© 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs237610. doi:10.1242/jcs.237610 RESEARCH ARTICLE SPECIAL ISSUE: CELL BIOLOGY OF THE IMMUNE SYSTEM Loss of Ca2+ entry via Orai–TRPC1 induces ER stress, initiating immune activation in macrophages Viviane Nascimento Da Conceicao1, Yuyang Sun1, Emily K. Zboril1, Jorge J. De la Chapa2 and Brij B. Singh1,* ABSTRACT interacts with proteins at the plasma membrane Ca2+ influx channels Activation of cellular stresses is associated with inflammation; (Liao et al., 2008; Shaw and Feske, 2012a). 2+ however, the mechanisms are not well identified. Here, we provide SOCE in immune cells is mediated by the highly Ca -selective 2+ 2+ evidence that loss of Ca2+ influx induces endoplasmic reticulum (ER) Ca release-activated Ca (CRAC) channel and is essential for stress in primary macrophages and in murine macrophage cell line the proper immune response (Shaw and Feske, 2012a; Vaeth et al., 2+ Raw 264.7, in which the unfolded protein response is initiated to 2015). An increase in [Ca ]i is initiated by agonist binding to cell- modulate cytokine production, thereby activating the immune surface receptors, which generates second messenger IP3, leading to 2+ 2+ response. Stressors that initiate the ER stress response block store- the release of Ca stored in the ER. Release of ER Ca allows ER dependent Ca2+ entry in macrophages prior to the activation of the protein STIM1 to aggregate, and also to interact with Orai1 (Feske unfolded protein response. The endogenous Ca2+ entry channel is et al., 2006; Liao et al., 2008) and transient receptor potential dependent on the Orai1–TRPC1–STIM1 complex, and the presence canonical 1 (TRPC1) channels (Asanov et al., 2015; Liao et al., 2+ of ER stressors decreased expression of TRPC1, Orai1 and STIM1. 2008), thus increasing the [Ca ]i that is essential for cellular Additionally, blocking Ca2+ entry with SKF96365 also induced functions (Parekh and Putney, 2005; Putney et al., 2017). ER stress, promoted cytokine production, activation of autophagy, Importantly, immune cell activation is also dependent on SOCE, increased caspase activation and induced apoptosis. Furthermore, and loss of Orai1 or STIM1 has been shown to lead to an impaired ER stress inducers inhibited cell cycle progression, promoted immune function (Shaw and Feske, 2012b). Another mechanism that the inflammatory M1 phenotype, and increased phagocytosis. also affects the innate and adaptive immune response is ER stress, Mechanistically, restoration of Orai1–STIM1 expression inhibited with chronic ER stress contributing to abnormal physiological the ER stress-mediated loss of Ca2+ entry that prevents ER stress and processes involved in disease pathogenesis and progression (Islam inhibits cytokine production, and thus induced cell survival. These et al., 2006; Kaufman, 1999; Kim et al., 2013; Todd et al., 2008). results suggest an unequivocal role of Ca2+ entry in modulating ER Moreover, abnormal immune activation caused by chronic ER stress stress and in the induction of inflammation. is also associated with autoimmune and inflammatory disorders such as diabetes, atherosclerosis, myositis and inflammatory bowel KEY WORDS: SOCE channels, Ca2+ modulation, TRPC1, ER stress, disease (Arruda and Hotamisligil, 2015). Thus, establishing the Immune activation mechanism(s) by which ER stress induces immune activation is essential for research into potential treatments for these diseases. INTRODUCTION The ER not only plays an important role in Ca2+ signaling but is The endoplasmic reticulum (ER) is a vital organelle responsible for also essential for protein synthesis and proper protein folding and various cellular functions, such as lipid and protein synthesis and modification (Braakman and Hebert, 2013). More than half of regulation of Ca2+ homeostasis (Braakman and Hebert, 2013; Parekh newly synthesized proteins translocate to the lumen of the ER, for and Putney, 2005). Importantly, cellular stress provokes the release folding and targeting to various cellular organelles or to be of pro-inflammatory cytokines that stimulate the inflammatory transported to the surface of the cell. (Görlach et al., 2006; response (Liu et al., 2017; Pinton et al., 2008); however, the Kaufman, 1999; Lawless and Greene, 2012; Zhang and Kaufman, mechanisms by which ER stress leads to immune activation are not 2008). Ca2+ is required for the chaperone activity of many ER well known. In non-excitable cells, change in intracellular Ca2+ proteins, and loss of this vital function induces ER stress. In 2+ 2+ ([Ca ]i) levels directly related to the release of Ca stores from the addition, genetic or chemical inhibition can also activate the ER ER, is due to activation of the store-operated Ca2+ entry (SOCE) stress response, through a common signaling mechanism. ER stress mechanism (Tojyo et al., 2014). Once ER Ca2+ stores are depleted, triggers a cascade of signaling networks referred to as the unfolded stromal interaction molecule 1 (STIM1), which acts as an ER sensor, protein response (UPR) to reduce stress and restore homeostasis. The UPR activates the cellular response that leads to the transcription of 1Department of Periodontics, School of Dentistry, University of Texas Health San molecular chaperones to alleviate this stress response. Importantly, Antonio, San Antonio, TX 78229, USA. 2Department of Comprehensive Dentistry, three major ER proteins, inositol-requiring enzyme 1α (IRE1α,also School of Dentistry, University of Texas Health San Antonio, San Antonio, TX 78229, known as ERN1), the PKR-like ER kinases (PERK), and activating USA. transcription factor 6α (ATF6α) have been shown to bind to *Author for correspondence ([email protected]) misfolded or unfolded proteins to initiate the UPR. Activation of the UPR is necessary to reduce stress; however, insufficient clearance of B.B.S., 0000-0003-0535-5997 these misfolded proteins or prolonged activation of the pathways that This is an Open Access article distributed under the terms of the Creative Commons Attribution induce ER stress result in apoptotic cell death (Chen et al., 2015; License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. Endo et al., 2006; Ron and Walter, 2007). ER stress activates the inflammatory processes, especially in Received 5 August 2019; Accepted 25 October 2019 macrophages, which are the sentinels of the immune systems Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs237610. doi:10.1242/jcs.237610 (Ho et al., 2012). These specialized cells, called first responders, production, which regulates inflammation. These results not play major roles in the maintenance of tissue integrity or tissue only help in understanding the physiology of ER stress-related damaging signals, host defense and inflammation. Disturbances in disorders but could help in the development of novel therapeutic this delicate balance cause the cells to be more susceptible to modalities for ER stress-related disorders by improving our activation of inflammatory processes, leading to immune diseases comprehension of the relationship between ER stress and the and disorders (Franken et al., 2016; Gautier et al., 2012; Lavin et al., UPR processes. 2015). Macrophages are key components of the innate immune system and are crucial parts of a system that senses and responds to RESULTS tissue invasion by infectious microorganisms and tissue injury Treatment with tunicamycin or brefeldin A induces ER through various scavenger, pattern recognition and phagocytic stress, affecting cell proliferation of macrophages receptors (Lavin et al., 2015). Although the exact mechanism for the ER stress response could trigger an inflammatory response, activation of macrophages under ER stress is not entirely therefore causing metabolic issues in the cell (Cao and Kaufman, understood, Ca2+ could be the vital link. In addition, switching of 2014; Grootjans et al., 2016). Thus, we initiated our study by the macrophages to the pro-inflammatory M1 phenotype is the key assessing the response to well-known ER stress inducers to chronic immune activation. tunicamycin (Tuni), a known glycosylation suppressor, and The aim of this study was to identify the pathways that involve brefeldin A (BFA), an inhibitor of Golgi complex transportation ER stress and lead to immune activation. Our data show that Ca2+ in the macrophage cell line. Raw 264.7 cells were incubated at homeostasis is vital to macrophages activation. We report for the different concentrations of both Tuni and BFA (5 µM and 10 µM) first time that ER stress inducers lead to the loss of Ca2+ entry, for 6 and 12 h. After treatment, proteins were extracted, and western which corresponds to a decrease in ER Ca2+ levels, and precede the blots were performed, using β-actin as positive control. Importantly, unfolded protein response. Loss of ER and intracellular Ca2+ was cells treated with 5 µM Tuni for 12 h showed an increase in the primarily due to the decrease in expression of Orai1, TRPC1 and expression of ER stress markers, whereas, treatment for 6 h did STIM1. Orai1 is a pore-forming component of ICRAC, and STIM1 not show a similar increase in CHOP (also known as DDIT3) or is an ER-Ca2+ sensor, which activates SOCE. TRPC1 and Orai1, other ER stress proteins (Fig. S1A). Expression of molecular together with STIM1, produce Ca2+ signals that consequently