In this reporting period we have made significant progress in our studies. 1. Resolving the specific functional contributions of TRPC1 and Orai1 channels in polarized secretory epithelial cells. Earlier we reported that basolateraly localized TRPC1 is a major determinant of Ca2+ entry in acinar cells and required for sustained saliva flow. While Orai1 is localized near the apical region of the cell, its contribution to salivary gland function is not known. We have now determined the individual contributions of TRPC1 and Orai1 in Ca2+-dependent activation of Kca channels, basolateral NKCC1, and apical TMEM16A, all of which are critical for fluid secretion. We report that report TRPC1 provides necessary Ca2+ to regulate all these mechanisms while Orai1 has minimal direct contributions to regulation fluid secretion. Importantly, we now show that the function of residual Orai1 in TRPC1-/- cells activates NFAT1. Furthermore, adenovirus-mediated overexpression of Orai1 and STIM1 in vivo in TRPC1-/- cells resulted in basolateral expression of Orai1 together with recovery of fluid secretion and Ca2+ entry. We suggest that the localization of the channel within the cells, and likely the amount expressed, determines its contribution to fluid secretion. Together these findings suggest that TRPC1-mediated Ca2+ entry via the basolateral region is key to regulation of fluid secretion while Orai1 has a specific role in Ca2+-dependent gene expression. The latter could be of importance in salivary gland pathology and inflammation. 2. Fast endocytic recycling determines TRPC1-STIM1 clustering in ER-PM junctions and plasma membrane function of channel. Clustering of TRPC1 with STIM1 and Orai1 in ER-PM junctions is critical since Orai1-mediated Ca2+ entry triggers surface expression of TRPC1 while STIM1 gates the channel. Thus, plasma membrane function of TRPC1 depends on the delivery of the channel to the sites where STIM1 puncta are formed. We report that TRPC1 is internalized by Arf6-dependent pathway, sorted to Rab5-containing early endosomes, and trafficked to ER-PM junctions by Rab4-dependent fast recycling. Importantly, expression of Rab4, but not STIM1, with Rab5 rescued surface expression and function of TRPC1. Together, these data demonstrate that trafficking via fast recycling endosomes determines TRPC1-STIM1 clustering within ER-PM junctions following ER-Ca2+ store depletion which is critical for the surface expression and function of the channel. Ca2+ influx mediated by TRPC1 modifies Ca2+-dependent physiological response of cells. 3. STIM2 enhances receptor-stimulated Ca2+ signaling by promoting recruitment of STIM1 to the endoplasmic reticulum-plasma membrane junctions. A central component of receptor-evoked Ca2+ signaling is store-operated Ca2+ entry (SOCE), which is activated by the assembly of STIM1-Orai1 channels in endoplasmic reticulum (ER) and plasma membrane (PM) (ER-PM) junctions in response to depletion of ER Ca2+. We report that STIM2 enhances agonist-mediated activation of SOCE by promoting STIM1 clustering in ER-PM junctions at low stimulus intensities. Targeted deletion of STIM2 in mouse salivary glands diminished fluid secretion in vivo and SOCE activation in dispersed salivary acinar cells stimulated with low concentrations of muscarinic receptor agonists. STIM2 knockdown in human embryonic kidney (HEK) 293 cells diminished agonist-induced Ca2+ signaling and nuclear translocation of NFAT (nuclear factor of activated T cells). STIM2 lacking five carboxyl-terminal amino acid residues did not promote formation of STIM1 puncta at low concentrations of agonist, whereas coexpression of STIM2 with the STIM1 mutant resulted in co-clustering of both proteins. Together, our findings suggest that STIM2 recruits STIM1 to ER-PM junctions at low stimulus intensities when ER-Ca2+ stores are mildly depleted, thus increasing the sensitivity of Ca2+ signaling to agonists. 4. Fast remodeling of the cytoskeleton and plasma membrane PIP2 contribute to assembly and regulation of store-operated calcium entry. In this study, we identified 155 specific STIM1 binding partners using a shotgun proteomic approach using SILAC with a STIM1 immunoprecipitated complex from HSG cell lysates. Based on results, we focused on the role of cytoskeletal regulatory proteins in SOCE. We have identified proteins related to fast actin remodeling that regulate SOCE, such as septin, CDC42, N-WASP, WAVE and ARP2/3 complex. These proteins are associated with remodeling of plasma membrane PIP2. In addition to proteins like zyxin and N-VASP that mediate slow remodeling of actin contribute to a lesser extent in SOCE. Together, our data suggest that the fast cytoskeletal remodeling likely respond to plasma membrane changes in PIP2 and regulate the assembly Orai1-STIM1 complex. 5. P3R deficit underlies loss of salivary fluid secretion in Sjgren's Syndrome. The autoimmune exocrinopathy, Sjgrens syndrome (SS), is associated with secretory defects in patients, including individuals with mild lymphocytic infiltration and minimal glandular damage. The mechanism(s) underlying the secretory dysfunction is not known. We have used minor salivary gland biopsies from SS patients and healthy individuals to assess acinar cell function in morphologically intact glandular areas. We report that agonist-regulated intracellular Ca2+ release, critically required for Ca2+ entry and fluid secretion, is defective in acini from SS patients. Importantly, these acini displayed reduction in IP3R2 and IP3R3, but not AQP5 or STIM1. Similar decreases in IP3R and carbachol (CCh)-stimulated elevation in cytosolic Ca2+ were detected in acinar cells from IL-14α transgenic (TG) mice, a model for (SS). Treatment of salivary glands from healthy individuals with lymphotoxin-α (LTα), a cytokine linked to disease progression in SS and IL14α mice, reduced Ca 2+ signaling. Together, our findings reveal novel IP3R deficits in acinar cells that underlie secretory dysfunction in SS patients. 6. Link between TRPM2 and mitochondria in radiation-induced salivary gland dysfunction: We have explored the mechanism of IR-induced irreversible loss of salivary gland function. We show that SOCE is attenuated following IR and protected in TRPM2-/- cells. In addition, IR-induced Ca2+ entry via TRPM2 led to increases in mitochondrial Ca2+ and ROS together with depolarization and activation of caspase 3 and decrease in cell viability. All these changes were prevented by suppressing expression of mitochondrial Ca2+ uptake protein (MCU) or blocking TRPM2 activity. More importantly inhibition of TRPM2 or MCU protected against IR-induced loss of store-operated Ca2+ entry. The loss of SOCE was associated with caspase-mediated cleavage of STIM1, which was protected by blocking TRPM2 or MCU activity. Together these finding reveal a novel link between TRPM2 and MCU that underlies IR-induced irreversible salivary gland dysfunction. We suggest that mitochondria can be targeted therapeutically for the protection and possibly for the recovery of salivary gland function.

Project Start
Project End
Budget Start
Budget End
Support Year
29
Fiscal Year
2015
Total Cost
Indirect Cost
Name
Dental & Craniofacial Research
Department
Type
DUNS #
City
State
Country
Zip Code
Subedi, Krishna Prasad; Ong, Hwei Ling; Son, Ga-Yeon et al. (2018) STIM2 Induces Activated Conformation of STIM1 to Control Orai1 Function in ER-PM Junctions. Cell Rep 23:522-534
Ambudkar, Indu (2018) Calcium signaling defects underlying salivary gland dysfunction. Biochim Biophys Acta :
Liu, Xibao; Ong, Hwei Ling; Ambudkar, Indu (2018) TRP Channel Involvement in Salivary Glands-Some Good, Some Bad. Cells 7:
Liu, Xibao; Gong, Baijuan; de Souza, Lorena Brito et al. (2017) Radiation inhibits salivary gland function by promoting STIM1 cleavage by caspase-3 and loss of SOCE through a TRPM2-dependent pathway. Sci Signal 10:
Subedi, Krishna P; Ong, Hwei Ling; Ambudkar, Indu S (2017) Assembly of ER-PM Junctions: A Critical Determinant in the Regulation of SOCE and TRPC1. Adv Exp Med Biol 981:253-276
Ambudkar, Indu; VanHook, Annalisa M (2017) Science Signaling Podcast for 6 June 2017: Calcium signaling and dry mouth. Sci Signal 10:
Ambudkar, Indu S; de Souza, Lorena Brito; Ong, Hwei Ling (2017) TRPC1, Orai1, and STIM1 in SOCE: Friends in tight spaces. Cell Calcium 63:33-39
Ong, Hwei Ling; Ambudkar, Indu S (2017) STIM-TRP Pathways and Microdomain Organization: Contribution of TRPC1 in Store-Operated Ca2+ Entry: Impact on Ca2+ Signaling and Cell Function. Adv Exp Med Biol 993:159-188
Gallo, Alessia; Jang, Shyh-Ing; Ong, Hwei Ling et al. (2016) Targeting the Ca(2+) Sensor STIM1 by Exosomal Transfer of Ebv-miR-BART13-3p is Associated with Sjögren's Syndrome. EBioMedicine 10:216-26
Jang, Shyh-Ing; Ong, Hwei Ling; Liu, Xibao et al. (2016) Up-regulation of Store-operated Ca2+ Entry and Nuclear Factor of Activated T Cells Promote the Acinar Phenotype of the Primary Human Salivary Gland Cells. J Biol Chem 291:8709-20

Showing the most recent 10 out of 57 publications