Glucocorticoid-induced Leucine Zipper mRNA Research Articles

High expression of L-GILZ transcript variant 1 (GILZ TV 1) is associated with increased 30-day sepsis mortality, and a high expression ratio possibly contraindicates hydrocortisone administration

BackgroundSepsis presents a challenge due to its complex immune responses, where balance between inflammation and anti-inflammation is critical for survival. Glucocorticoid-induced leucine zipper (GILZ) is key protein in achieving this balance, suppressing inflammation and mediating glucocorticoid response. This study aims to investigate GILZ transcript variants in sepsis patients and explore their potential for patient stratification and optimizing glucocorticoid therapy.MethodsSepsis patients meeting the criteria outlined in Sepsis-3 were enrolled, and RNA was isolated from whole blood samples. Quantitative mRNA expression of GILZ transcript variants in both sepsis patient samples (n = 121) and the monocytic U937 cell line (n = 3), treated with hydrocortisone and lipopolysaccharides, was assessed using quantitative PCR (qPCR).ResultsElevated expression of GILZ transcript variant 1 (GILZ TV 1) serves as a marker for heightened 30-day mortality in septic patients. Increased levels of GILZ TV 1 within the initial day of sepsis onset are associated with a 2.2-[95% CI 1.2–4.3] fold rise in mortality, escalating to an 8.5-[95% CI 2.0–36.4] fold increase by day eight. GILZ TV1 expression is enhanced by glucocorticoids in cell culture but remains unaffected by inflammatory stimuli such as LPS. In septic patients, GILZ TV 1 expression increases over the course of sepsis and in response to hydrocortisone treatment. Furthermore, a high expression ratio of transcript variant 1 relative to all GILZ mRNA TVs correlates with a 2.3-fold higher mortality rate in patients receiving hydrocortisone treatment.ConclusionHigh expression of GILZ TV 1 is associated with a higher 30-day sepsis mortality rate. Moreover, a high expression ratio of GILZ TV 1 relative to all GILZ transcript variants is a parameter for identifying patient subgroups in which hydrocortisone may be contraindicated.

Effects of acute exercise and training status on glucocorticoid-induced leucine zipper (GILZ) expression in human skeletal muscle

Interactions between statin therapy and physical exercise complicate effective cardiovascular prevention. Emerging evidence suggests that muscle strain related changes in the expression of the glucocorticoid-induced leucine zipper (GILZ) may be involved. Therefore, we measured GILZ mRNA expression levels in M. vastus lateralis samples of 32 healthy individuals before and after a standardized bout of strength or endurance exercise. Overall, we found a highly significant downregulation of GILZ after exercise training (p < 0.001). Within-subgroup changes were statistically significant only after strength training, supporting the role of muscle (as opposed to cardiocirculatory) strain. If confirmed, this may help fitting training recommendations and medication.

GILZ regulates type I interferon release and sequesters STAT1

Glucocorticoids remain a mainstay of modern medicine due to their ability to broadly suppress immune activation. However, they cause severe adverse effects that warrant urgent development of a safer alternative. The glucocorticoid-induced leucine zipper (GILZ) gene, TSC22D3, is one of the most highly upregulated genes in response to glucocorticoid treatment, and reduced GILZ mRNA and protein levels are associated with increased severity of inflammation in systemic lupus erythematosus (SLE), Ulcerative Colitis, Psoriasis, and other autoimmune/autoinflammatory diseases. Here, we demonstrate that low GILZ permits expression of a type I interferon (IFN) signature, which is exacerbated in response to TLR7 and TLR9 stimulation. Conversely, overexpression of GILZ prevents IFN-stimulated gene (ISG) up-regulation in response to IFNα. Moreover, GILZ directly binds STAT1 and prevents its nuclear translocation, thereby negatively regulating IFN-induced gene expression and the auto-amplification loop of the IFN response. Thus, GILZ powerfully regulates both the expression and action of type I IFN, suggesting restoration of GILZ as an attractive therapeutic strategy for reducing reliance on glucocorticoids.

Gender May Influence the Immunosuppressive Actions of Prednisone in Young Patients With Inflammatory Bowel Disease.

Although the use of glucocorticoids (GC) is well established, the therapeutic response to these agents often shows important interindividual differences, in particular among young patients with inflammatory bowel diseases (IBD). Currently, GC resistance or dependence cannot be predicted by clinical or laboratory findings. The aim of this study was to investigate the association of gender and age with GC efficacy and with the expression of Glucocorticoid-Induced Leucine Zipper (GILZ). One hundred thirty patients (mean age at enrolment 12.6 years, 53 Crohn’s disease, 70 males) were enrolled in this retrospective study. IBD patients with active disease despite prednisone at a daily dose of up to 2 mg/kg over a period of 4 weeks were defined as steroid resistant. Patients who initially responded but relapsed upon dose reduction were considered steroid-dependent. Total RNA was extracted from biopsies of 14 patients (9 males) and the levels of GILZ mRNA were evaluated by real-time PCR. Association between clinical response to prednisone and the considered demographic variables was evaluated using logistic regression models. After 4 weeks of treatment, 112 patients were responders to prednisone and 18 were resistant; at this time-point, resistant patients were older than responders (p=0.032). After 12 weeks, 42, 71 and 12 patients were sensitive, dependent and resistant respectively; at this time-point, females were more prone than males to develop prednisone dependence vs a good response (p=0.028) while age had no effect. Age was associated with response both at 4 and 12 weeks in the subgroups of females: resistant patients were older than sensitive ones at 4 weeks (p=0.02). Likewise, at 12 weeks of therapy, dependent patients resulted older than sensitive ones (p=0.05). No association of age with prednisone response was found in males. In a subgroup of 14 patients (5 females), GILZ mRNA expression in intestinal biopsies was higher in males (p=0.0031). Patients with unfavorable response (7) presented lower GILZ expression at disease onset in comparison to the responder group (p=0.017). Older females with IBD have a higher incidence of prednisone unfavorable response and reduced intestinal expression of the GC pharmacodynamic marker GILZ.

Glucocorticoid-induced leucine zipper \u201cquantifies\u201d stressors and increases male susceptibility to PTSD

Post-traumatic stress disorder (PTSD) selectively develops in some individuals exposed to a traumatic event. Genetic and epigenetic changes in glucocorticoid pathway sensitivity may be essential for understanding individual susceptibility to PTSD. This study focuses on PTSD markers in the glucocorticoid pathway, spotlighting glucocorticoid-induced leucine zipper (GILZ), a transcription factor encoded by the gene Tsc22d3 on the X chromosome. We propose that GILZ uniquely “quantifies” exposure to stressors experienced from late gestation to adulthood and that low levels of GILZ predispose individuals to PTSD in males only. GILZ mRNA and methylation were measured in 396 male and female human blood samples from the Grady Trauma Project cohort (exposed to multiple traumatic events). In mice, changes in glucocorticoid pathway genes were assessed following exposure to stressors at distinct time points: (i) CRF-induced prenatal stress (CRF-inducedPNS) with, or without, additional exposure to (ii) PTSD induction protocol in adulthood, which induces PTSD-like behaviors in a subset of mice. In humans, the number of traumatic events correlated negatively with GILZ mRNA levels and positively with % methylation of GILZ in males only. In male mice, we observed a threefold increase in the number of offspring exhibiting PTSD-like behaviors in those exposed to both CRF-inducedPNS and PTSD induction. This susceptibility was associated with reduced GILZ mRNA levels and epigenetic changes, not found in females. Furthermore, virus-mediated shRNA knockdown of amygdalar GILZ increased susceptibility to PTSD. Mouse and human data confirm that dramatic alterations in GILZ occur in those exposed to a stressor in early life, adulthood or both. Therefore, GILZ levels may help identify at-risk populations for PTSD prior to additional traumatic exposures.

Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages.

Activation of toll-like receptors (TLRs) plays a pivotal role in the host defense against bacteria and results in the activation of NF-κB-mediated transcription of proinflammatory mediators. Glucocorticoid-induced leucine zipper (GILZ) is an anti-inflammatory mediator, which inhibits NF-κB activity in macrophages. Thus, we aimed to investigate the regulation and role of GILZ expression in primary human and murine macrophages upon TLR activation. Treatment with TLR agonists, e.g., Pam3CSK4 (TLR1/2) or LPS (TLR4) rapidly decreased GILZ mRNA and protein levels. In consequence, GILZ downregulation led to enhanced induction of pro-inflammatory mediators, increased phagocytic activity, and a higher capacity to kill intracellular bacteria (Salmonella enterica serovar typhimurium), as shown in GILZ knockout macrophages. Treatment with the TLR3 ligand polyinosinic: polycytidylic acid [Poly(I:C)] did not affect GILZ mRNA levels, although GILZ protein expression was decreased. This effect was paralleled by sensitization toward TLR1/2- and TLR4-agonists. A bioinformatics approach implicated more than 250 miRNAs as potential GILZ regulators. Microarray analysis revealed that the expression of several potentially GILZ-targeting miRNAs was increased after Poly(I:C) treatment in primary human macrophages. We tested the ability of 11 of these miRNAs to target GILZ by luciferase reporter gene assays. Within this small set, four miRNAs (hsa-miR-34b*,−222,−320d,−484) were confirmed as GILZ regulators, suggesting that GILZ downregulation upon TLR3 activation is a consequence of the synergistic actions of multiple miRNAs. In summary, our data show that GILZ downregulation promotes macrophage activation. GILZ downregulation occurs both via MyD88-dependent and -independent mechanisms and can involve decreased mRNA or protein stability and an attenuated translation.

Peripheral blood GILZ mRNA levels in depression and following electroconvulsive therapy

Dysregulation of the hypothalamic-pituitary-adrenocortical (HPA)-axis is commonly observed in patients with depression. The delayed feedback system that mediates inhibition of HPA-axis activation is regulated by glucocorticoid receptors (GRs) found in stress-responsive areas of the brain. Glucocorticoid-induced leucine zipper (GILZ) is a key molecule in glucocorticoid biology and is thought to mediate the downstream anti-inflammatory effects of GRs. Previous reports suggest that GILZ levels are altered in the blood and brains of patients with, and animal models of, depression. However, no study has yet investigated the effects of antidepressant treatment on GILZ. Therefore, our aim was to examine peripheral blood GILZ mRNA levels in patients with depression (n = 88) compared to age- and sex-matched healthy controls (n = 63), and in patients with depression following treatment with a course of electroconvulsive therapy (ECT). We also assessed the relationship between GILZ and mood and clinical outcomes following ECT. GILZ mRNA levels were assessed using qRT-PCR. GILZ levels were found to be significantly lower in patients with depression compared to controls (p < 0.002), and ECT further decreased GILZ levels (p = 0.05). Both of these results survived adjustment for potential covariates. However, we found no association between GILZ and mood scores. Overall, these results suggest that GILZ is involved in the pathophysiology of depression and the peripheral molecular response to ECT.

Mechanistic Multi-Tissue Modeling of Glucocorticoid-Induced Leucine Zipper Regulation: Integrating Circadian Gene Expression with Receptor-Mediated Corticosteroid Pharmacodynamics.

The glucocorticoid-induced leucine zipper (GILZ) is an important mediator of anti-inflammatory corticosteroid action. The pharmacokinetic/pharmacodynamic/pharmacogenomic effects of acute and chronic methylprednisolone (MPL) dosing on the tissue-specific dynamics of GILZ expression were examined in rats. A mechanism-based model was developed to investigate and integrate the role of MPL and circadian rhythms on the transcriptional enhancement of GILZ in multiple tissues. Animals received a single 50-mg/kg intramuscular bolus or a 7-day 0.3-mg/kg/h subcutaneous infusion of MPL and were euthanized at several time points. An additional group of rats were euthanized at several times and served as 24-hour light/dark (circadian) controls. Plasma MPL and corticosterone concentrations were measured by high-performance liquid chromatography. The expression of GILZ and glucocorticoid receptor (GR) mRNA was quantified in tissues using quantitative real-time reverse-transcription polymerase chain reaction. The pharmacokinetics of MPL were described using a two-compartment model. Mild-to-robust circadian oscillations in GR and GILZ mRNA expression were characterized in muscle, lung, and adipose tissues and modeled using Fourier harmonic functions. Acute MPL dosing caused significant down-regulation (40%-80%) in GR mRNA and enhancement of GILZ mRNA expression (500%-1080%) in the tissues examined. While GILZ returned to its rhythmic baseline following acute dosing, a new steady-state was observed upon enhancement by chronic dosing. The model captured the complex dynamics in all tissues for both dosing regimens. The model quantitatively integrates physiologic mechanisms, such as circadian processes and GR tolerance phenomena, which control the tissue-specific regulation of GILZ by corticosteroids. These studies characterize GILZ as a pharmacodynamic marker of corticosteroid actions in several tissues.

Induction of Glucocorticoid-induced Leucine Zipper (GILZ) Contributes to Anti-inflammatory Effects of the Natural Product Curcumin in Macrophages

GILZ (glucocorticoid-induced leucine zipper) is inducible by glucocorticoids and plays a key role in their mode of action. GILZ attenuates inflammation mainly by inhibition of NF-κB and mitogen-activated protein kinase activation but does not seem to be involved in the severe side effects observed after glucocorticoid treatment. Therefore, GILZ might be a promising target for new therapeutic approaches. The present work focuses on the natural product curcumin, which has previously been reported to inhibit NF-κB. GILZ was inducible by curcumin in macrophage cell lines, primary human monocyte-derived macrophages, and murine bone marrow-derived macrophages. The up-regulation of GILZ was neither associated with glucocorticoid receptor activation nor with transcriptional induction or mRNA or protein stabilization but was a result of enhanced translation. Because the GILZ 3'-UTR contains AU-rich elements (AREs), we analyzed the role of the mRNA-binding protein HuR, which has been shown to promote the translation of ARE-containing mRNAs. Our results suggest that curcumin treatment induces HuR expression. An RNA immunoprecipitation assay confirmed that HuR can bind GILZ mRNA. In accordance, HuR overexpression led to increased GILZ protein levels but had no effect on GILZ mRNA expression. Our data employing siRNA in LPS-activated RAW264.7 macrophages show that curcumin facilitates its anti-inflammatory action by induction of GILZ in macrophages. Experiments with LPS-activated bone marrow-derived macrophages from wild-type and GILZ knock-out mice demonstrated that curcumin inhibits the activity of inflammatory regulators, such as NF-κB or ERK, and subsequent TNF-α production via GILZ. In summary, our data indicate that HuR-dependent GILZ induction contributes to the anti-inflammatory properties of curcumin.

Low expression of the GILZ may contribute to adipose inflammation and altered adipokine production in human obesity

The glucocorticoid-induced leucine zipper (GILZ), a primary target of glucocorticoids, is expressed in human adipocytes, but its importance in adipocyte function is unknown. Because TNFα is increased in obese adipose tissue and antagonizes a number of glucocorticoid actions, we investigated the interplay of these pathways. GILZ knockdown increased and GILZ overexpression decreased interleukin-6 (IL-6) and leptin mRNA and protein secretion. GILZ knockdown increased the magnitude of the glucocorticoid effect on leptin secretion, but did not affect the glucocorticoid suppression of IL-6. Although GILZ silencing decreased adiponectin mRNA levels, it did not affect the amount of adiponectin secreted. GILZ negatively modulated pro-inflammatory signaling pathways, blocking basal and TNFα-stimulated (1 h) p65 nuclear factor κB nuclear translocation and transcriptional activity by binding to p65 in the cytoplasm. GILZ silencing increased basal ERK1/2 and JNK phosphorylation, and decreased MAPK phosphatase-1 protein levels. Longer term TNFα (4 h or 24 h) treatment decreased GILZ expression in human adipocytes. Furthermore, adipose tissue GILZ mRNA levels were reduced in proportion to the degree of obesity and expression of inflammatory markers. Overall, these results suggest that GILZ antagonizes the pro-inflammatory effects of TNFα in human adipocytes, and its downregulation in obesity may contribute to adipose inflammation and dysregulated adipokine production, and thereby systemic metabolism.

Glucocorticoid-induced leucine zipper: a critical factor in macrophage endotoxin tolerance.

Induction of glucocorticoid-induced leucine zipper (GILZ) by glucocorticoids plays a key role in their anti-inflammatory action. In activated macrophages, GILZ levels are downregulated via tristetraprolin-mediated GILZ mRNA destabilization. To assess the functional significance of GILZ downregulation, we generated myeloid-specific GILZ knockout (KO) mice. GILZ-deficient macrophages displayed a higher responsiveness toward LPS, as indicated by increased TNF-α and IL-1β expression. This effect was due to an activation of ERK, which was significantly amplified in GILZ KO cells. The LPS-induced activation of macrophages is attenuated upon pretreatment of macrophages with low-dose LPS, an effect termed endotoxin tolerance. In LPS-tolerant macrophages, GILZ mRNA was stabilized, whereas ERK activation was strongly decreased. In contrast, GILZ KO macrophages exhibited a strongly reduced desensitization. To explore the contribution of GILZ expression in macrophages to endotoxin tolerance in vivo, we treated GILZ KO mice with repeated i.p. injections of low-dose LPS followed by treatment with high-dose LPS. LPS pretreatment resulted in reduced proinflammatory mediator expression upon high-dose LPS treatment in serum and tissues. In contrast, cytokine induction was preserved in tolerized GILZ KO animals. In summary, our data suggest that GILZ is a key regulator of macrophage functions.

Quantitative tissue-specific dynamics of in vivo GILZ mRNA expression and regulation by endogenous and exogenous glucocorticoids

Glucocorticoids (GC) are steroid hormones, which regulate metabolism and immune function. Synthetic GCs, or corticosteroids (CS), have appreciable clinical utility via their ability to suppress inflammation in immune-mediated diseases like asthma and rheumatoid arthritis. Recent work has provided insight to novel GC-induced genes that mediate their anti-inflammatory effects, including glucocorticoid-induced leucine zipper (GILZ). Since GILZ comprises an important part of GC action, its regulation by both drug and hormone will influence CS therapy. In addition, GILZ expression is often employed as a biomarker of GC action, which requires judicious selection of sampling time. Understanding the in vivo regulation of GILZ mRNA expression over time will provide insight into both the physiological regulation of GILZ by endogenous GC and the dynamics of its enhancement by CS. A highly quantitative qRT-PCR assay was developed for measuring GILZ mRNA expression in tissues obtained from normal and CS-treated rats. This assay was applied to measure GILZ mRNA expression in eight tissues; to determine its endogenous regulation over time; and to characterize its dynamics in adipose tissue, muscle, and liver following treatment with CS. We demonstrate that GILZ mRNA is expressed in several tissues. GILZ mRNA expression in adipose tissue displayed a robust circadian rhythm that was entrained with the circadian oscillation of endogenous corticosterone; and is strongly enhanced by acute and chronic dosing. Single dosing also enhanced GILZ mRNA in muscle and liver, but the dynamics varied. In conclusion, GILZ is widely expressed in the rat and highly regulated by endogenous and exogenous GCs.

AB0073 Measurement of the Anti-Inflammatory Molecule Glucocorticoid-Induced Leucine Zipper (GILZ) in Rheumatoid Arthritis Patients

BackgroundRheumatoid arthritis (RA) affects 1% of populations worldwide. Approximately 30% of RA patients receive treatment with glucocorticoids (GC), which cause significant toxicity (1). Glucocorticoid-induced leucine zipper (GILZ) is a GC-induced...

Role of glucocorticoids and Toxoplasma gondii infection on murine intestinal epithelial cells

Glucocorticoids (GCs) are stress hormones secreted in response to perceived psychological and or physiological stress. GCs have been shown to reduce tissue inflammation by down-regulating the production of inflammatory chemokines produced by epithelial cells. The protozoan parasite Toxoplasma gondii is known to increase cytokine, chemokine, and Toll-like receptors (TLRs) expression in parasite infected mouse intestinal epithelial cells (IECs). We sought to analyze the role of an anti-inflammatory protein, glucocorticoid-induced leucine zipper (GILZ) in MODE-K cells during infection with T. gondii. GILZ expression in MODE-K cells was assessed by PCR and immunoblotting after stimulation with GCs (corticosterone, CORT) or T. gondii infection. GILZ mRNA was constitutively expressed in MODE-K cells but not its protein product. While infection and pre-exposure to CORT decreased GILZ isoforms of 28 and 17kD, the presence of CORT during infection increased levels of 17kD isoform. Infected cells treated with CORT had decreased expression of chemokines (IP-10/CXCL10, MCP-1/CCL2, MIP-2/CXCL8) while their expression was increased when endogenous GILZ was removed by siRNA treatment. GILZ up-regulation during infection may serve as a mechanism to decrease epithelial cell responses and facilitate parasite replication.

Downregulation of the glucocorticoid-induced leucine zipper (GILZ) promotes vascular inflammation

ObjectiveGlucocorticoid-induced leucine zipper (GILZ) represents an anti-inflammatory mediator, whose downregulation has been described in various inflammatory processes. Aim of our study was to decipher the regulation of GILZ in vascular inflammation. Approach and resultsDegenerated aortocoronary saphenous vein bypass grafts (n = 15), which exhibited inflammatory cell activation as determined by enhanced monocyte chemoattractrant protein 1 (MCP-1, CCL2) and Toll-like receptor 2 (TLR2) expression, showed significantly diminished GILZ protein and mRNA levels compared to healthy veins (n = 23). GILZ was also downregulated in human umbilical vein endothelial cells (HUVEC) and macrophages upon treatment with the inflammatory cytokine TNF-α in a tristetraprolin (ZFP36, TTP)- and p38 MAPK-dependent manner. To assess the functional implications of decreased GILZ expression, we determined NF-κB activation after GILZ knockdown by siRNA and found that NF-κB activity and inflammatory gene expression were significantly enhanced. Importantly, ZFP36 is induced in TNF-α-activated HUVEC as well as in degenerated vein bypasses. When atheroprotective laminar shear stress was employed, GILZ levels in HUVEC increased on mRNA and protein level. Laminar flow also counteracted TNF-α-induced ZFP36 expression and GILZ downregulation. MAP kinase phosphatase 1 (MKP-1, DUSP1), a negative regulator of ZFP36 expression, was distinctly upregulated under laminar shear stress conditions and downregulated in degenerated vein bypasses. ConclusionOur data show a diminished expression of the anti-inflammatory mediator GILZ in the inflamed vasculature and indicate that GILZ downregulation requires the mRNA binding protein ZFP36. We suggest that reduced GILZ levels play a role in cardiovascular disease.

Dexamethasone Inhibits Repair of Human Airway Epithelial Cells Mediated by Glucocorticoid-Induced Leucine Zipper (GILZ)

BackgroundGlucocorticoids (GCs) are a first-line treatment for asthma for their anti-inflammatory effects, but they also hinder the repair of airway epithelial injury. The anti-inflammatory protein GC-induced leucine zipper (GILZ) is reported to inhibit the activation of the mitogen-activated protein kinase (MAPK)-extracellular-signal-regulated kinase (ERK) signaling pathway, which promotes the repair of airway epithelial cells around the damaged areas. We investigated whether the inhibition of airway epithelial repair imposed by the GC dexamethasone (DEX) is mediated by GILZ.MethodsWe tested the effect of DEX on the expressions of GILZ mRNA and GILZ protein and the MAPK-ERK signaling pathway in human airway epithelial cells, via RT-PCR and Western blot. We further evaluated the role of GILZ in mediating the effect of DEX on the MAPK-ERK signaling pathway and in airway epithelium repair by utilizing small-interfering RNAs, MTT, CFSE labeling, wound-healing and cell migration assays.ResultsDEX increased GILZ mRNA and GILZ protein levels in a human airway epithelial cell line. Furthermore, DEX inhibited the phosphorylation of Raf-1, Mek1/2, Erk1/2 (components of the MAPK-ERK signaling pathway), proliferation and migration. However, the inhibitory effect of DEX was mitigated in cells when the GILZ gene was silenced.ConclusionsThe inhibition of epithelial injury repair by DEX is mediated in part by activation of GILZ, which suppressed activation of the MAPK-ERK signaling pathway, proliferation and migration. Our study implicates the involvement of DEX in this process, and furthers our understanding of the dual role of GCs.

Yersinia enterocolitica YopT and Clostridium difficile Toxin B Induce Expression of GILZ in Epithelial Cells

Glucocorticoid induced-leucine zipper (GILZ) has been shown to be induced in cells by different stimuli such as glucocorticoids, IL-10 or deprivation of IL-2. GILZ has anti-inflammatory properties and may be involved in signalling modulating apoptosis. Herein we demonstrate that wildtype Yersinia enterocolitica which carry the pYV plasmid upregulated GILZ mRNA levels and protein expression in epithelial cells. Infection of HeLa cells with different Yersinia mutant strains revealed that the protease activity of YopT, which cleaves the membrane-bound form of Rho GTPases was sufficient to induce GILZ expression. Similarly, Clostridium difficile toxin B, another bacterial inhibitor of Rho GTPases induced GILZ expression. YopT and toxin B both increased transcriptional activity of the GILZ promoter in HeLa cells. GILZ expression could not be linked to the inactivation of an individual Rho GTPase by these toxins. However, forced expression of RhoA and RhoB decreased basal GILZ promoter activity. Furthermore, MAPK activation proved necessary for profound GILZ induction by toxin B. Promoter studies and gel shift analyses defined binding of upstream stimulatory factor (USF) 1 and 2 to a canonical c-Myc binding site (E-box) in the GILZ promoter as a crucial step of its trans-activation. In addition we could show that USF-1 and USF-2 are essential for basal as well as toxin B induced GILZ expression. These findings define a novel way of GILZ promoter trans-activation mediated by bacterial toxins and differentiate it from those mediated by dexamethasone or deprivation of IL-2.

Glucocorticoid‐induced leucine zipper is downregulated in human alveolar macrophages upon Toll‐like receptor activation

Induction of the glucocorticoid-induced leucine zipper (GILZ) by glucocorticoids plays a role in their antiinflammatory action, whereas GILZ expression is reduced under inflammatory conditions. The mechanisms regulating GILZ expression during inflammation, however, have not yet been characterized. Here, we investigated GILZ expression in human alveolar macrophages (AMs) following Toll-like receptor (TLR) activation. Macrophages were shown to predominantly express GILZ transcript variant 2. Lipopolysaccharide-treated AMs, THP-1 cells, and lungs of lipopolysaccharide-exposed mice displayed decreased GILZ protein and mRNA levels. The effect was strictly dependent on the adapter molecule MyD88, as shown by using specific ligands or a knockdown strategy. Investigations on the functional significance of GILZ downregulation performed by GILZ knockdown revealed a proinflammatory response, as indicated by increased cytokine expression and NF-κB activity. We found that TLR activation reduced GILZ mRNA stability, which was mediated via the GILZ 3'-untranslated region. Finally, involvement of the mRNA-binding protein tristetraprolin (TTP) is suggested, since TTP overexpression or knockdown modulated GILZ expression and TTP was induced in a MyD88-dependent fashion. Taken together, our data show a MyD88- and TTP-dependent GILZ downreg-ulation in human macrophages upon TLR activation. Suppression of GILZ is mediated by mRNA destabilization, which might represent a regulatory mechanism in macrophage activation.

Corticosteroid‐induced gene expression in allergen‐challenged asthmatic subjects taking inhaled budesonide

Inhaled corticosteroids (ICS) are the cornerstone of asthma pharmacotherapy and, acting via the glucocorticoid receptor (GR), reduce inflammatory gene expression. While this is often attributed to a direct inhibitory effect of the GR on inflammatory gene transcription, corticosteroids also induce the expression of anti-inflammatory genes in vitro. As there are no data to support this effect in asthmatic subjects taking ICS, we have assessed whether ICS induce anti-inflammatory gene expression in subjects with atopic asthma. Bronchial biopsies from allergen-challenged atopic asthmatic subjects taking inhaled budesonide or placebo were subjected to gene expression analysis using real-time reverse transcriptase-PCR for the corticosteroid-inducible genes (official gene symbols with aliases in parentheses): TSC22D3 [glucocorticoid-induced leucine zipper (GILZ)], dual-specificity phosphatase-1 (MAPK phosphatase-1), both anti-inflammatory effectors, and FKBP5 [FK506-binding protein 51 (FKBP51)], a regulator of GR function. Cultured pulmonary epithelial and smooth muscle cells were also treated with corticosteroids before gene expression analysis. Compared with placebo, GILZ and FKBP51 mRNA expression was significantly elevated in budesonide-treated subjects. Budesonide also increased GILZ expression in human epithelial and smooth muscle cells in culture. Immunostaining of bronchial biopsies revealed GILZ expression in the airways epithelium and smooth muscle of asthmatic subjects. Expression of the corticosteroid-induced genes, GILZ and FKBP51, is up-regulated in the airways of allergen-challenged asthmatic subjects taking inhaled budesonide. Consequently, the biological effects of corticosteroid-induced genes should be considered when assessing the actions of ICS. Treatment modalities that increase or decrease GR-dependent transcription may correspondingly affect corticosteroid efficacy.

Chronic rhinosinusitis with and without nasal polyps is associated with decreased expression of glucocorticoid‐induced leucine zipper

Chronic rhinosinusitis without nasal polyps (CRSsNP) and with nasal polyps (CRSwNP) is characterized by persistent inflammation of sinonasal mucosa. Glucocorticoid-induced leucine zipper (GILZ) is a recently described anti-inflammatory mediator. Here we analysed the expression of GILZ in CRSsNP and CRSwNP, its association with response to surgery, and its cytokine-driven expression regulation in the upper airways. Methods The messenger RNA (mRNA) and protein expression of GILZ in 33 CRSsNP, 32 CRSwNP, and 11 control samples was assessed by means of a quantitative RT-PCR and immunohistochemistry, respectively. Nasal explant culture was used to investigate the effect of IFN-gamma, IL-4, IL-13, IL-1beta, and TNF-alpha on GILZ mRNA expression in normal sinonasal mucosa. The GILZ mRNA and protein expression was significantly suppressed in both CRSsNP and CRSwNP patients compared with controls. No significant difference in GILZ expression was found between CRSsNP and CRSwNP patients. Comparing patients responsive and patients recalcitrant to surgery, a significant further decrease of GILZ expression was found in recalcitrant patients both in the CRSsNP and in the CRSwNP group. IL-1beta, TNF-alpha, IL-4, and IL-13 reduced, whereas IFN-gamma enhanced GILZ mRNA levels in the sinonasal mucosa. Down-regulated expression of GILZ may contribute to the pathogenesis of CRSsNP and CRSwNP and associate with response to surgery. GILZ expression in the upper airways can be regulated differentially by different cytokines.