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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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 7

GILZ reduces Ras-driven transformation and tumorigenicity.

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GILZ reduces Ras-driven transformation and tumorigenicity. (A) NIH-3T3 c...
(A) NIH-3T3 cells were transiently transfected with HA-tagged RasV12 together with either control or myc-GILZ vector. After 14 days, the dishes were stained with 0.1% crystal violet. Quantification of the foci in each dish and the expression of transfected vectors are shown at right. The data represent an average from 3 dishes in 3 independent experiments each. (B) Lysates of NIH-3T3 cells transiently cotransfected with Myc-GILZ and either HA-RasV12 or wild-type Xp-Ras were immunoprecipitated with anti-HA and anti-Xp Abs, respectively. Immunoreactive proteins were revealed with anti-myc Ab. Whole-cell lysates were loaded to control plasmid expression. (C) NIH-3T3 cells were transiently transfected as described above and injected subcutaneously into SCID mice (5 per group). Tumor growth was scored at the indicated times as tumor diameter and tumor weight (left). Plasmid expression was evaluated 2 days after transfection by Western blot (right). (D) NIH-3T3 cells transfected with HA-RasV12 with or without Xp-GILZ were double-selected with antibiotics, and plates were photographed 2 weeks after transfection, at which time the cells were used to assess expression levels by Western blot with the Abs indicated in E. (E) The cells described in D were subcutaneously injected into SCID mice, and tumor growth was scored at the indicated times. After excision, representative tumors were photographed by a digital camera. *P < 0.05; **P < 0.01.