Azathioprine Suppresses Ezrin-Radixin-Moesin-Dependent T Cell-APC Conjugation through Inhibition of Vav Guanosine Exchange Activity on Rac Proteins

Abstract

We have shown recently that the azathioprine metabolite 6-Thio-GTP causes immunosuppression by blockade of GTPase activation in T lymphocytes. In the present study, we describe a new molecular mechanism by which 6-Thio-GTP blocks GTPase activation. Although 6-Thio-GTP could bind to various small GTPases, it specifically blocked activation of Rac1 and Rac2 but not of closely related Rho family members such as Cdc42 and RhoA in primary T cells upon stimulation with αCD28 or fibronectin. Binding of 6-Thio-GTP to Rac1 did not suppress Rac effector coupling directly but blocked Vav1 exchange activity upon 6-Thio-GTP hydrolysis, suggesting that 6-Thio-GTP loading leads to accumulation of 6-Thio-GDP-loaded, inactive Rac proteins over time by inhibiting Vav activity. In the absence of apoptosis, blockade of Vav-mediated Rac1 activation led to a blockade of ezrin-radixin-moesin dephosphorylation in primary T cells and suppression of T cell-APC conjugation. Azathioprine-generated 6-Thio-GTP thus prevents the development of an effective immune response via blockade of Vav activity on Rac proteins. These findings provide novel insights into the immunosuppressive effects of azathioprine and suggest that antagonists of the Vav-Rac signaling pathway may be useful for suppression of T cell-dependent pathogenic immune responses.

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“…The GEF Vav2 is unable to exchange 6-T-GDP, preventing reactivation of Rac1. These data indicate that 6-T-GDP may irreversibly inhibit Rac1, including its downstream signaling (11).…”

“…To date, there is limited information on the exact cellular mechanism underling the immunosuppressive properties of azathioprine in T cells (10,11) and monocytes/macrophages (33). Poppe et al (11) demonstrated that azathioprine and its metabolites 6-MP and 6-T-GTP have the ability to potently block the activity of Rac1 and Rac-2 in T cells.…”

“…To date, there is limited information on the exact cellular mechanism underling the immunosuppressive properties of azathioprine in T cells (10,11) and monocytes/macrophages (33). Poppe et al (11) demonstrated that azathioprine and its metabolites 6-MP and 6-T-GTP have the ability to potently block the activity of Rac1 and Rac-2 in T cells. These observations are in line with our results, showing that treatment of endothelial cells with 6-MP or 6-T-GTP strongly reduces Rac1 activity, comparable with the inhibition by Rac1 inhibitor ITX-3, which is an inhibitor of GEF Trio, the most relevant Rac1 targeting GEF in endothelial cells.…”

“…1A, 1B). Azathioprine, 6-MP and the downstream metabolite 6-T-GTP have been shown to decrease Rac1 activation in T cells (11), which could be the underlying mechanism in endothelial cells as well. Indeed, we recently revealed that TNF-a-induced Rac1 activation is impaired when endothelial cells are pretreated with 6-MP, by pulldown of active Rac1 (19).…”

“…It has been shown that 6-MP and its metabolite 6-T-GTP can target Rac1 in CD4 + T cells, blocking T cell activation (10). Interestingly, 6-T-GTP also can bind other small Rho-GTPases such as Cdc42, RhoA, and Rac2, yet, it can only block the activity of Rac1 and Rac2 (11). The GEF Vav2 is unable to exchange 6-T-GDP, preventing reactivation of Rac1.…”