Glucocorticoid-induced Leucine Zipper Research Articles

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.

A colonic mineralocorticoid receptor cell model expressing epithelial Na+ channels

In the distal colon, the epithelial sodium channel (ENaC) is rate limiting for sodium absorption. Progress in the molecular characterization of ENaC expression and trafficking in response to the mineralocorticoid aldosterone has been hampered, since no epithelial colonic cell line existed expressing functional ENaC stimulated by nanomolar aldosterone via mineralocorticoid receptor (MR). Here, we present a human colonic epithelial cell line inducibly expressing the MR (HT-29/B6-Tet-On-MR) which exhibits aldosterone-dependent ENaC-mediated sodium transport in the presence of the short-chain fatty acid butyrate. Butyrate was necessary for high-level expression of MR which allowed for aldosterone-dependent upregulation of β- and γ-ENaC expression. As butyrate alone was not capable of promoting ENaC-mediated sodium transport, aldosterone-induced GILZ (glucocorticoid-induced leucine zipper protein) was identified as a candidate factor increasing apical ENaC levels.

Cigarette Smoke Impaired Antiviral Responses of Airway Epithelial Cells to dsRNA are Associated with Upregulation of Glucocorticoid Induced Leucine Zipper (GILZ)

Cigarette Smoke Impaired Antiviral Responses of Airway Epithelial Cells to dsRNA are Associated with Upregulation of Glucocorticoid Induced Leucine Zipper (GILZ)

MicroRNA 18 and 124a Down-Regulate the Glucocorticoid Receptor: Implications for Glucocorticoid Responsiveness in the Brain

Glucocorticoids (GCs) exert profound effects on a variety of physiological processes, including adaptation to stress, metabolism, immunity, and neuronal development. Cellular responsiveness to GCs depends on numerous factors, including the amount of the glucocorticoid receptor (GR) protein. We tested the hypothesis that micro-RNAs (miRs), a recently discovered group of noncoding RNAs involved in mRNA translation, might control GR activity by reducing GR protein levels in neuronal tissues. We tested a panel of five miRs consisting of 124aa, 328, 524, 22, and 18. We found that miRs 18 and 124a reduced GR-mediated events in addition to decreasing GR protein levels. miR reporter assays revealed binding of miR-124a to the 3' untranslated region of GR. In correspondence, the activation of the GR-responsive gene glucocorticoid-induced leucine zipper was strongly impaired by miR-124a and -18 overexpression. Although miR-18 is expressed widely throughout the body, expression of miR-124a is restricted to the brain. Endogenous miR-124a up-regulation during neuronal differentiation of P19 cells was associated with a decreasing amount of GR protein levels and reduced activity of luciferase reporter constructs bearing GR 3' untranslated regions. Furthermore, we show that miR-124a expression varies over time during the stress hyporesponsive period, a neonatal period when GC signaling is modulated. Our findings demonstrate a potential role for miRs in the regulation of cell type-specific responsiveness to GCs, as may occur during critical periods of neuronal development. Ultimately, our results may provide a better understanding of the etiology of stress-related diseases as well as the efficacy of GC therapy.

Conditional Transgenic Mice for Studying the Role of the Glucocorticoid Receptor in the Renal Collecting Duct

The mineralocorticoid receptor (MR) is a major regulator of renal sodium reabsorption and body fluid homeostasis. However, little is known about glucocorticoid receptor (GR)-dependent renal effects. Glucocorticoids may activate both receptors, so it is difficult to distinguish between MR- and GR-mediated effects in vivo. To overcome this complexity, we used a transgenic mouse model allowing conditional GR overexpression (doxycycline inducible TetON system, Hoxb7 promoter) in the renal collecting duct (CD) to identify GR-regulated genes involved in sodium transport in the CD. In microdissected cortical CD, induction of GR expression led (after 2 d of doxycycline) to increased alpha-epithelial sodium channel and glucocorticoid-induced leucine zipper and decreased abundance of with-no-lysine kinase 4 transcripts, without modification of Na,K-ATPase, serum- and glucocorticoid-kinase-1, or MR expression. No changes occurred in the upstream distal and connecting tubules [distal convoluted tubule (DCT), connecting tubule (CNT)]. Sodium excretion was unaltered, but the urinary aldosterone concentration was reduced, suggesting compensation of transitory extracellular volume expansion that subsequently disappeared. At steady state, i.e. after 15 d of doxycycline administration, transcript abundance remained altered in the CD, whereas mirror changes appeared in the DCT and CNT. Plasma aldosterone or glucocorticoids and blood pressure were all unaffected. These experiments show that: 1) GR, in addition to MR, controls epithelial sodium channel- and glucocorticoid-induced leucine zipper expression in vivo in the CD; 2) with-no-lysine kinase 4 is negatively controlled by GR; and 3) the DCT and CNT compensate for these alterations to maintain normal sodium reabsorption and blood pressure. These results suggest that enhanced GR expression may contribute to enhanced sodium retention in some pathological situations.

Expression and regulation of glucocorticoid-induced leucine zipper in the developing anterior pituitary gland

The expression profile of glucocorticoid-induced leucine zipper (GILZ) in the anterior pituitary during the second half of embryonic development in the chick is consistent with in vivo regulation by circulating corticosteroids. However, nothing else has been reported about the presence of GILZ in the neuroendocrine system. We sought to characterize expression and regulation of GILZ in the chicken embryonic pituitary gland and determine the effect of GILZ overexpression on anterior pituitary hormone levels. Pituitary GILZ mRNA levels increased during embryogenesis to a maximum on the day of hatch, and decreased through the first week after hatch. GILZ expression was rapidly upregulated by corticosterone in embryonic pituitary cells. To determine whether GILZ regulates hormone gene expression in the developing anterior pituitary, we overexpressed GILZ in embryonic pituitary cells and measured mRNA for the major pituitary hormones. Exogenous GILZ increased prolactin mRNA above basal levels, but not as high as that in corticosterone-treated cells, indicating that GILZ may play a small role in lactotroph differentiation. The largest effect we observed was a twofold increase in FSH beta subunit in cells transfected with GILZ but not treated with corticosterone, suggesting that GILZ may positively regulate gonadotroph development in a manner not involving glucocorticoids. In conclusion, this is the first report to characterize avian GILZ and examine its regulation in the developing neuroendocrine system. We have shown that GILZ is upregulated by glucocorticoids in the embryonic pituitary gland and may regulate expression of several pituitary hormones.

Molecular Events in Human T Cells Treated with Diesel Exhaust Particles or Formaldehyde that Underlie Their Diminished Interferon-γ and Interleukin-10 Production

Background: A series of epidemiological and experimental studies have suggested that diesel exhaust particles (DEP) and formaldehyde (FA) may help trigger T helper type 2 (Th2)-mediated allergic responses. Methods: To identify the molecular events by which DEP and FA induce a Th2-skewed immune response, we stimulated T cells from healthy subjects with anti-CD3/anti-CD28 monoclonal antibodies and examined the effect of pretreatment with DEP or FA on mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB by Western blotting and colorimetric NF-κB assays. We also examined the mRNA expression profiles of the T cells by microarray and real-time PCR analyses. Results:FA selectively suppressed interferon (IFN)-γ and interleukin-10 mRNA expression and protein production in stimulated T cells, as we previously reported with DEP. In the present study, we found that both DEP and FA suppressed NF-κB signaling and activated MAPKs. Both also significantly suppressed the mRNA expression of T-bet, Txk and c-Maf. Microarrays revealed significant augmentation of the expression of 2 FoxO3a-dependent genes, namely glucocorticoid-induced leucine zipper and growth arrest and DNA damage-inducible gene 45α (Gadd45a), which are known to modulate T cell immune responses. Treatment with N-acetylcysteine reversed the augmented Gadd45a mRNA response and caused the suppressed IFN-γ mRNA response to recover. Conclusions:DEP and FA have similar transcriptional and nontranscriptional effects on T cell signaling that together promote a Th2-skewed immune response.

Glucocorticoid-Induced Leucine Zipper Is Protective in Th1-Mediated Models of Colitis

Inflammatory bowel diseases are relatively common diseases of the gastrointestinal tract. The relative therapeutic efficacy of glucocorticoids used in inflammatory bowel diseases resides in part in their capability to inhibit activity of nuclear factor kappaB (NF-kappaB), a transcription factor central to the inflammatory process, and the consequent production of T-helper 1 (Th1)-type cytokines. Previous studies indicate that increased expression in transgenic mice of glucocorticoid-induced leucine zipper (GILZ), a gene rapidly induced by glucocorticoids, inhibits NF-kappaB and Th1 activity. We performed experiments with the aim to test the susceptibility of GILZ transgenic (GILZ-TG) mice to dinitrobenzene sulfonic acid-induced colitis. Consistent with a decreased Th1 response, GILZ-TG mice were less susceptible to colitis induction as compared with wild-type littermates, while they were more susceptible to Th2-mediated colitis. The inhibition was comparable to that obtained with dexamethasone treatment. Moreover, diminished intestinal tissue damage, associated with inhibition of NF-kappaB nuclear translocation, interferon-gamma, tumor necrosis factor-alpha, and interleukin-1 production in CD4+ T lymphocytes of the lamina propria, was evident in GILZ-TG as compared with wild-type mice. In addition, inhibition of colitis development was also evident when GILZ fusion protein was delivered in vivo in dinitrobenzene sulfonic acid-treated WT animals as well as in interleukin-10 knockout mice. Together these results demonstrate that GILZ mimics the effects of glucocorticoids, suggesting a contribution of this protein to the anti-inflammatory activity of glucocorticoids in Th1-induced colitis.

Chromatin immunoprecipitation scanning identifies glucocorticoid receptor binding regions in the proximal promoter of a ubiquitously expressed glucocorticoid target gene in brain

While the actions of glucocorticoids on brain functions have been comprehensively studied, the underlying genomic mechanisms are poorly understood. In this study, we show that glucocorticoid-induced leucine zipper (GILZ) mRNA is strongly and ubiquitously induced in rat brain. To decipher the molecular mechanisms underlying these genomic effects, it is of interest to identify the regulatory sites in the promoter region. Alignment of the rat GILZ promoter with the well-characterized human promoter resulted in poor sequence homology. Consequently, we analyzed the rat 5' flanking sequence by Matrix REDUCE and identified two high-affinity glucocorticoid response elements (GRE) located 2 kb upstream of the transcription start site. These findings were corroborated using the glucocorticoid receptor (GR) expressing Ns-1 PC12 rat cell-line. In these cells, dexamethasone treatment leads to a progressive increase of GILZ mRNA expression levels via a GR-dependent mechanism. Subsequently, using chromatin immunoprecipitation assays we show that the two high-affinity GREs are located within the GR-binding regions. Lastly, we demonstrate using multiple tissue in situ hybridization a marked increase in mRNA expression levels in spleen, thymus, heart, lung, liver, muscle, testis, kidney, colon, ileum, as well as in brain and conclude that the GILZ gene can be used to study glucocorticoid effects in many additional rodent tissues.

276 Annexin-1 regulates cytokines and dexamethasone sensitivity via glucocorticoid-induced leucine zipper

276 Annexin-1 regulates cytokines and dexamethasone sensitivity via glucocorticoid-induced leucine zipper

Enhanced Resistance of Restraint-Stressed Mice to Sepsis

Sepsis remains a major health concern across the world. The effects of stress on host resistance to sepsis are still not very clear. To explore the effects of chronic stress on sepsis(') we examined the impact of restraint stress on the resistance of mice to sepsis. Interestingly, it was found that restraint stress enhanced the antisepsis resistance of mice and the concentrations of the proinflammatory cytokines IL-1, IL-6, IL-12, and TNF-alpha in the blood of stressed mice were dramatically reduced post Escherichia coli infection or LPS treatment as compared with that of controls (p < 0.05). In addition, the mRNA expressions of glucocorticoid-induced leucine zipper (GILZ) were up-regulated in the spleen and peritoneal macrophages of mice receiving restraint stress or dexamethasone treatment. These results demonstrate that restraint stress enhances the resistance of mice to sepsis, supporting corticotherapy for sepsis and proposing restraint-stressed mouse as an animal model to elucidate mechanisms of stress-associated, antisepsis resistance.

Glucocorticoid Receptor Phosphorylation Differentially Affects Target Gene Expression

The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site-specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA-AM, a chelator of intracellular divalent cations, and curcumin, a natural product with antiinflammatory properties, reduced hormone-dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (vitamin D receptor interacting protein 150). Interestingly, in U2OS cells expressing a nonphosphorylated GR mutant S211A, the expression of IGF-binding protein 1 and interferon regulatory factor 8, both MED14-dependent GR target genes, was reduced relative to cells expressing wild-type receptor across a broad range of hormone concentrations. In contrast, the induction of glucocorticoid-induced leucine zipper, a MED14-independent GR target, was similar in S211A- and wild-type GR-expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement, and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene-selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and, in some cases, dependent upon the amount of activated receptor.

Glucocorticoid sensitivity in fibromyalgia patients: Decreased expression of corticosteroid receptors and glucocorticoid-induced leucine zipper

In fibromyalgia (FM) patients, differences in glucocorticoid receptor (GR) affinity and disturbances associated with loss of hypothalamic-pituitary-adrenal (HPA) axis resiliency have been observed. Based on these studies, we investigated whether FM would be associated with abnormalities in glucocorticoid (GC) sensitivity. Salivary and blood samples were collected from 27 FM patients and 29 healthy controls. Total plasma cortisol and salivary free cortisol were quantified by ELISA and time-resolved fluorescence immunoassay, respectively. GR sensitivity to dexamethasone was evaluated through IL-6 inhibition in stimulated whole blood. The corticosteroid receptors, GR alpha and mineralocorticoid receptor, as well as the glucocorticoid-induced leucine zipper (GILZ) and the FK506 binding protein 5 mRNA expression were assessed in peripheral blood mononuclear cells (PBMCs) by real-time RT-PCR. Furthermore, the corticosteroid receptors were analysed for polymorphism. We observed lower basal plasma cortisol levels (borderline statistical significance) and a lower expression of corticosteroid receptors and GILZ in FM patients when compared to healthy controls. The MR rs5522 (I180V) minor allele was found more often in FM patients than in controls and this variant was recently associated with a mild loss of receptor function. The lower GR and MR expression and possibly the reduced MR function may be associated with an impaired function of the HPA axis in these patients which, compounded by lower anti-inflammatory mediators, may sustain some of symptoms that contribute to the clinical picture of the syndrome.

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.

Cross Talk of Signaling Pathways in the Regulation of the Glucocorticoid Receptor Function

Several posttranslational modifications including phosphorylation have been detected on the glucocorticoid receptor (GR). However, the interdependence and combinatorial regulation of these modifications and their role in GR functions are poorly understood. We studied the effects of c-Jun N-terminal kinase (JNK)-dependent phosphorylation of GR on its sumoylation status and the impact that these modifications have on GR transcriptional activity. GR is targeted for phosphorylation at serine 246 (S246) by the JNK protein family in a rapid and transient manner. The levels of S246 phosphorylation of endogenous GR increased significantly in cells treated with UV radiation that activates JNK. S246 GR phosphorylation by JNK facilitated subsequent GR sumoylation at lysines 297 and 313. GR sumoylation increased with JNK activation and was inhibited in cells treated with JNK inhibitor. GR sumoylation in cells with activated JNK was mediated preferentially by small ubiquitin-like modifier (SUMO)2 rather than SUMO1. An increase in GR transcriptional activity was observed after inhibition of JNK or SUMO pathways and suppression of GR transcriptional activity after activation of both pathways in cells transfected with GR-responsive reporter genes. Endogenous GR transcriptional activity was inhibited on endogenous target genes IGF binding protein (IGFBP) and glucocorticoid-induced leucine zipper (GILZ) when JNK and SUMO pathways were induced individually or simultaneously. Activation of both of these signals inhibited GR-mediated regulation of human inhibitor of apoptosis gene (hIAP), whereas simultaneous activation had no effect. We conclude that phosphorylation aids GR sumoylation and that cross talk of JNK and SUMO pathways fine tune GR transcriptional activity in a target gene-specific manner, thereby modulating the hormonal response of cells exposed to stress.

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.

Differential recruitment of glucocorticoid receptor phospho-isoforms to glucocorticoid-induced genes

The human glucocorticoid receptor (GR) is phosphorylated on its N-terminus at three major sites (S203, S211 and S226) within activation function 1 (AF1). Although GR has been shown to assemble at glucocorticoid responsive elements (GREs) in the presence of hormone, the timing and specificity of GR phospho-isoform recruitment to receptor target genes has not been established. Using chromatin immunoprecipitation (ChIP) and GR phosphorylation site-specific antibodies, we examined GR phospho-isoform recruitment to several glucocorticoid-induced genes including tyrosine aminotransferase ( tat) and sulfonyltransferase-1A1 ( sult) in rat hepatoma cells, and the glucocorticoid-induced leucine zipper ( gilz) gene in human U2OS cells. GR P-S211 and GR P-S226 isoforms were efficiently recruited to the tat, sult and gilz GREs in a hormone-dependent manner. In contrast, the GR P-S203 isoform displayed no significant recruitment to any GREs of the genes analyzed, consistent with its lack of nuclear accumulation. Interestingly, the kinetics of GR P-S211 and GR P-S226 recruitment differed among genes. Our findings indicate that GR phospho-isoforms selectively occupy GR target genes, and suggests gene specific requirements for GR phosphorylation in receptor-dependent transcriptional activation.

Differential Activities of Glucocorticoid-induced Leucine Zipper Protein Isoforms

Glucocorticoid-induced leucine zipper protein (GILZ) is expressed in both epithelial and immune tissues and modulates a variety of cellular functions, including proliferation and epithelial sodium channel (ENaC) activity. A number of reports have described various GILZ activities, focusing on a single isoform with molecular mass of approximately 17 kDa, now termed GILZ1. In GILZ immunoblots using a newly developed antiserum, we detected multiple species in extracts from cultured kidney cells. Mass spectrometric analysis revealed that one of these represented a previously uncharacterized distinct isoform of GILZ, GILZ2. Rapid amplification of cDNA ends was used to clone cDNAs corresponding to four isoforms, which, in addition to GILZ1 and GILZ2, included new isoforms GILZ3 and GILZ4. Heterologous expression of these four GILZ isoforms in cultured cells revealed striking functional differences. Notably, GILZ1 was the only isoform that significantly stimulated ENaC-mediated Na+ current in a kidney collecting duct cell line, although GILZ2 and GILZ3 also stimulated ENaC surface expression in HEK 293 cells. GILZ1 and GILZ3, and to a lesser extent GILZ2, inhibited ERK phosphorylation. Interestingly, GILZ4, which had no effect on either ENaC or ERK, potently suppressed cellular proliferation, as did GILZ1, but not GILZ2 or GILZ3. Finally, rat and mouse tissues all expressed multiple GILZ species but varied in the relative abundance of each. These data suggest that multiple GILZ isoforms are expressed in most cells and tissues and that these play distinct roles in regulating key cellular functions, including proliferation and ion transport. Furthermore, GILZ inhibition of ERK appears to play an essential role in stimulation of cell surface ENaC but not in inhibition of proliferation.

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.

Glucocorticoid‐induced leucine zipper (GILZ) mediates glucocorticoid action and inhibits inflammatory cytokine‐induced COX‐2 expression

Cyclooxygenase-2 (COX-2) plays an important role in rheumatoid arthritis and therefore, has been a major target for anti-arthritis therapies. The expression of COX-2 is induced by inflammatory cytokines such as TNF-alpha and IL-1beta, and inhibited by glucocorticoids. However, the molecular mechanisms underlying the anti-inflammatory and immune suppressive actions of glucocorticoids are not well defined. Here we report that glucocorticoid-induced leucine zipper (GILZ) mimics glucocorticoid action and inhibits inflammatory cytokine-induced COX-2 expression in bone marrow mesenchymal stem cells, the cells that have been recently implicated in the pathogenesis and progression of rheumatoid arthritis. Using a retrovirus-mediated gene expression approach we demonstrate that overexpression of GILZ inhibits TNF-alpha and IL-1beta-induced COX-2 mRNA and protein expression, and knockdown of GILZ by shRNA reduces glucocorticoid inhibition of cytokine-induced COX-2 expression. Consistent to these results, overexpression of GILZ also inhibits NF-kappaB-mediated COX-2 promoter activity. Finally, we show that GILZ inhibits COX-2 expression by blocking NF-kappaB nuclear translocation. Our results suggest that GILZ is a key glucocorticoid effect mediator and that GILZ may have therapeutic value for novel anti-inflammation therapies.