Date Published: October 24, 2012
Publisher: Public Library of Science
Author(s): Hwei Ling Ong, Shyh-Ing Jang, Indu Suresh Ambudkar, David Holowka. http://doi.org/10.1371/journal.pone.0047146
Regulation of critical cellular functions, including Ca2+-dependent gene expression, is determined by the temporal and spatial aspects of agonist-induced Ca2+ signals. Stimulation of cells with physiological concentrations of agonists trigger increases [Ca2+]i due to intracellular Ca2+ release and Ca2+ influx. While Orai1-STIM1 channels account for agonist-stimulated [Ca2+]i increase as well as activation of NFAT in cells such as lymphocytes, RBL and mast cells, both Orai1-STIM1 and TRPC1-STIM1 channels contribute to [Ca2+]i increases in human submandibular gland (HSG) cells. However, only Orai1-mediated Ca2+ entry regulates the activation of NFAT in HSG cells. Since both TRPC1 and Orai1 are activated following internal Ca2+ store depletion in these cells, it is not clear how the cells decode individual Ca2+ signals generated by the two channels for the regulation of specific cellular functions. Here we have examined the contributions of Orai1 and TRPC1 to carbachol (CCh)-induced [Ca2+]i signals and activation of NFAT in single cells. We report that Orai1-mediated Ca2+ entry generates [Ca2+]i oscillations at different [CCh], ranging from very low to high. In contrast, TRPC1-mediated Ca2+ entry generates sustained [Ca2+]i elevation at high [CCh] and contributes to frequency of [Ca2+]i oscillations at lower [agonist]. More importantly, the two channels are coupled to activation of distinct Ca2+ dependent gene expression pathways, consistent with the different patterns of [Ca2+]i signals mediated by them. Nuclear translocation of NFAT and NFAT-dependent gene expression display “all-or-none” activation that is exclusively driven by local [Ca2+]i generated by Orai1, independent of global [Ca2+]i changes or TRPC1-mediated Ca2+ entry. In contrast, Ca2+ entry via TRPC1 primarily regulates NFκB-mediated gene expression. Together, these findings reveal that Orai1 and TRPC1 mediate distinct local and global Ca2+ signals following agonist stimulation of cells, which determine the functional specificity of the channels in activating different Ca2+-dependent gene expression pathways.
Stimulation of cells with physiologically relevant agonists that target G protein- or tyrosine kinase-coupled receptors leads to increases in cytosolic [Ca2+] ([Ca2+]i) as a result of inositol 1,4,5-triphosphate (IP3)-induced Ca2+ release from intracellular Ca2+ stores via the IP3 receptors (IP3Rs) and Ca2+ influx via plasma membrane Ca2+ channels. The temporal and spatial pattern of [Ca2+]i signals generated in response to agonist stimulation are utilized by the cells to regulate various critical functions, such as gene expression, ion channel activation and fluid secretion , . High levels of agonist typically induce sustained elevations in baseline [Ca2+]i, whereas lower [agonist] elicit oscillatory [Ca2+]i responses , , . Two types of oscillations are seen; baseline oscillations that are usually seen at very low [agonist] or oscillations over a sustained elevation in baseline [Ca2+]i at relatively higher [agonist]. Such oscillatory responses have been proposed to represent the physiological mode of signaling in many cell types and have been observed in almost all cell types, including cell lines as well as primary cell preparations from various tissues , , , , . These oscillations primarily reflect repetitive cycles of Ca2+ release from the ER stores via IP3Rs, inhibition of Ca2+ release, and reuptake into the store due to SERCA pump activity. In several cell types, sustained [Ca2+]i oscillations require extracellular Ca2+ influx to achieve refilling of the ER store after every release event, thus priming it for the next release cycle. Even in cells where the oscillations are sustained for longer periods in the absence of external Ca2+, intracellular Ca2+ stores are eventually depleted and there is a run-down of [Ca2+]i oscillations.
In some cell types, including salivary gland cells, more than one channel contributes to agonist stimulated [Ca2+]i signals. It is not fully understood how cells decode [Ca2+]i signals originating from multiple sources for the regulation of specific Ca2+-dependent functions. Variations in the pattern of individual [Ca2+]i signals generated by the two channel types is most likely the primary determinant of the functional specificity of the channels in regulation of cell function. Here we have studied the contributions of endogenous TRPC1 and Orai1 to agonist-stimulated [Ca2+]i signals in a single HSG cell. We show that Ca2+ entry via each channel generates a specific pattern of [Ca2+]i elevation, with Orai1 controlling the generation of [Ca2+]i oscillations and TRPC1 mediating sustained [Ca2+]i elevation at higher [agonist] and contributing to the frequency of baseline [Ca2+]i oscillations. Even more significant is the finding that the channels display functional specificity in the activation 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 were exclusively dependent on Orai1-mediated Ca2+ entry, without any contribution of TRPC1. Our data suggest that NFAT is strictly regulated by the [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 did 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 which is strictly dependent on Orai1; if an insufficient number of Orai1 channels is activated, NFAT dephosphorylation is not completed and nuclear translocation does not occur. However, we cannot rule out the possibility that at even higher level of stimulus (e.g. a more potent agonist or involving different receptor pathways) or if more channels were expressed, this pattern could vary and sustained [Ca2+]i elevations could be induced by Orai1 (e.g. in lymphocytes or in HEK293 cells overexpressing Orai1+STIM1). In contrast to the regulation of NFAT, we show that NFκB is primarily regulated by TRPC1. Furthermore, we previously reported that Ca2+ entry via TRPC1, but not Orai1, is required for sustained activation of KCa in HSG cells as well as acinar cells isolated from mouse salivary glands . However, it remains to be fully understood whether local [Ca2+]i achieved near the TRPC1 channel or global [Ca2+]i changes mediated by TRPC1 are involved in the activation of NFκB and KCa channel. In aggregate, these findings provide conclusive evidence that Orai1 and TRPC1 generate functionally specific local and global [Ca2+]i signals.