title: High dose of dexamethasone upregulates TCR/CD3-induced calcium response independent of TCR zeta chain expression in human T lymphocytes.
abstract: Glucocorticoids are very potent anti-inflammatory and immunosuppressive agents that modulate cellular immune responses, although, the molecular mechanisms that impart their complex effects have not been completely defined. We have previously demonstrated that dexamethasone (Dex), a synthetic glucocorticoid, biphasically modulates the expression of TCR (T cell receptor) zeta chain in human T cells. At 10 nM, it induced the expression of TCR zeta chain whereas at 100 nM, it inhibited its expression. In parallel to the upregulation of TCR zeta chain, the TCR/CD3-mediated [Ca(2+)](i) response was enhanced in 10 nM Dex-treated cells. However, at 100 nM, Dex treatment enhanced TCR/CD3-mediated [Ca(2+)](i) response without the induction of TCR zeta chain 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 more sustained TCR/CD3-mediated [Ca(2+)](i) response was also observed in 100 nM Dex-treated cells in the presence of actinomycin D or cycloheximide suggesting that cellular transcription and/or de novo protein synthesis are not required for the induction. The TCR/CD3-mediated hyper [Ca(2+)](i) response in 100 nM Dex-treated cells was readily reversible by short-term culture in steroid-free medium. RU-486, a competitive antagonist of Dex, inhibited the increase in [Ca(2+)](i) response suggesting that the effect of Dex is mediated through the glucocorticoid receptor. Although the lipid-raft association of the TCR zeta chain was not significantly increased, high-dose of Dex increased the amount of ubiquitinated form of the TCR zeta chain in the cell membrane along with increased levels of actin. Fluorescence microscopy showed that high-dose of Dex alters the distribution of the TCR zeta chain and form more distinct clusters upon TCR/CD3 stimulation. These results suggest that high dose of Dex perturbs the membrane distribution of TCR zeta chain leading to more functional signaling clusters that result in increased TCR/CD3-mediated [Ca(2+)](i) response independent of TCR zeta chain expression.
authors.affiliation: Department of Cellular Injury, Walter Reed Army Institute of Research, Building 503, Robert Grant Road, Silver Spring, Maryland 20910-7500, USA.
authors.forename: M P
authors.initials: MP
authors.lastname: Nambiar
authors.forename: E J
authors.initials: EJ
authors.lastname: Enyedy
authors.forename: C U
authors.initials: CU
authors.lastname: Fisher
authors.forename: V G
authors.initials: VG
authors.lastname: Warke
authors.forename: G C
authors.initials: GC
authors.lastname: Tsokos
mesh_terms: D000199:Actins / Q000378:metabolism; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D003470:Culture Media / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D015151:Immunoblotting; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D015398:Signal Transduction; D013256:Steroids / Q000494:pharmacology; D013601:T-Lymphocytes / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic; D015854:Up-Regulation
chemical_list: D000199:Actins; D000893:Anti-Inflammatory Agents; D003470:Culture Media; D003902:Detergents; D004791:Enzyme Inhibitors; D005938:Glucocorticoids; D007476:Ionophores; D008565:Membrane Proteins; D011500:Protein Synthesis Inhibitors; D011948:Receptors, Antigen, T-Cell; D013256:Steroids; C054979:antigen T cell receptor, zeta chain; D003609:Dactinomycin; D015759:Ionomycin; D019284:Thapsigargin; D003907:Dexamethasone; D003513:Cycloheximide; D002118:Calcium