Faculty Bibliography

Eccrine sweat glands are essential for sweating and thermoregulation in humans. Loss-of-function mutations in the Ca2+ release-activated Ca2+ (CRAC) channel genes ORAI1 and STIM1 abolish store-operated Ca2+ entry (SOCE), and patients with these CRAC channel mutations suffer from anhidrosis and hyperthermia at high ambient temperatures. Here we have shown that CRAC channel-deficient patients and mice with ectodermal tissue-specific deletion of Orai1 (Orai1K14Cre) or Stim1 and Stim2 (Stim1/2K14Cre) failed to sweat despite normal sweat gland development. SOCE was absent in agonist-stimulated sweat glands from Orai1K14Cre and Stim1/2K14Cre mice and human sweat gland cells lacking ORAI1 or STIM1 expression. In Orai1K14Cre mice, abolishment of SOCE was associated with impaired chloride secretion by primary murine sweat glands. In human sweat gland cells, SOCE mediated by ORAI1 was necessary for agonist-induced chloride secretion and activation of the Ca2+-activated chloride channel (CaCC) anoctamin 1 (ANO1, also known as TMEM16A). By contrast, expression of TMEM16A, the water channel aquaporin 5 (AQP5), and other regulators of sweat gland function was normal in the absence of SOCE. Our findings demonstrate that Ca2+ influx via store-operated CRAC channels is essential for CaCC activation, chloride secretion, and sweat production in humans and mice.

T follicular helper (Tfh) cells promote affinity maturation of B cells in germinal centers (GCs), whereas T follicular regulatory (Tfr) cells limit the GC reaction. Store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels mediated by STIM and ORAI proteins is a fundamental signaling pathway in T lymphocytes. Conditional deletion of Stim1 and Stim2 genes in T cells abolished SOCE and strongly reduced antibody-mediated immune responses following viral infection caused by impaired differentiation and function of Tfh cells. Conversely, aging Stim1Stim2-deficient mice developed humoral autoimmunity with spontaneous autoantibody production due to abolished Tfr cell differentiation in the presence of residual Tfh cells. Mechanistically, SOCE controlled Tfr and Tfh cell differentiation through NFAT-mediated IRF4, BATF, and Bcl-6 transcription-factor expression. SOCE had a dual role in controlling the GC reaction by regulating both Tfh and Tfr cell differentiation, thus enabling protective B cell responses and preventing humoral autoimmunity.

The function of CD4+ T cells is dependent on Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels formed by ORAI proteins. To investigate the role of ORAI1 in proinflammatory Th1 and Th17 cells and autoimmune diseases, we genetically and pharmacologically modulated ORAI1 function. Immunization of mice lacking Orai1 in T cells with MOG peptide resulted in attenuated severity of experimental autoimmune encephalomyelitis (EAE). The numbers of T cells and innate immune cells in the CNS of ORAI1-deficient animals were strongly reduced along with almost completely abolished production of IL-17A, IFN-gamma, and GM-CSF despite only partially reduced Ca2+ influx. In Th1 and Th17 cells differentiated in vitro, ORAI1 was required for cytokine production but not the expression of Th1- and Th17-specific transcription factors T-bet and RORgammat. The differentiation and function of induced regulatory T cells, by contrast, was independent of ORAI1. Importantly, induced genetic deletion of Orai1 in adoptively transferred, MOG-specific T cells was able to halt EAE progression after disease onset. Likewise, treatment of wild-type mice with a selective CRAC channel inhibitor after EAE onset ameliorated disease. Genetic deletion of Orai1 and pharmacological ORAI1 inhibition reduced the leukocyte numbers in the CNS and attenuated Th1/Th17 cell-mediated cytokine production. In human CD4+ T cells, CRAC channel inhibition reduced the expression of IL-17A, IFN-gamma, and other cytokines in a dose-dependent manner. Taken together, these findings support the conclusion that Th1 and Th17 cell function is particularly dependent on CRAC channels, which could be exploited as a therapeutic approach to T cell-mediated autoimmune diseases.

Introduction: Ca²⁺ release-activated Ca²⁺ (CRAC) channels and their activators stromal interaction molecule (STIM) 1 and 2 are the main regulators of calcium entry in T Lymphocytes through a process known as store-operated Ca²⁺ entry (SOCE). SOCE results in the activation of calcineurin and other downstream signals with important effects on lymphocyte function. Notch-1 is a protein that is essential for T lymphocyte development. Activating mutations of Notch-1 occurs in about 60% of T-cell acute lymphoblastic leukemia (T-ALL). Introduction of constitutively active forms of Notch-1 in hematopoietic stem cells (HSC) induces T-ALL in mice, providing a useful animal model for the study of leukemia. Methods: To study the role of CRAC channels in T-ALL, we used a mouse model in which c-kit+ HSC from wild-type (WT) and STIM1/STIM2-deficient mice (DKO) were retrovirally transduced with the intracellular Notch-1 domain (ICN1). Transduced HSC were injected into lethally irradiated C57BL/6 mice. Following leukemia development, mice were analyzed for survival and cellular and molecular activity of leukemic cells using various techniques including histology, flow cytometry, RT-PCR and gene array expression analysis. In addition, we used the human T-ALL cell line CEM, in which we introduced a dominant negative form of the CRAC channel subunit ORAI1 (ORAI1-DN) that abolishes CRAC channel function and SOCE, for coculture with the human bone marrow stromal cell line HS5. Results: Mice injected with wild-type HSC transduced with ICN1 succumbed from T-ALL characterized by the presence of CD4+ CD8+ leukemic T cell blasts in the blood, bone marrow and infiltrating organs within 3 to 4 weeks after transfer of HSC. By contrast, mice that had received ICN1 transduced STIM1/2 deficient HSC lived approximately twice as long. The survival benefit was not due to differences in leukemic cell numbers or in proliferation and apoptosis of leukemic cells. Histologies of the bone marrow and spleen of WT leukemic mice showed necrotic lesions, pronounced neutrophil infiltration, the presence of histiocytes engulfing red blood cells (RBC) indicative of severe inflammation. No signs of necrosis and inflammation were present in DKO leukemic mice. Paralleling the inflammation and destruction of the bone marrow environment, WT leukemic mice showed greatly diminished presence of erythroid precursors (EP) in the bone marrow whereas EP frequencies in DKO leukemic mice were similar to those in non-leukemic mice. In line with findings in mice, we observed that human leukemic CEM T cells reduced the viability of HS5 stromal cells in a contact-dependent manner. This cytotoxic effect of CEM cells depended on CRAC channel function as CEM cells transduced with ORAI1-DN had little effect on HS5 viability. Conclusion: These results suggest that CRAC channels are important for the function of T-ALL cells and their effects on the organs they infiltrate, most notably the bone marrow. Inhibition of CRAC channel function prolongs survival of mice with T-ALL potentially by attenuating the cytotoxic effects of leukemic T cells on their environment and on hematopoiesis. Further studies are underway to understand the mechanisms by which CRAC channels regulate leukemic T cell function

Dental enamel formation requires large quantities of Ca(2+) yet the mechanisms mediating Ca(2+) dynamics in enamel cells are unclear. Store-operated Ca(2+) entry (SOCE) channels are important Ca(2+) influx mechanisms in many cells. SOCE involves release of Ca(2+) from intracellular pools followed by Ca(2+) entry. The best-characterized SOCE channels are the Ca(2+) release-activated Ca(2+) (CRAC) channels. As patients with mutations in the CRAC channel genes STIM1 and ORAI1 show abnormal enamel mineralization, we hypothesized that CRAC channels might be an important Ca(2+) uptake mechanism in enamel cells. Investigating primary murine enamel cells, we found that key components of CRAC channels (ORAI1, ORAI2, ORAI3, STIM1, STIM2) were expressed and most abundant during the maturation stage of enamel development. Furthermore, inositol 1,4,5-trisphosphate receptor (IP3R) but not ryanodine receptor (RyR) expression was high in enamel cells suggesting that IP3Rs are the main ER Ca(2+) release mechanism. Passive depletion of ER Ca(2+) stores with thapsigargin resulted in a significant raise in [Ca(2+)]i consistent with SOCE. In cells pre-treated with the CRAC channel blocker Synta-66 Ca(2+) entry was significantly inhibited. These data demonstrate that enamel cells have SOCE mediated by CRAC channels and implicate them as a mechanism for Ca(2+) uptake in enamel formation.

Ca2+ release-activated Ca2+ (CRAC) channels mediate a specific form of Ca2+ influx called store-operated Ca2+ entry (SOCE) that contributes to the function of many cell types. CRAC channels are composed of ORAI1 proteins located in the plasma membrane, which form its ion-conducting pore. ORAI1 channels are activated by stromal interaction molecule (STIM) 1 and STIM2 located in the endoplasmic reticulum. Loss- and gain-of-function gene mutations in ORAI1 and STIM1 in human patients cause distinct disease syndromes. CRAC channelopathy is caused by loss-of-function mutations in ORAI1 and STIM1 that abolish CRAC channel function and SOCE; it is characterized by severe combined immunodeficiency (SCID)-like disease, autoimmunity, muscular hypotonia, and ectodermal dysplasia, with defects in sweat gland function and dental enamel formation. The latter defect emphasizes an important role of CRAC channels in tooth development. By contrast, autosomal dominant gain-of-function mutations in ORAI1 and STIM1 result in constitutive CRAC channel activation, SOCE, and increased intracellular Ca2+ levels that are associated with an overlapping spectrum of diseases, including nonsyndromic tubular aggregate myopathy (TAM) and York platelet and Stormorken syndromes. The latter two syndromes are defined, besides myopathy, by thrombocytopenia, thrombopathy, and bleeding diathesis. The fact that myopathy results from both loss- and gain-of-function mutations in ORAI1 and STIM1 highlights the importance of CRAC channels for Ca2+ homeostasis in skeletal muscle function. The cellular dysfunction and clinical disease spectrum observed in mutant patients provide important information about the molecular regulation of ORAI1 and STIM1 proteins and the role of CRAC channels in human physiology.

Activated T cells engage aerobic glycolysis and anabolic metabolism for growth, proliferation, and effector functions. We propose that a glucose-poor tumor microenvironment limits aerobic glycolysis in tumor-infiltrating T cells, which suppresses tumoricidal effector functions. We discovered a new role for the glycolytic metabolite phosphoenolpyruvate (PEP) in sustaining T cell receptor-mediated Ca2+-NFAT signaling and effector functions by repressing sarco/ER Ca2+-ATPase (SERCA) activity. Tumor-specific CD4 and CD8 T cells could be metabolically reprogrammed by increasing PEP production through overexpression of phosphoenolpyruvate carboxykinase 1 (PCK1), which bolstered effector functions. Moreover, PCK1-overexpressing T cells restricted tumor growth and prolonged the survival of melanoma-bearing mice. This study uncovers new metabolic checkpoints for T cell activity and demonstrates that metabolic reprogramming of tumor-reactive T cells can enhance anti-tumor T cell responses, illuminating new forms of immunotherapy.

Dental enamel formation is an intricate process tightly regulated by ameloblast cells. The correct spatiotemporal patterning of enamel matrix protein (EMP) expression is fundamental to orchestrate the formation of enamel crystals, which depend on a robust supply of Ca2+. In the extracellular milieu, Ca2+-EMP interactions occur at different levels. Despite its recognized role in enamel development, the molecular machinery involved in Ca2+ homeostasis in ameloblasts remains poorly understood. A common mechanism for Ca2+ influx is store-operated Ca2+ entry (SOCE). We evaluated the possibility that Ca2+ influx in enamel cells might be mediated by SOCE and the Ca2+ release-activated Ca2+ (CRAC) channel, the prototypical SOCE channel. Using ameloblast-like LS8 cells, we demonstrate that these cells express Ca2+-handling molecules and mediate Ca2+ influx through SOCE. As a rise in the cytosolic Ca2+ concentration is a versatile signal that can modulate gene expression, we assessed whether SOCE in enamel cells had any effect on the expression of EMPs. Our results demonstrate that stimulating LS8 cells or murine primary enamel organ cells with thapsigargin to activate SOCE leads to increased expression of Amelx, Ambn, Enam, Mmp20. This effect is reversed when cells are treated with a CRAC channel inhibitor. These data indicate that Ca2+ influx in LS8 cells and enamel organ cells is mediated by CRAC channels and that Ca2+ signals enhance the expression of EMPs. Ca2+ plays an important role not only in mineralizing dental enamel but also in regulating the expression of EMPs.

Store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels is essential for immunity to infection. CRAC channels are formed by ORAI1 proteins in the plasma membrane and activated by stromal interaction molecule (STIM)1 and STIM2 in the endoplasmic reticulum. Mutations in ORAI1 and STIM1 genes that abolish SOCE cause severe immunodeficiency with recurrent infections due to impaired T cell function. SOCE has also been observed in cells of the innate immune system such as macrophages and dendritic cells (DCs) and may provide Ca2+ signals required for their function. The specific role of SOCE in macrophage and DC function, as well as its contribution to innate immunity, however, is not well defined. We found that nonselective inhibition of Ca2+ signaling strongly impairs many effector functions of bone marrow-derived macrophages and bone marrow-derived DCs, including phagocytosis, inflammasome activation, and priming of T cells. Surprisingly, however, macrophages and DCs from mice with conditional deletion of Stim1 and Stim2 genes, and therefore complete inhibition of SOCE, showed no major functional defects. Their differentiation, FcR-dependent and -independent phagocytosis, phagolysosome fusion, cytokine production, NLRP3 inflammasome activation, and their ability to present Ags to activate T cells were preserved. Our findings demonstrate that STIM1, STIM2, and SOCE are dispensable for many critical effector functions of macrophages and DCs, which has important implications for CRAC channel inhibition as a therapeutic strategy to suppress pathogenic T cells while not interfering with myeloid cell functions required for innate immunity.