Rapid immunosuppressive effects of glucocorticoids mediated through Lck and Fyn.
Glucocorticoids (GCs) are effective immunosuppressive agents and mediate well-defined transcriptional effects via GC receptors. There is increasing evidence that GCs also initiate rapid nongenomic signaling events. Using activated human CD4(+) lymphocytes and a peptide array containing 1176 different kinase consensus substrates, we generated a comprehensive profile of GC-induced rapid effects on signal transduction. The results show marked early differences in phosphorylation between GC-pretreated cells and control cells, including impaired phosphorylation of p56lck/p59fyn (Lck/Fyn) consensus substrates. Immunoprecipitation and in vitro kinase assays reveal rapid GC-induced down-modulation of Lck and Fyn kinases using SAM68 (Src [pp60c-src]-associated in mitosis 68 kDa) as a substrate. Additionally, immunoprecipitation experiments revealed reduced Lck-CD4 and Fyn-CD3 associations, suggesting GC inhibited recruitment of these kinases to the T-cell receptor complex. Western blot analysis revealed reduced phosphorylation of a series of downstream signaling intermediates following GC treatment, including protein kinase B (PKB), protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). Experiments with GC receptor-negative Jurkat cells and a pharmacologic GC receptor ligand (RU486) indicated that rapid inhibition of Lck and Fyn kinases is GC receptor dependent. Parallel experiments conducted following the application of GCs in healthy individuals confirmed suppression of Lck/Fyn in T cells within 1 hour in vivo. These results identify the inhibition of Lck and Fyn kinases as rapid targets of GCs, mediated via a GC receptor-dependent pathway.
Löwenberg
Mark
M
Laboratory of Experimental Internal Medicine, Academic Medical Center, Meibergdreef 9, NL-1105 AZ Amsterdam, The Netherlands. m.lowenberg@amc.uva.nl
Tuynman
Jurriaan
J
Bilderbeek
Joyce
J
Gaber
Timo
T
Buttgereit
Frank
F
van Deventer
Sander
S
Peppelenbosch
Maikel
M
Hommes
Daniel
D
D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis / Q000187:drug effects; D003907:Dexamethasone / Q000494:pharmacology; D005938:Glucocorticoids / Q000276:immunology / Q000494:pharmacology*; D006801:Humans; D007165:Immunosuppression Therapy; D007166:Immunosuppressive Agents / Q000276:immunology / Q000494:pharmacology*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000187:drug effects / Q000276:immunology*; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000276:immunology*; D051076:Proto-Oncogene Proteins c-fyn; D015398:Signal Transduction / Q000276:immunology; D013329:Structure-Activity Relationship; D019061:src-Family Kinases / Q000187:drug effects / Q000276:immunology*
D016207:Cytokines; D005938:Glucocorticoids; D007166:Immunosuppressive Agents; D011518:Proto-Oncogene Proteins; D003907:Dexamethasone; C494957:FYN protein, human; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases