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)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. We have elucidated the molecular basis for TRPC1 activation and its contribution to SOCE by demonstrating that Orai1 triggers recruitment of TRPC1 to the plasma membrane 1. to determine the mechanism by which Orai1 and TRPC1 regulate distinct Ca2+-dependent cell function, we have examined the contributions of Orai1 and TRPC1 to carbachol (CCh)-induced Ca2+i signals and Ca2+ dependent gene expression in single cells. We report that endogenous Orai1 and TRPC1 channels contribute to distinct local and global Ca2+i signals following agonist stimulation. In HSG cells, Ca2+ entry via Orai1 controls the generation of Ca2+i oscillations at all CCh tested. In contrast, TRPC1 mediates sustained Ca2+i elevation at relatively high agonist and contributes to the frequency of baseline Ca2+i oscillations at lower agonist. Importantly, the channels display functional specificity in the regulation of Ca2+-dependent transcription factors and gene expression. Consistent with the oscillatory Ca2+i signals generated by Orai1, NFAT translocation and NFAT-dependent gene expression are exclusively dependent on Orai1-mediated Ca2+ entry, without any contribution of TRPC1. Our data suggest that NFAT is regulated by Ca2+i achieved locally near the Orai1 channel, likely due to localization of calmodulin-calcineurin-NFAT within the Orai1-associated microdomain, such that the Ca2+ entering via Orai1 can be locally sensed by the calcium sensor. Moreover, since Ca2+ entering into this microdomain via Orai1 rapidly rises to concentrations that exceed a threshold level required for activation, NFAT activation does not reflect global Ca2+i changes achieved at the various stimulus intensities. We also show that NFAT activation follows an all-or-none mode of activation;if an insufficient number of Orai1 channels is activated, NFAT dephosphorylation is not completed and nuclear translocation does not occur. In contrast to the regulation of NFAT, NFB is primarily regulated by TRPC1. The exact Ca2+ sensors that decode the two individual Ca2+ signals and the exact amplitude of the signals remain to be determined 2. Primary Sjgrens syndrome (pSS) is a chronic autoimmune disease affecting exocrine glands, primarily salivary and lacrimal glands, resulting in gland destruction, xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes). There are no conclusive data that establish the molecular basis for the disease, either in the salivary gland itself or systemically in cells such as lymphocytes. The late disease onset and the diverse genetic background of affected individuals complicate study of the disease mechanism and pathogenesis and no adequate animal models are available that fully recapitulate the disease. We report here that mice with T-cell-targeted deletion of STIM and STIM2 develop spontaneous and severe pSS-like autoimmune disease. Further, we have identified deficiency of STIM1 and STIM2 proteins and consequent defects in Ca2+ signaling in T cells from patients with pSS. Diffuse lymphocytic infiltration was seen in submandibular glands, a major target of pSS, by 6 weeks progressing to severe inflammation by 12 weeks of age in the knockout mice. pSS-specific autoantibodies (SSA/Ro and SSB/La) were detected in the serum, with progressive salivary gland destruction and loss of fluid secretion. Importantly, PMBCs as well as lymphocytic infiltrates in submandibular glands from pSS patients demonstrated significant reduction in STIM1 and STIM2 proteins as a function of the level of inflammation. Store-operated calcium entry was also reduced in PBMCs from pSS patients compared to those from healthy individuals. Together our findings reveal a novel link between STIM1 and STIM2 proteins and pSS. 3. Radiation treatment (IR) causes significant acute and long-term, either direct or by-stander, effects on other healthy tissues that are in the field of treatment. Debilitating side effects of IR in head and neck cancer patients include oral mucositis and xerostomia, which continue to exist post therapy. Currently, there are no adequate therapies to protect against, or reverse, IR-induced salivary hypofunction. Transient Receptor Potential Melastatin-like 2 (TRPM2) is a Ca2+-permeable nonselective cation channel that acts as a redox-sensor and is activated by ROS generation in cells. TRPM2 has been implicated in the amplification of ROS-induced signaling, cell damage, and chronic inflammation. We report that functional TRPM2 channel is present in salivary gland cells and is activated in reponse to IR. Importantly, loss of TRPM2, or suppression of function, protects against IR-induced irreversible salivary gland dysfunction. Our data show that salivary gland acinar cells display constitutive activation of TRPM2, 24 hours after mice receive head and neck IR. Following 15 Gy IR (1 dose) TRPM2+/+ mice show an irreversible loss of salivary gland fluid secretion within 10 days while mice lacking TRPM2 display a transient loss that recovers to >70% of the normal saliva flow within a month after treatment. Treatment of mice prior to IR with TPL, a free radical scavenger, or 3-AB, a PARP1 inhibitor, exerts significant protection of salivary gland function and reduces activation of TRPM2 by IR (Figure 4). These data demonstrate that IR-activation of TRPM2 is mediated via generation of ROS as well as by PARP1 activation in response to DNA damage. Further, TPL induced complete recovery of function within 30 days in TRPM2-/- mice. Recovery was also seen in CCh-stimulated salivary volume decrease that underlies loss of fluid secretion. Together, our findings provide evidence that TRPM2 contributes to IR-induced irreversible salivary gland dysfunction. Thus, TRPM2 can be proposed as a novel target for attenuating the effects of IR on healthy salivary gland tissues.
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