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。尽管它对于了解炎症性肠病（IBD）和结直肠癌（CRC）等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。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多种功能的调节中发挥着关键作用，包括细胞增殖、炎症和促进结直肠癌。细胞外钙敏感受体（CaSR）是GPCR家族的成员，主要由胃肠道（GI）上皮细胞表达，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仍然未知。我们证明细胞外 Ca2+ 的 CaSR 刺激可抑制结肠来源的上皮细胞的增殖。我们对新型 CaSR 肠道特异性敲除小鼠进行的新的初步研究表明，CaSR 的基因消除显着增加了结肠隐窝增殖。对培养的结肠源细胞的进一步初步结果表明，CaSR 刺激显着降低 2-连环蛋白 Ser-552 和 Ser-675 的磷酸化，这两个氨基酸残基调节 2-连环蛋白定位和转录活性。 CaSR 介导的 Ser-552 和 Ser-675 处的 2-连环蛋白去磷酸化与其从细胞核易位到质膜以及 2-连环蛋白介导的转录抑制相一致。根据我们的初步结果，该提议的总体假设是 CaSR 信号传导通过下调 β-连环蛋白信号传导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 胃肠道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)鉴定响应结肠源性上皮细胞中CaSR刺激而介导B-连环蛋白Ser-552和Ser-675磷酸化减少的信号转导途径。 3）。表征结肠来源上皮细胞中对 CaSR 刺激的 β-连环蛋白亚细胞分布和转录活性。我们预计，对培养的转基因小鼠和人类上皮细胞进行的机制研究将证明 CaSR 通过与 β-连环蛋白信号传导的负串扰抑制结肠上皮细胞增殖。这些研究还将建立强有力的理论基础来探索 CaSR 激活（例如通过饮食中的 Ca2+）对预防结直肠癌的保护作用，结直肠癌是普通人群以及美国退伍军人中最常见的恶性肿瘤之一。