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Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a detrimental intraoral lesion that occurs in patients with long-term or high-dose use of anti-resorptive agents such as bisphosphonates. Tooth extraction is a known risk factor for BRONJ, and such intervention is often performed to eliminate existing pathological inflammatory conditions. Previously, we determined that ligature-induced periodontitis (LIP) is a risk factor for the development of osteonecrosis in mice, but it remains unclear whether the chronicity of LIP followed by extraction influences osteonecrosis development. In this study, we assess the effect of short-term and long-term LIP (ligature placed for 3 weeks [S-LIP] or 10 weeks [L-LIP], respectively) on osteonecrosis development in mice receiving 250 μg/kg/week zoledronic acid (ZOL). When compared to S-LIP, L-LIP caused 70% (p ≤ 0.0014) more bone loss without altering microbe composition. In the presence of ZOL, bone loss mediated by LIP was prevented and bone necrosis was induced. When the ligated tooth was extracted, histologic hallmarks of osteonecrosis including empty lacunae and necrotic bone were increased by 88% (p = 0.0374) and 114% (p = 0.0457), respectively, in L-LIP compared to S-LIP. We also observed significant increases in serum platelet factor 4 (PF4) and macrophage inflammatory factor 1 γ (MIP1γ) in mice that received ZOL treatment and had tooth extractions compared to controls, which may be systemic markers of inflammation-associated osteonecrosis development. Additionally, CD3+ T cells were identified as the major immune population in both health and disease, and we observed a 116% (p = 0.0402) increase in CD3+IL23R+ T cells in L-LIP compared to S-LIP lesions following extraction. Taken together, our study reveals that extracting a periodontally compromised tooth increases the formation of necrotic bone compared to extracting a periodontally healthy tooth and that osteonecrosis may be associated with the duration of the preexisting pathological inflammatory conditions. © 2022 American Society for Bone and Mineral Research (ASBMR).

T cell activation stimulates substantially increased protein synthesis activity to accumulate sufficient biomass for cell proliferation. The protein synthesis is fueled by the amino acids transported from the environment. Steroid nuclear receptor coactivator 2 (SRC2) is a member of a family of transcription coactivators. Here, we show that SRC2 recruited by c-Myc enhances CD4 + T cell activation to stimulate immune responses via upregulation of amino acid transporter Slc7a5. Mice deficient of SRC2 in T cells ( SRC2 fl/fl /CD4 Cre ) are resistant to the induction of experimental autoimmune encephalomyelitis (EAE) and susceptible to Citrobacter rodentium ( C. rodentium ) infection. Adoptive transfer of naive CD4 + T cells from SRC2 fl/fl /CD4 Cre mice fails to elicit EAE and colitis in Rag1 / recipients. Further, CD4 + T cells from SRC2 fl/fl /CD4 Cre mice display defective T cell proliferation, cytokine production, and differentiation both in vitro and in vivo. Mechanically, SRC2 functions as a coactivator to work together with c-Myc to stimulate the expression of amino acid transporter Slc7a5 required for T cell activation. Slc7a5 fails to be up-regulated in CD4 + T cells from SRC2 fl/fl /CD4 Cre mice, and forced expression of Slc7a5 rescues proliferation, cytokine production, and the ability of SRC2 fl/fl /CD4 Cre CD4 + T cells to induce EAE. Therefore, SRC2 is essential for CD4 + T cell activation and, thus, a potential drug target for controlling CD4 + T cell-mediated autoimmunity.

Activation of the Ca2+ release–activated Ca2+ (CRAC) channel is crucial for T cell functions. It was recently shown that naked cuticle homolog 2 (NKD2), a signaling adaptor molecule, orchestrates trafficking of ORAI1, a pore subunit of the CRAC channels, to the plasma membrane for sustained activation of the CRAC channels. However, the physiological role of sustained Ca2+ entry via ORAI1 trafficking remains poorly understood. Using NKD2 as a molecular handle, we show that ORAI1 trafficking is crucial for sustained Ca2+ entry and cytokine production, especially in inflammatory Th1 and Th17 cells. We find that murine T cells cultured under pathogenic Th17-polarizing conditions have higher Ca2+ levels that are NKD2-dependent than those under nonpathogenic conditions. In vivo, deletion of Nkd2 alleviated clinical symptoms of experimental autoimmune encephalomyelitis in mice by selectively decreasing effector T cell responses in the CNS. Furthermore, we observed a strong correlation between NKD2 expression and proinflammatory cytokine production in effector T cells. Taken together, our findings suggest that the pathogenic effector T cell response demands sustained Ca2+ entry supported by ORAI1 trafficking.

RORγt is known to instruct the differentiation of T helper 17 (T

Acute immune responses to microbial insults in the oral cavity often progress to chronic inflammatory diseases such as periodontitis and apical periodontitis. Chronic oral inflammation causes destruction of the periodontium, potentially leading to loss of the dentition. Previous investigations have demonstrated that the composition of oral immune cells, rather than the overall extent of cellular infiltration, determines the pathological development of chronic inflammation. The role of T lymphocyte populations, including Th1, Th2, Th17, and Treg cells, has been extensively described. Studies now propose pathogenic Th17 cells as a distinct subset, uniquely classifiable from traditional Th17 populations. In situ differentiation of pathogenic Th17 cells has been verified as a source of destructive inflammation, which critically drives pathogenesis in chronic inflammatory diseases such as diabetes, rheumatoid arthritis, and inflammatory bowel disease. Pathogenic Th17 cells resemble a Th1 penotype and produce not only interleukin 17 (IL-17) but also γ-interferon (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The proinflammatory cytokine-specific mechanisms known to induce IL-17 expression in Th17 cells are well characterized; however, differentiation mechanisms that lead to pathogenic Th17 cells are less understood. Recently, Ca2+ signaling through Ca2+ release-activated Ca2+ channels (CRAC) in T cells has been uncovered as a major signaling axis involved in the regulation of T-cell-mediated chronic inflammation. In particular, pathogenic Th17 cell–mediated immunological diseases appear to be effectively targeted via such Ca2+ signaling pathways. Pathogenic plasticity of Th17 cells has been extensively illustrated in autoimmune and chronic inflammatory diseases. Although their specific causal relationship to oral infection-induced chronic inflammatory diseases is not fully established, pathogenic Th17 cells may be involved in the underlining mechanism. This review highlights the current understanding of T-cell phenotype regulation, calcium signaling pathways in this event, and the potential role of pathogenic Th17 cells in chronic inflammatory disorders of the oral cavity.

Steroid nuclear receptor coactivator 2 (SRC2) is a member of a family of transcription coactivators. While SRC1 inhibits the differentiation of regulatory T cells (T regs ) critical for establishing immune tolerance, we show here that SRC2 stimulates T reg differentiation. SRC2 is dispensable for the development of thymic T regs , whereas naive CD4 + T cells from mice deficient of SRC2 specific in T regs ( SRC2 fl/fl /Foxp3 YFP-Cre ) display defective T reg differentiation. Furthermore, the aged SRC2 fl/fl /Foxp3 YFP-Cre mice spontaneously develop autoimmune phenotypes including enlarged spleen and lung inflammation infiltrated with IFNγ-producing CD4 + T cells. SRC2 fl/fl /Foxp3 YFP-Cre mice also develop severer experimental autoimmune encephalomyelitis (EAE) due to reduced T regs . Mechanically, SRC2 recruited by NFAT1 binds to the promoter and activates the expression of Nr4a2 , which then stimulates Foxp3 expression to promote T reg differentiation. Members of SRC family coactivators thus play distinct roles in T reg differentiation and are potential drug targets for controlling immune tolerance.

Activation of the Ca(2+) release-activated Ca(2+) (CRAC) channel is crucial for T cell functions. It was recently shown that naked cuticle homolog 2 (NKD2), a signaling adaptor molecule, orchestrates trafficking of ORAI1, a pore subunit of the CRAC channels, to the plasma membrane for sustained activation of the CRAC channels. However, the physiological role of sustained Ca(2+) entry via ORAI1 trafficking remains poorly understood. Using NKD2 as a molecular handle, we show that ORAI1 trafficking is crucial for sustained Ca(2+) entry and cytokine production, especially in inflammatory Th1 and Th17 cells. We find that murine T cells cultured under pathogenic Th17-polarizing conditions have higher Ca(2+) levels that are NKD2-dependent than those under non-pathogenic conditions. In vivo, deletion of Nkd2 alleviated clinical symptoms of experimental autoimmune encephalomyelitis (EAE) in mice by selectively decreasing effector T cell responses in the central nervous system. Furthermore, we observed a strong correlation between NKD2 expression and pro-inflammatory cytokine production in effector T cells. Together, our findings suggest that the pathogenic effector T cell response demands sustained Ca(2+) entry supported by ORAI1 trafficking.

CRAC channel regulator 2 A (CRACR2A) is a large Rab GTPase that is expressed abundantly in T cells and acts as a signal transmitter between T cell receptor stimulation and activation of the Ca2+-NFAT and JNK-AP1 pathways. CRACR2A has been linked to human diseases in numerous genome-wide association studies, however, to date no patient with damaging variants in CRACR2A has been identified. In this study, we describe a patient harboring biallelic variants in CRACR2A [paternal allele c.834 gaG> gaT (p.E278D) and maternal alelle c.430 Aga > Gga (p.R144G) c.898 Gag> Tag (p.E300*)], the gene encoding CRACR2A. The 33-year-old patient of East-Asian origin exhibited late onset combined immunodeficiency characterised by recurrent chest infections, panhypogammaglobulinemia and CD4+ T cell lymphopenia. In vitro exposure of patient B cells to a T-dependent stimulus resulted in normal generation of antibody-secreting cells, however the patient’s T cells showed pronounced reduction in CRACR2A protein levels and reduced proximal TCR signaling, including dampened SOCE and reduced JNK phosphorylation, that contributed to a defect in proliferation and cytokine production. Expression of individual allelic mutants in CRACR2A-deleted T cells showed that the CRACR2AE278D mutant did not affect JNK phosphorylation, but impaired SOCE which resulted in reduced cytokine production. The truncated double mutant CRACR2AR144G/E300* showed a pronounced defect in JNK phosphorylation as well as SOCE and strong impairment in cytokine production. Thus, we have identified variants in CRACR2A that led to late-stage combined immunodeficiency characterized by loss of function in T cells.

ORAI1 and STIM1 are the critical mediators of store-operated Ca2+ entry by acting as the pore subunit and an endoplasmic reticulum-resident signaling molecule, respectively. In addition to Ca2+ signaling, STIM1 is also involved in regulation of a cytosolic nucleic acid sensing pathway. Using ORAI1 and STIM1 knockout cells, we examined their contribution to the host response to SARS-CoV-2 infection. STIM1 knockout cells showed strong resistance to SARS-CoV-2 infection due to enhanced type I interferon response. On the contrary, ORAI1 knockout cells showed high susceptibility to SARS-CoV-2 infection as judged by increased expression of viral proteins and a high viral load. Mechanistically, ORAI1 knockout cells showed reduced homeostatic cytoplasmic Ca2+ concentration and severe impairment in tonic interferon signaling. Transcriptome analysis showed downregulation of multiple cellular defense mechanisms, including antiviral signaling pathways in ORAI1 knockout cells, which are likely due to reduced expression of the Ca2+-dependent transcription factors of the activator protein 1 (AP-1) family and MEF2C. Our results identify a novel role of ORAI1-mediated Ca2+ signaling in regulating the baseline type I interferon level, which is a determinant of host resistance to SARS-CoV-2 infection.

Sustained activation of the Ca2+ release-activated Ca2+ (CRAC) channel is pivotal for effector T cell responses. The mechanisms underlying this sustainability remain poorly understood. We find that plasma membrane localization of ORAI1, the pore subunit of CRAC channels, is limited in effector T cells, with a significant fraction trapped in intracellular vesicles. From a targeted screen, we have identified an essential component of ORAI1+ vesicles, naked cuticle homolog 2 (NKD2). Mechanistically, NKD2, an adaptor molecule activated by signaling pathways downstream of T cell receptors, orchestrates trafficking and insertion of ORAI1+ vesicles to the plasma membrane. Together, our findings suggest that TCR stimulation-dependent insertion of ORAI1 into the plasma membrane is essential for sustained Ca2+ signaling and cytokine production in T cells.

Ca2+ release-activated Ca2+ (CRAC) channels elevate cytoplasmic Ca2+ concentration, which is essential for T cell activation, differentiation and effector functions. T cell receptor stimulation induces depletion of the endoplasmic reticulum (ER) Ca2+ stores, which is sensed by stromal interaction molecule 1 (STIM1). STIM1 translocates to the ER-plasma membrane (PM) junctions to interact with ORAI1, the pore subunit of the CRAC channels. Here, we show that two members of the extended synaptotagmin (E-Syt) family, E-Syt1, and the short isoform of E-Syt2 (E-Syt2S), contribute to activation of CRAC channels in T cells. Knockdown or deletion of both ESYT1 and ESYT2 reduced store-operated Ca2+ entry (SOCE) and ORAI1-STIM1 clustering in Jurkat T cells. Further, depletion of E-Syts in primary T cells decreased Ca2+ entry and cytokine production. While the ER-PM junctions were reduced in both HeLa and Jurkat T cells deleted for ESYT1 and ESYT2, SOCE was impaired only in Jurkat T cells, suggesting that the membrane-tethering function of E-Syts is distinct from their role in SOCE. Mechanistically, E-Syt2S, the predominant isoform of E-Syt2 in T cells, recruited STIM1 to the junctions via a direct interaction. This study demonstrates a membrane-tethering-independent role of E-Syts in activation of CRAC channels in T cells.

SRC3, a highly conserved member of the steroid receptor coactivator (SRC) family, is recruited by transcription factors to regulate cellular function. Previously, we demonstrated that SRC1, another highly conserved member of the SRC family, interacts with RORγt to regulate Th17 differentiation. However, the relationship between SRC1 and SRC3 in the regulation of Th17 cell function remains unknown. In this study, we demonstrate that mouse SRC3 interacts with RORγt in Th17 cells but not in thymocytes. In addition, Src3−/− mice exhibited defective Th17 differentiation and induction of experimental autoimmune encephalomyelitis but normal thymocyte development. Furthermore, a K313 to arginine mutation of RORγt (RORγt-K313R), which disrupts the interaction of RORγt with SRC3 but not with SRC1, impairs Th17 differentiation but not thymocyte development. These data suggest that SRC3 works with SRC1 to regulate RORγt-dependent Th17 differentiation but is not essential for RORγt-dependent thymocyte development.

Ca2+ release–activated Ca2+ channel regulator 2A (CRACR2A) is expressed abundantly in T cells and acts as a signal transmitter between TCR stimulation and activation of the Ca2+/NFAT and JNK/AP1 pathways. CRACR2A has been linked to human diseases in numerous genome-wide association studies and was shown to be one of the most sensitive targets of the widely used statin drugs. However, the physiological role of CRACR2A in T cell functions remains unknown. In this study, using transgenic mice for tissue-specific deletion, we show that CRACR2A promotes Th1 responses and effector function of Th17 cells. CRACR2A was abundantly expressed in Th1 and Th17 cells. In vitro, deficiency of CRACR2A decreased Th1 differentiation under nonpolarizing conditions, whereas the presence of polarizing cytokines compensated this defect. Transcript analysis showed that weakened TCR signaling by deficiency of CRACR2A failed to promote Th1 transcriptional program. In vivo, conditional deletion of CRACR2A in T cells alleviated Th1 responses to acute lymphocytic choriomeningitis virus infection and imparted resistance to experimental autoimmune encephalomyelitis. Analysis of CNS from experimental autoimmune encephalomyelitis–induced mice showed impaired effector functions of both Th1 and Th17 cell types, which correlated with decreased pathogenicity. Collectively, our findings demonstrate the requirement of CRACR2A-mediated TCR signaling in Th1 responses as well as pathogenic conversion of Th17 cells, which occurs at the site of inflammation.

Transcription factor retinoid acid–related orphan receptor (ROR)γt transcriptionally regulates the genes required for differentiation of Th17 cells that mediate both protective and pathogenic immunity. However, little is known about the function of posttranslational modifications in the regulation of RORγt activity. Mass spectrometric analysis of immunoprecipitated RORγt from Th17 cells identified multiple phosphorylation sites. Systematic mutation analysis of the identified phosphorylation sites found that phosphorylation of S376 enhances whereas phosphorylation of S484 inhibits Th17 differentiation. IκB kinase (IKK)α binds and phosphorylates RORγt at S376 but not S484. Knockdown of IKKα, dominant-negative IKKα, and RORγt mutants incapable of interacting with IKKα all decrease Th17 differentiation. Furthermore, nonphosophorylatable RORγt mutant (S376A) impairs whereas phosphomimetic mutant (S376E) stimulates Th17 differentiation independent of IKKα. Therefore, IKKα-dependent phosphorylation of S376 stimulated whereas IKKα-independent phosphorylation of S484 inhibited RORγt function in Th17 differentiation.

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.

Histone Lys-specific demethylases (KDMs) play a key role in many biological processes through epigenetic mechanisms. However, the role of KDMs in inflammatory responses to oral bacterial infection is poorly understood. Here, we show a novel regulatory role of KDM3C in inflammatory responses to oral bacterial infection. KDM3C expression is transiently suppressed in human and mouse macrophages exposed to LPS from Porphyromonas gingivalis (Pg LPS). Loss of KDM3C in both human and mouse macrophages led to notable induction of proinflammatory cytokines in response to Pg LPS stimulation. Also, KDM3C depletion led to strong induction of p65 phosphorylation and accelerated nuclear translocation in cells exposed to Pg LPS. Kdm3C knockout (KO) in mice led to increased alveolar bone destruction upon induction of experimental periodontitis or pulp exposure compared with those of the wild-type (WT) littermates. The Kdm3C KO mice also revealed an increased number of osteoclasts juxtaposed to the bony lesions. We also confirmed enhanced osteoclastogenesis by bone marrow-derived macrophages isolated from the Kdm3C KO compared with the WT controls. These findings suggest an anti-inflammatory function of KDM3C in regulating the inflammatory responses against oral bacterial infection through suppression of NF-κB signaling and osteoclastogenesis.-Lee, J. Y., Mehrazarin, S., Alshaikh, A., Kim, S., Chen, W., Lux, R., Gwack, Y., Kim, R. H., Kang, M. K. Histone Lys demethylase KDM3C demonstrates anti-inflammatory effects by suppressing NF-κB signaling and osteoclastogenesis.

The current optogenetic toolkit lacks a robust single-component Ca2+-selective ion channel tailored for remote control of Ca2+ signaling in mammals. Existing tools are either derived from engineered channelrhodopsin variants without strict Ca2+ selectivity or based on the stromal interaction molecule 1 (STIM1) that might crosstalk with other targets. Here, we describe the design of a light-operated Ca2+ channel (designated LOCa) by inserting a plant-derived photosensory module into the intracellular loop of an engineered ORAI1 channel. LOCa displays biophysical features reminiscent of the ORAI1 channel, which enables precise optical control over Ca2+ signals and hallmark Ca2+-dependent physiological responses. Furthermore, we demonstrate the use of LOCa to modulate aberrant hematopoietic stem cell self-renewal, transcriptional programming, cell suicide, as well as neurodegeneration in a Drosophila model of amyloidosis., Existing optogenetic methods to induce calcium mobilisation lack selectivity and specificity. Here, the authors design and engineer a single-component light-operated calcium channel to provide optical control over calcium signals and calcium-dependent physiological responses: LOCa.

Emerging evidence indicates that Orai1, a key calcium channel for store-operated Ca2+ entry, is associated with human cancer. However, the underlying mechanism by which Orai1 regulates cancer progression remains unknown. Here we report that intracellular level of Orai1 is increased in a stepwise manner during oral/oropharyngeal carcinogenesis and highly expressed in cancer stem-like cell (CSC)-enriched populations of human oral/oropharyngeal squamous cell carcinoma (OSCC). Ectopic Orai1 expression converted non-tumorigenic immortalized oral epithelial cells to malignant cells that showed CSC properties, e.g., self-renewal capacity, increased ALDH1HIGH cell population, increased key stemness transcription factors, and enhanced mobility. Conversely, inhibition of Orai1 suppressed tumorigenicity and CSC phenotype of OSCC, indicating that Orai1 could be an important element for tumorigenicity and stemness of OSCC. Mechanistically, Orai1 activates its major downstream effector molecule, NFATc3. Knockdown of NFATc3 in the Orai1-overexpressing oral epithelial cells abrogates the effect of Orai1 on CSC phenotype. Moreover, antagonist of NFAT signaling also decreases CSC phenotype, implying the functional importance of Orai1/NFAT axis in OSCC CSC regulation. Our study identifies Orai1 as a novel molecular determinant for OSCC progression by enhancing cancer stemness, suggesting that inhibition of Orai1 signaling may offer an effective therapeutic modality against OSCC.

More than 60 Rab GTPases exist in the human genome to regulate vesicle trafficking between organelles. Rab GTPases are members of the Ras GTPase superfamily that broadly control budding, uncoating, motility and fusion of vesicles in most cell types. Rab proteins interconvert between active, GTP-bound form and inactive, GDP-bound form. In their active conformation, they interact with various effector molecules to carry out diverse functions. Rab GTPases are usually small containing only a GTPase domain with a C-terminal prenylation site for membrane anchoring. Recently, we identified a large G protein, CRACR2A (CRAC channel regulator 2A), which uncovers novel functions of Rab GTPases. First, CRACR2A encodes a large Rab GTPase containing multiple functional domains contrary to small Rab GTPases. Second, CRACR2A plays an unexpected role in regulating intracellular signaling pathways important for T cell activation, unlike the canonical role of small Rab GTPases. Vesicles containing CRACR2A bud out from the proximal Golgi area and translocate into the immunological synapse to activate these signaling pathways. Third, instead of recycling, CRACR2A is consumed by a unidirectional pathway. These events are sequentially regulated by prenylation, GTP binding, protein interaction with a signaling adaptor Vav1, and degradation. Together, our findings reveal a novel function of a large Rab GTPase in intracellular signaling pathways, which may be shared by other large Rab GTPases, Rab44 and Rab45.

Orai1 is a pore-subunit of store-operated Ca(2+) release-activated Ca(2+) (CRAC) channel that mediates Ca(2+) influx in most non-excitable cells via store-operated Ca(2+) entry (SOCE) mechanism. We previously demonstrated that Orai1 is involved in mediating osteogenic potential of mesenchymal stem cells (MSCs), but the underlying mechanism of this function remains unknown. Here, we report that Orai1 mediates osteogenic differentiation via bone morphogenic protein (BMP) signaling pathway in bone marrow MSCs (BMSCs). In osteogenic conditions, BMSCs derived from wild-type mice underwent osteoblastic differentiation and induced mineralization as demonstrated by increased alkaline phosphatase activity and alizarin red S staining, respectively. The expression of Runx2, a master regulator of osteoblast differentiation, and osteogenic differentiation markers were markedly increased in wild-type BMSCs under osteogenic conditions. In contrast, osteogenic conditions failed to induce such effects in BMSCs derived from Orai1-deficient (Orai1(-/-)) mice, indicating that Orai1 is, in part, necessary for osteogenic differentiation of MSCs. We also found that BMP2 successfully induced phosphorylation of Smad1/5/8, the immediate effector molecules of BMP signaling, in wild-type BMSCs, but failed to do so in Orai1(-/-) BMSCs. Downstream target genes of BMP signaling pathway were consistently increased by osteogenic conditions in wild-type BMSCs, but not in Orai1(-/-) BMSCs, suggesting a novel molecular link between Orai1 and BMP signaling pathway in the osteogenic differentiation process. Further functional studies demonstrated that activation of BMP signaling rescues osteogenic differentiation capacity of Orai1(-/-) BMSCs. In conclusion, Orai1 regulates osteogenic differentiation through BMP signaling, and the Orai1-BMP signaling may be a possible therapeutic target for treating bone-related diseases.

SUMMARY Sustained activation of the Ca2+-release-activated Ca2+ (CRAC) channel is pivotal for effector T cell responses. The mechanisms underlying this sustainability remain poorly understood. We find that plasma membrane localization of ORAI1, the pore subunit of CRAC channels, is limited in effector T cells, with a significant fraction trapped in intracellular vesicles. From a targeted screen, we identify an essential component of ORAI1+ vesicles, naked cuticle homolog 2 (NKD2). Mechanistically, NKD2, an adaptor molecule activated by signaling pathways downstream of T cell receptors, orchestrates trafficking and insertion of ORAI1 + vesicles to the plasma membrane. Together, our findings suggest that T cell receptor (TCR)-stimulation-dependent insertion of ORAI1 into the plasma membrane is essential for sustained Ca2+ signaling and cytokine production in T cells., Graphical abstract, In brief Wu et al. address the mechanism underlying sustained activation of the Ca2+-release-activated Ca2+ (CRAC) channel that is pivotal for effector T cell responses. They report that NKD2-mediated insertion of ORAI1 into the plasma membrane after TCR stimulation is essential for sustained Ca2+ signaling and cytokine production in T cells.

Loss of function or null mutations of Stromal interaction molecule 1 (STIM1) are known to cause early-onset combined immunodeficiency (CID) disease with recurrent and chronic infections, autoimmunity, haemolytic anaemia, ectodermal dysplasia, muscular weakness and myalgia. here we report of novel STIM1 deficiency and 6 year follow up of two previous cases associated with mild immunological phenotype

Th17 cells are major players in multiple autoimmune diseases and are developmentally contingent on reciprocal functionality between the transcription factor Retineic acid receptor-related orphan nuclear receptor gamma (RORγt) and Forkhead box protein P3 (Foxp3). Here we deciphered a previously unappreciated role of Steroid receptor coactivator 1 (SRC1) in defining the lineage decision for the development of Th17 versus induced T-regulatory (iTreg) cells. We demonstrate that SRC1 functions as a critical coactivator for RORγt in vivo to promote the functional dominance of RORγt over Foxp3 and thus establishing an unopposed Th17 differentiation program. In the absence of SRC1, T cell polarization resulted in decreased IL-17+ and increased Foxp3+ cells during both in vitro differentiation and in vivo development of experimental autoimmune encephalomyelitis. Mechanistically, T cell receptor (TCR) signaling molecule protein kinase C theta (PKC-θ)–mediated phosphorylation of SRC1 is important for inducing enhanced RORγt–SRC1 interaction, stable DNA binding, and resultant IL-17A transcription. Furthermore, phospho-SRC1–mediated recruitment of CARM1 induced prominent asymmetric dimethylation of H3R17 while preventing repressive H3K9 trimethylation and hence further modifying the IL-17 locus for optimal transcription. Moreover, binding of phospho-SRC1 to RORγt displaced bound Foxp3, leading to prompt degradation of the dissociated Foxp3 via a ubiquitin–proteosomal pathway and hence reversing the inhibitory action of Foxp3 on RORγt activity. Thus, SRC1 acts as a crucial molecular mediator to integrate positive PKC-θ–dependent TCR signals to induce peak RORγt activity and establish phenotypic dominance of Th17 over the iTreg pathway.

Engagement of T cell receptors (TCRs) with cognate antigens triggers cascades of signaling pathways in helper T cells. TCR signaling is essential for the effector function of helper T cells including proliferation, differentiation and cytokine production. It also modulates effector T cell fate by inducing cell death, anergy (non-responsiveness), exhaustion and generation of regulatory T cells. One of the main axes of TCR signaling is the Ca(2+)-calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway. Stimulation of TCRs triggers depletion of intracellular Ca(2+) store and in turn, activates store-operated Ca(2+) entry (SOCE) to raise the intracellular Ca(2+) concentration. SOCE in T cells is mediated by the Ca(2+) release-activated Ca(2+) (CRAC) channels, which have been very well characterized in terms of their electrophysiological properties. Identification of STIM1 as a sensor to detect depletion of the endoplasmic reticulum (ER) Ca(2+) store and Orai1 as the pore subunit of CRAC channels has dramatically advanced our understanding of the regulatory mechanism of Ca(2+) signaling in T cells. In this review, we discuss our current understanding of Ca(2+) signaling in T cells with specific focus on the mechanism of CRAC channel activation and regulation via protein interactions. In addition, we will discuss the role of CRAC channels in effector T cells based on the analyses of genetically modified animal models.

Store-operated Ca2+ entry (SOCE) is a ubiquitous mechanism in eukaryotic cells to elevate the intracellular Ca2+ concentration and stimulate downstream signaling pathways. SOCE is especially important for Ca2+ entry in cells with immune receptors, including T cells, B cells, and mast cells. Under resting conditions, cytoplasmic Ca2+ concentration ([Ca2+]) is very low (∼100 nM) in T cells, while that in the endoplasmic reticulum (ER), which serves as an intracellular Ca2+ store, is much higher (∼4,000–10,000-fold higher—0.4–1.0 mM) [1,2]. Extracellular [Ca2+] reaches almost 2 mM concentration, establishing a huge [Ca2+] gradient between the extracellular space and the cytoplasm (∼20,000-fold). Therefore, dynamic regulation of Ca2+ flow occurs constantly to maintain these gradients even in resting T cells. When T cells are activated, there is a sustained increase in intracellular Ca2+ concentration ([Ca2+]i), which is initiated by emptying of the ER Ca2+ stores. The increase in cytoplasmic [Ca2+] by depletion of ER Ca2+ stores can be minor especially in T cells, due to small volume of the ER. Instead, ER Ca2+ depletion induces Ca2+ entry via store-operated Ca2+ (SOC) channels, which raises [Ca2+]i up to micromolar concentrations [3–6]. Therefore, SOCE via the Ca2+ release-activated Ca2+ (CRAC) channels is a primary mechanism for activation of Ca2+ signaling in T cells. Upon pathogen infection or self-peptide presentation in autoimmunity, antigen-presenting cells (APCs, e.g., dendritic cells or B cells) present antigens on their surface together with major histocompatibility complex class II to activate CD4+ helper T cells. Antigen engagement of T cell receptors (TCRs) triggers a conformational change of TCRαβ chain, which induces a cascade of tyrosine phosphorylation events mediated by CD3ζ chain-ZAP70 (zeta chain-associated protein kinase 70) complexes [7,8]. This results in phosphorylation of a signaling adaptor Lat, which dissociates from CD3ζ chain-ZAP70 complex and activates phospholipase C-γ (PLCγ). In turn, PLCγ hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol trisphosphate (IP3) and diacylglycerol. IP3 binds to the IP3 receptor on the ER membrane and releases Ca2+ from the ER into the cytoplasm, and this depletion leads to activation of CRAC channels. The CRAC channel is a prototype SOC channel, well characterized in immune cells. Because ER Ca2+ store especially in T cells is limited, SOCE via CRAC channels is important to maintain elevated levels of [Ca2+]i, which are required for activation of downstream signaling pathways including the protein kinase C, extracellular signal-regulated kinases, or nuclear factor of activated T cell (NFAT) pathways to affect the transcriptional programs for generating a productive immune response (see Chapter 5). Defective function or lack of expression of the CRAC channel components causes severe combined immune deficiency in humans [9]. Hence, an in-depth understanding of CRAC channel-mediated Ca2+ signaling in T cells is crucial for developing drug therapies for immune deficiency or inflammatory disorders.Identification of essential components of CRAC channels revealed a unique mechanism of its activation, which is mediated by protein interactions. Genome-wide RNAi screens identified Orai1 as a pore subunit of the CRAC channels [10–13]. Prior to identification of Orai1, limited RNAi screens in Drosophila and HeLa cells had identified STIM1, a Ca2+-binding protein localized predominantly in the ER as an important regulator of SOCE [14–16]. STIM1 senses [Ca2+]ER via its N-terminal EF-hands and gates Orai1 by direct interaction. The EF-hand of STIM1 has a low affinity for Ca2+, between 0.2 and 0.6 mM [17], and remains Ca2+ bound at rest. Under resting conditions, Orai1 and STIM1 are homogeneously distributed at the plasma membrane (PM) and the ER membrane, respectively. Upon ER Ca2+ depletion triggered by TCR stimulation, STIM1 loses Ca2+ binding, multimerizes, translocates to the ER-PM junctions, mediates clustering of Orai1 on the PM, and stimulates Ca2+ entry (Figure 4.1a) [14–16]. Detailed studies have identified a minimal domain of STIM1 necessary for activation of Orai1 as the CRAC activation domain (CAD)/STIM1-Orai1 activating region (SOAR) that directly binds to the cytosolic N and C termini of Orai1 [18,19] (see Chapter 2). This region, containing coiled-coil (CC) domains 2 and 3 of STIM1 is located in its cytoplasmic C terminus (Figure 4.1a). Further studies showed that Ca2+-bound STIM1 under resting conditions exhibits a folded structure mediated by intramolecular protein interaction between the positively charged residues within its CAD/SOAR domain and the negatively charged, autoinhibitory region preceding the CAD/SOAR domain, located in the CC1 region [20]. STIM1 activation requires unfolding of this intramolecular interaction to allow the basic residues within the CAD/SOAR domain to interact with the acidic residues within the C terminus of Orai1 [20]. While Orai1 and STIM1 are the major components of CRAC channels, multiple auxiliary proteins have been shown to regulate CRAC channel function. Some of these have been reviewed in detail before [21,22] and are only briefly summarized in Table 4.1. In this chapter, we focus on the recently identified molecules regulating the function of Orai1 and STIM1.

TRPV1 is a Ca(2+)-permeable channel studied mostly as a pain receptor in sensory neurons. However, its role in other cell types is poorly understood. Here we found that TRPV1 was functionally expressed in CD4(+) T cells, where it acted as a non-store-operated Ca(2+) channel and contributed to T cell antigen receptor (TCR)-induced Ca(2+) influx, TCR signaling and T cell activation. In models of T cell-mediated colitis, TRPV1 promoted colitogenic T cell responses and intestinal inflammation. Furthermore, genetic and pharmacological inhibition of TRPV1 in human CD4(+) T cells recapitulated the phenotype of mouse Trpv1(-/-) CD4(+) T cells. Our findings suggest that inhibition of TRPV1 could represent a new therapeutic strategy for restraining proinflammatory T cell responses.

CRAC channel regulator 2A (CRACR2A) is expressed abundantly in T cells and acts as a signal transmitter between T cell receptor stimulation and activation of the Ca(2+)-NFAT and JNK-AP1 pathways. CRACR2A has been linked to human diseases in numerous genome-wide association studies and was shown to be one of the most sensitive targets of the widely used statin drugs. However, the physiological role of CRACR2A in T cell functions remains unknown. In this study, using transgenic mice for tissue-specific deletion, we show that CRACR2A promotes Th1 responses and effector function of Th17 cells. CRACR2A was abundantly expressed in Th1 and Th17 cells. In vitro, deficiency of CRACR2A decreased Th1 differentiation under non-polarizing conditions, while presence of polarizing cytokines compensated this defect. Transcript analysis showed that weakened TCR signaling by deficiency of CRACR2A failed to promote Th1 transcriptional program. In vivo, conditional deletion of CRACR2A in T cells ameliorated Th1 responses to acute lymphocytic choriomeningitis virus infection and imparted resistance to experimental autoimmune encephalomyelitis. Analysis of central nervous system from experimental autoimmune encephalomyelitis-induced mice showed impaired effector functions of both Th1 and Th17 cell types, which correlated with decreased pathogenicity. Collectively, our findings demonstrate the requirement of CRACR2A-mediated TCR signaling in Th1 responses as well as pathogenic conversion of Th17 cells, that occurs at the site of inflammation.

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER) signaling adaptor that is essential for the type I interferon response to DNA pathogens. Aberrant activation of STING is linked to the pathology of autoimmune and autoinflammatory diseases. The rate-limiting step for the activation of STING is its translocation from the ER to the ER-Golgi intermediate compartment. Here, we found that deficiency in the Ca2+ sensor stromal interaction molecule 1 (STIM1) caused spontaneous activation of STING and enhanced expression of type I interferons under resting conditions in mice and a patient with combined immunodeficiency. Mechanistically, STIM1 associated with STING to retain it in the ER membrane, and coexpression of full-length STIM1 or a STING-interacting fragment of STIM1 suppressed the function of dominant STING mutants that cause autoinflammatory diseases. Furthermore, deficiency in STIM1 strongly enhanced the expression of type I interferons after viral infection and prevented the lethality of infection with a DNA virus in vivo. This work delineates a STIM1-STING circuit that maintains the resting state of the STING pathway.

T cell receptor (TCR) stimulation plays a crucial role in development, homeostasis, proliferation, cell death, cytokine production, and differentiation of T cells. Thus, in depth understanding of TCR signalling is crucial for development of therapy targeting inflammatory diseases, improvement of vaccination efficiency, and cancer therapy utilizing T cell-based strategies. TCR activation turns on various signalling pathways, one of the important one being the Ca(2+)-calcineurin-nuclear factor of activated T cells (NFAT) signalling pathway. Stimulation of TCRs triggers depletion of intracellular Ca(2+) store and in turn, initiates store-operated Ca(2+) entry (SOCE), one of the major mechanisms to raise the intracellular Ca(2+) concentrations in T cells. Ca(2+)-release-activated-Ca(2+) (CRAC) channels are a prototype of store-operated Ca(2+) (SOC) channels in immune cells that are very well characterized. Recent identification of STIM1 as the endoplasmic reticulum (ER) Ca(2+) sensor and Orai1 as the pore subunit has dramatically advanced the understanding of CRAC channels and provides a molecular tool to investigate the physiological outcomes of Ca(2+) signalling during immune responses. In this review, we focus on our current understanding of CRAC channel activation, regulation, and downstream calcineurin-NFAT signaling pathway.

Podocyte, the gatekeeper of the glomerular filtration barrier, is a primary target for growth factor and Ca

Store-operated Ca(2+) (SOC) entry is one of the major mechanisms to raise intracellular Ca(2+) concentration in non-excitable cells. Ca(2+)-release-activated Ca(2+) (CRAC) channels are a subtype of SOC channels that are extensively characterized in immune cells. Identification of STIM1 as an endoplasmic reticulum Ca(2+) sensor and Orai1 as the pore subunit has dramatically advanced the molecular understanding of CRAC channels. Recent efforts have focused on understanding the physiological aspects of CRAC channels at an organism level using transgenic animal models and at a molecular level using electrophysiological and biochemical tools. In this review, we summarize our current understanding of the interacting partners of Orai and STIM proteins in the regulation of CRAC channel activity and other non-CRAC channel-related functions.

DRAK2 is a serine/threonine kinase highly enriched in lymphocytes that raises the threshold for T cell activation and maintains T cell survival following productive activation. T cells lacking DRAK2 are prone to activation under suboptimal conditions and exhibit enhanced calcium responses to AgR stimulation. Despite this, mice lacking DRAK2 are resistant to organ-specific autoimmune diseases due to defective autoreactive T cell survival. DRAK2 kinase activity is induced by AgR signaling, and in this study we show that the induction of DRAK2 activity requires Ca2+ influx through the Ca2+ release-activated Ca2+ channel formed from Orai1 subunits. Blockade of DRAK2 activity with the protein kinase D (PKD) inhibitor Gö6976 or expression of a kinase-dead PKD mutant prevented activation of DRAK2, whereas a constitutively active PKD mutant promoted DRAK2 function. Knockdown of PKD in T cells strongly blocked endogenous DRAK2 activation following TCR ligation, implicating PKD as an essential intermediate in the activation of DRAK2 by Ca2+ influx. Furthermore, we identify DRAK2 as a novel substrate of PKD, and demonstrate that DRAK2 and PKD physically interact under conditions that activate PKD. Mitochondrial generation of reactive oxygen intermediates was necessary and sufficient for DRAK2 activation in response to Ca2+ influx. Taken together, DRAK2 and PKD form a novel signaling module that controls calcium homeostasis following T cell activation.

Orai1 and STIM1 are critical components of Ca(2+) release-activated Ca(2+) (CRAC) channels that mediate store-operated Ca(2+) entry (SOCE) in immune cells. Although it is known that Orai1 and STIM1 co-cluster and physically interact to mediate SOCE, the cytoplasmic machinery modulating these functions remains poorly understood. We sought to find modulators of Orai1 and STIM1 using affinity protein purification and identified a novel EF-hand protein, CRACR2A (also called CRAC regulator 2A, EFCAB4B or FLJ33805). We show that CRACR2A interacts directly with Orai1 and STIM1, forming a ternary complex that dissociates at elevated Ca(2+) concentrations. Studies using knockdown mediated by small interfering RNA (siRNA) and mutagenesis show that CRACR2A is important for clustering of Orai1 and STIM1 upon store depletion. Expression of an EF-hand mutant of CRACR2A enhanced STIM1 clustering, elevated cytoplasmic Ca(2+) and induced cell death, suggesting its active interaction with CRAC channels. These observations implicate CRACR2A, a novel Ca(2+) binding protein that is highly expressed in T cells and conserved in vertebrates, as a key regulator of CRAC channel-mediated SOCE.

The proteins STIM1 and Orai1 are the long sought components of the store-operated channels required in T-cell activation. However, little is known about the interaction of these proteins in T-cells after engagement of the T-cell receptor. We found that T-cell receptor engagement caused STIM1 and Orai1 to colocalize in puncta near the site of stimulation and accumulate in a dense structure on the opposite side of the T-cell. FRET measurements showed a close interaction between STIM1 and Orai1 both in the puncta and in the dense cap-like structure. The formation of cap-like structures did not entail rearrangement of the entire endoplasmic reticulum. Cap formation depended on TCR engagement and tyrosine phosphorylation, but not on channel activity or Ca2+influx. These caps were very dynamic in T-cells activated by contact with superantigen pulsed B-cells and could move from the distal pole to an existing or a newly forming immunological synapse. One function of this cap may be to provide preassembled Ca2+channel components to existing and newly forming immunological synapses.

The transcription factor retinoid acid–related orphan receptor γ t (RORγt) directs the differentiation of Th17 cells. Th17 cells mediate pathological immune responses responsible for autoimmune diseases, including psoriasis and multiple sclerosis. Previous studies focused on RORγt target genes and their function in Th17 differentiation. In this study, we assessed posttranscriptional regulation of RORγt and identified a functional ubiquitination site, K446. Mutation of K446 to arginine to prevent ubiquitination greatly enhanced recruitment of steroid receptor coactivator 1 (SRC1), a coactivator critical for RORγt activity. Correspondingly, the K446 to arginine mutation potentiated Th17 differentiation. We also showed that ubiquitin-specific protease (USP)15 interacted with RORγt, removed ubiquitin from K446, and stimulated RORγt activity by enhancing coactivator SRC1 recruitment. Knockdown of USP15 or expression of inactive USP15 impaired Th17 differentiation, suggesting a positive role for USP15-mediated deubiquitination of RORγt in Th17 differentiation. Therefore, ubiquitination of K446 limits RORγt-mediated Th17 differentiation by inhibiting the recruitment of coactivator SRC1. Our study will inform the development of treatments that target RORγt ubiquitination pathways to limit Th17-mediated autoimmunity.

The major function of the lymphatic system is to drain interstitial fluid from tissue. Functional drainage causes increased fluid flow that triggers lymphatic expansion, which is conceptually similar to hypoxia-triggered angiogenesis. Here, we have identified a mechanotransduction pathway that translates laminar flow-induced shear stress to activation of lymphatic sprouting. While low-rate laminar flow commonly induces the classic shear stress responses in blood endothelial cells and lymphatic endothelial cells (LECs), only LECs display reduced Notch activity and increased sprouting capacity. In response to flow, the plasma membrane calcium channel ORAI1 mediates calcium influx in LECs and activates calmodulin to facilitate a physical interaction between Krüppel-like factor 2 (KLF2), the major regulator of shear responses, and PROX1, the master regulator of lymphatic development. The PROX1/KLF2 complex upregulates the expression of DTX1 and DTX3L. DTX1 and DTX3L, functioning as a heterodimeric Notch E3 ligase, concertedly downregulate NOTCH1 activity and enhance lymphatic sprouting. Notably, overexpression of the calcium reporter GCaMP3 unexpectedly inhibited lymphatic sprouting, presumably by disturbing calcium signaling. Endothelial-specific knockouts of Orai1 and Klf2 also markedly impaired lymphatic sprouting. Moreover, Dtx3l loss of function led to defective lymphatic sprouting, while Dtx3l gain of function rescued impaired sprouting in Orai1 KO embryos. Together, the data reveal a molecular mechanism underlying laminar flow-induced lymphatic sprouting.

Orai1 and stromal interaction molecule 1 (STIM1) mediate store-operated Ca(2+) entry (SOCE) in immune cells. STIM1, an endoplasmic reticulum (ER) Ca(2+) sensor, detects store depletion and interacts with plasma membrane (PM)-resident Orai1 channels at the ER-PM junctions. However, the molecular composition of these junctions in T cells remains poorly understood. Here, we show that junctophilin-4 (JP4), a member of junctional proteins in excitable cells, is expressed in T cells and localized at the ER-PM junctions to regulate Ca(2+) signaling. Silencing or genetic manipulation of JP4 decreased ER Ca(2+) content and SOCE in T cells, impaired activation of the nuclear factor of activated T cells (NFAT) and extracellular signaling-related kinase (ERK) signaling pathways, and diminished expression of activation markers and cytokines. Mechanistically, JP4 directly interacted with STIM1 via its cytoplasmic domain and facilitated its recruitment into the junctions. Accordingly, expression of this cytoplasmic fragment of JP4 inhibited SOCE. Furthermore, JP4 also formed a complex with junctate, a Ca(2+)-sensing ER-resident protein, previously shown to mediate STIM1 recruitment into the junctions. We propose that the junctate-JP4 complex located at the junctions cooperatively interacts with STIM1 to maintain ER Ca(2+) homeostasis and mediate SOCE in T cells.

More than 60 members of the Rab family of guanosine triphosphatases (GTPases) exist in the human genome. Rab GTPases are small proteins that are primarily involved in the formation, trafficking, and fusion of vesicles. We showed thatCRACR2A(Ca(2+) release-activated Ca(2+) channel regulator 2A) encodes a lymphocyte-specific large Rab GTPase that contains multiple functional domains, including EF-hand motifs, a proline-rich domain (PRD), and a Rab GTPase domain with an unconventional prenylation site. Through experiments involving gene silencing in cells and knockout mice, we demonstrated a role for CRACR2A in the activation of the Ca(2+) and c-Jun N-terminal kinase signaling pathways in response to T cell receptor (TCR) stimulation. Vesicles containing this Rab GTPase translocated from near the Golgi to the immunological synapse formed between a T cell and a cognate antigen-presenting cell to activate these signaling pathways. The interaction between the PRD of CRACR2A and the guanidine nucleotide exchange factor Vav1 was required for the accumulation of these vesicles at the immunological synapse. Furthermore, we demonstrated that GTP binding and prenylation of CRACR2A were associated with its localization near the Golgi and its stability. Our findings reveal a previously uncharacterized function of a large Rab GTPase and vesicles near the Golgi in TCR signaling. Other GTPases with similar domain architectures may have similar functions in T cells.

In cells of the immune system that are stimulated by antigen or antigen-antibody complexes, Ca(2+) entry from the extracellular medium is driven by depletion of endoplasmic reticulum Ca(2+) stores and occurs through specialized store-operated Ca(2+) channels known as Ca(2+)-release-activated Ca(2+) (CRAC) channels. The process of store-operated Ca(2+) influx is essential for short-term as well as long-term responses by immune-system cells. Short-term responses include mast cell degranulation and killing of target cells by effector cytolytic T cells, whereas long-term responses typically involve changes in gene transcription and include T and B cell proliferation and differentiation. Transcription downstream of Ca(2+) influx is in large part funneled through the transcription factor nuclear factor of activated T cells (NFAT), a heavily phosphorylated protein that is cytoplasmic in resting cells, but that enters the nucleus when dephosphorylated by the calmodulin-dependent serine/threonine phosphatase calcineurin. The importance of the Ca(2+)/calcineurin/NFAT signalling pathway for lymphocyte activation is underscored by the finding that the underlying defect in a family with a hereditary severe combined immune deficiency (SCID) syndrome is a defect in CRAC channel function, store-operated Ca(2+) entry, NFAT activation and transcription of cytokines, chemokines and many other NFAT target genes whose transcription is essential for productive immune defence. We recently used a two-pronged genetic approach to identify Orai1 as the pore subunit of the CRAC channel. On the one hand, we initiated a positional cloning approach in which we utilised genome-wide single nucleotide polymorphism (SNP) mapping to identify the genomic region linked to the mutant gene in the SCID family described above. In parallel, we used a genome-wide RNAi screen in Drosophila to identify critical regulators of NFAT nuclear translocation and store-operated Ca(2+) entry. These approaches, together with subsequent mutational and electrophysiological analyses, converged to identify human Orai1 as a pore subunit of the CRAC channel and as the gene product mutated in the SCID patients.