Faculty Bibliography

Polarized Ca(2+) signals in secretory epithelial cells are determined by compartmentalized localization of Ca(2+) signaling proteins at the apical pole. Recently the ER Ca(2+) sensor STIM1 (stromal interaction molecule 1) and the Orai channels were shown to play a critical role in store-dependent Ca(2+) influx. STIM1 also gates the transient receptor potential-canonical (TRPC) channels. Here, we asked how cell stimulation affects the localization, recruitment and function of the native proteins in polarized cells. Inhibition of Orai1, STIM1, or deletion of TRPC1 reduces Ca(2+) influx and frequency of Ca(2+) oscillations. Orai1 localization is restricted to the apical pole of the lateral membrane. Surprisingly, cell stimulation does not lead to robust clustering of native Orai1, as is observed with expressed Orai1. Unexpectedly, cell stimulation causes polarized recruitment of native STIM1 to both the apical and lateral regions, thus to regions with and without Orai1. Accordingly, STIM1 and Orai1 show only 40% colocalization. Consequently, STIM1 shows higher colocalization with the basolateral membrane marker E-cadherin than does Orai1, while Orai1 showed higher colocalization with the tight junction protein ZO1. TRPC1 is expressed in both apical and basolateral regions of the plasma membrane. Co-IP of STIM1/Orai1/IP(3) receptors (IP(3) Rs)/TRPCs is enhanced by cell stimulation and disrupted by 2-aminoethoxydiphenyl borate (2APB). The polarized localization and recruitment of these proteins results in preferred Ca(2+) entry that is initiated at the apical pole. These findings reveal that in addition to Orai1, STIM1 likely regulates other Ca(2+) permeable channels, such as the TRPCs. Both channels contribute to the frequency of [Ca(2+) ] oscillations and thus impact critical cellular functions

The immune cells named T lymphocytes circulate around the body fulfilling their role in immunosurveillance by monitoring the tissues for injury or infection. To migrate from the blood into the tissues, they make use of the integrin LFA-1 which is exclusively expressed by immune cells. These highly motile cells attach and migrate on substrates expressing the LFA-1 ligand ICAM-1. The molecular events signaling LFA-1 activation and adhesion are now reasonably well identified, but the process of detaching LFA-1 adhesions is less understood. The cysteine protease calpain is involved in turnover of integrin-mediated adhesions in less motile cell types. In this study we have explored the involvement of calpain in turnover of LFA-1-mediated adhesions of T lymphocytes. Using live cell imaging and immunohistochemistry, we demonstrate that turnover of adhesions depends on the Ca2+-dependent enzyme, calpain 2. Inhibition of calpain activity by means of siRNA silencing or pharmacological inhibition results in inefficient disassembly of LFA-1 adhesions causing T lymphocyte elongation and shedding of LFA-1 clusters behind the migrating T lymphocytes. We show that calpain 2 is distributed throughout the T lymphocyte, but is most active at the trailing edge as detected by expression of its fluorescent substrate CMAC,t-BOC-Leu-Met. Extracellular Ca2+ entry is essential for the activity of calpain 2 that is constantly maintained as the T lymphocytes migrate. Use of T cells from a patient with mutation in ORAI1 revealed that the major calcium-release-activated-calcium channel is not the ion channel delivering the Ca2+. We propose a model whereby Ca2+ influx, potentially through stretch activated channels, is sufficient to activate calpain 2 at the trailing edge of a migrating T cell and this activity is essential for the turnover of LFA-1 adhesions.

ORAI1 is the pore-forming subunit of the Ca(2+) release-activated Ca(2+) (CRAC) channel, which is responsible for store-operated Ca(2+) entry in lymphocytes. A role for ORAI1 in T cell function in vivo has been inferred from in vitro studies of T cells from human immunodeficient patients with mutations in ORAI1 and Orai1(-/-) mice, but a detailed analysis of T cell-mediated immune responses in vivo in mice lacking functional ORAI1 has been missing. We therefore generated Orai1 knock-in mice (Orai1(KI/KI)) expressing a nonfunctional ORAI1-R93W protein. Homozygosity for the equivalent ORAI1-R91W mutation abolishes CRAC channel function in human T cells resulting in severe immunodeficiency. Homozygous Orai1(KI/KI) mice die neonatally, but Orai1(KI/KI) fetal liver chimeric mice are viable and show normal lymphocyte development. T and B cells from Orai1(KI/KI) mice display severely impaired store-operated Ca(2+) entry and CRAC channel function resulting in a strongly reduced expression of several key cytokines including IL-2, IL-4, IL-17, IFN-gamma, and TNF-alpha in CD4(+) and CD8(+) T cells. Cell-mediated immune responses in vivo that depend on Th1, Th2, and Th17 cell function were severely attenuated in ORAI1-deficient mice. Orai1(KI/KI) mice lacked detectable contact hypersensitivity responses and tolerated skin allografts significantly longer than wild-type mice. In addition, T cells from Orai1(KI/KI) mice failed to induce colitis in an adoptive transfer model of inflammatory bowel disease. These findings reaffirm the critical role of ORAI1 for T cell function and provide important insights into the in vivo functions of CRAC channels for T cell-mediated immunity

T-cell function is dependent on store-operated Ca(2+) influx that is activated by the stromal interaction molecules (STIM) 1 and 2. We show that mice with T-cell-specific deletion of STIM1 or STIM2 are protected from EAE, a mouse model of multiple sclerosis (MS). While STIM1- and STIM2-deficient T cells could be successfully primed by autoantigen, they failed to produce the proinflammatory cytokines IL-17 and IFN-gamma. STIM1-deficient T cells showed reduced expression of IL-23R, required for Th17 cell homeostasis, and had impaired chemokine-dependent T-cell migration caused by a lack of chemokine-induced Ca(2+) influx. Autoantigen-specific STIM1- or STIM2-deficient T cells failed to expand and accumulate in the CNS and lymph nodes following adoptive transfer to passively induce EAE, suggesting that autoantigen-specific restimulation or homeostasis of STIM1- and STIM2-deficient T cells are impaired. Combined deletion of both STIM1 and STIM2, previously shown to impair Treg development and function, completely protected mice from EAE. This indicates that, in the absence of Ca(2+) influx, autoreactive T cells are severely dysfunctional rendering Treg dispensable for the prevention of CNS inflammation. Our findings demonstrate that both STIM1 and STIM2 are critical for T-cell function and autoimmunity in vivo

Classic Kaposi sarcoma (KS) is exceedingly rare in children from the Mediterranean Basin, despite the high prevalence of human herpesvirus-8 (HHV-8) infection in this region. We hypothesized that rare single-gene inborn errors of immunity to HHV-8 may underlie classic KS in childhood. We investigated a child with no other unusually severe infectious or tumoral phenotype who died from disseminated KS at two years of age. Whole-exome sequencing in the patient revealed a homozygous splice-site mutation in STIM1, the gene encoding stromal interaction molecule 1, which regulates store-operated Ca(2+) entry. STIM1 mRNA splicing, protein production, and Ca(2+) influx were completely abolished in EBV-transformed B cell lines from the patient, but were rescued by the expression of wild-type STIM1. Based on the previous discovery of STIM1 deficiency in a single family with a severe T cell immunodeficiency and the much higher risk of KS in individuals with acquired T cell deficiencies, we conclude that STIM1 T cell deficiency precipitated the development of lethal KS in this child upon infection with HHV-8. Our report provides the first evidence that isolated classic KS in childhood may result from single-gene defects and provides proof-of-principle that whole-exome sequencing in single patients can decipher the genetic basis of rare inborn errors

Ca(2+) signals through store-operated Ca(2+) (SOC) channels, activated by the depletion of Ca(2+) from the endoplasmic reticulum, regulate various physiological events. Orai1 is the pore-forming subunit of the Ca(2+) release-activated Ca(2+) (CRAC) channel, the best characterized SOC channel. Orai1 is activated by stromal interaction molecule (STIM) 1, a Ca(2+) sensor located in the endoplasmic reticulum. Orai1 and STIM1 are crucial for SOC channel activation, but the molecular mechanisms regulating Orai1 function are not fully understood. In this study, we demonstrate that protein kinase C (PKC) suppresses store-operated Ca(2+) entry (SOCE) by phosphorylation of Orai1. PKC inhibitors and knockdown of PKCbeta both resulted in increased Ca(2+) influx. Orai1 is strongly phosphorylated by PKC in vitro and in vivo at N-terminal Ser-27 and Ser-30 residues. Consistent with these results, substitution of endogenous Orai1 with an Orai1 S27A/S30A mutant resulted in increased SOCE and CRAC channel currents. We propose that PKC suppresses SOCE and CRAC channel function by phosphorylation of Orai1 at N-terminal serine residues Ser-27 and Ser-30.

Store-operated Ca2+ entry (SOCE) is an important Ca2+ influx pathway in many non-excitable and some excitable cells. It is regulated by the filling state of intracellular Ca2+ stores, notably the endoplasmic reticulum (ER). Reduction in [Ca2+]ER results in activation of plasma membrane Ca2+ channels that mediate sustained Ca2+ influx which is required for many cell functions as well as refilling of Ca2+ stores. The Ca2+ release activated Ca2+ (CRAC) channel is the best characterized SOC channel with well-defined electrophysiological properties. In recent years, the molecular components of the CRAC channel, long mysterious, have been defined. ORAI1 (or CRACM1) acts as the pore-forming subunit of the CRAC channel in the plasma membrane. Stromal interaction molecule (STIM) 1 is localized in the ER, senses [Ca2+]ER, and activates the CRAC channel upon store depletion by binding to ORAI1. Both proteins are widely expressed in many tissues in both human and mouse consistent with the widespread prevalence of SOCE and CRAC channel currents in many cells types. CRAC channelopathies in human patients with mutations in STIM1 and ORAI1 are characterized by abolished CRAC channel currents, lack of SOCE and-clinically-immunodeficiency, congenital myopathy, and anhydrotic ectodermal dysplasia. This article reviews the role of ORAI and STIM proteins for SOCE and CRAC channel function in a variety of cell types and tissues and compares the phenotypes of ORAI1 and STIM1-deficient human patients and mice with targeted deletion of Orai and Stim genes

Several lines of evidence have demonstrated B cell intrinsic activation defects in patients with common variable immunodeficiency (CVID). The rapid increase of intracellular free calcium concentrations after engagement of the BCR represents one crucial element in this activation process. The analysis of 53 patients with CVID for BCR-induced calcium flux identified a subgroup of patients with significantly reduced Ca2+ signals in primary B cells. This subgroup strongly corresponded to the class Ia of the Freiburg classification. Comparison at the level of defined B cell subpopulations revealed reduced Ca2+ signals in all mature B cell populations of patients with CVID class Ia when compared with healthy individuals and other groups of patients with CVID but not in circulating transitional B cells. BCR-induced Ca2+ responses were the lowest in CD21low B cells in patients as well as healthy donors, indicating an additional cell-specific mechanism inhibiting the Ca2+ flux. Although proximal BCR signaling events are unperturbed in patients' B cells, including normal phospholipase Cgamma2 phosphorylation and Ca2+ release from intracellular stores, Ca2+ influx from the extracellular space is significantly impaired. CD22, a negative regulator of calcium signals in B cells, is highly expressed on CD21low B cells from patients with CVID Ia and might be involved in the attenuated Ca2+ response of this B cell subpopulation. These data from patients with CVID suggest that a defect leading to impaired BCR-induced calcium signaling is associated with the expansion of CD21low B cells, hypogammaglobulinemia, autoimmune dysregulation, and lymphadenopathy.

Lymphocyte activation requires Ca(2+) influx through specialized Ca(2+) channels in the plasma membrane. In T cells the predominant Ca(2+) channel is the Ca(2+) release activated Ca(2+) (CRAC) channel encoded by the gene ORAI1. ORAI1 is activated by stromal interaction molecule (STIM) 1 that is localized in the ER where it senses the concentration of stored Ca(2+). Following antigen binding to immunoreceptors such as the TCR, ER Ca(2+) stores are depleted, STIM1 is activated and ORAI1-CRAC channels open resulting in what is referred to as store-operated Ca(2+) entry (SOCE). Mutations in ORAI1 and STIM1 genes in human patients that lead to expression of non-functional ORAI1 or complete lack of ORAI1 or STIM1 protein are associated with a unique clinical phenotype that is characterized by immunodeficiency, muscular hypotonia and anhydrotic ectodermal dysplasia, as well as, in the case of STIM1 deficiency, autoimmunity and lymphoproliferative disease. The immunodeficiency in these patients is due to a severe defect in T cell activation but not in lymphocyte development. This review describes the immunological and non-immunological phenotypes of patients with defects in SOCE and CRAC channel function and discusses them in the context of similar immunodeficiency diseases and animal models of ORAI1 and STIM1 function