The volume-regulated anion channel as a regulator of secretory activity in the intestinal epithelium

容量调节阴离子通道作为肠上皮分泌活性的调节剂

• 批准号: 10557101
• 负责人: John Thomas Gebert
• 金额: $ 4.81万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10557101
• 关键词: Ablation; Adenosine Diphosphate; Age; Anions; Antidiarrheals; Biological Assay; Biosensor; CRISPR/Cas technology; Calcium; Calcium Oscillations; Calcium Signaling; Cells; Child; Data; Diarrhea; Discipline; Dysentery; Enterocytes; Epithelial Cells; Epithelium; Equilibrium; Event; Fluids and Secretions; Frequencies; Gastrointestinal tract structure; Genes; Genetic; Health; Homeostasis; Human; Image; Infection; Intestines; Ions; Knock-out; Knowledge; Life; Mediating; Mission; Morbidity - disease rate; Mus; Nonstructural Protein; Organoids; Paracrine Communication; Pathogenesis; Pathologic Processes; Pathway interactions; Phenotype; Physiologic pulse; Play; Protein Kinase; Proteins; Recombinants; Regulation; Research; Rho-associated kinase; Role; Rotavirus; Rotavirus Infections; Severities; Signal Pathway; Signal Transduction; Signaling Molecule; Swelling; Testing; Therapeutic; United States National Institutes of Health; Variant; Viral; Work; burden of illness; clinically relevant; in vivo; insight; intercellular communication; intestinal epithelium; knock-down; mortality; mouse model; new therapeutic target; novel; optogenetics; pathogenic virus; pharmacologic; purinoceptor P2Y1; rho; small hairpin RNA; stressor; targeted treatment; tool

PROJECT SUMMARY The intestinal epithelium relies on extensive cell-cell signaling to maintain a tight balance between absorptive and secretory activity, but the intricacies of this signaling remain uncharacterized. We recently discovered that cells infected with rotavirus, the most common cause of life-threatening diarrhea worldwide, release hundreds of pulses of adenosine diphosphate (ADP) causing intercellular calcium waves that dysregulate uninfected cells. Blocking the ADP receptor P2Y1 significantly reduces diarrhea severity in rotavirus-infected mice, suggesting that this signaling is integral in regulating secretory activity. While the mechanism through which rotavirus triggers the release of ADP has not been confirmed, our preliminary data show it relies on activation of the volume- regulated anion channel (VRAC). VRAC is increasingly recognized as a conduit for paracrine signals associated with a variety of intracellular stressors, but it has yet to be studied within the epithelium. Further, the mechanisms regulating its activation in any context remain poorly understood. Therefore, the objective of this proposal is to use rotavirus as a tool to study the role and activation of VRAC in the intestinal epithelium. Given that rotavirus-induced intercellular calcium waves are inhibited by knock down of rotavirus non-structural protein 4 (NSP4), a Ca2+-conducting channel, my central hypothesis is that NSP4-induced elevations of cytosolic Ca2+ activate host pathways that trigger ADP release through VRAC, amplifying secretory activity throughout the epithelium. I will test this hypothesis by using long-term live calcium imaging in conjunction with novel fluorescent Rho biosensors and CRISPR-Cas9 based gene editing to [Aim 1] determine the intracellular pathway mediating VRAC activation in RV-infected cells. Given ADP release is reduced upon pharmacological inhibition of Rho kinase or knockdown of the calcium-augmenting rotavirus protein NSP4, we expect both calcium and Rho are involved in VRAC activation. Using both human intestinal enteroids and the mouse model of rotavirus diarrhea, I will [Aim 2] determine the effect of VRAC activation on secretory activity in the intestinal epithelium. This work will identify novel therapeutic targets for the treatment of secretory diarrhea, a leading cause of mortality among children worldwide. Furthermore, our findings will address gaps in knowledge surrounding VRAC activation and its role in paracrine signaling. Given that VRAC has been implicated in an increasing variety of pathological processes, using this clinically relevant approach will yield information translatable to both human health and other scientific disciplines.

项目概要 肠上皮依靠广泛的细胞间信号传导来维持吸收和吸收之间的紧密平衡。 和分泌活动，但这种信号传导的复杂性仍未得到表征。我们最近发现 感染轮状病毒的细胞释放出数百 二磷酸腺苷 (ADP) 脉冲引起细胞间钙波，使未感染的细胞失调。 阻断 ADP 受体 P2Y1 可显着降低轮状病毒感染小鼠的腹泻严重程度，这表明 该信号传导对于调节分泌活动是不可或缺的。轮状病毒的触发机制 ADP的释放尚未得到确认，我们的初步数据显示它依赖于卷的激活- 调节阴离子通道（VRAC）。 VRAC 越来越被认为是旁分泌信号的管道 与多种细胞内应激源相关，但尚未在上皮内进行研究。此外， 在任何情况下调节其激活的机制仍然知之甚少。因此，本次活动的目的 建议是使用轮状病毒作为工具来研究VRAC在肠上皮中的作用和激活。给定 通过敲低轮状病毒非结构蛋白来抑制轮状病毒诱导的细胞间钙波 4 (NSP4)，一种 Ca2+ 传导通道，我的中心假设是 NSP4 诱导的细胞溶质升高 Ca2+ 激活宿主途径，通过 VRAC 触发 ADP 释放，从而增强分泌活性 遍及上皮。我将通过结合使用长期活钙成像来检验这个假设 使用新型荧光 Rho 生物传感器和基于 CRISPR-Cas9 的基因编辑来 [目标 1] 确定 RV 感染细胞中介导 VRAC 激活的细胞内途径。鉴于 ADP 释放量减少 通过药理学抑制 Rho 激酶或敲低钙增强轮状病毒蛋白 NSP4，我们 预计钙和 Rho 都参与 VRAC 激活。使用人类肠内肠素和 轮状病毒腹泻小鼠模型，我将[目标 2] 确定 VRAC 激活对轮状病毒腹泻小鼠分泌活性的影响 肠上皮。这项工作将确定治疗分泌性腹泻的新治疗靶点， 是全世界儿童死亡的主要原因。此外，我们的研究结果将解决知识差距 围绕 VRAC 激活及其在旁分泌信号传导中的作用。鉴于 VRAC 已涉及 病理过程的多样性不断增加，使用这种临床相关方法将产生信息 可转化为人类健康和其他科学学科。