Molecular Mechanisms Regulating Calcium Flux In Salivary Glands

调节唾液腺钙通量的分子机制

基本信息

批准号：
9555606
负责人：
INDU S. AMBUDKAR
金额：
$ 251.17万
依托单位：
NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
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/9555606
关键词：
ANGPTL2 gene AQP1 gene Acinar Cell Actins Address Affect Agonist Animal Model Attenuated Binding C-terminal CASP3 gene CDC42 gene Calcium Channel Calcium Signaling Carbachol Caspase Cations Cell Line Cell Volumes Cell membrane Cells Complex Coupling Data Defect Disease EF-Hand Domain Endoplasmic Reticulum Ensure Event Family Fluids and Secretions Fluorescence Resonance Energy Transfer Functional disorder Genes Gland Goals HTATIP2 gene Impairment Interphase Cell Ion Channel Lead Length Link Manuscripts Mediating Membrane Potentials Mitochondria Molecular Molecular Conformation Mus N-terminal Neurotransmitters Pathogenesis Pathway interactions Patients Permeability Phosphatidylinositol 4,5-Diphosphate Phosphoric Monoester Hydrolases Physiological Physiology Play Preparation Protein Biosynthesis Proteins Radiation Radiation therapy Reactive Oxygen Species Recovery Recruitment Activity Regulation Reporting Research Role SAM Domain STIM1 gene Salivary Salivary Glands Signal Pathway Signal Transduction Sjogren&apos s Syndrome TRPC1 protein Time Wasps Water adenoviral-mediated attenuation calcium uniporter cell growth endoplasmic reticulum stress gene therapy genetic regulatory protein human subject in vivo inhibitor/antagonist interest knock-down member mitochondrial membrane mutant novel prevent protein biomarkers protein folding receptor release of sequestered calcium ion into cytoplasm response saliva secretion sensor sulfated glycoprotein 2 trafficking treatment strategy

项目摘要

Research Advances 1. STIM2 mediates STIM1 conformational switch to promote STIM1/Orai1 activation at minimal ER-Ca2+ depletion: There is considerable evidence that, at low ER-Ca2+, Ca2+ dissociates from the N-terminus of STIM1, which initiates STIM1-STIM1 association and disruption of the intramolecular C-terminal CC1-CC3 interaction, and promotes the activated conformation required for gating Orai. These conformational changes in STIM1 are also critical for its clustering in ER-PM junctions with Orai1. We have now examined how STIM2 facilitates STIM1 clustering and gating of Orai1 at relatively high ER-Ca2+, i.e. when the STIM1-EF hand domain is expected to be loaded with Ca2+. We find that STIM2 expression causes recruitment and activation of endogenous Orai1 in resting cells. Co-expression of STIM1 with STIM2 (wild type; or gating- deficient mutant, STIM2-L485H) promoted assembly of STIM1/STIM2/Orai1 and increased Orai1 function (Figure 1B). In contrast, STIM1-F394H (lacking the ability to gate Orai1) or STIM1-R426L (with stabilized CC1-CC3 interactions) did not increase Orai1 function when expressed with STIM2. Importantly, STIM1-ES122 (with stabilized N-terminal EF-SAM domain) induced similar Orai1 activation as did STIM1. Thus, STIM2 interaction with STIM1 causes changes in the STIM1-CC1-CC3 domains that lead to Orai1 activation. We confirmed this by using the STIM1 conformational sensor (C-OASF-Y)-FRET. STIM2, but not STIM2-SOAR, decreased the FRET of C-OASF-Y. Together these findings reveal that under conditions in which ER-Ca2+ depletion is insufficient to trigger a response in STIM1, a critical STIM2-STIM1 interaction controls STIM1 conformation as well as its assembly with, and activation of, Orai1. Our results show that at relatively high ER-Ca2+, STIM2-STIM1 interaction promotes the active STIM1 conformation required for Orai1 activation. 2. PIP2-dependent cytoskeletal remodeling is involved in assembly and function of Orai1-STIM1 complexes: Reports suggest that PIP2 is essential for Orai1-STIM1 assembly in ER-PM junctions and coordination of cytoskeletal remodeling that might impact SOCE. Further, septin has been linked with organization of PIP2 in these junctions. Our studies reveal that a PIP2-dependent actin remodeling complex containing CDC42/N-WASP/WAVE/ARP2/3 is recruited to ER-PM junctions during SOCE, and affects the assembly and function of STIM1-Orai1. Depletion of PIP2 or knockdown of septin impaired STIM1-Orai1 assembly, SOCE and NFAT activation, together with a decrease in recruitment of CDC42. Knockdown of CDC42 and its interacting proteins, N-WASP/WAVE and ARP2 reduced STIM1-Orai1 clustering and function, but not Orai1 activation by STIM1 C-terminus or STIM1K. Importantly, TIRFM demonstrates a distinct reorganization of actin around STIM1/Orai1 clusters, which was dependent on CDC42 and ARP2/3. These findings suggest that PIP2 mediates recruitment of an actin remodeling complex within ER-PM junctions, which promotes/stabilizes a functional STIM1-Orai1 complex. The effects of cytoskeletal remodeling on STIM2/Orai1 assembly/regulation as well as TRPC1 trafficking need to be determined. 3. STIM2 promotes coupling of SOCE to the activation of NFAT: There is much interest in mechanisms that regulate the assembly and stability of SOCE components in ER-PM junctions. Our current studies examine the role of STIM2 in the assembly of Orai1/STIM1 in ER-PM junctions. By using an ER-marker protein to denote ER-PM junctions, we find that Orai1 does not induce ER recruitment into ER-PM junctions on its own, while clustering of STIM1, STIM2 or STIM1+STIM2 following store-depletion cause recruitment of ER into the clusters. In the case of STIM2, this is achieved at low agonist. Plasma membrane PIP2 have been shown to be importance for the recruitment of STIMs to ER-PM junctions and for SOCE. Phosphatase-induced depletion of PIP2 levels in the plasma membrane significantly decreased SOCE and clustering of both STIM1 and STIM2. Knocking down endogenous STIM2 also significantly reduced SOCE and NFAT activation, whereas loss of STIM1 abolished both. While co-expression of both Orai1 and STIM1 was sufficient to generate ICRAC, STIM2 is required for slow Ca2+-dependent inactivation of ICRAC. Similar to full-length STIM1, co-expression of Orai1 with STIM1K (mutant with polybasic domain deleted) also generated ICRAC and SOCE. However, the Orai1+ STIM1K-mediated Ca2+ entry did not activate NFAT. When STIM2 was co-expressed, it colocalized with Orai1 and STIM1K clusters, and also rescued NFAT activation. In summary, our data suggest that STIM2 plays an important role in modulating Orai1 channel function and coupling Orai1-mediated Ca2+ entry to the NFAT signaling pathway. Ong et al., manuscript under preparation. 4. Radiation triggers a TRPM2-dependent mitochondrial pathway to promote STIM1 cleavage by Caspase 3 and loss of SOCE. We have now addressed two major questions: (a) What is the underlying defect in acinar cells that causes loss of salivary fluid secretion? and (b) Is TRPM2 function associated with this defect? Our findings elucidate for the first time that SOCE, which is critical for salivary gland fluid secretion, is compromised by IR. Further, acinar cells from TRPM2+/+, but not TRPM2-/-, mice display an increase in Ca2+mt and ROSmt, a decrease in mitochondrial membrane potential and activation of Caspase 3, that is associated with persistent reduction in STIM1, SOCE and loss of salivary gland fluid secretion. In a salivary gland cell line, silencing Mitochondrial Calcium Uniporter (MCU) or treatment with TRPM2 or Caspase 3 inhibitors prevented IR-induced loss of STIM1 and SOCE. Importantly, adenovirus-mediated expression of STIM1 in vivo in salivary glands of IR-mice increased SOCE and fluid secretion. Thus, IR triggers a TRPM2-dependent mitochondrial pathway to promote STIM1 cleavage by Caspase 3 and loss of SOCE. These data provide the first mechanism that can fully account for IR-induced loss of salivary gland function. (Liu et al. 2017, Sci Signal. 2017 Jun 6;10,482). 5. Adenovirus mediated expression of hAQP1 in irradiated mouse salivary glands increases fluid secretion by enhancing carbachol-induced cell volume decrease. Agonist-stimulated cell volume decrease is a read-out for the secretory activity of acinar cells and correlates well with saliva secretion in animal models and human subjects. Compared to the sustained decrease in volume of acinar cells from non-IR mice, CCh induced a transient and attenuated decrease in cell volume of salivary gland acinar cells from IR-mice (2 and 8 months post-IR). The attenuation of CCh-induced cell volume change can be accounted for by the defect in CCh-induced Ca2+i increase (described above). Despite this defect, expression of hAQP1 in glands of mice post-IR, led to recovery of CCh-induced volume changes, with increase in the initial rates of cell volume reduction, but not the amplitude. Together the data suggest that expression of hAQP1 increases the water permeability of acinar cells, which underlies the recovery of fluid secretion in salivary glands functionally compromised by IR. Although Ca2+ signaling defects in acinar cells might preclude full recovery of saliva secretion, this is still a useful strategy for treatment. These data establish the physiological basis for the AdCMV-AQP1 gene therapy strategy. (Teos et al. Gene Ther. 2016 Jul;23(7):572-9).

研究进展 1. STIM2 介导 STIM1 构象转换，以在 ER-Ca2+ 最小消耗的情况下促进 STIM1/Orai1 激活：有大量证据表明，在 ER-Ca2+ 较低时，Ca2+ 从 STIM1 的 N 末端解离，从而启动 STIM1-STIM1 结合和破坏分子内 C 端 CC1-CC3 相互作用，并促进门控 Orai 所需的激活构象。 STIM1 的这些构象变化对于其在 ER-PM 与 Orai1 连接中的聚集也至关重要。我们现在已经研究了 STIM2 如何在相对较高的 ER-Ca2+ 下促进 STIM1 聚类和门控 Orai1，即当 STIM1-EF 手域预计会加载 Ca2+ 时。我们发现 STIM2 表达会导致静息细胞中内源性 Orai1 的募集和激活。 STIM1 与 STIM2（野生型；或门控缺陷突变体，STIM2-L485H）的共表达促进 STIM1/STIM2/Orai1 的组装并增强 Orai1 功能（图 1B）。相比之下，STIM1-F394H（缺乏门控Orai1的能力）或STIM1-R426L（具有稳定的CC1-CC3相互作用）在与STIM2一起表达时不会增加Orai1功能。重要的是，STIM1-ES122（具有稳定的 N 端 EF-SAM 结构域）诱导了与 STIM1 类似的 Orai1 激活。因此，STIM2 与 STIM1 的相互作用会导致 STIM1-CC1-CC3 结构域发生变化，从而导致 Orai1 激活。我们通过使用 STIM1 构象传感器 (C-OASF-Y)-FRET 证实了这一点。 STIM2（而非 STIM2-SOAR）降低了 C-OASF-Y 的 FRET。这些发现共同表明，在 ER-Ca2+ 消耗不足以触发 STIM1 反应的条件下，关键的 STIM2-STIM1 相互作用控制 STIM1 构象及其与 Orai1 的组装和激活。我们的结果表明，在相对较高的 ER-Ca2+ 下，STIM2-STIM1 相互作用促进 Orai1 激活所需的活性 STIM1 构象。 2. PIP2依赖性细胞骨架重塑参与Orai1-STIM1复合物的组装和功能：报告表明，PIP2对于ER-PM连接中的Orai1-STIM1组装以及可能影响SOCE的细胞骨架重塑的协调至关重要。此外，septin 与这些连接处 PIP2 的组织有关。我们的研究表明，在 SOCE 过程中，包含 CDC42/N-WASP/WAVE/ARP2/3 的 PIP2 依赖性肌动蛋白重塑复合物被招募到 ER-PM 连接处，并影响 STIM1-Orai1 的组装和功能。 PIP2 的耗尽或 septin 的敲低会损害 STIM1-Orai1 组装、SOCE 和 NFAT 激活，同时减少 CDC42 的募集。 CDC42 及其相互作用蛋白、N-WASP/WAVE 和 ARP2 的敲低会降低 STIM1-Orai1 聚类和功能，但不会降低 STIM1 C 末端或 STIM1K 激活的 Orai1。重要的是，TIRFM 证明了 STIM1/Orai1 簇周围肌动蛋白的明显重组，这依赖于 CDC42 和 ARP2/3。这些发现表明，PIP2 介导 ER-PM 连接内肌动蛋白重塑复合物的募集，从而促进/稳定功能性 STIM1-Orai1 复合物。需要确定细胞骨架重塑对 STIM2/Orai1 组装/调节以及 TRPC1 运输的影响。 3. STIM2 促进 SOCE 与 NFAT 激活的偶联：人们对调节 ER-PM 连接中 SOCE 组件的组装和稳定性的机制非常感兴趣。我们目前的研究探讨了 STIM2 在 ER-PM 连接中 Orai1/STIM1 组装中的作用。通过使用 ER 标记蛋白来表示 ER-PM 连接，我们发现 Orai1 本身不会诱导 ER 募集到 ER-PM 连接中，而 STIM1、STIM2 或 STIM1+STIM2 在储存耗尽后聚集会导致 ER 募集进入集群。对于 STIM2，这是在低激动剂的情况下实现的。质膜 PIP2 已被证明对于招募 STIM 至 ER-PM 连接和 SOCE 非常重要。磷酸酶诱导的质膜中 PIP2 水平的消耗显着降低了 STIM1 和 STIM2 的 SOCE 和聚类。敲低内源性 STIM2 也显着降低了 SOCE 和 NFAT 的激活，而 STIM1 的缺失则消除了这两者。虽然 Orai1 和 STIM1 的共表达足以产生 ICRAC，但 STIM2 是 ICRAC 缓慢 Ca2+ 依赖性失活所必需的。与全长 STIM1 类似，Orai1 与 STIM1K（删除多碱基结构域的突变体）共表达也产生 ICRAC 和 SOCE。然而，Orai1+ STIM1K 介导的 Ca2+ 进入不会激活 NFAT。当 STIM2 共表达时，它与 Orai1 和 STIM1K 簇共定位，并且还挽救了 NFAT 激活。总之，我们的数据表明 STIM2 在调节 Orai1 通道功能以及将 Orai1 介导的 Ca2+ 进入 NFAT 信号通路耦合方面发挥着重要作用。 Ong 等人，手稿正在准备中。 4. 辐射触发 TRPM2 依赖性线粒体途径，促进 Caspase 3 裂解 STIM1 并导致 SOCE 丢失。我们现在已经解决了两个主要问题：（a）导致唾液分泌丧失的腺泡细胞的潜在缺陷是什么？ (b) TRPM2 功能是否与该缺陷相关？我们的研究结果首次阐明了对唾液腺液分泌至关重要的 SOCE 受到 IR 的损害。此外，来自 TRPM2+/+ 而非 TRPM2-/- 的腺泡细胞显示 Ca2+mt 和 ROSmt 增加、线粒体膜电位降低和 Caspase 3 激活，这与 STIM1、SOCE 和唾液腺液分泌丧失。在唾液腺细胞系中，沉默线粒体钙单向转运蛋白 (MCU) 或用 TRPM2 或 Caspase 3 抑制剂治疗可防止 IR 诱导的 STIM1 和 SOCE 丢失。重要的是，IR 小鼠唾液腺中腺病毒介导的 STIM1 体内表达增加了 SOCE 和液体分泌。因此，IR 触发 TRPM2 依赖性线粒体途径，促进 Caspase 3 裂解 STIM1 并导致 SOCE 丢失。这些数据提供了第一个可以充分解释红外线引起的唾液腺功能丧失的机制。（Liu 等人，2017 年，Sci Signal。2017 年 6 月 6 日；10,482）。 5.在受辐射的小鼠唾液腺中，腺病毒介导的hAQP1表达通过增强卡巴胆碱诱导的细胞体积减少来增加液体分泌。激动剂刺激的细胞体积减少是腺泡细胞分泌活性的读数，并且与动物模型和人类受试者的唾液分泌密切相关。与非 IR 小鼠的腺泡细胞体积持续减少相比，CCh 诱导 IR 小鼠唾液腺腺泡细胞的细胞体积出现短暂且减弱的减少（IR 后 2 个月和 8 个月）。 CCh 诱导的细胞体积变化的减弱可以通过 CCh 诱导的 Ca2+i 增加的缺陷来解释（如上所述）。尽管存在这种缺陷，IR后小鼠腺体中hAQP1的表达导致CCh诱导的体积变化恢复，细胞体积减少的初始速率增加，但幅度没有增加。总之，数据表明 hAQP1 的表达增加了腺泡细胞的水渗透性，这是 IR 功能受损的唾液腺液体分泌恢复的基础。尽管腺泡细胞中的 Ca2+ 信号传导缺陷可能会妨碍唾液分泌的完全恢复，但这仍然是一种有用的治疗策略。这些数据为 AdCMV-AQP1 基因治疗策略奠定了生理学基础。（Teos 等人 Gene Ther。2016 年 7 月；23(7):572-9）。