Ca 2+ entry via Orai1 store-operated Ca 2+ channels in the plasma membrane is critical to cell function, and Orai1 loss causes severe immunodeficiency and developmental defects. The tetraspanins are a superfamily of transmembrane proteins that interact with specific ‘partner proteins’ and regulate their trafficking and clustering. The aim of this study was to functionally characterize tetraspanin Tspan18. We show that Tspan18 is expressed by endothelial cells at several-fold higher levels than most other cell types analyzed. Tspan18-knockdown primary human umbilical vein endothelial cells have 55-70% decreased Ca 2+ mobilization upon stimulation with the inflammatory mediators thrombin or histamine, similar to Orai1-knockdown. Tspan18 interacts with Orai1, and Orai1 cell surface localization is reduced by 70% in Tspan18-knockdown endothelial cells. Tspan18 overexpression in lymphocyte model cell lines induces 20-fold activation of Ca 2+ -responsive nuclear factor of activated T cell (NFAT) signaling, in an Orai1-dependent manner. Tspan18-knockout mice are viable. They lose on average 6-fold more blood in a tail-bleed assay. This is due to Tspan18 deficiency in non-hematopoietic cells, as assessed using chimeric mice. Tspan18-knockout mice have 60% reduced thrombus size in a deep vein thrombosis model, and 50% reduced platelet deposition in the microcirculation following myocardial ischemia-reperfusion injury. Histamine- or thrombin-induced von Willebrand factor release from endothelial cells is reduced by 90% following Tspan18-knockdown, and histamine-induced increase of plasma von Willebrand factor is reduced by 45% in Tspan18-knockout mice. These findings identify Tspan18 as a novel regulator of endothelial cell Orai1/Ca 2+ signaling and von Willebrand factor release in response to inflammatory stimuli.
The tetraspanins are a superfamily of four-transmembrane proteins, which regulate the trafficking, lateral diffusion and clustering of the transmembrane proteins with which they interact. We have previously shown that tetraspanin Tspan9 is expressed on platelets. Here we have characterised gene-trap mice lacking Tspan9. The mice were viable with normal platelet numbers and size. Tspan9-deficient platelets were specifically defective in aggregation and secretion induced by the platelet collagen receptor GPVI, despite normal surface GPVI expression levels. A GPVI activation defect was suggested by partially impaired GPVI-induced protein tyrosine phosphorylation. In mechanistic experiments, Tspan9 and GPVI co-immunoprecipitated and co-localised, but super-resolution imaging revealed no defects in collagen-induced GPVI clustering on Tspan9-deficient platelets. However, single particle tracking using total internal reflection fluorescence microscopy showed that GPVI lateral diffusion was reduced by approximately 50% in the absence of Tspan9. Therefore, Tspan9 plays a fine-tuning role in platelet activation by regulating GPVI membrane dynamics.
The interplay between thrombosis and inflammation, termed thrombo-inflammation, causes acute organ damage in diseases such as ischaemic stroke and venous thrombosis. We have recently identified tetraspanin Tspan18 as a novel regulator of thrombo-inflammation. The tetraspanins are a family of 33 membrane proteins in humans that regulate the trafficking, clustering, and membrane diffusion of specific partner proteins. Tspan18 partners with the store-operated Ca 2+ entry channel Orai1 on endothelial cells. Orai1 appears to be expressed in all cells and is critical in health and disease. Orai1 mutations cause human immunodeficiency, resulting in chronic and often lethal infections, while Orai1-knockout mice die at around the time of birth. Orai1 is a promising drug target in autoimmune and inflammatory diseases, and Orai1 inhibitors are in clinical trials. The focus of this review is our work on Tspan18 and Orai1 in Tspan18-knockout mice and Tspan18-knockdown primary human endothelial cells. Orai1 trafficking to the cell surface is partially impaired in the absence of Tspan18, resulting in impaired Ca 2+ signaling and impaired release of the thrombo-inflammatory mediator von Willebrand factor following endothelial stimulation. As a consequence, Tspan18-knockout mice are protected in ischemia-reperfusion and deep vein thrombosis models. We provide new evidence that Tspan18 is relatively highly expressed in endothelial cells, through the analysis of publicly available single-cell transcriptomic data. We also present new data, showing that Tspan18 is required for normal Ca 2+ signaling in platelets, but the functional consequences are subtle and restricted to mildly defective platelet aggregation and spreading induced by the platelet collagen receptor GPVI. Finally, we generate structural models of human Tspan18 and Orai1 and hypothesize that Tspan18 regulates Orai1 Ca 2+ channel function at the cell surface by promoting its clustering.
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