Molecular Mechanisms Regulating Calcium Flux In Salivary Glands

调节唾液腺钙通量的分子机制

• 批准号: 10246729
• 负责人: INDU S. AMBUDKAR
• 金额: $ 277.02万
• 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/10246729
• 关键词: Affinity; Aftercare; Agonist; Autoimmunity; Birth; CRISPR/Cas technology; Calcineurin; Cell Line; Cell Nucleus; Cell physiology; Cells; Characteristics; Complex; Coupling; Cyclic AMP; Data; Defect; Deglutition Disorders; Dental caries; Development; EF-Hand Domain; Estrogen receptor positive; Fluids and Secretions; Forskolin; Functional disorder; Gene Expression; Generations; Gland; Goals; Hydrolysis; ITPR1 gene; Immunologic Deficiency Syndromes; Ions; Knock-in; Link; Liquid substance; Location; Maintenance; Mediating; Molecular; Molecular Conformation; Mus; Nature; Neurotransmitters; Nuclear Translocation; Oral health; Pathway interactions; Patients; Pattern; Phosphatidylinositol 4,5-Diphosphate; Physiological; Production; Proteins; Regulation; Reporting; Research; Rest; Role; STIM1 gene; Saliva; Salivary; Salivary Glands; Signal Transduction; Site; Standardization; Stimulus; System; T-Cell Activation; T-Lymphocyte; Techniques; Thymidine Kinase; Xerostomia; base; cell type; cytokine; knock-down; mouse model; mutant; novel; nuclear factors of activated T-cells; oral infection; overexpression; promoter; protein expression; receptor; recruit; release of sequestered calcium ion into cytoplasm; response; scaffold; sensor; synaptotagmin; Affinity; Aftercare; Agonist; Autoimmunity; Birth; CRISPR/Cas technology; Calcineurin; Cell Line; Cell Nucleus; Cell physiology; Cells; Characteristics; Complex; Coupling; Cyclic AMP; Data; Defect; Deglutition Disorders; Dental caries; Development; EF-Hand Domain; Estrogen receptor positive; Fluids and Secretions; Forskolin; Functional disorder; Gene Expression; Generations; Gland; Goals; Hydrolysis; ITPR1 gene; Immunologic Deficiency Syndromes; Ions; Knock-in; Link; Liquid substance; Location; Maintenance; Mediating; Molecular; Molecular Conformation; Mus; Nature; Neurotransmitters; Nuclear Translocation; Oral health; Pathway interactions; Patients; Pattern; Phosphatidylinositol 4,5-Diphosphate; Physiological; Production; Proteins; Regulation; Reporting; Research; Rest; Role; STIM1 gene; Saliva; Salivary; Salivary Glands; Signal Transduction; Site; Standardization; Stimulus; System; T-Cell Activation; T-Lymphocyte; Techniques; Thymidine Kinase; Xerostomia; base; cell type; cytokine; knock-down; mouse model; mutant; novel; nuclear factors of activated T-cells; oral infection; overexpression; promoter; protein expression; receptor; recruit; release of sequestered calcium ion into cytoplasm; response; scaffold; sensor; synaptotagmin

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. Key role for STIM2 in regulation of NFAT1 signaling: Activation of nuclear factor of activated T-cells (NFAT) 1-dependent gene expression in the nucleus is a vital cell function that is regulated by SOCE. Defects in this pathway or aberrant function of either Orai1 or STIM1 proteins are associated with immunodeficiency and autoimmunity in patients and mouse models. STIM1 is the primary activator of Orai1 as loss of the protein eliminates Orai1 activation and consequently, regulation of downstream Ca2+-dependent cell functions. STIM2 is reported to induce relatively weak activation of the channel. Intriguingly, although STIM2 is not a major contributor to SOCE in most cell types, it appears to have a unique physiological relevance. Mice lacking the protein displayed developmental defects and died 4-6 weeks after birth. Further, knockdown of the protein in T-cells caused small decrease in SOCE, in contrast to STIM1-deficient T cells that had almost complete loss of SOCE. Importantly, STIM2-deficient cells display significantly greater decrease in cytokine production due to a defect in nuclear translocation of NFAT1 (15). Together, these previous findings suggest a specific role for STIM2 in SOCE-dependent NFAT activation. We have examined the contribution of STIM2 to NFAT1 activation in response to Ca2+ entry via Orai1/STIM1 channel. Our findings demonstrate that while clustering of STIM1 with Orai1 in ER-PM junctions induces sufficient increase in both local and global Ca2+i to trigger NFAT1 activation, assembly of Orai1/STIM1 with STIM2 is essential for targeting the channel to specific ER-PM junctions which are permissive for assembly of Orai1 with the AKAP79 signaling complex. Interaction of Orai1 with AKAP79 is an essential step in coupling Orai1 function with NFAT1 activation since it allows local Ca2+ increases mediated by the channel to be utilized for activating calcineurin that is scaffolded by AKAP79. In aggregate, our findings establish a critical, and essential, role for STIM2 in targeting Orai1/STIM1 to specific ER-PM junctions that permit assembly of the channel with AKAP79 signaling complex. Compartmentalization of Orai1/STIM1 by STIM2 underlies its important physiological contribution to Ca2+ dependent gene expression and cytokine release. 3. Assessment of dynamic status of endogenous STIM/Orai proteins: Till date all studies reporting clustering patterns of the proteins within ER-PM junctions, have been done using overexpression systems utilizing tagged proteins. Most of these use strong promoter based expression systems. Thus the status of endogenous proteins and how they respond to store-depletion is not known. For example, the temporal characteristics of their mobilization, the size/nature of endogenous STIM clusters within ER-PM junctions for all three components of SOCE have not yet been studied. We have utilized CRISPR/Cas9 technique to knock-in a fluorescent tag (mVenus) into the endogenous STIM2-N terminus in HEK293 and HEK293T cells. We also use a weak (Thymidine kinase) promoter for protein expression and have standardized conditions to express protein at levels <2-fold over endogenous. We find that endogenous STIM2 localizes in the ER-PM junction (pre-clustered) under resting conditions in the cell. Analysis of TIRFM data reveal that majority of STIM2 at ER-PM junctions are unstable (mobile/dynamic) and that the number of stable STIM2 clusters (Immobile) increases after treatment with agonist. This report aims to elucidate the cellular factors that govern STIM2 pre-clustering. Immobile STIM2 clusters are the critical sites where STIM1 and Orai1 are recruited under low agonist stimulus. In the absence of Extended-Synaptotagmins (E-Syt2/3), ER-PM tethers, STIM2 does not appear to precluster under basal stimulus, although few dynamic or immobile clusters appears under TIRF plane. In such a condition, STIM2 does not cluster with STIM1/Orai1 in response to low CCh and substantial loss of SOCE was observed. We have further found an important link between the localization and function of IP3R and STIM2 pre-clustering. Low level expression of IP3R demonstrated presence of stable pre-clusters of the ER Ca2+ channel in ER-PM-junctions. In cells expressing IP3R and STIM2, we noted that STIM2 co-localized with stable IP3R. Use of IP3R-triple KO cell lines demonstrated that stable STIM2 pre-clusters are determined by the presence and localization of IP3R. cAMP can phosphorylate IP3Rs to enhance their function under ambient IP3 and Ca2+. Treatment of cells expressing WTIP3R1 with forskolin potentiated Stim2 clustering. STIM2 expression in cells with IP3RKO, or expressing mutant receptors (P-deficient and gating deficient) did not respond to forskolin. Finally, IP3R clustering was abolished by si ESyt2 and 3. Together our data elucidate novel characteristics of STIM2 and a critical role for IP3R in assembly of STIM/Orai1 complexes. The location of IP3R and STIM2 in ER-PM junctions, and the site of the latter, is most likely determined by the proximity to where receptor-mediated PIP2 hydrolysis occurs and IP3 is generated. IP3R can rapidly sense local IP3 and respond causing depletion of ER-Ca2+. STIM2 localized within the clusters or recruited after IP3R activation can sense local decrease in ER-Ca2+ and initiate the assembly of Orai1/STIm1 complex and activation Thus, ER-PM junctions where IP3R and STIM2 are localized are extremely relevant physiologically. Based on other findings from our lab, we can also propose that regulation of downstream Ca2+-dependent functions is also coordinated within these microdomains.

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。STIM2在NFAT1信号调节中的关键作用：活化T细胞（NFAT）1依赖性基因表达的核因子的激活是由SOCE调节的重要细胞功能。 ORAI1或Stim1蛋白的这种途径中的缺陷或异常功能与患者和小鼠模型的免疫缺陷和自身免疫有关。 STIM1是ORAI1的主要激活剂，因为蛋白质的丧失消除了Orai1激活，因此，调节下游Ca2+依赖性细胞功能。据报道，STIM2诱导通道的相对较弱的激活。有趣的是，尽管STIM2并不是大多数细胞类型中SOCE的主要因素，但它似乎具有独特的生理相关性。 缺乏蛋白质的小鼠出现发育缺陷，出生后4-6周死亡。此外，T细胞中蛋白质的敲低导致SOCE降低，而STIM1缺陷型T细胞几乎完全丧失了SOCE。重要的是，由于NFAT1的核易位缺陷，刺激性缺陷的细胞在细胞因子产生中显示出明显更大的降低（15）。总之，这些先前的发现表明了STIM2在SOCE依赖性NFAT激活中的特定作用。我们已经检查了STIM2对通过ORAI1/Stim1通道对Ca2+进入的响应NFAT1激活的贡献。我们的发现表明，在ER-PM连接中使用oRAI1聚集的stim1时，局部和全局Ca2+i的足够增加以触发NFAT1激活，而Orai1/stim1的组装对于将通道定位到特定的ER-PM连接的通道至关重要，这些通道允许ORAI1允许ORAI1与Akap79信号复合物组成。 ORAI1与AKAP79的相互作用是耦合ORAI1函数与NFAT1激活的重要步骤，因为它允许使用该通道介导的局部CA2+增加，用于激活由AKAP79支付的钙调神经素。总体而言，我们的发现在靶向ORAI1/STIM1靶向特定的ER-PM连接中建立了至关重要的，至关重要的作用，该连接允许通道与AKAP79信号传导复合物组装。 STIM2对ORAI1/Stim1的隔室化是其对Ca2+依赖基因表达和细胞因子释放的重要生理贡献的基础。 3.内源性刺激/ORAI蛋白的动态状态的评估：直到迄今为止，使用使用标记蛋白的过表达系统进行了ER-PM连接中蛋白质的聚类模式的研究。 其中大多数使用强启动子基于启动子的表达系统。因此，尚不清楚内源性蛋白质的状态及其对储存量的反应。例如，尚未研究其动员的时间特征，ER-PM连接中内源性刺激簇的大小/性质尚未被研究。我们已经利用CRISPR/CAS9技术将HEK293和HEK293T细胞中的内源性STIM2-N末端敲入荧光标签（Mvenus）。我们还使用弱（胸苷激酶）启动子进行蛋白质表达，并且具有标准化的条件，以<2倍的内源性表达蛋白质。 我们发现，内源性STIM2在细胞中的静止条件下定位在ER-PM结（预聚类）中。 TIRFM数据的分析表明，ER-PM连接处的大多数Stim2是不稳定的（移动/动态），并且用激动剂处理后稳定的STIM2簇（不动）数量增加。该报告旨在阐明控制刺激前聚类的细胞因子。 Immbile STIM2簇是在低激动剂刺激下募集stim1和orai1的关键部位。在没有扩展的突出磁蛋白（E-SYT2/3）的情况下，ER-PM tethers，STIM2似乎并未在基础刺激下群集群集，尽管很少有动态或不动簇在TIRF平面下出现。在这种情况下，STIM2不会响应低CCH与STIM1/ORAI1聚集，并且观察到SOCE的大量损失。我们进一步发现了IP3R和STIM2预聚类的本地化和功能之间的重要联系。 IP3R的低水平表达表明在ER-PM界面中存在ER Ca2+通道的稳定前群体。在表达IP3R和Stim2的细胞中，我们注意到STIM2与稳定的IP3R共定位。 IP3R-Triple KO细胞系的使用表明，稳定的STIM2前群体由IP3R的存在和定位确定。 CAMP可以磷酸化IP3R，以在环境IP3和Ca2+下增强其功能。用孔螺林增强的刺激聚类来处理表达WTIP3R1的细胞。 IP3RKO细胞中的STIM2表达或表达突变受体（缺乏p和门控不足）对福斯科林没有反应。最后，通过Si esyt2和3废除了IP3R聚类。我们的数据阐明了STIM2的新型特征，而IP3R在IP3R中的关键作用在IMT/ORAI1复合物组装中的关键作用。 IP3R和STIM2在ER-PM连接中的位置以及后者的位置，很可能取决于受体介导的PIP2水解发生并产生IP3的位置。 IP3R可以迅速感知本地IP3并响应导致ER-CA2+的耗竭。 IP3R激活后本地化或募集的STIM2可以感觉到ER-CA2+的局部降低，并启动Orai1/Stim1复合物的组装并激活，因此，IP3R和Stim2本地化的ER-PM连接在生理上非常相关。基于我们实验室的其他发现，我们还可以提出，在这些微区内，下游Ca2+依赖性功能的调节也是协调的。