Molecular Mechanisms Regulating Calcium Flux 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需要鉴定其分子成分并确定其调控。TRPC（瞬时受体电位规范）蛋白被认为是储存操作钙进入（SOCE）通道的分子候选者。SOCE普遍存在于所有细胞中，并调节多种细胞功能，包括唾液腺液体分泌和炎症。此外，其他钙通道，包括TRP通道，参与调节各种其他细胞功能，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）和体积调节Ca2+通道（TRPV4）的分子组分。我们还利用TRPC1（-/-）小鼠提供了证据，表明TRPC1占SMG腺泡和导管中SOCE的90%以上，并且是匹罗卡品刺激的唾液流动所必需的。此外，我们已经报道了TRPC1功能需要Orai1和STIM1， TRPC1- soce需要功能性Orai1。本报告所述期间的主要调查结果如下：

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 and 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. Our major findings in this reporting period are as follows: 1. Previously, we established that TRPC1 is essential for SG gland fluid secretion and that CRAC channel components Orai1 and STIM1 are required for TRPC1 function. We have now elucidated the molecular basis for TRPC1 activation and its contribution to SOCE. Thus our studies have made significant advancement in identifying the molecular components, their assembly, and early molecular mechanism(s) that are involved in regulation of SOCE channels in salivary gland cells. Our data demonstrate that Orai1-dependent recruitment of TRPC1 within ER/PM junctional domains provides a mechanism for rapid modulation of SOCE-associated Ca2+ signaling microdomain. By coordinating the regulation of ion channel activities and downstream signaling pathways, CRAC channel serves as a key regulator of Ca2+ signaling. We propose that Orai1 and STIM1 are central and versatile components that coordinate the regulation of SOCE-dependent critical cellular functions. By induction of auxiliary Ca2+ entry channels, and other regulatory pathways, CRAC channels determine the generation and modulation of local and global Ca2+ signals that differentially impact cell function. 2. Our previous studies demonstrated that the store-operated calcium channels, TRPC1 and Orai1, regulate distinct Ca2+-dependent cellular functions. Orai1 was involved in regulating NFAT activation while TRPC1 was the primary contributor for KCa activation. We hypothesized that distinct spatiotemporal characteristics of the Ca2+i signals generated by these two channels during store-operated calcium entry (SOCE) might account for the specificity in the functions that are regulated. Our findings show that Ca2+ entry via TRPC1 and Orai1 channels have distinct contributions to agonist-stimulated Ca2+i responses. While Orai1 determines the oscillations, TRPC1 determines sustained Ca2+ elevation. We suggest that these channels could thus generate very different Ca2+i microdomains in the cell. Such temporally and spatially distinct Ca2+ signatures account for the distinct functional input of the channels in SG cells. Studies to establish this hypothesis and extend the findings to a physiologically relevant system (mouse salivary glands) are now ongoing. 3. Sjgrens syndrome is a systemic autoimmune disease predominantly involving salivary and lacrimal glands that leads to progressive irreversible loss of function and destruction of tissue. The mechanism underlying this disease is still poorly understood. In a recent study, targeted knockout of Stromal Interaction Molecule (STIM) 1 and STIM2 in mouse T cells and fibroblasts was shown to induce a lymphoproliferative phenotype with infiltration in lung and liver tissues, in addition to a selective decrease in regulatory T cell numbers. STIM1 and STIM2 are newly identified ER proteins that regulate store-operated calcium entry, a ubiquitously present calcium entry pathway that regulates a variety of cellular functions including T-cell activation and proliferation as well as fluid secretion in salivary gland acinar cells. Here we report that T-cell targeted knockout of STIM1 and STIM2 leads to spontaneous and relatively rapidly progressing submandibular gland inflammation in mice. The pathology appears, in histopathological analysis, to be similar to those of salivary gland samples from patients diagnosed with Sjgrens Syndrome. Conversely, PBMCs isolated from patients with Sjogren's Syndrome patients, but not those with lupus, display reduced STIM1 expression as well as decreased function. 4. The underlying mechanism of IR-induced injury to the salivary glands is still unknown. TRPM2 is a nonselective cation channel that is suggested to play a central role in oxidative stress-mediated cell death and inflammation. We have tested our hypothesis that TRPM2 plays a critical role in radiation-mediated salivary gland damage. TRPM2 is a calcium-permeable cation channel that is activated as a result of ADP-ribose generated by the effects of reactive oxygen species in cells. We have examined cell lines and mice lacking TRPM2 to demonstrate that radiation treatment leads to activation of TRPm2 and that ca2+ entry via this channel is key to the cell injury process that leads to salivary gland dysfunction. Salivary gland function is protected in mice lacking this channel. thus TRPM2 provides and attractive target for pharmacological intervention to protect against radiation-induced salivary gland damage. Together, 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 as well as the involvement of calcium channels and signaling components in salivary gland disease and dysfunction.