| Store-operated Ca2+ entry (SOCE) is an evolutionarily conserved mechanism for generating cellular Ca2+ signals. Store-operated Ca2+ release-activated Ca2+ (CRAC) channels play a vital role in driving transcriptional, proliferative and effector cell programs in many different cell-types. CRAC channels are activated by the ER-resident STIM (stromal interaction molecule) family of proteins (STIM1-2) and are encoded by the Orai family of proteins (Orai1-3). Patients with mutation in the CRAC channel proteins STIM1 or Orai1 suffer from a form of severe combined immunodeficiency and show increased susceptibility to respiratory infections, the etiology of which is incompletely understood. Epithelial cells lining the conducting airways play an important role in host defense. Airway epithelial cells (AECs) release a host of inflammatory mediators in response to a variety of pathophysiological stimuli and coordinate the recruitment and activation of other immune cells, thus helping to shape the nature of immune responses in infections and diseases like asthma, chronic bronchitis and lung injury. Activation of AECs occurs by several mechanisms, of which mobilization of cytosolic Ca2+ is a key signaling event. Yet, the properties and physiological role of this potentially important signaling pathway in the inflammatory responses of AECs are unknown.;In my thesis dissertation, I investigate the hypothesis that CRAC channels regulate production of inflammatory mediators from AECs. I find that CRAC channels encoded by STIM1 and Orai1 are a major route of Ca2+ influx in AECs and regulate the production of several key inflammatory mediators from AECs, in part by regulating gene expression through the transcription factor NFAT (Nuclear factor of activated T-cells). Furthermore, purinergic and PAR2 agonists, both important regulators of airway inflammation, generate Ca2+ signals in AECs by activating CRAC channels. Finally, I show that purinergic signaling activates mitochondrial ROS (reactive oxygen species) through CRAC channels and induces PGE2 (prostaglandin E2) production in AECs. These findings highlight a potentially important role for CRAC channels as a regulatory check point in the generation of inflammatory mediators from the airway epithelium. Additionally, I find that an arginine residue in the CC3 domain of STIM1 (R429) regulates STIM1 oligomerization and STIM1-Orai1 interaction. Furthermore, I also find a mechanistic role for the Orai1 N-terminus in STIM-Orai binding. These results add new insights into the basic molecular mechanisms of CRAC channel function. |
