High dose of dexamethasone upregulates TCR/CD3-induced calcium response independent of TCR zeta chain expression in human T lymphocytes

Glucocorticoids are very potent anti-inflammatory and immunosuppressive age nts that modulate cellular immune responses, although, the molecular mechan isms that impart their complex effects have not been completely defined. We have previously demonstrated that dexamethasone (Dex), a synthetic glucoco rticoid, biphasically modulates the expression of TCR (T cell receptor) zet a chain in human T cells. At 10 nM, it induced the expression of TCR zeta c hain whereas at 100 nM it inhibited its expression. In parallel to the upre gulation of TCR zeta chain, theTCR/CD3-mediaLed [Ca2+](i) response was enha nced in 10 nM Dex-treated cells. However, at 100 nM, Dex treatment enhanced TCR/CD3-mediated [Ca2+](i) response without the induction of TCR zeta chai n expression. Because the classical transcriptional model of glucocorticoid action cannot account for the effects of high dose of Dex, here we studied alternative mechanisms of action. We show that, increased and morel sustai ned TCR/CD3-mediated [Ca2+](i) response was also observed in 100 nM Dex-tre ated cells in the presence of actinomycin D or cycloheximide suggesting tha t cellular transcription and/or de novo protein synthesis are not required for the induction. The TCR/CD3-mediated hyper [Ca2+](i) response in 100 nM Dex-treated cells was readily reversible by short-term culture in steroid-f ree medium. RU-486, a competitive antagonist of Dex, inhibited the increase in [Ca2+](i) response suggesting that the effect of Dex is mediated throug h the glucocorticoid receptor. Although the lipid-raft association of the T CR chain was not significantly increased, high-close of Dex increased the a mount of ubiquitinated form of the TCR zeta chain in the cell membrane alon g with increased levels of actin. Fluorescence microscopy showed that high- dose of Dex alters the distribution of the TCR zeta chain and form more dis tinct clusters upon TCR/CD3 stimulation. These results suggest that high do se of Dex perturbs the membrane distribution of TCR zeta chain leading to m ore functional signaling clusters that result in increased TCR/CD3-mediated [Ca2+](i) response independent of TCR zeta chain expression. J. Cell. Bioc hem. 83: 401-413, 2001. (C) 2001 Wiley-Liss, Inc.