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Tspan18 regulates Orai1 in endothelial cells
Introduction
The tetraspanins are a superfamily of proteins contain- ing four transmembrane regions that interact with and regulate the trafficking, lateral mobility and clustering of specific ‘partner proteins’. These include signaling recep- tors, adhesion molecules and metalloproteinases.1-3 Recently, the first crystal structure of a tetraspanin, CD81, demonstrated a cone-shaped structure with a cholesterol-binding cavity within the transmembranes.4 Molecular dynamics simulations suggest that cholesterol removal causes a dramatic conformational change, whereby the main extracellular region swings upwards.4 This raises the possibility that tetraspanins function as ‘molecular switches’ to regulate partner protein function via conformational change, and suggests that tetraspanins are viable future drug targets.
Tetraspanin Tspan18 was previously studied in chick embryos, in which it stabilizes expression of the homophilic adhesion molecule cadherin 6B to maintain adherens junctions between premigratory epithelial cra- nial neural crest cells.5,6 Transcriptional Tspan18 down- regulation is required for loss of cadherin 6B expression, breakdown of epithelial junctions, and neural crest cell migration. However, Tspan18 knockdown has no major effect on chick embryonic development.5,6 The function of Tspan18 in humans or mice has still not been studied.
Store-operated Ca2+ entry (SOCE) through the plasma membrane Ca2+ channel Orai1 is essential for the healthy function of most cell types.7 Loss of SOCE results in severe immunodeficiency that requires a bone marrow transplant for survival. Further symptoms include ecto- dermal dysplasia and impaired development of skeletal muscle.7 The process of SOCE is biphasic. The first step is initiated following the generation of the second mes- senger inositol trisphosphate (IP3) from upstream tyro- sine kinase or G protein-coupled receptor signaling. IP3 induces the transient release of Ca2+ from endoplasmic reticulum (ER) stores via IP3 receptor channels.8 Depletion of Ca2+ is detected by the ER-resident dimeric Ca2+-sensor protein STIM1, which then undergoes a con- formational change and interacts with Orai1 hexamers in the plasma membrane.9,10 STIM1 binding induces Orai1 channel opening and clustering via a mechanism that is not fully understood, allowing Ca2+ entry across the plas- ma membrane.9,10 The resulting increase in intracellular Ca2+ concentration is relatively large and sustained, suffi- cient to activate a variety of signaling proteins, including the widely-expressed nuclear factor of activated T-cell (NFAT) transcription factors.8
Endothelial cells line all blood and lymphatic vessels and play a central role in hemostasis and in thrombo- inflammation, in which inflammatory cells contribute to thrombosis.11,12 In the thrombo-inflammatory disease deep vein thrombosis, blood flow stagnation induced by prolonged immobility, for example, is the trigger for endothelial cells to exocytose Weibel-Palade storage bodies via a mechanism involving Ca2+ signaling.13,14 This releases the multimeric glycoprotein von Willebrand fac- tor (vWF) and the adhesion molecule P-selectin, which recruit platelets and leukocytes, respectively. vWF-bound platelets provide a pro-coagulant surface for activation of clotting factors and thrombin generation, neutrophils release neutrophil extracellular traps, and mast cells release endothelial-activating substances.15-17 This series
of thrombo-inflammatory events leads to formation of a blood clot which occludes the vein, and can cause death by pulmonary thromboembolism.
The aim of this study was to determine the function of tetraspanin Tspan18 in humans and mice. We found that Tspan18 is highly expressed by endothelial cells, inter- acts with Orai1, and is required for its cell surface expres- sion and SOCE function. As a consequence, Tspan18- deficient endothelial cells have impaired Ca2+ mobiliza- tion and release of vWF upon activation induced by inflammatory mediators, and Tspan18-knockout mice are protected from deep vein thrombosis and myocardial ischemia-reperfusion injury, and have defective hemo- stasis.
Methods
Ethicsstatement
Procedures in Birmingham were approved by the UK Home Office according to the Animals (Scientific Procedures) Act 1986, and those in Würzburg by the district government of Lower Frankonia (Bezirksregierung Unterfranken).
Mice
Tspan18-/- mice were generated by Genentech/Lexicon Pharmaceuticals on a mixed genetic background of 129/SvEvBrd and C57BL/6J.18 They were purchased from the Mutant Mouse Regional Resource Center and bred as heterozy- gotes to generate litter-matched Tspan18-/- and Tspan18+/+ pairs. Radiation fetal liver chimeric mice were generated as described.19
Antibodies
Anti-epitope tag antibodies were mouse anti-Myc 9B11 and rabbit anti-Myc 71D10 (Cell Signaling Technology), mouse anti-FLAG M2 and rabbit anti-FLAG (Sigma). Other antibodies were mouse anti-human calnexin AF18 (Abcam), rat anti- mouse CD16/32 (BioLegend), CD41 (eBioscience) and panen- dothelial cell antigen MECA-32 (BD Pharmingen), mouse anti- ERK1/2 and rabbit anti-phospho-ERK1/2 (Cell Signaling Technology) and rabbit anti-human vWF (GE Healthcare). Biotinylated isolectin GS-IB4 glycoprotein was from ThermoFisher Scientific.
Expression constructs
The NFAT/AP1-luciferase transcriptional reporter construct has been described previously.20,21 pEF6/Myc-His (mock) and pEF6/Myc-His/lacZ were from Invitrogen. N-terminal FLAG- tagged tetraspanin constructs were generated in pEF6/Myc-His as described.22,23 pcDNA3.1 Myc-His-tagged human Orai1 and MO70-FLAG-tagged human Orai1 E106Q were from Addgene24 and the dominant-active calcineurin was as described.25
Cell culture and transfections
Detailed information on cell cultures is provided in the Online Supplementary Appendix. Wild-type and IP3 receptor-deficient DT40 chicken B-cell lines,26 and Jurkat human T-cell line, were transfected by electroporation.21 Human embryonic kidney (HEK)-293T (HEK-293 cells expressing the large T-antigen of simian virus 40) and the human HeLa epithelial cell line were transfected using polyethylenimine (Sigma)27 and Lipofectamine 2000 (Invitrogen),28 respectively. Human umbilical vein endothelial cells (HUVEC)29 were transfected with 10 nM
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