GILZ mediates the antiproliferative activity of glucocorticoids by negative regulation of Ras signaling
Emira Ayroldi, … , Rosa Di Virgilio, Carlo Riccardi
Emira Ayroldi, … , Rosa Di Virgilio, Carlo Riccardi
Published June 1, 2007
Citation Information: J Clin Invest. 2007;117(6):1605-1615. https://doi.org/10.1172/JCI30724.
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Research Article Immunology

GILZ mediates the antiproliferative activity of glucocorticoids by negative regulation of Ras signaling

Abstract

Tsc22d3 coding for glucocorticoid-induced leucine zipper (GILZ) was initially identified as a dexamethasone-responsive gene involved in the control of T lymphocyte activation and apoptosis. However, the physiological role of this molecule and its function in the biological activity of glucocorticoids (GCs) has not been clarified. Here, we demonstrate that GILZ interacts directly with Ras in vitro and in vivo as shown by GILZ and Ras coimmunoprecipitation and colocalization upon PMA activation in primary mouse spleen T lymphocytes and thymus cells. The analysis of GILZ mutants showed that they bound Ras through the tuberous sclerosis complex box (TSC) and, depending on the Ras activation level, formed a trimeric complex with Ras and Raf, which we previously identified as a GILZ binder. As a consequence of these interactions, GILZ diminished the activation of Ras and Raf downstream targets including ERK1/2, AKT/PKB serine/threonine kinase, and retinoblastoma (Rb) phosphorylation and cyclin D1 expression, leading to inhibition of Ras- and Raf-dependent cell proliferation and Ras-induced NIH-3T3 transformation. GILZ silencing resulted in an increase in concanavalin A–induced T cell proliferation and, most notably, inhibition of dexamethasone antiproliferative effects. Together, these findings indicate that GILZ serves as a negative regulator of Ras- and Raf-induced proliferation and is an important mediator of the antiproliferative effect of GCs.

Authors

Emira Ayroldi, Ornella Zollo, Alessandra Bastianelli, Cristina Marchetti, Massimiliano Agostini, Rosa Di Virgilio, Carlo Riccardi

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Figure 2

GILZ interacts mainly with Ras in the active form.

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GILZ interacts mainly with Ras in the active form. (A) COS-7 cells were ...
(A) COS-7 cells were cotransfected with the indicated vectors. After 48 hours, the cells were treated with PMA (100 ng/ml) for 20 minutes. Total cell lysates were subjected to immunoprecipitation with anti-myc Ab and analyzed by immunoblot for the indicated Abs. Whole-cell lysates were loaded to control plasmid expression. (B) COS-7 cells were cotransfected with the indicated vectors, immunoprecipitated, and analyzed by Western blot as described in A. Dom-neg Ras, dominant-negative Ras. (C) Splenic T lymphocytes were activated by cross-linked anti-CD3 Ab for 30 minutes. Cell lysates were immunoprecipitated by anti-Ras Ab and analyzed by immunoblot for anti-GILZ Ab. (D) 3DO cells were treated with PMA (100 ng/ml) for 20 minutes, and pulldown assays were performed by incubating total lysates with GST-GILZ fusion protein or GST alone for 18 hours at 4°C. Immunoblot was analyzed by anti-Ras Ab. (E) Confocal analysis of Ras (red) and GILZ (green) localization in myc-GILZ/pUSEamp-Ras wild-type–cotransfected COS-7 cells treated with PMA for 20 minutes. PMA treatment produced iuxta-membrane accumulation of Ras and an increase in the colocalization with GILZ in the same areas (inset). IF, immunofluorescence. Scale bar, 10 μm. Original magnification, ×5 (top inset); ×6 (bottom inset).