The authors studied the role of endothelial cells (EC) in the development of T cell resistance to the immunosuppressive drug cyclosporin A (CsA). T cell activation triggers a series of intracellular events leading to cytokine production and cell proliferation. Critical for this process is the synthesis and translocation of several transcription factors including AP-1, NF-kappaB and factors belonging to the NFAT family. The activity of NFAT proteins is regulated by calcineurin and GSK-3beta (glycogen synthase kinase-3beta). The authors studied whether T cell NFAT becomes localized in the nucleus during EC-mediated CsA resistance, and if so, by what mechanism. Therefore, human T cells were nucleofected with a HA tagged GSK-3 beta expression plasmid and incubated 4 hours in the presence or absence of EC. Tagged GSK-3beta was then immunoprecpitated with anti-HA, and its activity was assessed by kinase assay. GSK-3beta activity was inhibited by 30-70% in the presence of EC, demonstrating that T cell GSK-3beta is regulated by EC costimulatory signals.
T cells resistant to the immunosuppressive drug cyclosporin A (CsA) may be important mediators of chronic graft rejection. We previously reported that T cells activated in the presence of endothelial cells (EC) develop resistance to CsA, and initiate IL-2 secretion within 8-12 h of triggering. CsA normally blocks the phosphatase, calcineurin, thus preventing nuclear translocation of the transcription factor, NFAT. We find that in the presence but not the absence of EC, NFAT1 can be detected in the nuclei of CsA-treated T cells within 8 h of triggering, reaching a maximal level of 60% of control by 24 h. Glycogen synthase kinase-3beta (GSK-3beta), which rephosphorylates NFAT and promotes nuclear export, is inhibited by EC costimulation. GSK-3beta is a component of the wnt signaling pathway, and EC express wnt-5a and T cells express frizzled-5, a wnt-5a receptor. Wnt-5a promotes T cell NFAT nuclear accumulation in the presence of CsA, an effect mimicked by Li(+), a potent inhibitor of GSK-3beta. The protein kinase C agonist PMA dramatically synergizes with both EC and wnt-5a in stimulating T cell IL-2 synthesis, and inhibition of either protein kinase C by Ro-31-8425 or G-proteins by pertussis toxin effectively blocks the actions of wnt-5a on T cells. Finally, a secreted, dominant-negative form of frizzled-5 blocks EC-mediated CsA resistance. Thus, EC promote CsA-resistant nuclear localization of NFAT and subsequent IL-2 synthesis through a noncanonical wnt-dependent pathway.