Glucocorticoid-induced Leucine Zipper Expression Research Articles

Dual regulation of glucocorticoid-induced leucine zipper ( GILZ) by the glucocorticoid receptor and the PI3-kinase/AKT pathways in multiple myeloma

Glucocorticoids (GCs) are effective therapeutics commonly used in multiple myeloma (MM) treatment. Clarifying the pathway of GC-induced apoptosis is crucial to understanding the process of drug resistance and to the development of new targets for MM treatment. We have previously published results of a micro-array identifying glucocorticoid-induced leucine zipper ( GILZ) as GC-regulated gene in MM.1S cells. Consistent with those results, GCs increased GILZ in MM cell lines and patient samples. Reducing the levels of GILZ with siRNA decreased GC-induced cell death suggesting GILZ may mediate GC-killing. We conducted a screen to identify other pathways that affect GILZ regulation and report that inhibitors of PI3-kinase/AKT enhanced GILZ expression in MM cell lines and clinical samples. The combination of dexamethasone (Dex) and LY294002, wortmannin, triciribine, or AKT inhibitor VIII dramatically up regulated GILZ levels and enhanced apoptosis. Addition of interleukin-6 (IL-6) or insulin-like growth factor (IGF1), both which activate the PI3-kinase/AKT pathway and inhibit GC killing, blocked up regulation of GILZ by GC and PI3-kinase/AKT inhibitors. In summary, these results identify GILZ as a mediator of GC killing, indicate a role of PI3-kinase/AKT in controlling GILZ regulation and suggest that the combination of PI3-kinase/AKT inhibitors and GCs may be a beneficial MM treatment.

Regulation of Mesenchymal Stem Cell Osteogenic Differentiation by Glucocorticoid-induced Leucine Zipper (GILZ)

Mesenchymal stem cells (MSCs) can differentiate into multiple cell lineages, including osteoblasts and adipocytes. We reported previously that glucocorticoid-induced leucine zipper (GILZ) inhibits peroxisome proliferator-activated receptor gamma-2 (Ppargamma2) expression and blocks adipocyte differentiation. Here we show that overexpression of GILZ in mouse MSCs, but not MC3T3-E1 osteoblasts, increases alkaline phosphatase activity and enhances mineralized bone nodule formation, whereas knockdown of Gilz reduces MSC osteogenic differentiation capacity. Consistent with these observations, real-time reverse transcription-PCR analysis showed that both basal and differentiation-induced transcripts of the lineage commitment gene Runx2/Cbfa1, as well as osteoblast differentiation marker genes including alkaline phosphatase, type I collagen, and osteocalcin, were all increased in GILZ-expressing cells. In contrast, the mRNA levels of adipogenic Ppargamma2 and C/ebpalpha were significantly reduced in GILZ-expressing cells under both osteogenic and adipogenic conditions. Together, our results demonstrate that GILZ functions as a modulator of MSCs and that overexpression of GILZ shifts the balance between osteogenic and adipogenic differentiation of MSCs toward the osteogenic pathway. These data suggest that GILZ may have therapeutic value for stem cell-based therapies of metabolic bone diseases, such as fracture repair.

Glucocorticoid-induced leucine zipper: A key protein in the sensitization of monocytes to lipopolysaccharide in alcoholic hepatitis

Glucocorticoid-induced leucine zipper (GILZ), a recently identified protein induced by glucocorticoids (GCs), inhibits the nuclear factor kappaB pathway and the activation of monocytes/macrophages by lipopolysaccharides (LPS). This study aimed to elucidate the contribution of GILZ to the pathogenesis of alcoholic hepatitis (AH): we (1) assessed GILZ expression in the livers of patients with AH and (2) treated patients with severe AH with GCs (prednisolone 40 mg/day) and studied the effect of GILZ modulation on circulating monocyte function. We quantified GILZ expression in the livers of 42 consecutive alcoholic patients (21 with and 21 without AH). GILZ messenger RNA (mRNA) levels were lower in the livers of patients with AH versus those without AH (P < 0.05). We collected circulating monocytes from patients with severe AH before and 48 hours after GC treatment to quantify GILZ expression and cytokine secretion. GC treatment induced significantly higher levels of GILZ mRNA than that observed before treatment and impaired LPS-induced tumor necrosis factor-alpha (TNF-alpha) and regulated upon activation, normal T cell-expressed secretion (RANTES) by these monocytes. We transfected circulating monocytes with GILZ small interfering RNA (siRNA), specifically blocking GILZ expression, to demonstrate the role of GILZ in mediating GC effect. GILZ siRNA abrogated the effect of GC treatment on LPS-induced TNF-alpha and RANTES secretion. Low expression of GILZ may contribute to liver inflammation in AH. GCs enhance GILZ expression, abrogating macrophage sensitivity to LPS and proinflammatory cytokine secretion. These findings may explain the beneficial effect of GC treatment in patients with severe AH.

Localization of glucocorticoid-induced leucine zipper (GILZ) expressing neurons in the central nervous system and its relationship to the stress response

The glucocorticoid-induced leucine zipper (GILZ) is a 137 amino acid protein, which was originally identified as a dexamethasone-inducible gene, and is characterized by a leucine zipper domain, an N-terminal domain and a C-terminal proline and glutamic acid rich domain. In this study, we performed in situ hybridization analyses and found that many neurons from the olfactory bulb to the spinal cord express GILZ mRNA, suggesting that GILZ is involved in fundamental and common cellular events of neurons. In addition, the predominant expression of GILZ mRNA in the motor nuclei indicated a strong relationship between GILZ and the motor nervous system. On the other hand, real-time RT-PCR analyses of brains of animals exposed to water-immersion restraint stress (WRS) showed that WRS significantly up-regulated GILZ expression in the medial prefrontal cortex (mPFC) and hippocampus, which are known to be mental stress-related areas, suggesting the multiple roles of GILZ in the brain. This up-regulation was not observed in adrenalectomized mice, confirming its dependency on hypothalamic–pituitary–adrenal axis activation.

The Anti-Inflammatory Effect of Ciglitazone is Partially Mediated by Glucocorticoid Induced Leucine Zipper in Human Airway Epithelial Cells

RATIONALE: PPARγ activation has been shown to inhibit airway inflammation but the mechanisms are not fully understood; we therefore assessed whether the anti-inflammatory protein Glucocorticoid-Induced Leucine Zipper (GILZ), which we have shown is upregulated by the PPARγ agonist ciglitazone, is involved in this process. METHODS: The expression of GILZ was assessed by immunoblotting in A549 and normal human bronchial epithelial cells (NHBE) at baseline and following ciglitazone treatment. RNA silencing was used to knock down GILZ protein levels in A549 cells in order to determine whether GILZ mediates the anti-inflammatory effects of ciglitazone. GILZ siRNA- and Control siRNA-treated A549 cells were stimulated for 20 hours with ciglitazone followed by IL-1beta for 4 hours. Total RNA was isolated and IL-8 mRNA levels were assessed by quantitative real-time PCR. RESULTS: GILZ levels were upregulated by ciglitazone in A549 and NHBE cells. Ciglitazone potently inhibited the induction of IL-8 mRNA by IL-1beta in control siRNA cells, whereas in GILZ siRNA cells this effect was partially abrogated. CONCLUSION: Here we demonstrate the induction of GILZ, an anti-inflammatory protein known to inhibit NF-κB and AP-1, by ciglitazone in human airway epithelial cells. Further we demonstrate that GILZ in part mediates the anti-inflammatory effects of agonist-bound PPARγ. These findings help elucidate a mechanism via which PPARγ agonists reduce airway inflammation, and suggest the potential of the thiazolidinediones in the treatment of asthma.

Glucocorticoid Induced Leucine Zipper Mediates the Anti-Inflammatory Effects of Dexamethasone in Airway Epithelial Cells

RATIONALE: Since the anti-inflammatory protein glucocorticoid induced leucine zipper (GILZ) is known to be induced by steroids in hematopoietic cells and can regulate NF-kappaB activity in lymphocytes, we examined the regulation of GILZ expression in airway epithelial cells and investigated whether GILZ mediates part of the anti-inflammatory effect of corticosteroids.

The anti-inflammatory effect of glucocorticoids is mediated by glucocorticoid-induced leucine zipper in epithelial cells

Nuclear factor kappaB (NF-kappaB) plays a key role in the pathogenesis of asthma, being linked to the production of inflammatory cytokines that drive inflammation. A recently described anti-inflammatory protein, glucocorticoid-induced leucine zipper (GILZ), interferes with NF-kappaB-mediated gene transcription in T cells and macrophages. We sought to analyze the regulation of GILZ expression in airway epithelial cells and determine whether GILZ mediates part of the anti-inflammatory effect of corticosteroids. GILZ expression was assessed by means of PCR and immunoblotting in human epithelial cells at baseline and after stimulation with dexamethasone or cytokines (IL-1beta, TNF-alpha, and IFN-gamma). The effect of GILZ on LPS-, IL-1beta-, and polyinosinic:polycytidylic acid-induced NF-kappaB activation was assessed in BEAS-2B cells overexpressing GILZ. The requirement for GILZ in the inhibitory action of dexamethasone was assessed by knocking down GILZ expression by means of small interfering RNA (siRNA) technology. GILZ is constitutively expressed by human airway epithelial cells, and its levels are increased by dexamethasone and decreased by inflammatory cytokines. Overexpression of GILZ in BEAS-2B cells significantly inhibited the ability of IL-1beta, LPS, and polyinosinic:polycytidylic acid to activate NF-kappaB, whereas knockdown of GILZ inhibited the ability of dexamethasone to suppress IL-1beta-induced chemokine expression. This study demonstrates the expression of GILZ in human airway epithelial cells, its induction by dexamethasone, its suppression by inflammatory cytokines, and its role in mediating the anti-inflammatory effects of dexamethasone. Therapeutic upregulation of GILZ may be a novel strategy for the treatment of asthma.

Dexamethasone and IL‐10 stimulate glucocorticoid‐induced leucine zipper synthesis by human mast cells

Glucocorticoids (GCs) decrease tissue mast cell (MC) number and prevent their activation via their high-affinity IgE receptor. Glucocorticoid-induced leucine zipper (GILZ) is one of the GC-induced genes, which inhibits the functions of the transcriptional activators AP-1 and NF-kappaB. GILZ appears to be a critical actor in the anti-inflammatory and immunosuppressive effects of GCs in human T lymphocytes, macrophages and dendritic cells. We investigated whether GILZ was produced by human MCs and whether GILZ synthesis was stimulated by GCs. We also investigated whether GILZ production was modulated by (i) IL-10, because of its common immunosuppressive properties with GCs, (ii) histamine because of its pro-inflammatory properties and (iii) IL-4 and IL-5 because of their ability to favour MC survival and proliferation with SCF. The human MC lines HMC-1 5C6 and LAD-2, and cord blood-derived MCs (CB-MCs) were cultured alone or in the presence of GCs, IL-10, histamine, IL-4 or IL-5. The expression of GILZ was evaluated by using RT-PCR, Western blotting or immunocytochemistry. We found that human MC lines and CB-MCs constitutively produce GILZ. We also show that GCs and IL-10 stimulate GILZ production by human MCs. Our present results indicate that histamine, IL-4 and IL-5 alone or in combination with SCF do not downregulate GILZ production by MCs. These results show that GCs and IL-10 stimulate GILZ production by human MCs. As GILZ mediates anti-inflammatory effects of GCs in immune cells, we speculate that GILZ could account for the deactivation of MCs by GCs and IL-10.

Genomic and non-genomic effects of different glucocorticoids on mouse thymocyte apoptosis

Glucocorticoids, widely used therapeutic agents for several pathologies, act upon diverse cells and tissues, including the lympho-haemopoietic system. Glucocorticoid-mediated apoptosis has been described as one of the mechanisms underlying their pharmacological and physiological effects. Glucocorticoids induce apoptosis in thymocytes through genomic and non-genomic signals. We tested thymocyte apoptosis rates as induced by a panel of glucocorticoids. Using four glucocorticoids that are widely adopted in clinical practice we compared their induction of thymocyte apoptosis and activation of non-genomic and genomic signals, including phosphatidylinositol–specific phospholipase C (PI–PLC), caspase-8, -9 and -3, and Glucocorticoid-Induced Leucine Zipper (GILZ). GILZ is a protein that is rapidly induced by glucocorticoids treatment and involved in apoptosis modulation. Results indicate different glucocorticoids have different apoptotic activity which is related to their ability to induce both genomic, evaluated as caspases activation and GILZ expression, and non-genomic effects, evaluated as PI–PLC phosphorylation.

FoxO3 Mediates Antagonistic Effects of Glucocorticoids and Interleukin-2 on Glucocorticoid-Induced Leucine Zipper Expression

We have analyzed the promoter of human gilz (glucocorticoid-induced leucine zipper), a dexamethasone-inducible gene that is involved in regulating apoptosis, and identified six glucocorticoid (GC)-responsive elements and three Forkhead responsive elements (FHREs). Promoter deletion analysis and point mutations showed that individual mutation of the GC-responsive elements does not affect GC-induced transcription and that FHRE-1 and FHRE-3 elements contribute to the effects of GCs. Furthermore, overexpression of the Forkhead transcription factor FoxO3 enhances GC-induced gilz mRNA expression. The functional significance of the interaction between FoxO3 and GC receptor was established in T lymphocytes. Indeed, we show that GCs failed to induce GILZ expression in the presence of IL-2, a cytokine known to antagonize GC effects in T cells. Using a constitutive active mutant of protein kinase B that inactivates FoxO3 or a FoxO3 mutant that cannot be inactivated by protein kinase B, we demonstrate that IL-2 inhibitory effects on GILZ expression are mediated through inhibition of FoxO3 transcriptional activity. Therefore, FoxO3 appears to be a key factor mediating GC and IL-2 antagonism for gilz regulation in T lymphocytes. This regulation of GILZ expression was placed in a meaningful context in evaluating the effects of GILZ on GC-induced apoptosis in T lymphocytes.

GILZ, a new target for the transcription factor FoxO3, protects T lymphocytes from interleukin-2 withdrawal–induced apoptosis

AbstractInterleukin-2 (IL-2) withdrawal is a physiologic process inducing cell death in activated T lymphocytes. Glucocorticoid-induced leucine zipper (GILZ) has recently been identified as a protein modulating T-cell receptor activation by repressing various signaling pathways. We report here that IL-2 deprivation leads to expression of GILZ in T lymphocytes. We then characterized the human gilz promoter and showed that FoxO3 (Forkhead box class O3) binding to the Forkhead responsive elements identified in the promoter is necessary for induction of gilz expression upon IL-2 withdrawal. To assess the functional consequences of this induction, we used 2 strategies, GILZ overexpression and GILZ silencing in murine IL-2–dependent CTLL-2 cells. GILZ overexpression protects CTLL-2 cells from IL-2 withdrawal–induced apoptosis, whereas cell death is accelerated in cells unable to express GILZ. Concomitantly, the expression of Bim is inhibited in GILZ-overexpressing cells and enhanced when GILZ expression is impaired. Furthermore, GILZ inhibits FoxO3 transcriptional activity that leads to inhibition of Bim expression but also to down-regulation of GILZ itself. Therefore, GILZ is a transiently expressed protein induced upon IL-2 withdrawal that protects T cells from the onset of apoptosis.

Synthesis of glucocorticoid-induced leucine zipper (GILZ) by macrophages: an anti-inflammatory and immunosuppressive mechanism shared by glucocorticoids and IL-10

Glucocorticoids and interleukin 10 (IL-10) prevent macrophage activation. In murine lymphocytes, glucocorticoids induce expression of glucocorticoid-induced leucine zipper (GILZ), which prevents the nuclear factor kappaB (NF-kappaB)-mediated activation of transcription. We investigated whether GILZ could account for the deactivation of macrophages by glucocorticoids and IL-10. We found that GILZ was constitutively produced by macrophages in nonlymphoid tissues of humans and mice. Glucocorticoids and IL-10 stimulated the production of GILZ by macrophages both in vitro and in vivo. Transfection of the macrophagelike cell line THP-1 with the GILZ gene inhibited the expression of CD80 and CD86 and the production of the proinflammatory chemokines regulated on activation normal T-cell expressed and secreted (CCL5) and macrophage inflammatory protein 1alpha (CCL3). It also prevented toll-like receptor 2 production induced by lipopolysaccharide, interferongamma, or an anti-CD40 mAb, as well as NF-kappaB function. In THP-1 cells treated with glucocorticoids or IL-10, GILZ was associated with the p65 subunit of NF-kappaB. Activated macrophages in the granulomas of patients with Crohn disease or tuberculosis do not produce GILZ. In contrast, GILZ production persists in tumor-infiltrating macrophages in Burkitt lymphomas. Therefore, GILZ appears to play a key role in the anti-inflammatory and immunosuppressive effects of glucocorticoids and IL-10. Glucocorticoid treatment stimulates GILZ production, reproducing an effect of IL-10, a natural anti-inflammatory agent. The development of delayed-type hypersensitivity reactions is associated with the down-regulation of GILZ gene expression within lesions. In contrast, the persistence of GILZ gene expression in macrophages infiltrating Burkitt lymphomas may contribute to the failure of the immune system to reject the tumor.

Modulation of T-cell activation by the glucocorticoid-induced leucine zipper factor via inhibition of nuclear factor κB

AbstractPreviously a novel gene was identified that encodes a glucocorticoid-induced leucine zipper (GILZ) whose expression is up-regulated by dexamethasone. This study analyzed the role of GILZ in the control of T-cell activation and its possible interaction with nuclear factor κB (NF-κB). Results indicate that GILZ inhibits both T-cell receptor (TCR)–induced interleukin-2/interleukin-2 receptor expression and NF-κB activity. In particular, GILZ inhibits NF-κB nuclear translocation and DNA binding due to a direct protein-to-protein interaction of GILZ with the NF-κB subunits. Moreover, GILZ-mediated modulation of TCR-induced responses is part of a circuit because TCR triggering down-regulates GILZ expression. These results identify a new molecular mechanism involved in the dexamethasone-induced regulation of NF-κB activity and T-cell activation.

A New Dexamethasone-Induced Gene of the Leucine Zipper Family Protects T Lymphocytes from TCR/CD3-Activated Cell Death

By comparing mRNA species expressed in dexamethasone (DEX)–treated and untreated murine thymocytes, we have identified a gene, glucocorticoid-induced leucine zipper (GILZ), encoding a new member of the leucine zipper family. GILZ was found expressed in normal lymphocytes from thymus, spleen, and lymph nodes, whereas low or no expression was detected in other nonlymphoid tissues, including brain, kidney, and liver. In thymocytes and peripheral T cells, GILZ gene expression is induced by DEX. Furthermore, GILZ expression selectively protects T cells from apoptosis induced by treatment with anti-CD3 monoclonal antibody but not by treatment with other apoptotic stimuli. This antiapoptotic effect correlates with inhibition of Fas and Fas ligand expression. Thus, GILZ is a candidate transcription factor involved in the regulation of apoptosis of T cells.