Molecular Regulaton Calcium Flux In Salivary Glands

唾液腺钙通量的分子调节

• 批准号: 7146105
• 负责人: INDU S. AMBUDKAR
• 金额: --
• 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/7146105
• 关键词: MDCK cell; animal tissue; biological signal transduction; calcium flux; cell line; human tissue; membrane channels; membrane proteins; protein protein interaction; protein structure function; salivary glands; transfection /expression vector

This project is aimed towards understanding the mechanisms which mediate and regulate calcium signaling in salivary gland cells. Neurotransmitter stimulation of fluid secretion in salivary glands is mediated via a biphasic elevation in cytosolic [Ca-2+]; an initial transient increase due to internal release and a latter sustained increase due to Ca-2+ influx. Sustained fluid secretion is directly dependent upon the sustained elevation of [Ca-2+] and thus on neurotransmitter-stimulated calcium influx. In the past 8-10 years, our efforts have been focused on the neurotransmitter-stimulated calcium influx mechanism in salivary gland cells. Recent data from our laboratory and others demonstrate that two types of calcium entry mechanisms can contribute to this calcium signal; store-operated calcium entry (which is activated by the depletion of calcium in the intracellular calcium store) and receptor-or second messenger-operated calcium entry (which is activated directly by receptor signaling via second messengers such as diacylglycerol that are generated in response to neurotransmitter-stimulated phosphatidyl inositol bisphosphate hydrolysis). These calcium entry pathways are ubiquitously present in excitable and non-excitable cells and critically affect a number cellular functions. The molecular components or regulatory mechanism(s) of these calcium entry pathways have not yet been established in any cell type. Members of the transient receptor potential (TRPC) family of ion channel proteins have been proposed as molecular components of the neurotransmitter-activated calcium influx channels. All TRPCs have the ability to be activated by agonist-stimulation of phosphatidyl inositol bisphosphate hydrolysis and contribute to both types of calcium entry. The physiological function(s) and regulation of the presently identified TRPCs have not yet been fully established. Further, how they contribute to both store-operated and store-independent calcium entry pathways is not known. By expressing TRPC1 in vivo in rat SMG by using an adenovirus encoding hTrp1 (AdHA-hTrp1) and by examining the role of native and mutant TRPC1 in the HSG cell line, we had previously reported that TRPC1 is involved in the regulation of store-operated calcium influx in salivary gland cells. In the past year we have continued to characterize agonist-regulated calcium entry and identify the role of TRPC channels in this mechanism in salivary gland cells. Our previous studies have been largely carried out with HSG cells in which we have demonstrated conclusively that TRPC1 is the primary store-operated calcium entry channel component. Towards characterizing this channel in other salivary gland cells, we have now studied calcium entry in HSY (human parotid gland cell line) cells. Our previous results demonstrated that distinct store-operated calcium channels are present in HSG (human submandibular gland cell line) and HSY cells. Both of these channels were different from the channel in rat basophilic leukemia cells, the components of which are presently unknown. Although the physiological relevance of these different SOCE channels is not presently clear, we have now examined the molecular components of the channel in HSY cells. Interestingly, while the HSG channel appears to primarily depend on TRPC1, the HSY channel appears to be formed by the co-assembly of TRPC1 and TRPC3. Further, TRPC1-TRPC3 interactions are mediated via their N-terminal domains. Expression of the N-terminal domain of either TRPC1 or TRPC3, or a dominant negative mutant of TRPC1, disrupts calcium entry in HSY cells. This effect on calcium entry is due to the competetive interaction of the expressed proteins with endogenous TRPC proteins and disruption of channel assembly. Thus, we propose that TRPCs can assemble as homomers or heteromers to form store-operated calcium channels and that the channel properties are defined by the specific TRPC components that are involved. We have previously hypothesized that the type of channel formed in a cell will depend on the physiological function it serves in that particular cell. To further assess the physiological relevance of TRPC channels in salivary glands we have examined their routing in polarized epithelial cells. We have established stable TRPC expression in MDCK (canine kidney cell line) and rat salivary epithelial (SMIE) cells, both of which form high resistance monolayers when cultured on Transwell filters. We have observed that TRPCs have distinct cellular localization. TRPC3 is apically localized, TRPC1, TRPC5, and TRPC2 are basally localized while TRPC6 is found in both apical and basal regions of the cell. Further, endogenous TRPC3, TRPC1, and TRPC6 were found at the same locale. We have also studied the regulation of the TRP channels in these cellular regions. Consistent with previous reports, calcium signaling proteins were also predominantly localized in the apical region of these cells. Further, TRPC3 was assembled in a complex with TRPC6, but not TRPC1, and key calcium signaling proteins like inositol trisphosphate receptor, G-proteins, and phospholipase C. Importantly, we showed that TRPC3/TRPC6 channels can mediate apical calcium uptake and transepithelial calcium transport in polarized epithelial cells. These data demonstrate a novel role for TRPC3/TRPC6 channels; i.e. agonist-stimulated apical calcium uptake. Consistent with this, we detected localization of TRPC3 and TRPC6 in the apical regions of rat submandibular gland and kidney ducts. Further, addition of OAG (a diacylglycerol analogue) induced calcium entry in isolated rat submandibular gland ducts. These data indicate that non-store-operated channels are formed by TRPC3-TRPC6 interaction in the luminal membrane of salivary gland ducts. Studies are ongoing to determine how these apically localized channels are regulated and what is their physiological function. In another study members of SPS closely collaborated with Dr. Barker?s lab (NINDS) to demonstrate that TRPC1 regulates fibroblast growth factor (FGF) receptor-mediated growth of rat embryonic neuronal stem cells. We performed all the electrophysiology as well as studies showing the interaction and localization of the FGF receptor as well as TRPC1. These data add to the emerging concept that TRPC channels contribute to critical cellular functions such as secretion, proliferation, and cell death. In the coming fiscal year we will continue our studies along these directions. A major focus will be directed towards determining novel TRPC interacting proteins to help us to understand their function and regulation. We will also continue to study the trafficking of TRPC channels and the mechanisms involved in their assembly and multimerization. Studies will also be directed towards identifying specific channels generated by different TRPC combinations. In new studies that are ongoing we are also looking at the role of osmosensing TRPV channels in the regulation of water permeability and cell volume in salivary gland cells. Another study is directed towards identifying the internal calcium store that is coupled to the plasma membrane channel and the mechanism relaying signals between these two cellular membranes. We further plan to use various mouse models as well as in vivo adenoviral directed expression of TRPC channels or RNAi to further establish the role of these channels in salivary gland function. We believe that together our studies will provide substantial information about the mechanisms regulating salivary gland fluid secretion.

该项目旨在了解介导和调节唾液腺细胞中钙信号传导的机制。神经递质对唾液腺液体分泌的刺激是通过胞质 [Ca-2+] 的双相升高介导的；最初由于内部释放而短暂增加，后来由于 Ca-2+ 流入而持续增加。持续的液体分泌直接依赖于 [Ca-2+] 的持续升高，从而依赖于神经递质刺激的钙流入。在过去的8-10年里，我们的努力主要集中在唾液腺细胞中神经递质刺激的钙内流机制。我们实验室和其他实验室的最新数据表明，两种类型的钙进入机制可以促成这种钙信号：钙库操纵的钙进入（通过细胞内钙库中钙的耗尽而激活）和受体或第二信使操纵的钙进入（通过第二信使（例如响应于神经递质刺激的磷脂酰肌醇二磷酸水解而产生的二酰甘油）的受体信号传导直接激活）。这些钙进入途径普遍存在于兴奋性和非兴奋性细胞中，并且严重影响许多细胞功能。这些钙进入途径的分子成分或调节机制尚未在任何细胞类型中建立。离子通道蛋白瞬时受体电位 (TRPC) 家族的成员已被提议作为神经递质激活的钙流入通道的分子成分。所有 TRPC 都能够被磷脂酰肌醇二磷酸水解的激动剂刺激激活，并有助于两种类型的钙进入。目前确定的 TRPC 的生理功能和调节尚未完全确定。此外，它们如何促进钙池操纵和钙池非依赖性钙进入途径尚不清楚。通过使用编码 hTrp1 (AdHA-hTrp1) 的腺病毒在大鼠 SMG 体内表达 TRPC1，并通过检查天然和突变 TRPC1 在 HSG 细胞系中的作用，我们之前报道过 TRPC1 参与唾液腺细胞中钙池操纵的钙内流的调节。 在过去的一年中，我们继续表征激动剂调节的钙进入，并确定 TRPC 通道在唾液腺细胞的这一机制中的作用。我们之前的研究主要是用 HSG 细胞进行的，其中我们最终证明 TRPC1 是主要的钙池操纵的钙进入通道成分。为了表征其他唾液腺细胞中的该通道，我们现在研究了 HSY（人腮腺细胞系）细胞中的钙进入。我们之前的结果表明，HSG（人颌下腺细胞系）和 HSY 细胞中存在不同的钙池操纵钙通道。这两个通道都与大鼠嗜碱性白血病细胞中的通道不同，其成分目前尚不清楚。尽管这些不同 SOCE 通道的生理相关性目前尚不清楚，但我们现在已经检查了 HSY 细胞中通道的分子成分。有趣的是，虽然 HSG 通道似乎主要依赖于 TRPC1，但 HSY 通道似乎是由 TRPC1 和 TRPC3 共同组装形成的。此外，TRPC1-TRPC3 相互作用是通过其 N 端结构域介导的。 TRPC1 或 TRPC3 或 TRPC1 显性失活突变体的 N 端结构域的表达会破坏 HSY 细胞中的钙进入。这种对钙进入的影响是由于表达蛋白与内源 TRPC 蛋白的竞争性相互作用以及通道组装的破坏所致。因此，我们提出 TRPC 可以作为同聚体或异聚体组装以形成钙库操纵的钙通道，并且通道特性由所涉及的特定 TRPC 组件定义。 我们之前假设细胞中形成的通道类型将取决于它在该特定细胞中发挥的生理功能。为了进一步评估唾液腺中 TRPC 通道的生理相关性，我们检查了它们在极化上皮细胞中的路径。我们已经在 MDCK（犬肾细胞系）和大鼠唾液上皮（SMIE）细胞中建立了稳定的 TRPC 表达，这两种细胞在 Transwell 过滤器上培养时形成高电阻单层。我们观察到 TRPC 具有独特的细胞定位。 TRPC3 位于细胞顶端，TRPC1、TRPC5 和 TRPC2 位于细胞基底，而 TRPC6 存在于细胞的顶端和基底区域。此外，在同一区域还发现了内源性 TRPC3、TRPC1 和 TRPC6。我们还研究了这些细胞区域中 TRP 通道的调节。与之前的报道一致，钙信号蛋白也主要位于这些细胞的顶端区域。此外，TRPC3 与 TRPC6（而非 TRPC1）以及关键钙信号蛋白（如肌醇三磷酸受体、G 蛋白和磷脂酶 C）组装成复合物。重要的是，我们表明 TRPC3/TRPC6 通道可以介导极化上皮细胞中的顶端钙摄取和跨上皮钙转运。这些数据证明了 TRPC3/TRPC6 通道的新作用；即激动剂刺激的顶端钙摄取。与此一致的是，我们在大鼠颌下腺和肾管的顶端区域检测到 TRPC3 和 TRPC6 的定位。此外，添加 OAG（一种二酰基甘油类似物）可诱导钙进入离体大鼠颌下腺导管。这些数据表明非储存操纵通道是由唾液腺管腔膜中的 TRPC3-TRPC6 相互作用形成的。正在进行研究以确定这些顶端局部通道是如何调节的以及它们的生理功能是什么。 在另一项研究中，SPS 成员与 Barker 博士的实验室 (NINDS) 密切合作，证明 TRPC1 调节成纤维细胞生长因子 (FGF) 受体介导的大鼠胚胎神经元干细胞的生长。我们进行了所有电生理学以及显示 FGF 受体和 TRPC1 相互作用和定位的研究。这些数据进一步证实了 TRPC 通道有助于分泌、增殖和细胞死亡等关键细胞功能的新兴概念。 在下一个财政年度，我们将继续沿着这些方向进行研究。主要重点将是确定新型 TRPC 相互作用蛋白，以帮助我们了解它们的功能和调节。我们还将继续研究 TRPC 通道的运输及其组装和多聚化所涉及的机制。研究还将针对识别不同 TRPC 组合产生的特定通道。在正在进行的新研究中，我们还在研究渗透感应 TRPV 通道在调节唾液腺细胞的水渗透性和细胞体积中的作用。另一项研究旨在确定与质膜通道耦合的内部钙储存以及在这两个细胞膜之间传递信号的机制。我们进一步计划使用各种小鼠模型以及体内腺病毒定向表达 TRPC 通道或 RNAi，以进一步确定这些通道在唾液腺功能中的作用。我们相信，我们的研究将提供有关调节唾液腺液分泌机制的大量信息。