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

描述(由申请人提供)： 结肠粘膜上皮细胞的增殖、谱系特异性分化、迁移和死亡是一个严格调控的过程，受广泛的调节肽、分化信号和腔刺激的调控。尽管它对理解包括炎症性肠病(IBD)和结直肠癌(CRC)在内的人类疾病的发病机制具有重要意义，但其涉及的信号机制仍不完全清楚。在此背景下，GTP结合(G)蛋白偶联受体(GPCRs)及其细胞内信号转导通路在调节消化系统的多种功能中发挥着关键作用，包括细胞增殖、炎症和促进结直肠癌的发生。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。我们证明了细胞外钙离子对CaSR的刺激抑制了结肠来源的上皮细胞的增殖。我们用一种新的CaSR肠道特异性基因敲除小鼠进行的初步研究表明，CaSR的遗传消融显著增加了结肠隐窝的增殖。进一步的结肠来源细胞培养的初步结果表明，CaSR刺激显著降低了Ser-552和Ser-675上的2-连环蛋白磷酸化，这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Ser-552和Ser-675上的2-catenin去磷酸化与其从细胞核到质膜的转位和2-catenin介导的转录抑制相一致。根据我们的初步结果，这一建议的首要假设是CaSR信号通过下调b-catenin信号来抑制结肠上皮细胞的增殖。我们计划通过追求以下具体目标来探索这一假说：1)。利用一种新的CaSR GI区域特异性敲除小鼠模型，研究CaSR在结肠上皮细胞增殖、分化和癌症中的作用；2)确定在CaSR刺激下，介导Ser-552和Ser-675上b-catenin磷酸化降低的信号转导通路。3)。研究结肠来源上皮细胞中b-连环蛋白亚细胞分布和转录活性对CaSR刺激的响应。我们预计，对转基因小鼠和培养的人上皮细胞进行的机制研究将证明，CaSR通过与b-catenin信号的负干扰来抑制结肠上皮细胞的增殖。这些研究还将建立一个强有力的理论基础，以探索激活CaSR(例如通过饮食中的钙离子)在预防CRC方面的保护作用。CRC是普通人群以及美国退伍军人中最常见的恶性肿瘤之一。

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.