ADAM10-Interacting Tetraspanins Tspan5 and Tspan17 Regulate VE-Cadherin Expression and Promote T Lymphocyte Transmigration

Abstract

The recruitment of blood leukocytes across the endothelium to sites of tissue infection is central to inflammation, but also promotes chronic inflammatory diseases. A disintegrin and metalloproteinase 10 (ADAM10) is a ubiquitous transmembrane molecular scissor that is implicated in leukocyte transmigration by proteolytically cleaving its endothelial substrates. These include VE-cadherin, a homotypic adhesion molecule that regulates endothelial barrier function, and transmembrane chemokines CX3CL1 and CXCL16, which have receptors on leukocytes. However, a definitive role for endothelial ADAM10 in transmigration of freshly isolated primary leukocytes under flow has not been demonstrated, and the relative importance of distinct ADAM10 substrates is unknown. Emerging evidence suggests that ADAM10 can be regarded as six different molecular scissors with different substrate specificities, depending on which of six TspanC8 tetraspanins it is associated with, but TspanC8s remain unstudied in leukocyte transmigration. In the current study, ADAM10 knockdown on primary HUVECs was found to impair transmigration of freshly isolated human peripheral blood T lymphocytes, but not neutrophils or B lymphocytes, in an in vitro flow assay. This impairment was due to delayed transmigration rather than a complete block, and was overcome in the presence of neutrophils. Transmigration of purified lymphocytes was dependent on ADAM10 regulation of VE-cadherin, but not CX3CL1 and CXCL16. Tspan5 and Tspan17, the two most closely related TspanC8s by sequence, were the only TspanC8s that regulated VE-cadherin expression and were required for lymphocyte transmigration. Therefore endothelial Tspan5- and Tspan17-ADAM10 complexes may regulate inflammation by maintaining normal VE-cadherin expression and promoting T lymphocyte transmigration.

Tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

“…Thus, Tspan15 and ADAM10 appear to be each required for the normal expression of the other. Indeed, it is well established that TspanC8s are important for ADAM10 maturation and trafficking to the cell surface (Dornier et al 2012; Haining et al 2012; Prox et al 2012; Zhou et al 2014; Jouannet et al 2016; Noy et al 2016; Virreira Winter et al 2016; Reyat et al 2017; Brummer et al 2018; Seipold et al 2018; Shah et al 2018; Brummer et al 2019), and we extended these observations by showing that ADAM10 surface expression was partially reduced in the absence of Tspan15 in A549, HEK-293T and HUVECs. Expression of other TspanC8s by these cells (Haining et al 2012; Matthews et al 2017) is likely to be the reason that the reduction is only partial.…”

“…To create the immunogen for mouse anti-human Tspan15 mAb generation, ADAM10-knockout MEFs were transduced with lentivirus packaged with FLAG-tagged human Tspan15, produced in HEK-293T cells using a previously described protocol (Reyat et al 2017). Clonal cells stably overexpressing Tspan15 were selected with 1 μg/ml puromycin and validated by anti-FLAG western blotting.…”

“…RNA was extracted using the RNeasy Mini kit (Qiagen, Manchester, UK) from cells homogenised with QIAshredder columns (Qiagen). Complementary DNA was generated using the High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific) and subjected to TaqMan qPCR assays (Thermo Fisher Scientific) for Tspan15 (Hs00202548_m1) and GAPDH (Hs02758991_g1) and analysed as described (Reyat et al 2017). All qPCR data were normalised to GAPDH as the internal loading control.…”

“…The TspanC8s comprise Tspan5, 10, 14, 15, 17 and 33, and are so-called because of eight cysteines within their large extracellular loop. Different cell types express distinct TspanC8 repertoires (Matthews et al 2017; Matthews et al 2018) and emerging evidence suggests that each TspanC8 may cause ADAM10 to cleave specific substrates (Dornier et al 2012; Jouannet et al 2016; Noy et al 2016; Reyat et al 2017; Saint-Pol et al 2017; Seipold et al 2018; Brummer et al 2019). However, there is an urgent need to generate monoclonal antibodies (mAbs) to all TspanC8s, to define fully their expression profiles and functional mechanisms.…”

Proteolytic processing of platelet receptors

“…In nucleated cells, members of the TspanC8 subgroup are required for correct enzymatic maturation and trafficking of ADAMs to the cell surface. In certain cell types, there is evidence that cells can target the ADAMs to distinct substrates and this may involve different TspanC8s and iRhoms . Roles for iRhoms and tetraspanins (in particular Tspan14) in regulating platelet ADAMs activity are exciting avenues of research enquiry that are likely to explain differential cleavage of GPVI and GPIbα in circulating platelets.…”