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Tspan18 regulates Orai1 in endothelial cells
Tspan18-knockout mice have impaired histamine-induced release of endothelial von Willebrand factor and impaired thrombo-inflammatory responses
To determine whether Tspan18 has a role in vWF release in vivo, Tspan18-knockout mice were intra-peri- toneally injected with histamine, and plasma vWF levels were analyzed by ELISA. Induced plasma vWF release was reduced by approximately 50% in the absence of Tspan18 (Figure 8A). Basal plasma vWF was normal in Tspan18-knockout mice (Figure 8A), indicating a require- ment for Tspan18 in regulated, but not basal, vWF release.
To investigate the role of Tspan18 in thrombosis, two arterial thrombosis models and two thrombo-inflammato- ry models were used. In a platelet-driven aorta injury arte- rial thrombosis model,38 no difference in time to complete occlusion of the vessel between Tspan18-knockout and wild-type littermate control mice was observed (Figure 8B). Similarly, there was no thrombosis defect in mesen- teric arterioles following application of FeCl3 (Figure 8C), which is also a platelet-driven model,38 but shows reduced platelet deposition and thrombus formation in the com- plete absence of vWF.53,54 In a deep vein thrombosis throm- bo-inflammatory model that is dependent on endothelial vWF,13 thrombus length and weight were reduced by approximately 60% in the Tspan18-knockout mice, com- pared to wild-type littermate controls (Figure 8D). Moreover, 4 of 9 Tspan18-knockout mice failed to develop a thrombus compared to 100% thrombus formation in wild-type mice (Figure 8D). Macroscopically, thrombi from Tspan18-knockout mice had similar red and white parts to those from wild-type mice (data not shown). Finally, in a vWF-dependent myocardial ischemia-reperfusion throm- bo-inflammatory model,55,56 platelet deposition and aggre- gate size in the microcirculation were reduced by approxi- mately 50% (Figure 8E). The reduction in severity in the two thrombo-inflammatory models is consistent with the requirement of Tspan18 for endothelial vWF release in response to inflammatory mediators.
Discussion
We have discovered that Tspan18 is expressed by endothelial cells and interacts with the SOCE channel Orai1. Tspan18-knockdown endothelial cells had reduced Orai1 expression at the cell surface and impaired Ca2+ signaling. This is consistent with the established role of tetraspanins in interacting with specific partner pro- teins in the ER, and promoting their trafficking to the cell surface,1,50,51 albeit via mechanisms that are yet to be defined. Tspan18 is not particularly related to any of the other 32 mammalian tetraspanins,22 suggesting that it may be unique amongst tetraspanins in regulating Orai1. Indeed, none of the five tetraspanins that were selected as controls interacted with Orai1, or induced Ca2+-respon- sive NFAT activation, when over-expressed.
At the cell surface, tetraspanins can regulate the lateral diffusion and clustering of their partner proteins.2,3 A question that arises from the present study is whether Tspan18 regulates Orai1 clustering at the endothelial cell surface. Interestingly, a unimolecular coupling model of Orai1 activation was recently proposed, whereby one molecule of a STIM1 dimer is sufficient to induce opening of the Orai1 hexamer channel.10 This would enable the
other STIM1 molecule in the dimer to cross-link with a second Orai1 hexamer and form a lattice of clustered Orai1 channels. The degree of cluster formation could dictate the kinetics of channel activation and could con- centrate Ca2+ influx to particular regions of the plasma membrane.10 This could affect the extent to which down- stream effectors are activated. Tetraspanins have been reported to exist as nanodomains of approximately ten tetraspanins of a single type,57 therefore Tspan18 may cluster Orai1 into pre-formed nanodomains, so modulat- ing Orai1 lattice formation by STIM1. This may provide a means by which endothelial cells fine-tune SOCE and downstream functional responses. It remains to be deter- mined whether Tspan18 also regulates Orai2 and Orai3, but we found no role for these Orai family members in inflammatory mediator-induced HUVEC Ca2+ mobiliza- tion, consistent with other studies.48,49
The inflammatory mediators thrombin and histamine activate G protein-coupled receptors to induce down- stream Ca2+ mobilization and the release of vWF from Weibel-Palade bodies.14 Consistent with impaired Ca2+ signaling, Tspan18-knockout endothelial cells had impaired inflammatory mediator-induced vWF release in vitro and in vivo. In contrast, basal release of vWF appeared to be normal, because basal plasma vWF levels were unaffected in Tspan18-knockout mice. We hypoth- esize that impaired vWF release, in response to inflamma- tory mediators, explains the in vivo phenotypes observed in Tspan18-knockout mice. The protection from deep vein thrombosis is consistent with the central role of vWF in this disease.13 Furthermore, the reduced platelet depo- sition in the microcirculation during myocardial ischemia-reperfusion injury is consistent with the role of vWF in this process.55,56 The hemostasis defect still needs to be explained, because although a tail bleeding pheno- type has been demonstrated in endothelial-specific vWF- knockout mice, these animals also had low plasma vWF,58,59 unlike Tspan18 knockouts. The tail bleeding assay measures blood loss following excision of the tip of the tail, which contains the two lateral veins, the dorsal vein, and the ventral artery. We speculate that endothe- lial cells in the veins and artery, adjacent to the site of excision, are activated and release vWF via Ca2+-depen- dent signaling. The vWF may trap platelets, facilitating their aggregation and preventing excessive blood loss from the site of tail injury. Therefore, our data suggest that acute release of vWF adjacent to a site of injury might be important for hemostasis, at least for some types of injury. Finally, the lack of a phenotype in the two arterial thrombosis models is consistent with the importance of platelets in these models,38 and we found no defect in aggregation in vitro for Tspan18-knockout platelets.
In summary, we have identified Tspan18 as a novel reg- ulator of endothelial Orai1 and SOCE. Our in vivo data show that Tspan18 regulates inflammation-induced vWF release but not basal release, and promotes hemostasis and thrombo-inflammatory processes but not arterial thrombosis.
Acknowledgments
We are grateful to Carl Blobel, Chris Bunce, Dean Kavanagh, Neil Morgan and Steve Publicover for their helpful comments on this project. We thank the Birmingham Biomedical Sciences Unit for maintaining mice, and the Birmingham Advanced Light Microscope Facility for imaging expertise.
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