Molecular Mechanisms Regulating Calcium Flux In Salivary Glands

调节唾液腺钙通量的分子机制

• 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
• 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' 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

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构象转换以在最小ER-CA2+消耗时促进strip1/Orai1激活：有相当大的证据表明，在低ER-CA2+，Ca2+，Ca2+在STIM1的N末端分离出来，从奥莱。 STIM1中的这些构象变化对于与Orai1的ER-PM连接中的聚类也至关重要。现在，我们已经检查了STIM2如何在相对较高的ER-CA2+下促进ORAI1的刺激和门控，即何时何时使用CA2+加载STIM1-EF手动域。 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；或门控缺乏突变体，STIM2-L485H）的共表达促进了STIM1/STIM2/ORAI1的组装以及增加的ORAI1功能（图1B）。相比之下，STIM1-F394H（缺乏Gate Orai1的能力）或STIM1-R426L（具有稳定的CC1-CC3相互作用）在用STIM2表达时不会增加ORAI1的功能。重要的是，STIM1-ES122（具有稳定的N末端EF-SAM结构域）与STIM1诱导类似的ORAI1激活。因此，STIM2与STIM1的相互作用会导致导致ORAI1激活的STIM1-CC1-CC3结构域的变化。我们通过使用STIM1构象传感器（C-OASF-Y）-FRET来证实这一点。 stim2，但不是stim2-soar，降低了c-oasf-y的fret。这些发现共同表明，在ER-CA2+耗竭的条件下，不足以触发STIM1中的响应，关键的STIM2-stim1相互作用控制STIM1构象以及其与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相互作用促进了ORAI1激活所需的活性STIM1构象。 2。PIP2依赖性细胞骨架重塑与Orai1-Stim1复合物的组装和功能有关：报告表明，PIP2对于ER-PM连接中的Orai1-stim1组装至关重要，并且对可能影响SOCE的细胞骨架重塑的配位和骨骼骨骼重塑的配位是必不可少的。此外，Septin与这些连接处的PIP2组织有关。我们的研究表明，在SOCE期间募集了含有CDC42/N-WASP/WAVE/ARP2/3的PIP2依赖性肌动蛋白重塑复合物，并影响stim1-orai1的组装和功能。 PIP2的耗竭或SEPTIN损坏的stim1-orai1组件，SOCE和NFAT激活，以及CDC42募集的减少。 CDC42及其相互作用的蛋白的敲低，N-WASP/WAVE和ARP2降低了stim1-orai1聚类和功能，但不是通过stim1 c-terminus或stim1k激活ORAI1。重要的是，TIRFM证明了肌肉围绕STIM1/ORAI1簇的明显重组，这取决于CDC42和ARP2/3。这些发现表明，PIP2介导了ER-PM连接中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/调控以及TRPC1运输的影响。 3。STIM2促进了SOCE与NFAT激活的耦合：对调节ER-PM连接中SOCE成分的组装和稳定性的机制有很大的兴趣。我们目前的研究检查了stim2在ORAI1/stim1组装中的作用在ER-PM连接中。通过使用ER标记蛋白来表示ER-PM连接，我们发现Orai1不会自行诱导ER募集到ER-PM连接中，而在商店depletion之后，将ER募集到簇中。就STIM2而言，这是在低激动剂中实现的。质膜PIP2已被证明对于将刺激募集到ER-PM连接和SOCE中至关重要。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。击倒内源性STIM2还显着降低了SOCE和NFAT激活，而STIM1的损失则消除了。虽然ORAI1和STIM1的共表达足以产生ICRAC，​​但STIM2对于慢速Ca2+依赖性ICRAC的失活是必需的。与全长stim1相似，与stim1k（删除多重域的突变体）共表达也产生了ICRAC和SOCE。但是，Orai1+ stim1k介导的Ca2+进入未激活NFAT。当STIM2共表达时，它将与Orai1和stim1k簇共定位，并挽救了NFAT激活。总而言之，我们的数据表明STIM2在调节ORAI1通道函数和耦合ORAI1介导的Ca2+进入NFAT信号通路方面起着重要作用。 Ong等人，手稿准备。 4。辐射触发trpm2依赖性的线粒体途径，以通过caspase 3和SOCE丢失来促进刺激裂解。我们现在已经解决了两个主要问题：（a）腺泡细胞中导致唾液液分泌丧失的基本缺陷是什么？ （b）TRPM2功能是否与此缺陷关联？我们的发现首次阐明了SOCE对唾液腺液体分泌至关重要的SOCE受到IR的损害。此外，来自TRPM2+/+的腺泡细胞，但不是TRPM2 - / - ，小鼠显示出Ca2+MT和ROSMT的增加，这是线粒体膜电位的降低和caspase 3的激活，这与Stim1，SOCE的持续减少以及溶性腺体的损失有关。在唾液腺细胞系中，使线粒体钙钙（MCU）或用TRPM2或caspase 3抑制剂进行沉默，阻止了IR诱导的STIM1和SOCE的损失。重要的是，腺病毒介导的IR-唾液腺体内STIM1的表达增加了SOCE和液体分泌。因此，IR触发了TRPM2依赖性线粒体途径，以通过caspase 3和SOCE丢失来促进刺激裂解。这些数据提供了可以完全解释IR诱导的唾液腺功能丧失的第一种机制。 （Liu etal。2017，SciSignal。20176月6日； 10,482）。 5。腺病毒介导的HAQP1在辐照小鼠唾液腺中的表达通过增强卡尔巴霍尔诱导的细胞体积减少，从而增加液体分泌。激动剂刺激的细胞体积减小是腺泡细胞分泌活性的读出，并且与动物模型和人类受试者中的唾液分泌良好相关。与非IR小鼠的腺泡细胞体积持续减少相比，CCH诱导了来自IR-Mice的唾液腺腺泡细胞细胞体积的短暂和减弱（IR后2和8个月）。 CCH诱导的细胞体积变化的衰减可以通过CCH诱导的Ca2+I增加（如上所述）的缺陷来解释。尽管存在这种缺陷，但HAQP1在IR后IR的腺体中的表达导致CCH诱导的体积变化的恢复，而细胞体积降低的初始速率降低，但幅度却没有增加。数据一起表明，HAQP1的表达增加了腺泡细胞的水渗透性，这是IR在功能上损害的唾液腺中液体分泌的恢复的基础。尽管腺泡细胞中的Ca2+信号传导缺陷可能排除了唾液分泌的完全恢复，但这仍然是治疗的有用策略。这些数据建立了ADCMV-AQP1基因治疗策略的生理基础。 （Teos等人，GeneTher。2016Jul; 23（7）：572-9）。