提出的机械研究将表明，CASR通过负串扰与B-catenin信号传导抑制结肠上皮细胞增殖。这些研究还将建立一个强大的理由，以探索CASR激活的保护作用（例如，通过饮食CA2+）预防CRC，CRC是普通人群中最常见的恶性肿瘤之一，也是美国退伍军人人口。