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 dexamethasoneresponsive 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.

Glucocorticoid-Induced Leucine Zipper (GILZ) Over-Expression in T Lymphocytes Inhibits Inflammation and Tissue Damage in Spinal Cord Injury

“…Moreover, GILZ can interact with Ras-dependent mitogen activated protein kinase pathway, in a monomeric conformation and through its NH2 terminal domain, with consequent inhibition of the downstream pathways, including NF-κB activation [22]. Thus, GILZ inhibits NF-κB by multiple mechanisms and acts as an anti-inflammatory molecule [15,[20][21][22][23][24].…”

L-GILZ binds p53 and MDM2 and suppresses tumor growth through p53 activation in human cancer cells

“…For pull-down assays, the fusion proteins GST-GILZ or GST-L-GILZ were loaded on glutathione-sepharose beads as previously described 24 and incubated with lysates from p53 À / À HCT116 cells transfected with HA-p53. Beads were washed extensively, suspended in sample buffer, and analyzed by SDS-PAGE and Western blotting with anti-HA antibodies.…”

“…For [ 3 H]-thymidine uptake, transfected cells were seeded in triplicate in 96-well plates in the presence of [ 3 H]-thymidine (2 mCi/well; Amersham Biosciences, Piscataway, NJ, USA), and the uptake assays were performed as previously described. 24 Cell cycle profiles were analyzed by flow cytometry to determine DNA content of cell nuclei stained with PI, as previously described. 24 RNA interference.…”

“…24 Cell cycle profiles were analyzed by flow cytometry to determine DNA content of cell nuclei stained with PI, as previously described. 24 RNA interference. The post-transcriptional silencing of the L-GILZ gene was induced by dsRNA interference using the Trilencer-27 siRNA knockdown duplexes commercial kit (Origene, Rockville, MD, USA) according the manufacturer's instructions.…”

“…[18][19][20][21][22][23] GILZ and L-GILZ bind Ras and contribute to cell differentiation and the control of tumorigenesis. 24,25 Crosstalk between p53 and GCs has been described as either antagonistic or synergistic depending on cell type and environmental context. 26 Some reports suggest that p53 activation is not necessary for GC-induced apoptotic cell death 27 but may instead increase resistance to GC therapy.…”