Faculty Bibliography

Calcium signaling is critical for lymphocyte function, and intracellular Ca2+ concentrations are regulated by store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels. In patients, loss-of-function mutations in CRAC channel components ORAI1 and STIM1 abolish SOCE and are associated with recurrent and chronic viral infections. Here, using mice with conditional deletion of Stim1 and its homolog Stim2 in T cells, we determined that both components are required for the maintenance of virus-specific memory CD8+ T cells and recall responses following secondary infection. In the absence of STIM1 and STIM2, acute viral infections became chronic. Early during infection, STIM1 and STIM2 were required for the differentiation of naive CD8+ T cells into fully functional cytolytic effector cells and mediated the production of cytokines and prevented cellular exhaustion in viral-specific CD8+ effector T cells. Importantly, memory and recall responses by CD8+ T cells required expression of STIM1 and STIM2 in CD4+ T cells. CD4+ T cells lacking STIM1 and STIM2 were unable to provide "help" to CD8+ T cells due to aberrant regulation of CD40L expression. Together, our data indicate that STIM1, STIM2, and CRAC channel function play distinct but synergistic roles in CD4+ and CD8+ T cells during antiviral immunity.

Calcium signals regulate many critical processes during vertebrate brain development including neurogenesis, neurotransmitter specification, and axonal outgrowth. However, the identity of the ion channels mediating Ca(2+) signaling in the developing nervous system is not well defined. Here, we report that embryonic and adult mouse neural stem/progenitor cells (NSCs/NPCs) exhibit store-operated Ca(2+) entry (SOCE) mediated by Ca(2+) release-activated Ca(2+) (CRAC) channels. SOCE in NPCs was blocked by the CRAC channel inhibitors La(3+), BTP2, and 2-APB and Western blots revealed the presence of the canonical CRAC channel proteins STIM1 and Orai1. Knock down of STIM1 or Orai1 significantly diminished SOCE in NPCs, and SOCE was lost in NPCs from transgenic mice lacking Orai1 or STIM1 and in knock-in mice expressing the loss-of-function Orai1 mutant, R93W. Therefore, STIM1 and Orai1 make essential contributions to SOCE in NPCs. SOCE in NPCs was activated by epidermal growth factor and acetylcholine, the latter occurring through muscarinic receptors. Activation of SOCE stimulated gene transcription through calcineurin/NFAT (nuclear factor of activated T cells) signaling through a mechanism consistent with local Ca(2+) signaling by Ca(2+) microdomains near CRAC channels. Importantly, suppression or deletion of STIM1 and Orai1 expression significantly attenuated proliferation of embryonic and adult NPCs cultured as neurospheres and, in vivo, in the subventricular zone of adult mice. These findings show that CRAC channels serve as a major route of Ca(2+) entry in NPCs and regulate key effector functions including gene expression and proliferation, indicating that CRAC channels are important regulators of mammalian neurogenesis.

Mushroom dendritic spine structures are essential for memory storage, and the loss of mushroom spines may explain memory defects in Alzheimer's disease (AD). Here we show a significant reduction in the fraction of mushroom spines in hippocampal neurons from the presenilin-1 M146V knockin (KI) mouse model of familial AD (FAD). The stabilization of mushroom spines depends on STIM2-mediated neuronal store-operated calcium influx (nSOC) and continuous activity of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). We demonstrate that STIM2-nSOC-CaMKII pathway is compromised in KI neurons, in aging neurons, and in sporadic AD brains due to downregulation of STIM2 protein. We further establish that overexpression of STIM2 rescues synaptic nSOC, CaMKII activity, and mushroom spine loss in KI neurons. Our results identify STIM2-nSOC-CaMKII synaptic maintenance pathway as a novel potential therapeutic target for treatment of AD and age-related memory decline.

Background: The aim of this study was to determine the role of stromal interacting molecule-1 (STIM1) in the regulation of vascular function using tissue-specific STIM1 knockout mice either in endothelial cells or in smooth muscle cells. Methods & results: We used male C57/BL6 homozygous and heterozygous mice lacking STIM1 specifically in either smooth muscle cells (STIM1SMC-/-, STIM1SMC-/+) or in endothelial cells (STIM1EC-/-, STIM1EC-/+). Systolic blood pressure and glucose levels were similar in all mice, but body weight was significantly reduced in STIM1EC-/- and STIM1SMC-/- mice. The contraction of resistance and conductance arteries in response to phenylephrine was significantly reduced in STIM1SMC-/- mice only. However, contraction to thromboxane and potassium chloride was similar in all groups. The endothelium-dependent relaxation was significantly impaired in STIMEC-/+ and drastically reduced in STIM1EC-/- mice while the endothelium-independent vasorelaxation was similar among all groups. Acute down regulation of STIM1 in resistance arteries reduced endothelium-dependent relaxation to acetylcholine and the contractile response to phenylephrine, while the contractile response to thromboxane was not affected. NADPH oxidase activity was only increased in STIMEC-/+ and STIMEC-/- mice. Calcium Ca²⁺ entry in microvascular endothelial cells stimulated with thrombin and histamine, two vasoactive compounds that cause NO production, had the pharmacological features of store-operated Ca²⁺ entry (SOCE) and was dependent on STIM1 expression. Conclusions: STIM1 plays opposing roles in vascular smooth muscle vs. endothelial cells in the regulation of vascular reactivity. We conclude that STIM1 is a critical factor in the basic mechanisms of vascular endothelium-dependent relaxation and the contractility in response to sympathetic activity but not in the intrinsic contractility of smooth muscle

Ca²⁺ influx is an important signal transduction mechanism that regulates the function of immune cells. In T cells, antigen binding by the TCR results in a [Ca²⁺]i rise that is due to Ca²⁺ release from the ER and Ca²⁺ influx across the plasma membrane through Ca²⁺ release-activated Ca²⁺ (CRAC) channels. Opening of CRAC channels is required for sustained [Ca²⁺]i increases, activation of Ca²⁺ dependent signaling molecules and T cell effector functions. The pore of the CRAC channel is formed by ORAI1 proteins in the plasma membrane, which are activated by two proteins located in the ER, stromal interaction molecules (STIM) 1 and STIM2. This process is called store-operated Ca²⁺ entry (SOCE) and constitutes the predominant Ca²⁺ influx pathway in lymphocytes. The importance of CRAC channels for adaptive immunity is emphasized by loss-of-function and null mutations in the human ORAI1 and STIM1 genes that abolish Ca²⁺ influx in T cells and cause severe combined immunodeficiency (SCID). Similarly, abolishing murine Stim1 or Orai1 gene function severely impairs SOCE and the function of T cells, evident in the inability of STIM1- and ORAI1-deficient CD4+ T cells to mediate inflammation in animal models of autoimmune disease. The role of SOCE in CD8+ T cell-mediated immunity in vivo is less well defined. SOCE-deficient patients with mutations in STIM1 or ORAI1 genes are susceptible to recurrent viral infections, potentially due to impaired CD8+ T cell function and elimination of virus infected cells. Using mice with conditional, T cell-specific deletion of Stim1 and Stim2 genes, we find that SOCE is essential for the maintenance of virus-specific memory CD8+ T cells and recall responses to secondary infection. Accordingly, in the absence of SOCE, acute viral infections become chronic. Detailed analyses reveal that SOCE plays distinct but synergistic roles in CD4+ and CD8+ T cells during !

Store-operated Ca(2+) entry (SOCE) is a major mechanism of Ca(2) (+) import from extracellular to intracellular space, involving detection of Ca(2+) store depletion in endoplasmic reticulum (ER) by stromal interaction molecule (STIM) proteins, which then translocate to plasma membrane and activate Orai Ca(2+) channels there. We found that STIM1 and Orai1 isoforms were abundantly expressed in human melanoma tissues and multiple melanoma/melanocyte cell lines. We confirmed that these cell lines exhibited SOCE, which was inhibited by knockdown of STIM1 or Orai1, or by a pharmacological SOCE inhibitor. Inhibition of SOCE suppressed melanoma cell proliferation and migration/metastasis. Induction of SOCE was associated with activation of extracellular-signal-regulated kinase (ERK), and was inhibited by inhibitors of calmodulin kinase II (CaMKII) or Raf-1, suggesting that SOCE-mediated cellular functions are controlled via the CaMKII/Raf-1/ERK signaling pathway. Our findings indicate that SOCE contributes to melanoma progression, and therefore may be a new potential target for treatment of melanoma, irrespective of whether or not Braf mutation is present.

Entry of lymphocytes into secondary lymphoid organs (SLOs) involves intravascular arrest and intracellular calcium ion ([Ca2+ ]i ) elevation. TCR activation triggers increased [Ca2+ ]i and can arrest T-cell motility in vitro. However the requirement for [Ca2+ ]i elevation in arresting T cells in vivo has not been tested. Here, we have manipulated the Ca2+ release-activated Ca2+ (CRAC) channel pathway required for [Ca2+ ]i elevation in T cells through genetic deletion of stromal interaction molecule (STIM) 1 or by expression of a dominant negative ORAI1 channel subunit (ORAI1-DN). Interestingly, the absence of CRAC did not interfere with homing of naive CD4+ T cells to SLOs and only moderately reduced crawling speeds in vivo. T cells expressing ORAI1-DN lacked TCR activation induced [Ca2+ ]i elevation, yet arrested motility similar to control T cells in vitro. In contrast, antigen specific ORAI1-DN T cells had a two-fold delayed onset of arrest following injection of OVA peptide in vivo. CRAC channel function is not required for homing to SLOs, but enhances spatiotemporal coordination of TCR signaling and motility arrest

Store-operated calcium entry (SOCE) through Ca(2+) release-activated Ca(2+) (CRAC) channels regulates the function of many immune cells. Patients with loss-of-function mutations in the CRAC channel genes ORAI1 or STIM1 are immunodeficient and are prone to develop virus-associated tumours. This and the reported role of Ca(2+) signals in cytotoxic lymphocyte function suggest that SOCE may be critical for tumour immune surveillance. Using conditional knock out mice lacking STIM1 and its homologue STIM2, we find that SOCE in CD8(+) T cells is required to prevent the engraftment of melanoma and colon carcinoma cells and to control tumour growth. SOCE is essential for the cytotoxic function of CTLs both in vivo and in vitro by regulating the degranulation of CTLs, their expression of Fas ligand and production of TNF-alpha and IFN-gamma. Our results emphasize an important role of SOCE in antitumour immunity, which is significant given recent reports arguing in favour of CRAC channel inhibition for cancer therapy.