Molecular Mechanisms Regulating Calcium Flux In Salivary Glands

调节唾液腺钙通量的分子机制

• 批准号: 8929666
• 负责人: INDU S. AMBUDKAR
• 金额: $ 251.01万
• 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/8929666
• 关键词: Accounting; Acinar Cell; Acinus organ component; Affect; Agonist; Apical; Area; Attenuated; Autoimmune Diseases; Back; Binding; Biopsy; CDC42 gene; Calcium Channel; Calcium Signaling; Carbachol; Cations; Cell Death; Cell membrane; Cell physiology; Cells; Complex; Data; Disease; Endocytosis; Endoplasmic Reticulum; Ensure; Epithelial Cells; Event; Family; Fluids and Secretions; Functional disorder; GTP-Binding Proteins; Generations; Gland; Goals; Hour; ITPR1 gene; Individual; Infiltration; Inflammation; Knock-out; Lead; Location; Lymphocyte; Mediating; Membrane Potentials; Minor salivary gland structure; Mitochondria; Molecular; Mus; Neurotransmitters; Nuclear Translocation; Pathogenesis; Pathway interactions; Patients; Phospholipase; Physiological; Physiology; Play; Prevention; Process; Protein Biosynthesis; Proteins; Proteome; Proteomics; Radiation; Reactive Oxygen Species; Recovery; Recruitment Activity; Recycling; Regulation; Reporting; Research; Rest; Role; STIM1 gene; Saliva; Salivary; Salivary Glands; Shotguns; Signal Transduction; Stimulus; Stress; Surface; Syndrome; TRP channel; Tissues; Up-Regulation; Xerostomia; ZYX gene; attenuation; cell growth; cellular targeting; cytokine; endoplasmic reticulum stress; genetic regulatory protein; healthy volunteer; in vivo; inhibitor/antagonist; interest; irradiation; member; mitochondrial membrane; novel; overexpression; patient population; prevent; protein folding; protein protein interaction; radiation effect; receptor; release of sequestered calcium ion into cytoplasm; response; saliva secretion; salivary cell; sulfated glycoprotein 2; tempol; trafficking

In this reporting period we have made significant progress in our studies. 1. Resolving the specific functional contributions of TRPC1 and Orai1 channels in polarized secretory epithelial cells. Earlier studies have shown that basolateraly localized TRPC1 is a major determinant of Ca2+ entry in acinar cells and thus required for sustained saliva flow. While Orai1 is localized near the apical region of the cell, its contribution to salivary gland function is not known. Here we have determined the individual contributions of TRPC1 and Orai1 in Ca2+-dependent activation of Kca channels, basolateral NKCC1, and apical TMEM16A, all of which are critical for fluid secretion. We report that report TRPC1 provides necessary Ca2+I in both apical and basolateral region of the cell to regulate these mechanisms while Orai1 has minimal direct contributions to regulation fluid secretion. Consistent with its basolateral localization, CCh-stimulated upregulation of NKCC1 was decreased >80% in acini from TRPC1-/- mice. Importantly, Ca2+ required for sustained activation of apically localized TMEM16A was also dependent on TRPC1-mediated Ca2+ entry. However, Orai1 is critical for fluid secretion since it is required for activation of TRPC1. Knockout of Orai1 within salivary glands of Oraifl/fl or TRPC1-/-/Orai1fl/fl mice by delivery of adCRE induced loss of fluid secretion and CCh-stimulated Ca2+ entry in acini to the same extent as in TRPC1-/- mice. Furthermore, overexpression of Orai1 and STIM1 in vivo in salivary glands of TRPC1-/- mice resulted in basolateral expression of Orai1 in acinar cells together with recovery of fluid secretion and Ca2+ entry. Thus the location, and possibly the expression level, of Orai1 in acinar cells determines its contribution to saliva flow. Together these findings suggest that TRPC1-mediated Ca2+ entry via the basolateral region is key to regulation of both apical and basal mechanisms involved in fluid secretion. 2. Mechanisms regulating plasma membrane recycling of TRPC1 channels: TRPC1 is the predominant channel contributing to SOCE in salivary gland cells. Previous studies revealed that TRPC1 is recruited to the plasma membrane upon Ca2+store depletion, resulting in enhancement of Ca2+entry. However, the mechanisms involved in the trafficking of TRPC1 to the cellular surface have not yet been identified. We show that endocytosis of TRPC1 is dependent on a mechanism that involves Arf6 and Rab5. Further, recycling back to the plasma membrane depends of Rab4. Together, our data suggest that Arf6, Rab5 and Rab4 play a critical, and specific, role in the endocytic recycling of TRPC1 and determine the level and activity of TRPC1 in the plasma membrane. Modulators of this mechanism can be suggested to have significant impact on salivary gland function. 3. STIM2 tunes receptor-stimulated Ca2+ signaling by escorting STIM1-Orai1 to the ER-PM junctional domains: We report that STIM2 tunes agonist-activation of SOCE by recruiting STIM1 to these microdomains at physiologically relevant intensities of agonist stimuli. Targeted deletion of STIM2 in mouse salivary glands diminished agonist-stimulated fluid secretion in vivo and SOCE activation in dispersed acinar cells. Interestingly, the effect of in vivo knockdown of STIM2 is particularly prominent at low agonist. To further investigate how STIM2 modulates Ca2+ signals induced by a range of agonist, we looked agonist-induced Ca2+ responses in single HEK293 cells. Loss of STIM2 decreased the agonist-sensitivity of SOCE activation and nuclear translocation of NFAT. Remarkably, deletion of the STIM2 polybasic domain impaired the recruitment of STIM1 and Orai1 into ER-PM junctional domains at low agonist. Together, our findings suggest that the major physiological role of STIM2 is to escort STIM1 into ER-PM junctional domains at low stimulus intensities when ER-Ca2+ stores are mildly depleted, thus increasing the agonist-sensitivity of Ca2+ signaling. 4. Recruitment of a cytoskeletal-regulatory complex with Orai1 and STIM1 during activation of SOCE: Little is known about other protein-protein interactions involving STIM1 (both in the resting state as well the changes as the activation process occurs). In this study, we identified 155 specific STIM1 binding partners using a shotgun proteomic approach using SILAC with a STIM1 immunoprecipitated complex from HSG cell lysates. Our analysis revealed several interesting changes in the STIM1 proteome upon stimulation with Tg (although, as expected, a majority of the binding partners were not affected by activation). As a result of the SILAC findings, our research has now focused on the role and interactions that cytoskeletal regulatory proteins appear to associate the SOCE core complex. For example proteins such as CD42, ARP2, N-WASP, septin, and zyxin interact with the SOCE proteins STIM1 and Orai-1. Knockdown of these results in decreases SOCE in the order CDC42>Septin>ARP2/3>N-WASP. We are presently trying to understand the interactions of these with cytoskeletal changes and assembly of Orai1-STIM1 channels. 5. Involvement of mitochondria in salivary gland damage in IR: Here we have explored the mechanism of IR-induced permanent damages of salivary gland cells by assessing mitochondria, as the primary cellular target for IR. Mitochondria reactive oxygen species (mROS) in HSG cells were elevated after IR. The elevation was attenuated by maintaining cells in Ca2+-free medium during IR, or by treatment with Tempol. Further, mitochondrial membrane potential (mΨ) was decreased (depolarization) and mitochondrial Ca2+ (mCa2+) was significantly increased in irradiated cells. Both of these were blocked by inhibition of the mitochondrial Ca2+ uniporter (MCU) with Ru360, treating cells with siMCU, or removing external Ca2+ during IR. More importantly cells treated with either Ru360 or siMCU showed greater viability than control cell 24 and 96 hours after irradiation. Our data suggest that IR causes pathophysiological changes in mitochondria that lead to loss of cell function and cell death. 6. Characterization of Ca2+ signaling in salivary gland biopsies from SS patients: Sjgrens syndrome (SS) is an autoimmune disease associated with lymphocytic infiltration and reduced fluid secretion in salivary glands that results in xerostomia. Intriguingly, there is substantial loss of gland function with minimal infiltration or tissue damage. Here we have examined single acinar cell function in minor salivary gland biopsies from SS patients and healthy volunteers. Glands from SS patients with low inflammation showed acinar destruction within the small areas of infiltration; while a large part of the tissue was relatively intact. Importantly, acinar cells in these areas of the glands displayed significant attenuation of Carbachol (Cch) -stimulated intracellular Ca2+ release and Ca2+ entry; as compared to those from healthy volunteers. Consistent with this, agonist-stimulated volume decrease, representing fluid secretion was also lower in these acini. In contrast to the poor correlation between inflammation and saliva flow, there was significant correlation between CCh-stimulated cellular responses and saliva secretion within the patient population. IP3R2 & IP3R3, but not AQP5 or STIM1, were decreased in the intact areas of the gland which can account for the disruption in Ca2+ signaling and consequently fluid secretion. Together, our findings reveal that IP3R deficit in acinar cells underlies the loss of fluid secretion in SS patients who display significant xerostomia.

在此报告期间，我们在研究中取得了重大进展。 1。解决极化分泌上皮细胞中TRPC1和ORAI1通道的特定功能贡献。 较早的研究表明，基底侧侧局部局部TRPC1是腺泡细胞中Ca2+进入的主要决定因素，因此需要持续的唾液流动。尽管Orai1位于细胞的顶端区域附近，但其对唾液腺功能的贡献尚不清楚。 在这里，我们确定了TRPC1和ORAI1在Ca2+依赖性激活KCA通道，基底外侧NKCC1和Apical TMEM16A中的个体贡献，所有这些对流体分泌至关重要。我们报告说，报告TRPC1在细胞的顶端和基底外侧区域提供了必要的Ca2+I，以调节这些机制，而ORAI1对调节流体分泌的直接贡献最少。与其基底外侧定位一致，从TRPC1 - / - 小鼠中，CCH刺激的NKCC1的上调降低了> 80％。 重要的是，持续激活顶端局部TMEM16A所需的Ca2+也取决于TRPC1介导的Ca2+进入。但是，ORAI1对于流体分泌至关重要，因为它是激活TRPC1所必需的。 ORAIFL/FL或TRPC1 - / - /ORAI1FL/FL小鼠在ADCRE诱导的液体分泌损失和CCH刺激的Ca2+进入ACINI中的Ca2+进入与TRPC1 - / - 小鼠相同的程度的敲除Oraifl/fl或TRPC1 - / - /ORAI1FL/FL小鼠的唾液腺内的ORAI1的敲除。 此外，trpc1 - / - 小鼠唾液腺中ORAI1和Stim1在体内的过表达导致腺泡细胞中Orai1的基底外侧表达，并恢复流体分泌和Ca2+进入。因此，腺泡细胞中Orai1的位置，可能是表达水平决定了其对唾液流动的贡献。 这些发现一起表明，通过基底外侧区域的TRPC1介导的Ca2+进入是调节流体分泌所涉及的顶端和基础机制的关键。 2。调节TRPC1通道的质膜回收的机制：TRPC1是导致唾液腺细胞中SOCE的主要通道。先前的研究表明，在Ca2+存储耗竭后，将TRPC1募集到质膜上，从而增强Ca2+进入。但是，尚未确定将TRPC1运输到细胞表面的机制。我们表明，TRPC1的内吞作用取决于涉及ARF6和RAB5的机制。此外，回收回到质膜取决于Rab4。总之，我们的数据表明，ARF6，RAB5和RAB4在TRPC1的内吞回收中起着至关重要的作用，并确定质膜中TRPC1的水平和活性。该机制的调节剂可以建议对唾液腺功能产生重大影响。 3。STIM2通过将STIM1-ORAI1送到ER-PM连接域来调节受体刺激的Ca2+信号传导：我们报告说，刺激刺激通过将stim1募集到这些微域中的这些微域通过生理相关的激动剂刺激来调节SOCE的激动剂激活。小鼠唾液腺中STIM2的靶向缺失减少了激动剂刺激的体内流体分泌，并在分散的腺泡细胞中激活了SOCE激活。有趣的是，在低激动剂中，STIM2体内敲低的效果尤为突出。为了进一步研究STIM2如何调节一系列激动剂诱导的Ca2+信号，我们看上去是激动剂诱导的单个HEK293细胞中的Ca2+响应。 STIM2的损失降低了NFAT的SOCE激活和核转运的激动剂敏感性。值得注意的是，STIM2多重基质结构域的缺失损害了sim1和orai1在低激动剂处的ER-PM连接域中的募集。总之，我们的发现表明，当ER-CA2+存储中有轻度耗尽时，STIM2的主要生理作用是在低刺激强度下将STIM1护送到ER-PM连接域中，从而增加Ca2+信号的激动剂敏感性。 4。在SOCE激活期间，募集与Orai1和stim1的细胞骨架调节复合物：对涉及刺激的其他蛋白质蛋白相互作用知之甚少（在静止状态下，以及激活过程的变化）。 在这项研究中，我们使用SILAC使用SILAC和STIM1免疫沉淀复合物，从HSG细胞裂解物中鉴定出155个特定的STIM1结合伴侣。我们的分析揭示了用TG刺激时STIM1蛋白质组的几个有趣的变化（尽管正如预期的那样，大多数结合伙伴不受激活影响）。由于SILAC的发现，我们的研究现在集中在细胞骨架调节蛋白似乎与SOCE核心复合物相关联的作用和相互作用上。例如，诸如CD42，ARP2，N-WASP，SEPTIN和Zyxin之类的蛋白质与SOCE蛋白STIM1和ORAI-1相互作用。敲除这些结果减少了cdc42> septin> arp2/3> n-wasp的SOCE。我们目前正在尝试了解这些相互作用与Orai1-Stim1通道的细胞骨架变化和组装的相互作用。 5。线粒体参与IR中的唾液腺损伤：在这里，我们通过评估线粒体作为IR的主要细胞靶标探索了IR诱导的唾液腺细胞永久损害的机制。 IR后，HSG细胞中的线粒体活性氧（MRO）升高。通过在IR期间或用Tempol处理的无CA2+培养基中的细胞维持细胞来减弱升高。此外，在辐照细胞中，线粒体膜电位（Min）降低（去极化），线粒体Ca2+（MCA2+）显着增加。这两者都通过抑制用RU360的线粒体Ca2+ Uniporter（MCU）抑制，用SIMCU处理细胞，或在IR期间删除外部Ca2+。更重要的是，用RU360或SIMCU处理的细胞显示出比对照细胞24和96小时后的生存能力更大。我们的数据表明，IR会导致线粒体的病理生理变化，从而导致细胞功能和细胞死亡的丧失。 6。来自SS患者的唾液腺活检中Ca2+信号传导的表征：SJGRENS综合征（SS）是一种与淋巴细胞浸润有关的自身免疫性疾病，唾液腺中液体分泌减少，导致静脉炎的液体分泌。有趣的是，腺功能的大幅丧失，浸润最小或组织损伤。在这里，我们检查了来自SS患者和健康志愿者的次要唾液腺活检中的单个腺泡细胞功能。炎症低的SS患者的腺体在浸润小区域内显示出腺泡的破坏。虽然大部分组织相对完整。 重要的是，这些腺体这些区域中的腺泡细胞显示出明显的卡尔巴乔（CCH）刺激的细胞内Ca2+释放和Ca2+进入。与健康志愿者相比。与此相一致，在这些acini中，激动剂刺激的体积减小，代表流体分泌也较低。 与炎症与唾液流动之间的相关性差相反，患者人群中CCH刺激的细胞反应与唾液分泌之间存在显着相关性。 IP3R2和IP3R3（而不是AQP5或STIM1）在腺体的完整区域减少了，这可以解释Ca2+信号传导中的破坏以及因此流体分泌。总之，我们的发现表明，腺泡细胞中的IP3R赤字是表现出明显的静脉症的SS患者流体分泌丧失的基础。