Glucocorticoid-induced Leucine Zipper Research Articles

Characterization of gonadal glucocorticoid-induced leucine zipper (GILZ) protein expression during sex change in the protogynous orange-spotted grouper, Epinephelus coioides

Steroid hormones have been proven as a key drive of sex change in sequentially hermaphroditic organisms. However, the upstream mechanism of sex steroid hormones regulation that affect sex change remain unknown. The main glucocorticoid in teleost fish is cortisol, which both regulates steroidogenesis and has antistress action. Thus, cortisol might be one of the prime factors in sex change. In this study, the glucocorticoid-induced leucine zipper (GILZ) gene, was proven to have a dramatic effect in orange-spotted groupers (Epinephelus coioides) during sex change at the early stage of gonadal transition. The specific action of the GILZ protein is at the pouch-shaped proliferative spermatogonia instead of the degenerative oocyte at the onset of sex change. Immunohistochemical (IHC) evidence revealed that GILZ performs intensively at undifferentiated spermatogonia in the early testis stage. These results imply that cortisol provokes a rise of GILZ through regulation caused by steroid hormones leading to sex change.

Glucocorticoid-Induced Leucine Zipper as a Druggable Target in Inflammatory Bowel Diseases.

Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders with a complex pathogenesis, affecting people of all ages. They are characterized by alternating phases of clinical relapse and remission, depending on the fine balance between immune cells and the gut microbiota. The cross talk between cells of the immune system and the gut microbiota can result in either tolerance or inflammation, according to multifactorial triggers, ranging from environmental factors to genetic susceptibility. Glucocorticoid (GC) administration remains the first-line treatment for IBDs, although long-term use is limited by development of serious adverse effects. Recently, new alternative pharmacological therapies have been developed, although these are not always effective in IBD patients. There is a constant demand for effective new drug targets to guarantee total remission and improve the quality of life for IBD patients. The glucocorticoid-induced leucine zipper (GILZ) has been implicated as a promising candidate for this purpose, in view of its powerful anti-inflammatory effects that mimic those of GCs while avoiding their unwanted adverse reactions. Here we present and discuss the latest findings about the involvement of GILZ in IBDs.

Bcl-xL overexpression decreases GILZ levels and inhibits glucocorticoid-induced activation of caspase-8 and caspase-3 in mouse thymocytes

Glucocorticoids promote thymocyte apoptosis and modulate transcription of numerous regulators of thymic apoptosis. Among these, glucocorticoid-induced leucine zipper (GILZ) is strongly upregulated in the thymus. We have previously demonstrated that GILZ decreases Bcl-xL expression, activates caspase-8 and caspase-3, and augments apoptosis in mice thymocytes. To better understand the causal links between glucocorticoids, GILZ, Bcl-xL, caspase-8, and caspase-3, we analyzed the thymocytes of Bcl-xL-overexpressing transgenic mice with or without glucocorticoid stimulation in vitro. Overexpression of Bcl-xL inhibited the glucocorticoid-induced up-regulation of GILZ in murine thymocytes as well as the glucocorticoid-dependent activation of caspase-8 and caspase-3. By contrast, no appreciable change in caspase-9 activation was observed upon Bcl-xL overexpression. Thus, these experiments highlighted a novel thymocyte apoptotic pathway in which Bcl-xL overexpression inhibited the glucocorticoid-induced activation of caspase-8 and caspase-3, but not caspase-9, as well as the accumulation of GILZ protein. These findings, together with our previous results showing that caspase-8 protects GILZ from proteasomal degradation, suggest the presence of a glucocorticoid-induced apoptosis self-amplification loop in which GILZ decreases Bcl-xL expression with a subsequent activation of caspase-8 and caspase-3; caspase-8 activation then enhances the stability and accumulation of GILZ and ensures the unimpeded and irreversible progression of apoptosis. By contrast, inappropriate increases in Bcl-xL levels could have catastrophic effects on thymic apoptosis as it would shut down caspase-8/3 activation, diminish the expression of GILZ, and impair the fine control necessary for thymic generation of a healthy immune repertoire.

Synthetic Glucocorticoid-Induced Leucine Zipper Peptide Inhibits Lipopolysaccharide-Induced Ocular Inflammation in Rats

Purpose: To demonstrate the anti-inflammatory action of a synthetic glucocorticoid-induced leucine zipper (GILZ^98–134) peptide (GILZ-p) in a model of endotoxin-induced uveitis (EIU) in rats. Methods: The EIU model was induced in Sprague Dawley rats with an intravitreal injection of lipopolysaccharide (LPS). Synthetic GILZ-p was injected intravitreally 6 h after the LPS injection. To evaluate the anti-inflammatory effects of GILZ-p, the inflammatory response in the anterior chamber and iris of the rat eyes was evaluated with a slitlamp microscope on days 0, 1, 2, 3, and 4 after GILZ-p injection. The retinal expression of inflammatory cytokines was measured on days 0, 1, 2, 3, and 4 after GILZ-p injection. Müller cell gliosis was also detected at planned time points after GILZ-p injection. Results: Anterior segment inflammation peaked at 24 h after LPS injection in the EIU model. Compared with the controls, intravitreal GILZ-p significantly suppressed LPS-induced anterior segment inflammation in the EIU rats. The levels of retinal inflammatory factors IL-1β, TNF-α, MCP-1, and ICAM-1 were simultaneously reduced by the intravitreal GILZ-p injection. The expression of vimentin in the EIU retina was significantly reduced by GILZ-p, and the downregulated aquaporin 4 in the EIU retina was significantly restored by GILZ-p. Conclusion: The synthetic GILZ-p inhibited the inflammatory reaction in the EIU model and may have utility in the treatment of inflammatory ocular disease.

The Selective Progesterone Receptor Modulator Ulipristal Acetate Inhibits the Activity of the Glucocorticoid Receptor.

The selective progesterone modulator ulipristal acetate (ulipristal) offers a much-needed therapeutic option for the clinical management of uterine fibroids. Although ulipristal initially passed safety evaluations in Europe, postmarketing analysis identified cases of hepatic injury and failure, leading to restrictions on the long-term use of ulipristal. One of the factors potentially contributing to significant side effects with the selective progesterone modulators is cross-reactivity with other steroid receptors. To determine whether ulipristal can alter the activity of the endogenous glucocorticoid receptor (GR) in relevant cell types. Immortalized human uterine fibroid cells (UtLM) and hepatocytes (HepG2) were treated with the synthetic glucocorticoid dexamethasone and/or ulipristal. Primary uterine fibroid tissue was isolated from patients undergoing elective gynecological surgery and treated ex vivo with dexamethasone and/or ulipristal. In vivo ulipristal exposure was performed in C57Bl/6 mice to measure the effect on basal gene expression in target tissues throughout the body. Dexamethasone induced the expression of established glucocorticoid-target genes period 1 (PER1), FK506 binding protein 51 (FKBP5), and glucocorticoid-induced leucine zipper (GILZ) in UtLM and HepG2 cells, whereas cotreatment with ulipristal blocked the transcriptional response to glucocorticoids in a dose-dependent manner. Ulipristal inhibited glucocorticoid-mediated phosphorylation, nuclear translocation, and DNA interactions of GR. Glucocorticoid stimulation of PER1, FKBP5, and GILZ was abolished by cotreatment with ulipristal in primary uterine fibroid tissue. The expression of glucocorticoid-responsive genes was decreased in the lung, liver, and uterus of mice exposed to 2 mg/kg ulipristal. Interestingly, transcript levels of Fkbp5 and Gilz were increased in the hippocampus and pituitary. These studies demonstrate that ulipristal inhibits endogenous glucocorticoid signaling in human fibroid and liver cells, which is an important consideration for its use as a long-term therapeutic agent.

Glucocorticoids and Glucocorticoid-Induced-Leucine-Zipper (GILZ) in Psoriasis.

Psoriasis is a prevalent chronic inflammatory human disease initiated by impaired function of immune cells and epidermal keratinocytes, resulting in increased cytokine production and hyperproliferation, leading to skin lesions. Overproduction of Th1- and Th17-cytokines including interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-23, IL-17, and IL-22, is a major driver of the disease. Glucocorticoids (GCs) represent the mainstay protocol for treating psoriasis as they modulate epidermal differentiation and are potent anti-inflammatory compounds. The development of safer GC-based therapies is a high priority due to potentially severe adverse effects associated with prolonged GC use. Specific efforts have focused on downstream anti-inflammatory effectors of GC-signaling such as GC-Induced-Leucine-Zipper (GILZ), which suppresses Th17 responses and antagonizes multiple pro-inflammatory signaling pathways involved in psoriasis, including AP-1, NF-κB, STAT3, and ROR-γt. Here we review evidence regarding defective GC signaling, GC receptor (GR) function, and GILZ in psoriasis. We discuss seemingly contradicting data on the loss- and gain-of-function of GILZ in the imiquimod-induced mouse model of psoriasis. We also present potential therapeutic strategies aimed to restore GC-related pathways.

Glucocorticoid-induced leucine zipper protects noise-induced apoptosis in cochlear cells by inhibiting endoplasmic reticulum stress in rats.

Endoplasmic reticulum (ER) stress-mediated apoptosis has been reported to be involved in the noise-induced hearing loss (NIHL). Glucocorticoid-induced leucine zipper (GILZ) protein has been reported to have different regulatory effects on apoptosis according to cell types. However, whether GILZ regulates apoptosis in cochlear cells is unclear. Our study aimed to investigate the mechanism by which GILZ protected ER stress-mediated cochlear apoptosis induced by noise exposure. In our trials, forty-eight male Spraque-Dawley rats were randomized into the noise, OE-GILZ-rLV + noise (ON), shRNA-GILZ-rLV + noise (SN), and control group. Rats in noise and control groups were pre-treated by administration of Blank-rLV. Before and on days 1, 4, 14 after noise exposure, auditory brainstem response (ABR) and cochlear apoptosis were detected. Changes in GILZ, GRP78, CHOP, Bcl-xL, Bax, and cleaved caspase-3 levels were investigated. Noise exposure increased ABR threshold shifts and cochlear apoptosis in parallel with downregulation of Bcl-xL and upregulation of GRP78, CHOP, Bax and cleaved caspase-3. GILZ overexpression significantly reduced ABR threshold shifts and apoptotic cochlear cells owing to noise exposure. GILZ overexpression in the cochlea further increased GRP78 elevation, decreased expression of CHOP, Bax and cleaved caspase-3, and increased expression of Bcl-xL. GILZ silencing demonstrated the opposite effect on these effects. GILZ protects cochlea from ER stress-mediated apoptosis induced by noise exposure through reduction of CHOP and regulation of ER stress-associated apoptotic proteins.

Stress hormones as potential mediators of air pollutant effects on the brain: Rapid induction of glucocorticoid-responsive genes

Air pollution is associated with adverse effects on brain health including cognitive decline, dementia, anxiety, depression, and suicide. While toxicological studies have demonstrated the potential for repeated or chronic pollutant exposure to lead to disease states, characterisation of initial biological responses to exposure is needed to better understand underlying mechanisms. The brain is highly sensitive to glucocorticoids (primarily cortisol in humans, corticosterone in rodents), stress hormones that play important roles in cognition and mental health. We tested whether glucocorticoids could be implicated in central nervous system (CNS) effects of pollutant exposure by examining glucocorticoid-dependent signaling across brain regions after exposure to the common pollutant ozone. Male Fischer-344 rats were exposed for 4 h to air or 0.8 ppm ozone ± metyrapone (50 mg/kg), a drug that blocks corticosterone synthesis (n = 5/group). Key glucocorticoid-responsive genes (serum- and glucocorticoid-inducible kinase, SGK; glucocorticoid-inducible leucine zipper, GILZ), and a gene responsive to both glucocorticoids and oxidative stress (metallothionein (MT)-1), were increased by ozone in all brain regions (olfactory bulb, frontal lobe, cortex, midbrain, hippocampus, cerebellum, brainstem), correlating with plasma corticosterone levels. Metyrapone prevented the increase in SGK and GILZ, and reduced but did not eliminate the effect on MT-1, suggesting glucocorticoid-dependent and –independent regulation. Administering exogenous corticosterone (10 mg/kg) to air-exposed rats reproduced the ozone effects, confirming specificity. The results demonstrate that early pollutant effects include stress hormone-dependent signaling. As both ozone and particulate matter activate the hypothalamic-pituitary-adrenal axis, and elevated glucocorticoids are implicated in brain pathologies, stress hormones could contribute to CNS impacts of air pollutants.

Acute stress imposed during adolescence yields heightened anxiety in Sprague Dawley rats that persists into adulthood: Sex differences and potential involvement of the Medial Amygdala

Stressors experienced during adolescence have been demonstrated to have a long-lasting influence on affective behavior in adulthood. Notably, most studies to date have found these outcomes after chronic stress during adolescence. In the present study we tested how exposure to a single episode of acute footshock during early adolescence would modify subsequent adult anxiety- and depressive-like behaviors in male and female Sprague-Dawley rats. Adolescent rats were exposed to inescapable footshock (80 shocks, 5 s, 1.0 mA, 90 sec variable inter-trial interval (ITI)) at Post-natal day (PND) 29-30 and remained undisturbed until adulthood where they were evaluated with several behavioral assays for anxiety as well as depressive-like behavior via forced swim. In addition, gene expression changes were assessed immediately after a 30 min forced swim challenge in adulthood among several stress-related brain regions including the Central Amygdala (CeA), Medial Amygdala (MeA), ventral Hippocampus (vHPC), and Paraventricular Nucleus (PVN). Studies used real-time RT-PCR to examine the cytokines Interleukin-1β (IL-1β) and Interleukin-6 (IL-6), corticotropin-releasing hormone (CRH), the immediate early genes c-Fos, c-Jun, Egr1 and Arc, and several genes relating to corticosteroid receptor function (glucocorticoid and mineralocorticoid receptor (GR and MR, respectively), Gilz (glucocorticoid-induced leucine zipper), Sgk1 (Serum and Glucocorticoid regulated Kinase 1)). Behaviorally, males displayed signs of increased anxiety, most notably in the light-dark box, whereas females did not. No notable depressive-like behavior was observed in forced swim as a result of adolescent stress history, but adolescent footshock exacerbated the c-Fos response in the MeA produced by swim in both sexes. Forced swim led to increased IL-1β expression in the PVN regardless of adolescent stress history, whereas most HPA (hypothalamic-pituitaryadrenal) axis-related genes were largely unaffected in the vHPC. To determine the potential for β-adrenergic receptors to contribute to the male-specific anxiety-like behavior, two further studies applied a β-adrenergic agonist (isoproterenol) or antagonist (propranolol) in male rats. These studies found that propranolol administered 2 h after footshock led to a reduction in some anxiety-like behaviors as compared to controls. Overall, these findings suggest that exposure to a single, intense stress challenge imposed during adolescence may have sex-specific consequences across the lifespan and may implicate the MeA in developmental plasticity.

Implicating the Role of GILZ in Glucocorticoid Modulation of T-Cell Activation.

Glucocorticoid-induced leucine zipper (GILZ) is a protein with multiple biological roles that is upregulated by glucocorticoids (GCs) in both immune and non-immune cells. Importantly, GCs are immunosuppressive primarily due to their regulation of cell signaling pathways that are crucial for immune system activity. GILZ, which is transcriptionally induced by the glucocorticoid receptor (GR), mediates part of these immunosuppressive, and anti-inflammatory effects, thereby controlling immune cell proliferation, survival, and differentiation. The primary immune cells targeted by the immunosuppressive activity of GCs are T cells. Importantly, the effects of GCs on T cells are partially mediated by GILZ. In fact, GILZ regulates T-cell activation, and differentiation by binding and inhibiting factors essential for T-cell function. For example, GILZ associates with nuclear factor-κB (NF-κB), c-Fos, and c-Jun and inhibits NF-κB-, and AP-1-dependent transcription. GILZ also binds Raf and Ras, inhibits activation of Ras/Raf downstream targets, including mitogen-activated protein kinase 1 (MAPK1). In addition GILZ inhibits forkhead box O3 (FoxO3) without physical interaction. GILZ also promotes the activity of regulatory T cells (Tregs) by activating transforming growth factor-β (TGF-β) signaling. Ultimately, these actions inhibit T-cell activation and modulate the differentiation of T helper (Th)-1, Th-2, Th-17 cells, thereby mediating the immunosuppressive effects of GCs on T cells. In this mini-review, we discuss how GILZ mediates GC activity on T cells, focusing mainly on the therapeutic potential of this protein as a more targeted anti-inflammatory/immunosuppressive GC therapy.

Overexpression of Gilz Protects Mice Against Lethal Septic Peritonitis.

Sepsis in humans and experimental animals is characterized by an acute inflammatory response. glucocorticoids (GCs) are widely used for the treatment of many inflammatory disorders, yet their effectiveness in sepsis is debatable. One of the major anti-inflammatory proteins induced by GCs is glucocorticoid-induced leucine zipper (GILZ, coded by the TSC22D3 gene). We found that TSC22D3 mRNA expression is downregulated in white blood cells of human sepsis patients. Interestingly, transgenic GILZ-overexpressing mice (GILZ-tg) showed better survival rates in the cecal ligation and puncture (CLP) model of mouse sepsis. To our surprise, GILZ had only mild anti-inflammatory effects in this model, as the systemic proinflammatory response was not significantly reduced in GILZ-tg mice compared with control mice. During CLP, we observed reduced bacterial counts in blood of GILZ-tg mice compared with control mice. We found increased expression of Tsc22d3 mRNA specifically in peritoneal exudate cells in the CLP model, as well as increased capacity for bacterial phagocytosis of CD45 GILZ-tg cells compared with CD45 GILZ-wt cells. Hence, we believe that the protective effects of GILZ in the CLP model can be linked to a more efficient phagocytosis.

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.

Could GILZ Be the Answer to Glucocorticoid Toxicity in Lupus?

Glucocorticoids (GC) are used globally to treat autoimmune and inflammatory disorders. Their anti-inflammatory actions are mainly mediated via binding to the glucocorticoid receptor (GR), creating a GC/GR complex, which acts in both the cytoplasm and nucleus to regulate the transcription of a host of target genes. As a result, signaling pathways such as NF-κB and AP-1 are inhibited, and cell activation, differentiation and survival and cytokine and chemokine production are suppressed. However, the gene regulation by GC can also cause severe side effects in patients. Systemic lupus erythematosus (SLE or lupus) is a multisystem autoimmune disease, characterized by a poorly regulated immune response leading to chronic inflammation and dysfunction of multiple organs, for which GC is the major current therapy. Long-term GC use, however, can cause debilitating adverse consequences for patients including diabetes, cardiovascular disease and osteoporosis and contributes to irreversible organ damage. To date, there is no alternative treatment which can replicate the rapid effects of GC across multiple immune cell functions, effecting disease control during disease flares. Research efforts have focused on finding alternatives to GC, which display similar immunoregulatory actions, without the devastating adverse metabolic effects. One potential candidate is the glucocorticoid-induced leucine zipper (GILZ). GILZ is induced by low concentrations of GC and is shown to mimic the action of GC in several inflammatory processes, reducing immunity and inflammation in in vitro and in vivo studies. Additionally, GILZ has, similar to the GC-GR complex, the ability to bind to both NF-κB and AP-1 as well as DNA directly, to regulate immune cell function, while potentially lacking the GC-related side effects. Importantly, in SLE patients GILZ is under-expressed and correlates negatively with disease activity, suggesting an important regulatory role of GILZ in SLE. Here we provide an overview of the actions and use of GC in lupus, and discuss whether the regulatory mechanisms of GILZ could lead to the development of a novel therapeutic for lupus. Increased understanding of the mechanisms of action of GILZ, and its ability to regulate immune events leading to lupus disease activity has important clinical implications for the development of safer anti-inflammatory therapies.

Modeling Corticosteroid Pharmacokinetics and Pharmacodynamics, Part III: Estrous Cycle and Estrogen Receptor-Dependent Antagonism of Glucocorticoid-Induced Leucine Zipper (GILZ) Enhancement by Corticosteroids.

Our previous report examined the pharmacokinetics (PK) of methylprednisolone (MPL) and adrenal suppression after a 50 mg/kg IM bolus in male and female rats, and we described in detail the development of a minimal physiologically based pharmacokinetic/pharmacodynamic (mPBPK/PD) model. In continuation of such assessments, we investigated sex differences in genomic MPL responses (PD). Message expression of the glucocorticoid-induced leucine zipper (GILZ) was chosen as a multitissue biomarker of glucocorticoid receptor (GR)-mediated drug response. Potential time-dependent interplay between sex hormone and glucocorticoid signaling in vivo was assessed by comparing the enhancement of GILZ by MPL in the uterus [high estrogen receptor (ER) density] and in liver (lower ER density) from male and female rats dosed within the proestrus (high estradiol/progesterone) and estrus (low estradiol/progesterone) phases of the rodent estrous cycle. An expanded-systems PD model of MPL considering circadian rhythms, multireceptor (ER and GR) control, and estrous variations delineated the determinants controlling receptor/gene-mediated steroid responses. Hepatic GILZ response was ∼3-fold greater in females, regardless of estrous stage, compared with males, driven predominantly by increased MPL exposure in females and a negligible influence of estrogen interaction. In contrast, GILZ response in the uterus during proestrus in females was 60% of that observed in estrus-phased females, despite no PK or receptor differences, providing in vivo support to the hypothesis of estrogen-mediated antagonism of glucocorticoid signaling. The developed model offers a mechanistic platform to assess the determinants of sex and tissue specificity in corticosteroid actions and, in turn, reveals a unique PD drug-hormone interaction occurring in vivo. SIGNIFICANCE STATEMENT: Mechanisms relating to sex-based pharmacodynamic variability in genomic responses to corticosteroids have been unclear. Using combined experimental and systems pharmacology modeling approaches, sex differences in both pharmacokinetic and pharmacodynamic mechanisms controlling the enhancement of a sensitive corticosteroid-regulated biomarker, the glucocorticoid-induced leucine zipper (GILZ), were clarified in vivo. The multiscale minimal physiologically based pharmacokinetics/pharmacodynamic model successfully captured the experimental observations and quantitatively discerned the roles of the rodent estrous cycle (hormonal variation) and tissue specificity in mediating the antagonistic coregulation of GILZ gene synthesis. These findings collectively support the hypothesis that estrogens antagonize pharmacodynamic signaling of genomic corticosteroid actions in vivo in a time- and estrogen receptor-dependent manner.

Cholesterol Starvation and Hypoxia Activate the FVII Gene via the SREBP1-GILZ Pathway in Ovarian Cancer Cells to Produce Procoagulant Microvesicles.

Interaction between the transcription factors, hypoxia-inducible factor (HIF1α and HIF2α) and Sp1, mediates hypoxia-driven expression of FVII gene encoding coagulation factor VII (fVII) in ovarian clear cell carcinoma (CCC) cells. This mechanism is synergistically enhanced in response to serum starvation, a condition possibly associated with tumor hypoxia. This transcriptional response potentially results in venous thromboembolism, a common complication in cancer patients by producing procoagulant extracellular vesicles (EVs). However, which deficient serum factors are responsible for this characteristic transcriptional mechanism is unknown. Here, we report that cholesterol deficiency mediates synergistic FVII expression under serum starvation and hypoxia (SSH) via novel sterol regulatory element binding protein-1 (SREBP1)-driven mechanisms. Unlike conventional mechanisms, SREBP1 indirectly enhances FVII transcription through the induction of a new target, glucocorticoid-induced leucine zipper (GILZ) protein. GILZ expression induced in response to hypoxia by a HIF1α-dependent mechanism activates SREBP1 under SSH, suggesting reciprocal regulation between SREBP1 and GILZ. Furthermore, GILZ binds to the FVII locus. Xenograft tumor samples analyzed by chromatin immunoprecipitation confirmed that HIF1α-aryl hydrocarbon nuclear translocator and GILZ bind to the TSC22D3 (GILZ) and FVII gene loci, respectively, thereby potentially modulating chromatin function to augment FVII transcription. Thus, deficiency of both O2 and cholesterol, followed by interplay between HIFs, Sp1, and SREBP1-GILZ pathways synergistically induce fVII synthesis, resulting in the shedding of procoagulant EVs.

Glucocorticoid induced leucine zipper is inversely related with neuroinflammation in the brain

Abstract Glucocorticoids (GC) are steroid hormones secreted as the end-product of the neuroendocrine stress cascade. Absence or elevated GC mediate neurotoxic responses, suggesting that a narrow window ranging from physiological to slightly high GC mediate protective responses. The beneficial effects are attributed to the transactivation of regulatory proteins and inhibition mediated by glucocorticoid receptor interactions with other cofactors. Elevated GC accelerate beta amyloid (Aβ ) plaque formation and tau phosphorylation, aggregates of which upregulate reactive oxygen species in vulnerable neurons. The oxidative stress negatively regulates glucocorticoid receptor, preventing suppression of the transcriptional factor, nuclear factor-κ B (NF-κ B) by GC. The ensuing inflammatory distress enhance further Aβ accumulation initiating a vicious cycle. The glucocorticoid induced leucine zipper (GILZ) is a gene strongly upregulated by GC and mediates many of the anti-inflammatory and anti-proliferative effects of GC. Mechanistically GILZ inhibits NF-κ B by binding the p65 subunit of NF-κ B in the cytoplasm and thereby prevent transactivation of inflammatory and apoptosis mediators. Although GILZ is constitutively expressed in many tissues including the brain, the expression has been shown to occur with varying dynamics suggesting that the local milieu modulates its expression with consequent effects on cellular responses. Here we investigated the expression profile of GILZ in lipopolysaccharide mediated neuroinflammation model of Alzheimer’s disease. Our data suggest that the GILZ expression is downregulated in neuroinflammation correlating inversely with the pro-inflammatory cytokines and innate immune responses.

Sex-specific regulation of stress-induced fetal glucocorticoid surge by the mouse placenta.

Antenatal stress increases the prevalence of diseases in later life, which shows a strong sex-specific effect. However, the underlying mechanisms remain unknown. Maternal glucocorticoids can be elevated by stress and are potential candidates to mediate the effects of stress on the offspring sex-specifically. A comprehensive evaluation of dynamic maternal and placental mechanisms modulating fetal glucocorticoid exposure upon maternal stress was long overdue. Here, we addressed this gap in knowledge by investigating sex-specific responses to midgestational stress in mice. We observed increased levels of maternal corticosterone, the main glucocorticoid in rodents, along with higher corticosteroid-binding globulin levels at midgestation in C57Bl/6 dams exposed to sound stress. This resulted in elevated corticosterone in female fetuses, whereas male offspring were unaffected. We identified that increased placental expression of the glucocorticoid-inactivating enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2; Hsd11b2 gene) and ATP-binding cassette transporters, which mediate glucocorticoid efflux toward maternal circulation, protect male offspring from maternal glucocorticoid surges. We generated mice with an Hsd11b2 placental-specific disruption (Hsd11b2PKO) and observed moderately elevated corticosterone levels in offspring, along with increased body weight. Subsequently, we assessed downstream glucocorticoid receptors and observed a sex-specific differential modulation of placental Tsc22d3 expression, which encodes the glucocorticoid-induced leucine zipper protein in response to stress. Taken together, our observations highlight the existence of unique and well-orchestrated mechanisms that control glucocorticoid transfer, exposure, and metabolism in the mouse placenta, pinpointing toward the existence of sex-specific fetal glucocorticoid exposure windows during gestation in mice.

Glucocorticoid-Induced Leucine Zipper: A Novel Anti-inflammatory Molecule.

Glucocorticoids (GCs) are the most commonly used drugs for treatment of autoimmune and inflammatory diseases. Their efficacy is due to their ability to bind cytoplasmic receptors (glucocorticoid receptors, GR) and other cytoplasmic proteins, thus regulating gene expression. Although GCs are potent life-saving drugs, their therapeutic effects are transitory and chronic use of GCs is accompanied by serious side effects. Therefore, new drugs are needed to replace GCs. We have identified a gene, glucocorticoid-induced leucine zipper (GILZ or tsc22d3), that is rapidly and invariably induced by GCs. Human GILZ is a 135-amino acid protein that mediates many GC effects, including inhibition of the NF-κB and MAPK pathways. Similar to GCs, GILZ exerts anti-inflammatory activity in experimental disease models, including inflammatory bowel diseases and arthritis. While transgenic mice that overexpress GILZ are more resistant, GILZ knockout mice develop worse inflammatory diseases. Moreover, the anti-inflammatory effect of GCs is attenuated in GILZ-deficient mice. Importantly, in vivo delivery of recombinant GILZ protein cured colitis and facilitated resolution of lipopolysaccharide-induced inflammation without apparent toxic effects. A synthetic GILZ-derived peptide, corresponding to the GILZ region that interacts with NF-κB, was able to suppress experimental autoimmune encephalomyelitis. Collectively, these findings indicate that GILZ is an anti-inflammatory molecule that may serve as the basis for designing new therapeutic approaches to inflammatory diseases.

Formoterol counteracts the inhibitory effect of cigarette smoke on glucocorticoid-induced leucine zipper (GILZ) transactivation in human bronchial smooth muscle cells

Cigarette smokers with asthma and chronic obstructive pulmonary disease (COPD) are less responsive to glucocorticoids (GCs). The anti-inflammatory action of GCs depends also on their ability to transactivate genes such as GC-induced leucine zipper (GILZ). We investigated the effects of aqueous cigarette smoke extract (CSE) on GILZ transactivation evoked by 17-beclomethasone monopropionate (BMP) or fluticasone propionate (FP) in the presence or absence of the long acting β2-adrenoceptor agonist (LABA) bronchodilator formoterol or salmeterol in human primary cultures of human bronchial smooth muscle cells (HBSMC). We monitored GC receptor Ser211 phosphorylation by western blot analysis and GC receptor nuclear translocation by immunostaining followed high-content imaging analysis. BMP, as well as FP, induced GILZ expression in a concentration-dependent manner (EC50 of 0.87 and 0.16 nM respectively). Pre-incubation with CSE inhibited GC-evoked GILZ transactivation (>50%), GC receptor Ser211 phosphorylation and nuclear translocation. Both formoterol and salmeterol counteracted the effect of CSE on GC-induced GILZ expression but not on nuclear translocation or phosphorylation. The effect of formoterol was mimicked by the cAMP-elevating agent forskolin and blocked by ICI 118,551, a selective β2-adrenoceptor antagonist. Pre-incubation with TNF-α also reduced GC-evoked GILZ transactivation but was not counteracted by formoterol undercovering a different responsiveness to LABAs of TNF-α in comparison to CSE.In sum, CSE inhibits GC-evoked transactivation of GILZ and such effect is counteracted by LABAs, through β2-adrenoceptors and a cAMP-dependent mechanism. This study sheds light on a mechanism underlying complementary interactions between LABAs and inhaled GCs that could be relevant in smokers with asthma and COPD.

05 SUSTAINED EXPRESSION OF GLUCOCORTICOID-INDUCED LEUCINE ZIPPER (GILZ) MARKS A POPULATION OF FUNCTIONALLY IMMATURE INTESTINAL REGULATORY T CELLS IN FEMALE CROHN’S DISEASE PATIENTS

In addition to its classic roles in sexual development and function, estrogen (“E2”) is an important immune regulator. The impact of E2 in a given cell is determined by the relative contribution of signaling downstream of two ligand-activated nuclear receptors, estrogen receptors alpha and beta (ERa, ERb), with ERb generally mediating anti-inflammatory effects. Altered ER signaling likely contributes to the female sex bias observed for many autoimmune diseases including Crohn’s disease (CD). We observed reduced ERb expression in inflamed mucosal tissues and peripheral blood T cells from female CD patients, suggesting that reductions in ERb signaling may promote chronic intestinal inflammation in females. To investigate the contribution of ERb signaling in an experimental ileitis model, we backcrossed mice lacking ERb (ERb-KO) onto SAMP/YitFc (SAMP) mice, generating SAMP-ERb-KO mice lacking global ERb expression. CD patient T cells were FACS-sorted from heparinized peripheral blood from adult volunteers naïve to biologic therapies and corticosteroids. SAMP-ERb-KO mice demonstrated a female-specific exacerbation of ileitis, in which ileal inflammation developed at an earlier age and to a more severe extent in SAMP-ERb-KO female mice compared to SAMP-ERb-KO male mice or to native SAMP mice expressing ERb. Flow cytometric analysis of CD4+ T cells isolated from the mesenteric lymph node (MLN) revealed a decrease in regulatory T cell (Treg) frequency and suppressive function in SAMP-ERb-KO female mice, with a significant reduction in peripherally-induced Tregs. Next-generation RNA sequencing of intestinal Tregs from SAMP-ERb-KO male versus female mice revealed sex-based differences in the expression of several target genes. The most significant of these was Tsc22d3 (GILZ), a glucocorticoid-induced leucine zipper gene showing five-fold upregulation in Tregs from SAMP-ERb-KO female mice. We observed similar overexpression of GILZ in Tregs isolated from female CD patients. GILZ is an E2-regulated gene that affects several aspects of T cell function including peripheral Treg differentiation and Th17 responses. GILZ expression has not been previously observed in mature Tregs. Our data reveal that sustained expression of GILZ in SAMP-ERb-KO female Tregs correlates with impaired suppressive function, suggesting that ERb-mediated negative regulation of GILZ in peripheral Tregs represents an important immunoregulatory circuit contributing to peripheral Treg homeostasis. We show that female SAMP-ERb-KO mice and female CD patients exhibit reductions in anti-inflammatory ERb expression, leading to sustained expression of GILZ among peripheral Tregs. Sustained GILZ expression marks a population of immature, functionally insufficient Tregs that may contribute to the enhanced inflammation frequently observed in female CD patients.