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 6

GILZ impairs cell proliferation rate.

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GILZ impairs cell proliferation rate. (A) Empty vector– or GILZ-transfec...
(A) Empty vector– or GILZ-transfected 3DO stable clones (clones 1 and 2) were starved for 48 hours and then cultured for 24 hours in the presence of 10% FCS. Proliferation was evaluated by PI staining and cell cycle analysis (left) and [3H]-thymidine uptake assays (right). (B) NIH-3T3 cells were transfected, selected, and controlled for vector expression by Western blot as described in Figure 5, then starved for 24 hours (1% FCS). Growth curves (left) and [3H]-thymidine uptake (center) were quantified following FCS stimulation at the indicated times. Cell cycle analysis of PI-labeled cells (right) was performed 24 hours after FCS stimulation. (C) Inhibitory effect of GILZ on NIH-3T3 cell proliferation, transiently transfected with Raf-CAAX or RasV12 and reversion of GILZ-mediated inhibition by activated AKT or activated Raf. [3H]-thymidine uptake (left) and cell number (right) are shown. (D) Cell proliferation of NIH-3T3 cells transfected with RasV12 and full-length GILZ or GILZ mutant lacking the TSC or expressing only the TSC and LZ regions, as evaluated by [3H]-thymidine uptake. Data represent the average of 3 independent experiments; triplicate samples were counted at each time point. *P < 0.05; **P < 0.01.