Molecular Mechanisms Regulating Calcium Flux In Salivary Glands

调节唾液腺钙通量的分子机制

• 批准号: 10685865
• 负责人: INDU S. AMBUDKAR
• 金额: $ 260.95万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10685865
• 关键词: ANGPTL2 gene; Actins; Affect; Affinity; Agonist; Animals; Antioxidants; Attenuated; Binding Proteins; CASP3 gene; CDC42 gene; CRISPR/Cas technology; Calcium; Cell Line; Cell Nucleus; Cell membrane; Cell physiology; Cells; Characteristics; Communication; Couples; Cyclic AMP-Dependent Protein Kinases; Cytoskeleton; Data; Deglutition Disorders; Dental caries; Dose; EF-Hand Domain; Fluids and Secretions; Functional disorder; Generations; Gland; Goals; Hydrolysis; ITPR1 gene; Immobilization; Interphase Cell; Ions; Knock-in; Label; Liquid substance; Location; Maintenance; Mediating; Mitochondria; Molecular; Molecular Conformation; Mus; Neurotransmitters; Oral health; Phosphatidylinositol 4,5-Diphosphate; Physiological; Population; Proteins; Radiation; Regulation; Reporting; Research; Role; STIM1 gene; Saliva; Salivary Glands; Signal Transduction; Structure; Variant; Work; Xerostomia; attenuation; efficacy testing; irradiation; knock-down; mouse model; novel; oral infection; prevent; recruit; release of sequestered calcium ion into cytoplasm; response; saliva secretion; sensor

Research Advances Role of STIM2 in SOCE and Regulation of Cell Function: Previously we demonstrated the role of STIM2 in regulating SOCE and cell function. We showed that STIM2 facilitates (i) STIM1 clustering and (ii) STIM1-gating of Orai1 at relatively high ER-Ca2+, i.e. conditions that likely do not elicit a STIM1 response; (iii) remodeling STIM1 C-terminus into the active conformation. Our key findings in this fiscal year are as follows: 1. When exogenously expressed, STIM2, a low affinity ER-Ca2+-sensor protein, displays constitutive clustering within ER-PM junctions and when co-expressed with Orai1, it recruits the channel to these junctions. Consistent with this co-localization, expression of STIM2 leads to activation of endogenous Orai1 under basal conditions. Currently, there is no information about endogenous STIM2, whether it is pre-clustered or how it is regulated under basal and low intensity stimulations. In this study, we used CRISPR/Cas9 to knock-in mVenus into endogenous Stim2, tagging the protein at the N-terminus, and generated cell lines expressing fluorescently labeled endogenous STIM2. The endogenous STIM2 is pre-clustered in the ER-PM junctions of unstimulated cells. While majority of the STIM2 clusters are mobile, there is a small population of immobile STIM2 clusters. Importantly, the immobile clusters, detected in ER-PM junctions of resting cells, denote locations of local ER-Ca2+ decrease that is mediated by functional IP3Rs and sensed by the N-terminus of STIM2. In absence of added agonist, constitutive PLC-dependent PIP2 hydrolysis and PKA activity concertedly control IP3R function and pre-clustering of STIM2. Consequently, immobile STIM2 clusters increase following agonist stimulation and co-ordinate recruitment of Orai1/STIM1. Our data suggest that immobilization of STIM2 clusters in ER-PM junctions, an early response to IP3R-mediated local ER-Ca2+ store release, is a critical check-point for initiation of SOCE. Together, our data reveal a novel and critical mechanism of communication between the IP3R and STIM2 that couples ER-Ca2+ release with assembly of Orai1/STIM1 channels in basal conditions which is then amplified after agonist stimulation of cells . 2. We previously demonstrated that IR-induced salivary gland dysfunction is associated with an early transient increase in mitochondrial Ca2+ and ROSmt. The goal of this work was to examine the role of the early rise in ROSmt in radiation-induced loss of salivary gland function. We tested the efficacy of mito-ROS scavenger, MitoTEMPO, by treating mice shortly before and on several days after IR. We report that treatment of mice with the mitochondrial-targeted antioxidant, MitoTEMPO, resulted in almost complete protection of salivary gland secretion following either single (15 Gy) or fractionated (5x3 Gy) doses of irradiation. Salivary gland cells isolated from MitoTEMPO-treated, irradiated, mice displayed significant attenuation of the initial increases in ROSmt, (Ca2+mt, and activated caspase-3 as compared to cells from irradiated, but untreated, animals. Importantly, MitoTEMPO treatment prevented radiation-induced decrease in STIM1, consequently protecting store-operated Ca2+ entry which is critical for saliva secretion. Together, these findings identify the initial increase in ROSmt, that is induced by irradiation, as a critical driver of persistent salivary gland hypofunction. We suggest that the mitochondrially targeted antioxidant, MitoTEMPO, can be potentially important in preventing IR-induced salivary gland dysfunction. 3. Store-operated calcium entry (SOCE) is triggered by assembly of Orai1 with STIM proteins in ER-PM junctions. Plasma membrane PIP2 as well as PIP2-binding protein, SEPT4, significantly impact Orai1-STIM1 interaction. While septins and PIP2 can organize the actin cytoskeleton, it is unclear whether the status of actin within the junctions contributes to SOCE. We report herein that actin remodeling modulates STIM1 clustering. Our findings show that a PIP2- and SEPT4-dependent mechanism involving CDC42, WASP/WAVE, and ARP2 regulates actin remodeling into a ring-like structure around STIM1 puncta. CDC42 localization in the ER-plasma membrane region is enhanced following ER-Ca2+ store depletion. PIP2 depletion or knockdown of SEPT4 attenuate the recruitment of CDC42 to the ER-PM region. Importantly, knockdown of SEPT4, or CDC42+ARP2, disrupts the organization of actin as well as STIM1 clustering. Consequently, Orai1 recruitment to STIM1 puncta, SOCE, and NFAT translocation to the nucleus are all attenuated. Ca2+ influx induced by STIM1-C terminus is not affected by CDC42 knockdown. In aggregate, our findings reveal that PIP2 and SEPT4 affect Orai1/STIM1 clustering by coordinating actin remodeling within ER-PM junctions. This dynamic reorganization of actin has an important role in regulation of SOCE and downstream Ca2+-dependent effector functions.

研究进展 STIM2在SOCE和细胞功能调节中的作用：之前我们证明了STIM2在调节SOCE和细胞功能中的作用。我们表明，STIM2 促进 (i) STIM1 聚类和 (ii) 在相对较高的 ER-Ca2+ 下对 Orai1 进行 STIM1 门控，即可能不会引发 STIM1 反应的条件； (iii) 将 STIM1 C 末端重塑为活性构象。我们在本财年的主要调查结果如下： 1. 当外源表达时，STIM2（一种低亲和力 ER-Ca2+-传感器蛋白）在 ER-PM 连接内显示组成型聚类，当与 Orai1 共表达时，它会募集通向这些连接的通道。与这种共定位一致，STIM2 的表达导致基础条件下内源性 Orai1 的激活。目前，还没有关于内源性 STIM2 的信息，无论它是预聚集的还是在基础和低强度刺激下它是如何调节的。在本研究中，我们使用 CRISPR/Cas9 将 mVenus 敲入内源性 Stim2，在 N 末端标记蛋白质，并生成表达荧光标记的内源性 STIM2 的细胞系。内源性 STIM2 预先聚集在未刺激细胞的 ER-PM 连接处。虽然大多数 STIM2 簇是可移动的，但也有一小部分是不可移动的 STIM2 簇。 重要的是，在静息细胞的 ER-PM 连接处检测到的固定簇表示局部 ER-Ca2+ 减少的位置，该减少由功能性 IP3R 介导并由 STIM2 N 末端感知。在不添加激动剂的情况下，组成型 PLC 依赖性 PIP2 水解和 PKA 活性协同控制 IP3R 功能和 STIM2 的预聚集。因此，固定的 STIM2 簇在激动剂刺激后增加，并协调 Orai1/STIM1 的募集。我们的数据表明，STIM2 簇在 ER-PM 连接处的固定是对 IP3R 介导的局部 ER-Ca2+ 库释放的早期反应，是 SOCE 启动的关键检查点。 总之，我们的数据揭示了 IP3R 和 STIM2 之间的一种新颖且关键的通讯机制，该机制将 ER-Ca2+ 释放与基础条件下 Orai1/STIM1 通道的组装结合起来，然后在细胞激动剂刺激后放大。 2. 我们之前证明，IR 诱导的唾液腺功能障碍与线粒体 Ca2+ 和 ROSmt 的早期短暂增加有关。这项工作的目的是检查 ROSmt 早期升高在辐射引起的唾液腺功能丧失中的作用。我们通过在 IR 前不久和后几天治疗小鼠来测试 mito-ROS 清除剂 MitoTEMPO 的功效。我们报告说，用线粒体靶向抗氧化剂 MitoTEMPO 治疗小鼠，在单次 (15 Gy) 或分次 (5x3 Gy) 剂量照射后几乎完全保护唾液腺分泌。与来自经辐射但未处理的动物的细胞相比，从经 MitoTEMPO 处理、辐射的小鼠中分离的唾液腺细胞显示 ROSmt、（Ca2+mt 和活化的 caspase-3）的初始增加显着减弱。重要的是，MitoTEMPO 处理可防止辐射-诱导 STIM1 减少，从而保护对唾液分泌至关重要的钙池操纵的 Ca2+ 进入，这些发现共同确定了 ROSmt 的初始增加，即辐射引起的唾液腺功能障碍是持续性唾液腺功能减退的关键驱动因素，我们认为线粒体靶向抗氧化剂 MitoTEMPO 在预防红外线引起的唾液腺功能障碍方面可能具有潜在的重要作用。 3. 钙库操纵的钙内流 (SOCE) 由 Orai1 与 ER-PM 连接处的 STIM 蛋白组装触发。质膜 PIP2 以及 PIP2 结合蛋白 SEPT4 显着影响 Orai1-STIM1 相互作用。虽然 septin 和 PIP2 可以组织肌动蛋白细胞骨架，但目前尚不清楚连接处肌动蛋白的状态是否有助于 SOCE。我们在此报告肌动蛋白重塑调节 STIM1 聚类。我们的研究结果表明，涉及 CDC42、WASP/WAVE 和 ARP2 的 PIP2 和 SEPT4 依赖性机制调节肌动蛋白重塑为 STIM1 斑点周围的环状结构。 ER-Ca2+ 储备耗尽后，CDC42 在内质网质膜区域的定位增强。 PIP2 耗尽或 SEPT4 敲除会减弱 CDC42 向 ER-PM 区域的募集。重要的是，敲除 SEPT4 或 CDC42+ARP2 会破坏肌动蛋白的组织以及 STIM1 聚类。因此，Orai1 向 STIM1 点的募集、SOCE 和 NFAT 易位至细胞核的作用均减弱。 STIM1-C 末端诱导的 Ca2+ 流入不受 CDC42 敲低的影响。总的来说，我们的研究结果表明，PIP2 和 SEPT4 通过协调 ER-PM 连接内的肌动蛋白重塑来影响 Orai1/STIM1 聚类。肌动蛋白的这种动态重组在 SOCE 和下游 Ca2+ 依赖性效应器功能的调节中具有重要作用。