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和调节细胞功能中的作用：以前我们证明了STIM2在调节SOCE和细胞功能中的作用。我们表明stim2促进了（i）stim1聚类和（ii）在相对较高的ER-CA2+下对Orai1进行刺激，即可能不会引起stim1响应的条件； （iii）将刺激末端重塑为活性构象。我们在这个财政年度的主要发现如下： 1。当外源表达时，STIM2（低亲和力ER-CA2+传感器蛋白）将在ER-PM连接中显示组成群集，并且与Orai1共表达时，它将募集到这些连接的通道。与这种共定位的一致，STIM2的表达导致基础条件下的内源性ORAI1激活。当前，尚无有关内源性STIM2的信息，无论是预簇还是在基础和低强度刺激下如何调节它。在这项研究中，我们使用CRISPR/CAS9将Mvenus敲入内源性STIM2，在N末端标记蛋白质，并产生表达荧光标记为内源性刺激的细胞系。内源性STIM2被预先集中在未刺激细胞的ER-PM连接中。尽管大多数STIM2群集都是可移动的，但少量的固定IMTIM2簇。 重要的是，在静息细胞的ER-PM连接中检测到的固定簇表示局部ER-CA2+降低的位置，这些位置是由功能性IP3RS介导的，并由STIM2的N末端感测。在没有添加激动剂的情况下，构成依赖性PLC依赖性PIP2水解和PKA活性协同控制IP3R功能以及STIM2的群集。因此，在激动剂刺激和坐标ORAI1/STIM1的协调募集后，固定的STIM2簇增加了。我们的数据表明，ER-PM连接中STIM2簇的固定是对IP3R介导的本地ER-CA2+商店释放的早期响应，是SOCE启动的关键检查点。 总之，我们的数据揭示了IP3R和STIM2之间的一种新颖而关键的通信机制，该机制将ER-CA2+与Orai1/Stim1通道的组装释放，并在基础条件下释放，然后在细胞激动剂刺激后放大。 2。我们先前证明了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早期上升在辐射引起的唾液腺功能丧失中的作用。我们通过在IR后不久和几天对Mitotempo进行Mitotempo的Mito-Ros清除剂的功效。我们报告说，用线粒体靶向抗氧化剂Mitotempo治疗小鼠，在单个（15 Gy）或分离（5x3 Gy）辐照剂量后，几乎完全保护了唾液腺分泌。 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分泌。这些发现确定了Rosmt的最初增加，这是由辐射引起的，这是持续的唾液腺功能低下的关键驱动力。 3。商店经营的钙进入（SOCE）是由ORAI1与ER-PM连接中的刺激蛋白组装在一起的。质膜PIP2以及PIP2结合蛋白SEPT4显着影响Orai1-stim1相互作用。虽然SEPTINS和PIP2可以组织肌动蛋白细胞骨架，但尚不清楚连接处的肌动蛋白的状态是否有助于SOCE。我们在此报告，肌动蛋白重塑调节刺激群。我们的发现表明，涉及Cdc42，WASP/WAVE和ARP2的PIP2和SEPT4依赖性机制调节肌动蛋白重塑刺激刺激量周围的环状结构。 ER-CA2+存储耗竭后，CDC42在ER-铂膜区域中的定位得到增强。 PIP2耗尽或敲除9月4日，会使CDC42募集到ER-PM区域。重要的是，SEPT4或CDC42+ARP2的敲低破坏了肌动蛋白和刺激群的组织。因此，ORAI1募集到STIM1点，SOCE和NFAT转移到细胞核上都被减弱。由STIM1-C末端诱导的Ca2+流入不受CDC42敲低的影响。总体而言，我们的发现表明，PIP2和SEPT4通过在ER-PM连接中协调肌动蛋白重塑来影响ORAI1/STIM1。肌动蛋白的这种动态重组在调节SOCE和下游Ca2+依赖性效应子功能中具有重要作用。