The sheddase activity of ADAM17/TACE is regulated by the tetraspanin CD9

Abstract

ADAM17/TACE is a metalloproteinase responsible for the shedding of the proinflammatory cytokine TNF-α and many other cell surface proteins involved in development, cell adhesion, migration, differentiation, and proliferation. Despite the important biological function of ADAM17, the mechanisms of regulation of its metalloproteinase activity remain largely unknown. We report here that the tetraspanin CD9 and ADAM17 partially co-localize on the surface of endothelial and monocytic cells. In situ proximity ligation, co-immunoprecipitation, crosslinking, and pull-down experiments collectively demonstrate a direct association between these molecules. Functional studies reveal that treatment with CD9-specific antibodies or neoexpression of CD9 exert negative regulatory effects on ADAM17 sheddase activity. Conversely, CD9 silencing increased the activity of ADAM17 against its substrates TNF-α and ICAM-1. Taken together, our results show that CD9 associates with ADAM17 and, through this interaction, negatively regulates the sheddase activity of ADAM17.

Dual role of ALCAM in neuroinflammation and blood–brain barrier homeostasis

“…Interestingly, although the cytoplasmic tail of ALCAM contains clusters of positively charged amino acid residues that typically interact with members of the ezrin/radixin/moesin (ERM) family, a study performed with pulmonary microvascular ECs found no association between ALCAM and ERM protein family members, corroborating our results (53). Furthermore, Gilsanz et al (54) have recently reported that ALCAM forms a complex with the tetraspanin molecule CD9 and the "sheddase" ADAM17, which together regulate ALCAM expression and activity (55). Although CD9 is highly expressed on ECs (56), we were unable to confirm the link between ALCAM and CD9.…”

The ADAM family

“…ADAM17 has been shown to interact with the tetraspanin CD9 on leukocyte and endothelial cell membranes to regulate ADAM17 cleavage of TNF-a and ICAM-1, as well as with protein disulfide isomerase in cleavage of the same substrates. 39,40 The potential role of any molecules interacting with ADAM17 during Notch cleavage is unknown. Although homologs of ADAMs 10 and 17 are functionally redundant in C. elegans, 23 it is unknown if ADAM17 has a normal in vivo role in Notch cleavage in higher animals.…”

Functional interplay between tetraspanins and proteases

“…The functional relevance of the interaction between the c-secretase complex and proteins from the tetraspanin-enriched microdomains was also demonstrated using cells derived from CD81-and CD9-knockout mice where the c-secretase activity was significantly impaired. For some cellular processes, treatment with monoclonal antibodies against tetraspanin molecules has been shown to have either an agonist or antagonist activity [22,24,53]. In this study, c-secretase activity was also a target for modulation using antibodies against the CD81 and CD9 tetraspanins, which resulted in a disruption, more evident for CD9, of the Ab generation, thus implicating TEMs in the endogenous regulation of c-secretase [52].…”

“…MT1-MMP subcellular localization is also dependant on its association with CD44, which is in turn shedded by the protease, and is also a component of TEMs Functional interplay between tetraspanins and proteases 3329 ADAM17 [92,93], and elevated soluble levels of these proteins have been found in several inflammatory and tumoral pathologies [94,95]. Hence, CD9 silencing on stimulated HUVEC results in a marked decrease in the membrane level of ICAM-1 reflecting the release from the inhibitory effect exerted by CD9 on ADAM17 activity [24]. These results also provide a mechanism by which CD9 regulates the levels of ICAM-1 on endothelial cells and reinforces the subsequent consequences in terms of leukocyte adhesion and transmigration [91,96].…”

“…A possible explanation is that, on the cells employed, ADAM17 was expressed at a much lower level compared to ADAM10, making it difficult to reveal the interaction. Direct association of the tetraspanin CD9 with ADAM17 on the surface of leukocytes and endothelial cells has been recently described by our group [24]. This interaction was established using a combination of imaging and biochemical approaches based on co-localization, in situ proximity ligation assays, co-immunoprecipitation, cross linking, and pull-down experiments.…”