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

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. STIM2 在 NFAT1 信号传导调节中的关键作用：细胞核中激活 T 细胞核因子 (NFAT) 1 依赖性基因表达是一项重要的细胞功能，受 SOCE 调节。该通路的缺陷或 Orai1 或 STIM1 蛋白的异常功能与患者和小鼠模型的免疫缺陷和自身免疫相关。 STIM1 是 Orai1 的主要激活剂，因为该蛋白的丢失会消除 Orai1 的激活，从而消除下游 Ca2+ 依赖性细胞功能的调节。据报道，STIM2 诱导相对较弱的通道激活。有趣的是，尽管 STIM2 并不是大多数细胞类型中 SOCE 的主要贡献者，但它似乎具有独特的生理相关性。 缺乏这种蛋白质的小鼠表现出发育缺陷，并在出生后 4-6 周死亡。此外，T 细胞中该蛋白的敲低导致 SOCE 小幅下降，而 STIM1 缺陷型 T 细胞的 SOCE 几乎完全丧失。重要的是，由于 NFAT1 核易位缺陷，STIM2 缺陷细胞的细胞因子产生显着减少 (15)。总之，这些先前的发现表明 STIM2 在 SOCE 依赖性 NFAT 激活中具有特定作用。我们研究了 STIM2 对 NFAT1 激活的贡献，以响应 Ca2+ 通过 Orai1/STIM1 通道进入。我们的研究结果表明，虽然 STIM1 与 Orai1 在 ER-PM 连接中的聚集会诱导局部和整体 Ca2+i 的充分增加以触发 NFAT1 激活，但 Orai1/STIM1 与 STIM2 的组装对于将通道靶向特定的 ER-PM 连接至关重要，而特定的 ER-PM 连接允许 Orai1 与 AKAP79 信号复合物的组装。 Orai1 与 AKAP79 的相互作用是将 Orai1 功能与 NFAT1 激活耦合的重要步骤，因为它允许通道介导的局部 Ca2+ 增加用于激活由 AKAP79 支撑的钙调神经磷酸酶。总的来说，我们的研究结果确定了 STIM2 在将 Orai1/STIM1 靶向特定 ER-PM 连接点方面发挥着关键且重要的作用，从而允许通道与 AKAP79 信号复合物组装。 STIM2 对 Orai1/STIM1 的区室化是其对 Ca2+ 依赖性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 STIM/Orai 蛋白动态状态的评估：迄今为止，所有报告 ER-PM 连接内蛋白聚类模式的研究都是使用利用标记蛋白的过表达系统完成的。 其中大多数使用基于强启动子的表达系统。因此，内源蛋白的状态以及它们如何响应储存耗尽尚不清楚。例如，SOCE 的所有三个组成部分的动员的时间特征、ER-PM 连接内内源性 STIM 簇的大小/性质尚未研究。我们利用 CRISPR/Cas9 技术将荧光标签 (mVenus) 敲入 HEK293 和 HEK293T 细胞的内源性 STIM2-N 末端。我们还使用弱（胸苷激酶）启动子进行蛋白质表达，并具有标准化条件，以比内源性低 2 倍的水平表达蛋白质。 我们发现，在细胞静息条件下，内源性 STIM2 定位于 ER-PM 连接处（预簇）。 TIRFM 数据分析表明，ER-PM 连接处的大多数 STIM2 不稳定（移动/动态），并且稳定 STIM2 簇（不移动）的数量在激动剂治疗后增加。本报告旨在阐明控制 STIM2 预聚类的细胞因素。 固定的 STIM2 簇是 STIM1 和 Orai1 在低激动剂刺激下招募的关键位点。在没有扩展突触结合蛋白 (E-Syt2/3)、ER-PM 系链的情况下，STIM2 在基础刺激下似乎不会预聚集，尽管在 TIRF 平面下出现很少的动态或固定簇。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预聚类的定位和功能之间的重要联系。 IP3R 的低水平表达表明 ER-PM 连接处存在稳定的 ER Ca2+ 通道前簇。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-三重 KO 细胞系的使用表明，稳定的 STIM2 前簇是由 IP3R 的存在和定位决定的。 cAMP 可以磷酸化 IP3R，以增强其在环境 IP3 和 Ca2+ 下的功能。用毛喉素处理表达 WTIP3R1 的细胞可增强 Stim2 聚类。具有 IP3RKO 或表达突变受体（P 缺陷和门控缺陷）的细胞中的 STIM2 表达对毛喉素没有反应。最后，IP3R 聚类被 si ESyt2 和 3 废除。我们的数据共同阐明了 STIM2 的新特征以及 IP3R 在 STIM/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是由受体介导的 PIP2 水解发生和 IP3 生成的位置的接近程度决定的。 IP3R 可以快速感知本地 IP3 并做出响应，导致 ER-Ca2+ 耗尽。定位在簇内或在 IP3R 激活后招募的 STIM2 可以感知 ER-Ca2+ 的局部减少，并启动 Orai1/STIm1 复合物的组装和激活。因此，IP3R 和 STIM2 定位的 ER-PM 连接在生理上极为相关。根据我们实验室的其他发现，我们还可以提出下游 Ca2+ 依赖性功能的调节也在这些微域内协调。