Transcriptional regulation of kinases downstream of the T cell receptor: another immunomodulatory mechanism of glucocorticoids

Maria G razia Petrillo, Katia Fettucciari, Paolo Montuschi, Simona Ronchetti, Luigi Cari, Graziella Migliorati, Emanuela Mazzon, Oxana Bereshchenko, Stefano Bruscoli, Giuseppe Nocentini, Carlo Riccardi

Research output: Contribution to journalArticle

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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.

Original languageEnglish
Pages (from-to)35
Number of pages1
JournalBMC pharmacology & toxicology
Volume15
DOIs
Publication statusPublished - 2014

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T-Cell Antigen Receptor
Glucocorticoids
Thymocytes
Phosphotransferases
Dexamethasone
Lymphocytes
T-Lymphocytes
Apoptosis
Cell Differentiation
Up-Regulation
Lymphocyte Specific Protein Tyrosine Kinase p56(lck)
src-Family Kinases
Propidium
Hybridomas
Ribonucleases
Genes
Real-Time Polymerase Chain Reaction
Flow Cytometry
Western Blotting
Staining and Labeling

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Petrillo, M. G. R., Fettucciari, K., Montuschi, P., Ronchetti, S., Cari, L., Migliorati, G., ... Riccardi, C. (2014). Transcriptional regulation of kinases downstream of the T cell receptor: another immunomodulatory mechanism of glucocorticoids. BMC pharmacology & toxicology, 15, 35. https://doi.org/10.1186/2050-6511-15-35

Transcriptional regulation of kinases downstream of the T cell receptor : another immunomodulatory mechanism of glucocorticoids. / Petrillo, Maria G razia; Fettucciari, Katia; Montuschi, Paolo; Ronchetti, Simona; Cari, Luigi; Migliorati, Graziella; Mazzon, Emanuela; Bereshchenko, Oxana; Bruscoli, Stefano; Nocentini, Giuseppe; Riccardi, Carlo.

In: BMC pharmacology & toxicology, Vol. 15, 2014, p. 35.

Research output: Contribution to journalArticle

Petrillo, MGR, Fettucciari, K, Montuschi, P, Ronchetti, S, Cari, L, Migliorati, G, Mazzon, E, Bereshchenko, O, Bruscoli, S, Nocentini, G & Riccardi, C 2014, 'Transcriptional regulation of kinases downstream of the T cell receptor: another immunomodulatory mechanism of glucocorticoids', BMC pharmacology & toxicology, vol. 15, pp. 35. https://doi.org/10.1186/2050-6511-15-35
Petrillo, Maria G razia ; Fettucciari, Katia ; Montuschi, Paolo ; Ronchetti, Simona ; Cari, Luigi ; Migliorati, Graziella ; Mazzon, Emanuela ; Bereshchenko, Oxana ; Bruscoli, Stefano ; Nocentini, Giuseppe ; Riccardi, Carlo. / Transcriptional regulation of kinases downstream of the T cell receptor : another immunomodulatory mechanism of glucocorticoids. In: BMC pharmacology & toxicology. 2014 ; Vol. 15. pp. 35.
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AU - Petrillo, Maria G razia

AU - Fettucciari, Katia

AU - Montuschi, Paolo

AU - Ronchetti, Simona

AU - Cari, Luigi

AU - Migliorati, Graziella

AU - Mazzon, Emanuela

AU - Bereshchenko, Oxana

AU - Bruscoli, Stefano

AU - Nocentini, Giuseppe

AU - Riccardi, Carlo

PY - 2014

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N2 - 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.

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