Molecular Mechanisms Regulating Calcium Flux In Salivary Glands

调节唾液腺钙通量的分子机制

• 批准号: 7967039
• 负责人: INDU S. AMBUDKAR
• 金额: $ 186.27万
• 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/7967039
• 关键词: Accounting; Acinus organ component; Address; Agonist; Attenuated; Binding; Calcium; Calcium Channel; Calcium Signaling; Calcium-Binding Proteins; Carbachol; Cations; Cell Death; Cell Proliferation; Cell membrane; Cell physiology; Cells; Cholesterol; Complex; Data; Diffuse; Disease; Dissociation; Duct (organ) structure; Embryonic Development; Endoplasmic Reticulum; Ensure; Event; Family; Fluids and Secretions; Functional disorder; GTP-Binding Proteins; Goals; Growth and Development function; Human; Hydrolysis; ITPR1 gene; Inflammation; Injury; Knowledge; Lead; Mediating; Mediator of activation protein; Membrane Microdomains; Molecular; Morphogenesis; Mus; Mutation; N-terminal; Nature; Neurotransmitters; Pathogenesis; Pathway interactions; Pattern; Phosphatidylinositol 4,5-Diphosphate; Phospholipase; Physiological; Physiology; Pilocarpine; Process; Protein Biosynthesis; Proteins; Radiation; Reactive Oxygen Species; Regulation; Relative (related person); Reporting; Research; Role; STIM1 gene; Saliva; Salivary; Salivary Glands; Signal Transduction; Signaling Protein; Site; Sjogren' s Syndrome; Submandibular gland; Surface; Syndrome; TRP channel; TRPC3 ion channel; Tertiary Protein Structure; Thapsigargin; Tissues; caveolin 1; cell growth; cytokine; endoplasmic reticulum stress; gland development; irradiation; member; mutant; novel; overexpression; prevent; radiation effect; receptor; receptors for activated C kinase; release of sequestered calcium ion into cytoplasm; salivary cell; scaffold; sulfated glycoprotein 2; therapeutic target

A major step towards understanding the physiological function of agonist-stimulated calcium entry channels in salivary gland cells requires identification of their molecular components and defining their regulation. TRPC (transient receptor potential canonical) proteins have been suggested as molecular candidates for store-operated calcium entry (SOCE) channels. SOCE is ubiquitously present in all cells and regulates a variety of cellular functions including salivary gland fluid secretion and inflammation. In addition other calcium channels, including TRP channels, are involved in regulating various other cellular functions such as cell growth, development. Some channels are critical mediators of cellular dysfunction. Our long term goal is to define the components that mediate and regulate Ca2+ entry into salivary gland cells. Towards this goal, our studies determine cellular mechanisms which are involved in the activation and inactivation of SOCE and define the role of TRP channels in salivary gland function as well as dysfunction. Our previous findings suggested that TRP proteins are molecular components of SOCE (TRPC1) and volume regulated Ca2+ channels (TRPV4) in salivary gland cells. We also provided evidence using TRPC1(-/-) mouse that TRPC1 accounts for more than 90% of the SOCE in SMG acini and ducts and is required for pilocarpine-stimulated saliva flow. Further, we had reported that Orai1 and STIM1 are required for TRPC1 function and that functional Orai1 was required for TRPC1-SOCE. Thus our studies have made significant advancement in our understanding of the molecular components, their assembly, and mechanism(s) of regulation of SOCE channels in salivary gland cells. We have now further assessed the molecular mechanisms involved in regulating TRPC1. Our major findings are as follows: 1.It is now well established that store-operated Ca2+ entry (SOCE) is activated by redistribution of the calcium binding protein, STIM1, from relatively diffused localization in the endoplasmic reticulum into puncta in discrete domains near the cell periphery where it interacts with and activates SOCE channels The factors involved in precise targeting of the channels and their retention at these specific microdomains are not yet defined. We had earlier investigated the nature of the plasma membrane domains that determine the sites of STIM1 aggregation and reported that lipid rafts domains (LRD) function as centers for the assembly of signaling complexes. We have reported earlier that TRPC1 is assembled in a signaling complex with key Ca2+ signaling proteins from both the ER and plasma membrane and that intact LRD are required for activation of TRPC1-mediated SOCE. Thus, our findings demonstrate that STIM1-dependent activation of TRPC1 occurs within LRD. We now report that the cholesterol-binding LRD protein Caveolin-1 (Cav1) is a critical plasma membrane scaffold that retains TRPC1 within the regions where STIM1 puncta are localized following store depletion. This enables the interaction of TRPC1 with STIM1 that is required for the activation of TRPC1-SOCE. Silencing Cav1 in human submandibular gland cells (HSG) decreased plasma membrane retention of TRPC1, TRPC1-STIM1 clustering, and consequently reduced TRPC1-SOCE, without altering STIM1 puncta. Importantly, activation of TRPC1-SOCE was associated with an increase in TRPC1-STIM1 and a decrease in TRPC1-Cav1 clustering. Consistent with this, overexpression of Cav1 decreased TRPC1-STIM1 clustering and SOCE, both of which were recovered when STIM1 was expressed at higher levels relative to Cav1. Silencing STIM1 or expression of STIM mutants with disrupted interaction with TRPC1 (ERM-STIM1 or STIM1-KK/EE) prevented dissociation of TRPC1-Cav1 as well activation of TRPC1-SOCE. Further, conditions that promoted TRPC1-STIM1 clustering and TRPC1-SOCE elicited corresponding changes in SOCE-dependent NFkB activation and cell proliferation. Together these data demonstrate that Cav1 is a critical plasma membrane scaffold for inactive TRPC1. We suggest that activation of TRPC1-SOC by STIM1 mediates release of the channel from Cav1. These important data reveal the intricate processes that regulate store-operated calcium entry. 2. Activation of TRPC3 channels is concurrent with IP3R-mediated intracellular Ca2+ release and associated with PIP2 hydrolysis and recruitment to the plasma membrane. Here we report that interaction of TRPC3 with Receptor for Activated C-Kinase-1 (RACK1) not only determines plasma membrane localization of the channel but also interaction of IP3R with RACK1 and IP3-dependent intracellular Ca2+ release. We show that TRPC3 interacts with RACK1 via N-terminal residues E232, D233, E240, and E244. Carbachol (CCh)-stimulation of HEK293 cells expressing wt-TRPC3 induced recruitment of a ternary TRPC3-RACK1-IP3R complex and increased surface expression of TRPC3 and Ca2+ entry. Mutation of the putative RACK1-binding sequence in TRPC3 disrupted plasma membrane localization of the channel. CCh-stimulated recruitment of TRPC3-RACK1-IP3R complex as well as increased surface expression of TRPC3 and receptor-operated Ca2+ entry were also attenuated. Importantly, CCh-induced intracellular Ca2+ release was significantly reduced as was RACK1-IP3R association without any change in thapsigargin-stimulated Ca2+ release and entry. Knockdown of endogenous TRPC3 also decreased RACK1-IP3R association and decreased CCh-stimulated Ca2+ entry. Further, an oscillatory pattern of CCh-stimulated intracellular Ca2+ release was seen in these cells compared to the more sustained pattern seen in control cells. Similar oscillatory pattern of Ca2+ release was seen following CCh-stimulation of cells expressing the TRPC3 mutant. Together these data demonstrate a novel role for TRPC3 in regulation of IP3R function. We suggest TRPC3 controls agonist-stimulated intracellular Ca2+ release by mediating interaction between IP3R and RACK1.IP3R is a central and critical Ca2+ signaling protein in cells. It is assembled in a complex with both plasma membrane, cytosolic, and ER proteins. IP3R function is tightly controlled and the channel is involved in regulating both physiological and cell death pathways. Studies over the past several years have demonstrated that IP3Rs interact with plasma membrane TRPC channels and more recently, with other TRP channels such as polycystin2. While many of these earlier studies propose a role for IP3R in the regulation of TRPC channel function, to our knowledge the findings we have presented here provide the first demonstration that TRPC channels have a role in intracellular Ca2+ release via IP3R. Thus, our studies have made significant advancement in our understanding of the molecular components and molecular mechanism(s) that are involved in regulation of store operated calcium channels in salivary gland cells. Additionally, we have now identified a critical role for TRPM7 in salivary gland branching morphogenesis and a potentially important role for TRPM2 in salivary gland damage due to irradiation. Ongoing studies are focused on further resolving the exact functions of Orai1 and TRPc1 in SOCE and to determine how TRPC1 channels are assembled. Further, we are focusing our efforts on understanding the novel functions of TRPM2 and TRPM7 in salivary glands.

了解唾液腺细胞中激动剂刺激的钙进入通道的生理功能的一个重要步骤需要识别其分子成分并定义其调节。 TRPC（瞬时受体电位规范）蛋白已被建议作为钙池操纵钙进入（SOCE）通道的分子候选者。 SOCE 普遍存在于所有细胞中，调节多种细胞功能，包括唾液腺液分泌和炎症。此外，其他钙通道，包括TRP通道，参与调节各种其他细胞功能，例如细胞生长、发育。一些通道是细胞功能障碍的关键介质。我们的长期目标是确定介导和调节 Ca2+ 进入唾液腺细胞的成分。为了实现这一目标，我们的研究确定了参与 SOCE 激活和失活的细胞机制，并确定了 TRP 通道在唾液腺功能和功能障碍中的作用。我们之前的研究结果表明，TRP 蛋白是唾液腺细胞中 SOCE (TRPC1) 和容量调节 Ca2+ 通道 (TRPV4) 的分子成分。 我们还使用 TRPC1(-/-) 小鼠提供了证据，表明 TRPC1 占 SMG 腺泡和导管中 90% 以上的 SOCE，并且是毛果芸香碱刺激唾液流所必需的。此外，我们报道了TRPC1功能需要Orai1和STIM1，并且TRPC1-SOCE需要功能性Orai1。 因此，我们的研究在我们对唾液腺细胞中 SOCE 通道的分子成分、组装和调节机制的理解方面取得了重大进展。我们现在进一步评估了调节 TRPC1 的分子机制。 我们的主要发现如下： 1. 现在已经确定，钙库操纵的 Ca2+ 进入 (SOCE) 是通过钙结合蛋白 STIM1 的重新分布而激活的，从内质网中相对分散的定位到细胞周围附近离散区域的斑点中，在那里它与 和 相互作用激活 SOCE 通道 通道的精确定位及其在这些特定微域中的保留所涉及的因素尚未确定。我们之前研究了决定 STIM1 聚集位点的质膜结构域的性质，并报道了脂筏结构域 (LRD) 作为信号复合物组装中心的功能。我们之前报道过，TRPC1 与来自内质网和质膜的关键 Ca2+ 信号蛋白组装在信号复合物中，并且完整的 LRD 是激活 TRPC1 介导的 SOCE 所必需的。因此，我们的研究结果表明 TRPC1 的 STIM1 依赖性激活发生在 LRD 内。我们现在报道，胆固醇结合 LRD 蛋白 Caveolin-1 (Cav1) 是一种关键的质膜支架，可将 TRPC1 保留在 STIM1 点在储存耗尽后定位的区域内。这使得 TRPC1 与 STIM1 能够相互作用，这是激活 TRPC1-SOCE 所需的。沉默人颌下腺细胞 (HSG) 中的 Cav1 可减少 TRPC1、TRPC1-STIM1 簇的质膜保留，从而减少 TRPC1-SOCE，而不改变 STIM1 斑点。重要的是，TRPC1-SOCE 的激活与 TRPC1-STIM1 的增加和 TRPC1-Cav1 聚类的减少相关。 与此一致的是，Cav1 的过度表达降低了 TRPC1-STIM1 聚类和 SOCE，当 STIM1 相对于 Cav1 以更高水平表达时，这两者都得到恢复。沉默 STIM1 或与 TRPC1 相互作用被破坏的 STIM 突变体（ERM-STIM1 或 STIM1-KK/EE）的表达可防止 TRPC1-Cav1 解离以及 TRPC1-SOCE 激活。 此外，促进 TRPC1-STIM1 聚类和 TRPC1-SOCE 的条件引起 SOCE 依赖性 NFkB 激活和细胞增殖的相应变化。这些数据共同表明 Cav1 是非活性 TRPC1 的关键质膜支架。我们认为 STIM1 激活 TRPC1-SOC 介导 Cav1 通道的释放。这些重要数据揭示了调节商店经营的钙进入的复杂过程。 2. TRPC3 通道的激活与 IP3R 介导的细胞内 Ca2+ 释放同时发生，并与 PIP2 水解和募集到质膜相关。在此，我们报道 TRPC3 与激活 C-激酶-1 (RACK1) 受体的相互作用不仅决定通道的质膜定位，而且还决定 IP3R 与 RACK1 的相互作用以及 IP3 依赖性细胞内 Ca2+ 释放。我们发现 TRPC3 通过 N 端残基 E232、D233、E240 和 E244 与 RACK1 相互作用。卡巴胆碱 (CCh) 刺激表达 wt-TRPC3 的 HEK293 细胞诱导三元 TRPC3-RACK1-IP3R 复合物的募集，并增加 TRPC3 和 Ca2+ 进入的表面表达。 TRPC3 中假定的 RACK1 结合序列的突变破坏了通道的质膜定位。 CCh 刺激的 TRPC3-RACK1-IP3R 复合物募集以及 TRPC3 表面表达的增加和受体操作的 Ca2+ 进入也减弱。重要的是，CCh 诱导的细胞内 Ca2+ 释放显着减少，RACK1-IP3R 关联也显着减少，而毒胡萝卜素刺激的 Ca2+ 释放和进入没有任何变化。内源性 TRPC3 的敲低也减少了 RACK1-IP3R 关联并减少了 CCh 刺激的 Ca2+ 进入。此外，与对照细胞中观察到的更持续的模式相比，在这些细胞中观察到CCh刺激的细胞内Ca2+释放的振荡模式。 对表达 TRPC3 突变体的细胞进行 CCh 刺激后，观察到类似的 Ca2+ 释放振荡模式。这些数据共同证明了 TRPC3 在调节 IP3R 功能中的新作用。我们认为 TRPC3 通过介导 IP3R 和 RACK1 之间的相互作用来控制激动剂刺激的细胞内 Ca2+ 释放。IP3R 是细胞中重要且关键的 Ca2+ 信号蛋白。它与质膜、胞浆和 ER 蛋白组装成复合物。 IP3R 功能受到严格控制，该通道参与调节生理和细胞死亡途径。过去几年的研究表明，IP3R 与质膜 TRPC 通道相互作用，最近还与其他 TRP 通道（如多囊蛋白 2）相互作用。虽然许多早期研究提出 IP3R 在 TRPC 通道功能调节中发挥作用，但据我们所知，我们在此提出的研究结果首次证明 TRPC 通道在通过 IP3R 释放细胞内 Ca2+ 中发挥作用。 因此，我们的研究在我们对参与唾液腺细胞钙通道钙池调节的分子成分和分子机制的理解方面取得了重大进展。此外，我们现在已经确定了 TRPM7 在唾液腺分支形态发生中的关键作用，以及 TRPM2 在辐射引起的唾液腺损伤中的潜在重要作用。正在进行的研究重点是进一步解析 SOCE 中 Orai1 和 TRPc1 的确切功能，并确定 TRPC1 通道的组装方式。 此外，我们正致力于了解 TRPM2 和 TRPM7 在唾液腺中的新功能。