Faculty Bibliography

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

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.

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.

Store-operated Ca 2+ entry (SOCE) is an important Ca 2+ influx pathway, which is defined by the fact that depletion of intracellular Ca 2+ stores, mainly the endoplasmic reticulum (ER), triggers the opening of Ca 2+ channels in the plasma membrane. The best characterized SOC channel is the Ca 2+ release-activated Ca 2+ (CRAC) channel, which was first described in cells of the immune system but has since been reported in many different cell types. CRAC channels are multimers of ORAI family proteins, of which ORAI1 is the best characterized. They are activated by stromal interaction molecules (STIM) 1 and 2, which respond to the depletion of intracellular Ca 2+ stores with oligomerization and binding to ORAI proteins. The resulting SOCE is critical for the physiological function of many cell types including immune cells and platelets. Recent studies using cell lines, animal models and primary cells from human patients with defects in SOCE have highlighted the importance of this Ca 2+ entry mechanism in a variety of pathophysiological processes. This review focuses on the role of SOCE in immunity to infection, allergy, hemostasis and cancer.

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.

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

T cell receptor (TCR) signaling driven by interaction of the TCR with specific complexes of self-peptide and the major histocompatibility complex determines T cell fate in thymic development. However, the signaling pathway through which TCR signal strength regulates distinct T cell lineages remains unknown. Here we have used mice lacking the endoplasmic reticulum Ca2+ sensors stromal interaction molecule 1 (STIM1) and STIM2 to show that STIM-induced store-operated Ca2+ entry is not essential for thymic development of conventional TCRalphabeta+ T cells but is specifically required for the development of agonist-selected T cells (regulatory T cells, invariant natural killer T cells, and TCRalphabeta+ CD8alphaalpha+ intestinal intraepithelial lymphocytes). The severe impairment of agonist-selected T cell development is mainly due to a defect in interleukin-2 (IL-2) or IL-15 signaling. Thus, STIM1 and STIM2-mediated store-operated Ca2+ influx, leading to efficient activation of NFAT (nuclear factor of activated T cells), is critical for the postselection maturation of agonist-selected T cells.