title: Transcriptional regulation of kinases downstream of the T cell receptor: another immunomodulatory mechanism of glucocorticoids.
abstract: BACKGROUND
  Glucocorticoids affect peripheral immune responses, including modulation of T-cell activation, differentiation, and apoptosis. The quantity and quality of T-cell receptor (TCR)-triggered intracellular signals modulate T-cell function. Thus, glucocorticoids may affect T cells by interfering with the TCR signaling cascade. The purpose of the study was to search for glucocorticoid-modulated kinases downstream of the TCR.
  
  
  METHODS
  Gene modulation in lymphoid cells either treated with glucocorticoids or from glucocorticoid-treated mice was studied using a RNase protection assay, real-time PCR, and western blotting. The sensitivity of genetically modified thymocytes to glucocorticoid-induced apoptosis was studied by performing hypotonic propidium iodide staining and flow cytometry. The Student's t-test was employed for statistical evaluation.
  
  
  RESULTS
  We found that transcription of Itk, a non-receptor tyrosine kinase of the Tec family, was up-regulated in a mouse T-cell hybridoma by the synthetic glucocorticoid dexamethasone. In contrast, dexamethasone down-regulated the expression of Txk, a Tec kinase that functions redundantly with Itk, and Lck, the Src kinase immediately downstream of the TCR. We investigated the expression of Itk, Txk, and Lck in thymocytes and mature lymphocytes following in vitro and in vivo dexamethasone treatment at different time points and doses. Kinase expression was differentially modulated and followed distinct kinetics. Itk was up-regulated in all cell types and conditions tested. Txk was strongly up-regulated in mature lymphocytes but only weakly up-regulated or non-modulated in thymocytes in vitro or in vivo, respectively. Conversely, Lck was down-regulated in thymocytes, but not modulated or up-regulated in mature lymphocytes in the different experimental conditions. This complex behaviour correlates with the presence of both positive and negative glucocorticoid responsive elements (GRE and nGRE, respectively) in the Itk, Txk and Lck genes. To investigate the function associated with Itk up-regulation, dexamethasone-induced apoptosis of thymocytes from Itk-deficient mice was evaluated. Our results demonstrated that Itk deficiency causes increased sensitivity to dexamethasone but not to other pro-apoptotic stimuli.
  
  
  CONCLUSIONS
  Modulation of Itk, Txk, and Lck in thymocytes and mature lymphocytes is another mechanism by which glucocorticoids modulate T-cell activation and differentiation. Itk up-regulation plays a protective role in dexamethasone-treated thymocytes.
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Petrillo
authors.forename: Katia
authors.initials: K
authors.lastname: Fettucciari
authors.forename: Paolo
authors.initials: P
authors.lastname: Montuschi
authors.forename: Simona
authors.initials: S
authors.lastname: Ronchetti
authors.forename: Luigi
authors.initials: L
authors.lastname: Cari
authors.forename: Graziella
authors.initials: G
authors.lastname: Migliorati
authors.forename: Emanuela
authors.initials: E
authors.lastname: Mazzon
authors.forename: Oxana
authors.initials: O
authors.lastname: Bereshchenko
authors.forename: Stefano
authors.initials: S
authors.lastname: Bruscoli
authors.affiliation: Department of Medicine, University of Perugia, Perugia, Italy. giuseppe.nocentini@unipg.it.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Nocentini
authors.forename: Carlo
authors.initials: C
authors.lastname: Riccardi
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D015536:Down-Regulation / Q000187:drug effects; D005786:Gene Expression Regulation; D005938:Glucocorticoids / Q000494:pharmacology*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000235:genetics*; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D014158:Transcription, Genetic; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D000276:Adjuvants, Immunologic; D005938:Glucocorticoids; D011948:Receptors, Antigen, T-Cell; D011494:Protein Kinases; C089848:Tec protein-tyrosine kinase; C064626:Ltk protein, mouse; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)