Molecular Mechanisms Regulating Calcium Flux In Salivary Glands

National Institute Of Dental & Craniofacial Research

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Abstract

Research Advances Role of STIM2 in SOCE and Regulation of Cell Function: Previously we demonstrated the role of STIM2 in regulating SOCE and cell function. We showed that STIM2 facilitates (i) STIM1 clustering and (ii) STIM1-gating of Orai1 at relatively high ER-Ca2+, i.e. conditions that likely do not elicit a STIM1 response; (iii) remodeling STIM1 C-terminus into the active conformation. Our key findings in this fiscal year are as follows: 1. When exogenously expressed, STIM2, a low affinity ER-Ca2+-sensor protein, displays constitutive clustering within ER-PM junctions and when co-expressed with Orai1, it recruits the channel to these junctions. Consistent with this co-localization, expression of STIM2 leads to activation of endogenous Orai1 under basal conditions. Currently, there is no information about endogenous STIM2, whether it is pre-clustered or how it is regulated under basal and low intensity stimulations. In this study, we used CRISPR/Cas9 to knock-in mVenus into endogenous Stim2, tagging the protein at the N-terminus, and generated cell lines expressing fluorescently labeled endogenous STIM2. The endogenous STIM2 is pre-clustered in the ER-PM junctions of unstimulated cells. While majority of the STIM2 clusters are mobile, there is a small population of immobile STIM2 clusters. Importantly, the immobile clusters, detected in ER-PM junctions of resting cells, denote locations of local ER-Ca2+ decrease that is mediated by functional IP3Rs and sensed by the N-terminus of STIM2. In absence of added agonist, constitutive PLC-dependent PIP2 hydrolysis and PKA activity concertedly control IP3R function and pre-clustering of STIM2. Consequently, immobile STIM2 clusters increase following agonist stimulation and co-ordinate recruitment of Orai1/STIM1. Our data suggest that immobilization of STIM2 clusters in ER-PM junctions, an early response to IP3R-mediated local ER-Ca2+ store release, is a critical check-point for initiation of SOCE. Together, our data reveal a novel and critical mechanism of communication between the IP3R and STIM2 that couples ER-Ca2+ release with assembly of Orai1/STIM1 channels in basal conditions which is then amplified after agonist stimulation of cells . 2. We previously demonstrated that IR-induced salivary gland dysfunction is associated with an early transient increase in mitochondrial Ca2+ and ROSmt. The goal of this work was to examine the role of the early rise in ROSmt in radiation-induced loss of salivary gland function. We tested the efficacy of mito-ROS scavenger, MitoTEMPO, by treating mice shortly before and on several days after IR. We report that treatment of mice with the mitochondrial-targeted antioxidant, MitoTEMPO, resulted in almost complete protection of salivary gland secretion following either single (15 Gy) or fractionated (5x3 Gy) doses of irradiation. Salivary gland cells isolated from MitoTEMPO-treated, irradiated, mice displayed significant attenuation of the initial increases in ROSmt, (Ca2+mt, and activated caspase-3 as compared to cells from irradiated, but untreated, animals. Importantly, MitoTEMPO treatment prevented radiation-induced decrease in STIM1, consequently protecting store-operated Ca2+ entry which is critical for saliva secretion. Together, these findings identify the initial increase in ROSmt, that is induced by irradiation, as a critical driver of persistent salivary gland hypofunction. We suggest that the mitochondrially targeted antioxidant, MitoTEMPO, can be potentially important in preventing IR-induced salivary gland dysfunction. 3. Store-operated calcium entry (SOCE) is triggered by assembly of Orai1 with STIM proteins in ER-PM junctions. Plasma membrane PIP2 as well as PIP2-binding protein, SEPT4, significantly impact Orai1-STIM1 interaction. While septins and PIP2 can organize the actin cytoskeleton, it is unclear whether the status of actin within the junctions contributes to SOCE. We report herein that actin remodeling modulates STIM1 clustering. Our findings show that a PIP2- and SEPT4-dependent mechanism involving CDC42, WASP/WAVE, and ARP2 regulates actin remodeling into a ring-like structure around STIM1 puncta. CDC42 localization in the ER-plasma membrane region is enhanced following ER-Ca2+ store depletion. PIP2 depletion or knockdown of SEPT4 attenuate the recruitment of CDC42 to the ER-PM region. Importantly, knockdown of SEPT4, or CDC42+ARP2, disrupts the organization of actin as well as STIM1 clustering. Consequently, Orai1 recruitment to STIM1 puncta, SOCE, and NFAT translocation to the nucleus are all attenuated. Ca2+ influx induced by STIM1-C terminus is not affected by CDC42 knockdown. In aggregate, our findings reveal that PIP2 and SEPT4 affect Orai1/STIM1 clustering by coordinating actin remodeling within ER-PM junctions. This dynamic reorganization of actin has an important role in regulation of SOCE and downstream Ca2+-dependent effector functions.