Impact of SOCE Abolition by ORAI1 Knockout on the Proliferation, Adhesion, and Migration of HEK-293 Cells.

Store-operated calcium entry (SOCE) provided through channels formed by ORAI proteins is a major regulator of several cellular processes. In immune cells, it controls fundamental processes such as proliferation, cell adhesion, and migration, while in cancer, SOCE and ORAI1 gene expression are dysregulated and lead to abnormal migration and/or cell proliferation. In the present study, we used the CRISPR/Cas9 technique to delete the ORAI1 gene and to identify its role in proliferative and migrative properties of the model cell line HEK-293. We showed that ORAI1 deletion greatly reduced SOCE. Thereby, we found that this decrease and the absence of ORAI1 protein did not affect HEK-293 proliferation. In addition, we determined that ORAI1 suppression did not affect adhesive properties but had a limited impact on HEK-293 migration. Overall, we showed that ORAI1 and SOCE are largely dispensable for cellular proliferation, migration, and cellular adhesion of HEK-293 cells. Thus, despite its importance in providing Ca2+ entry in non-excitable cells, our results indicate that the lack of SOCE does not deeply impact HEK-293 cells. This finding suggests the existence of compensatory mechanism enabling the maintenance of their physiological function.

A novel STIM1-Orai1 gating interface essential for CRAC channel activation.

Calcium signalling through store-operated calcium (SOC) entry is of crucial importance for T-cell activation and the adaptive immune response. This entry occurs via the prototypic Ca2+ release-activated Ca2+ (CRAC) channel. STIM1, a key molecular component of this process, is located in the membrane of the endoplasmic reticulum (ER) and is initially activated upon Ca2+ store depletion. This activation signal is transmitted to the plasma membrane via a direct physical interaction that takes place between STIM1 and the highly Ca2+-selective ion channel Orai1. The activation of STIM1 induces an extended cytosolic conformation. This, in turn, exposes the CAD/SOAR domain and leads to the formation of STIM1 oligomers. In this study, we focused on a small helical segment (STIM1 α3, aa 400-403), which is located within the CAD/SOAR domain. We determined this segment's specific functional role in terms of STIM1 activation and Orai1 gating. The STIM1 α3 domain appears not essential for STIM1 to interact with Orai1. Instead, it represents a key domain that conveys STIM1 interaction into Orai1 channel gating. The results of cysteine crosslinking experiments revealed the close proximity of STIM1 α3 to a region within Orai1, which was located at the cytosolic extension of transmembrane helix 3, forming a STIM1-Orai1 gating interface (SOGI). We suggest that the interplay between STIM1 α3 and Orai1 TM3 allows STIM1 coupling to be transmitted into physiological CRAC channel activation.

Store-operated Ca2+ entry and Ca2+ responses to hypothalamic releasing hormones in anterior pituitary cells from Orai1-/- and heptaTRPC knockout mice.

Store operated Ca2+ entry (SOCE) is the most important Ca2+ entry pathway in non-excitable cells. However, SOCE can also play a pivotal role in excitable cells such as anterior pituitary (AP) cells. The AP gland contains five different cell types that release six major AP hormones controlling most of the entire endocrine system. AP hormone release is modulated by Ca2+ signals induced by different hypothalamic releasing hormones (HRHs) acting on specific receptors in AP cells. TRH and LHRH both induce Ca2+ release and Ca2+ entry in responsive cells while GHRH and CRH only induce Ca2+ entry. SOCE has been shown to contribute to Ca2+ responses induced by TRH and LHRH but no molecular evidence has been provided. Accordingly, we used AP cells isolated from mice devoid of Orai1 channels (noted as Orai1-/- or Orai1 KO mice) and mice lacking expression of all seven canonical TRP channels (TRPC) from TRPC1 to TRPC7 (noted as heptaTRPC KO mice) to investigate contribution of these putative channel proteins to SOCE and intracellular Ca2+ responses induced by HRHs. We found that thapsigargin-evoked SOCE is lost in AP cells from Orai1-/- mice but unaffected in cells from heptaTRPC KO mice. Conversely, while spontaneous intracellular Ca2+-oscillations related to electrical activity were not affected in the Orai1-/- mice, these responses were significantly reduced in heptaTRPC KO mice. We also found that Ca2+ entry induced by TRH and LHRH is decreased in AP cells isolated from Orai1-/-. In addition, Ca2+ responses to several HRHs, particularly TRH and GHRH, are decreased in the heptaTRPC KO mice. These results indicate that expression of Orai1, and not TRPC channel proteins, is necessary for thapsigargin-evoked SOCE and is required to support Ca2+ entry induced by TRH and LHRH in mouse AP cells. In contrast, TRPC channel proteins appear to contribute to spontaneous Ca2+-oscillations and Ca2+ responses induced by TRH and GHRH. We conclude that expression of Orai1 and TRPC channels proteins may play differential and significant roles in AP physiology and endocrine control.

Calcium store refilling and STIM activation in STIM- and Orai-deficient cell lines.

Mediated through the combined action of STIM proteins and Orai channels, store-operated Ca2+ entry (SOCE) functions ubiquitously among different cell types. The existence of multiple STIM and Orai genes has made it difficult to assign specific roles of each STIM and Orai homolog in mediating Ca2+ signals. Using CRISPR/Cas9 gene editing tools, we generated cells with both STIM or all three Orai homologs deleted and directly monitored store Ca2+ and Ca2+ signals. We found that unstimulated, SOCE null KO cells still retain 50~70% of ER Ca2+ stores of wildtype (wt) cells. After brief exposure to store-emptying conditions, acute refilling of ER Ca2+ stores was totally blocked in KO cells. However, after 24 h in culture, stores were eventually refilled. Thus, SOCE is critical for immediate refilling of ER Ca2+ but is dispensable for the maintenance of long-term ER Ca2+ homeostasis. Using the Orai null background triple Orai-KO cells, we examined the plasma membrane translocation properties of a series of truncated STIM1 variants. FRET analysis reveals that, even though PM tethering of STIM1 expedites the activation of STIM1 by facilitating its oligomerization, migration, and accumulation in ER-PM junctions, it is not required for the conformational switch, oligomerization, and clustering of STIM1. Even without overt puncta formation at ER-PM junctions, STIM11-491 and STIM11-666 could still rescue SOCE when expressed in STIM KO cells. Thus, ER-PM trapping and clustering of STIM molecules only facilitates the process of SOCE activation, but is not essential for the activation of Orai channels.

ORAI2 modulates store-operated calcium entry and T cell-mediated immunity.

Store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels is critical for lymphocyte function and immune responses. CRAC channels are hexamers of ORAI proteins that form the channel pore, but the contributions of individual ORAI homologues to CRAC channel function are not well understood. Here we show that deletion of Orai1 reduces, whereas deletion of Orai2 increases, SOCE in mouse T cells. These distinct effects are due to the ability of ORAI2 to form heteromeric channels with ORAI1 and to attenuate CRAC channel function. The combined deletion of Orai1 and Orai2 abolishes SOCE and strongly impairs T cell function. In vivo, Orai1/Orai2 double-deficient mice have impaired T cell-dependent antiviral immune responses, and are protected from T cell-mediated autoimmunity and alloimmunity in models of colitis and graft-versus-host disease. Our study demonstrates that ORAI1 and ORAI2 form heteromeric CRAC channels, in which ORAI2 fine-tunes the magnitude of SOCE to modulate immune responses.

Orai1-Mediated Antimicrobial Secretion from Pancreatic Acini Shapes the Gut Microbiome and Regulates Gut Innate Immunity.

The gut microbiome participates in numerous physiologic functions and communicates intimately with the host immune system. Antimicrobial peptides are critical components of intestinal innate immunity. We report a prominent role for antimicrobials secreted by pancreatic acini in shaping the gut microbiome that is essential for intestinal innate immunity, barrier function, and survival. Deletion of the Ca2+ channel Orai1 in pancreatic acini of adult mice resulted in 60%-70% mortality within 3 weeks. Despite robust activation of the intestinal innate immune response, mice lacking acinar Orai1 exhibited intestinal bacterial outgrowth and dysbiosis, ultimately causing systemic translocation, inflammation, and death. While digestive enzyme supplementation was ineffective, treatments constraining bacterial outgrowth (purified liquid diet, broad-spectrum antibiotics) rescued survival, feeding, and weight gain. Pancreatic levels of cathelicidin-related antimicrobial peptide (CRAMP) were reduced, and supplement of synthetic CRAMP prevented intestinal disease. These findings reveal a critical role for antimicrobial pancreatic secretion in gut innate immunity.

ORAI1 Activates Proliferation of Lymphatic Endothelial Cells in Response to Laminar Flow Through Krüppel-Like Factors 2 and 4.

RATIONALE: Lymphatic vessels function to drain interstitial fluid from a variety of tissues. Although shear stress generated by fluid flow is known to trigger lymphatic expansion and remodeling, the molecular basis underlying flow-induced lymphatic growth is unknown. OBJECTIVE: We aimed to gain a better understanding of the mechanism by which laminar shear stress activates lymphatic proliferation. METHODS AND RESULTS: Primary endothelial cells from dermal blood and lymphatic vessels (blood vascular endothelial cells and lymphatic endothelial cells [LECs]) were exposed to low-rate steady laminar flow. Shear stress-induced molecular and cellular responses were defined and verified using various mutant mouse models. Steady laminar flow induced the classic shear stress responses commonly in blood vascular endothelial cells and LECs. Surprisingly, however, only LECs showed enhanced cell proliferation by regulating the vascular endothelial growth factor (VEGF)-A, VEGF-C, FGFR3, and p57/CDKN1C genes. As an early signal mediator, ORAI1, a pore subunit of the calcium release-activated calcium channel, was identified to induce the shear stress phenotypes and cell proliferation in LECs responding to the fluid flow. Mechanistically, ORAI1 induced upregulation of Krüppel-like factor (KLF)-2 and KLF4 in the flow-activated LECs, and the 2 KLF proteins cooperate to regulate VEGF-A, VEGF-C, FGFR3, and p57 by binding to the regulatory regions of the genes. Consistently, freshly isolated LECs from Orai1 knockout embryos displayed reduced expression of KLF2, KLF4, VEGF-A, VEGF-C, and FGFR3 and elevated expression of p57. Accordingly, mouse embryos deficient in Orai1, Klf2, or Klf4 showed a significantly reduced lymphatic density and impaired lymphatic development. CONCLUSIONS: Our study identified a molecular mechanism for laminar flow-activated LEC proliferation.

Male infertility in mice lacking the store-operated Ca(2+) channel Orai1.

Store-operated calcium entry (SOCE) is an important Ca(2+) influx pathway in somatic cells. In addition to maintaining endoplasmic reticulum (ER) Ca(2+) stores, Ca(2+) entry through store-operated channels regulates essential signaling pathways in numerous cell types. Patients with mutations in the store-operated channel subunit ORAI1 exhibit defects in store-operated Ca(2+) influx, along with severe immunodeficiency, congenital myopathy and ectodermal dysplasia. However, little is known about the functional role of ORAI1 in germ cells and reproductive function in mice, or in men, since men with loss-of-function or null mutations in ORAI1 rarely survive to reproductive age. In this study, we investigated the role of ORAI1 in male reproductive function. We reveal that Orai1(-/-) male mice are sterile and have severe defects in spermatogenesis, with prominent deficiencies in mid- to late-stage elongating spermatid development. These studies establish an essential in vivo role for store-operated ORAI1 channels in male reproductive function and identify these channels as potential non-steroidal regulators of male fertility.