The glucocorticoid-induced leucine zipper mediates statin-induced muscle damage.

Statins, the most prescribed class of drugs for the treatment of hypercholesterolemia, can cause muscle-related adverse effects. It has been shown that the glucocorticoid-induced leucine zipper (GILZ) plays a key role in the anti-myogenic action of dexamethasone. In the present study, we aimed to evaluate the role of GILZ in statin-induced myopathy. Statins induced GILZ expression in C2C12 cells, primary murine myoblasts/myotubes, primary human myoblasts, and in vivo in zebrafish embryos and human quadriceps femoris muscle. Gilz induction was mediated by FOXO3 activation and binding to the Gilz promoter, and could be reversed by the addition of geranylgeranyl, but not farnesyl, pyrophosphate. Atorvastatin decreased Akt phosphorylation and increased cleaved caspase-3 levels in myoblasts. This effect was reversed in myoblasts from GILZ knockout mice. Similarly, myofibers isolated from knockout animals were more resistant toward statin-induced cell death than their wild-type counterparts. Statins also impaired myoblast differentiation, and this effect was accompanied by GILZ induction. The in vivo relevance of our findings was supported by the observation that gilz overexpression in zebrafish embryos led to impaired embryonic muscle development. Taken together, our data point toward GILZ as an essential mediator of the molecular mechanisms leading to statin-induced muscle damage.

Glucocorticoid-transactivated TSC22D3 attenuates hypoxia- and diabetes-induced Müller glial galectin-1 expression via HIF-1α destabilization.

Galectin-1/LGALS1, a newly recognized angiogenic factor, contributes to the pathogenesis of diabetic retinopathy (DR). Recently, we demonstrated that glucocorticoids suppressed an interleukin-1ß-driven inflammatory pathway for galectin-1 expression in vitro and in vivo. Here, we show glucocorticoid-mediated inhibitory mechanism against hypoxia-inducible factor (HIF)-1α-involved galectin-1 expression in human Müller glial cells and the retina of diabetic mice. Hypoxia-induced increases in galectin-1/LGALS1 expression and promoter activity were attenuated by dexamethasone and triamcinolone acetonide in vitro. Glucocorticoid application to hypoxia-stimulated cells decreased HIF-1α protein, but not mRNA, together with its DNA-binding activity, while transactivating TSC22 domain family member (TSC22D)3 mRNA and protein expression. Co-immunoprecipitation revealed that glucocorticoid-transactivated TSC22D3 interacted with HIF-1α, leading to degradation of hypoxia-stabilized HIF-1α via the ubiquitin-proteasome pathway. Silencing TSC22D3 reversed glucocorticoid-mediated ubiquitination of HIF-1α and subsequent down-regulation of HIF-1α and galectin-1/LGALS1 levels. Glucocorticoid treatment to mice significantly alleviated diabetes-induced retinal HIF-1α and galectin-1/Lgals1 levels, while increasing TSC22D3 expression. Fibrovascular tissues from patients with proliferative DR demonstrated co-localization of galectin-1 and HIF-1α in glial cells partially positive for TSC22D3. These results indicate that glucocorticoid-transactivated TSC22D3 attenuates hypoxia- and diabetes-induced retinal glial galectin-1/LGALS1 expression via HIF-1α destabilization, highlighting therapeutic implications for DR in the era of anti-vascular endothelial growth factor treatment.

Exclusive transmission of the embryonic stem cell-derived genome through the mouse germline.

Gene targeting in embryonic stem (ES) cells remains best practice for introducing complex mutations into the mouse germline. One aspect in this multistep process that has not been streamlined with regard to the logistics and ethics of mouse breeding is the efficiency of germline transmission: the transmission of the ES cell-derived genome through the germline of chimeras to their offspring. A method whereby male chimeras transmit exclusively the genome of the injected ES cells to their offspring has been developed. The new technology, referred to as goGermline, entails injecting ES cells into blastocysts produced by superovulated homozygous Tsc22d3 floxed females mated with homozygous ROSA26-Cre males. This cross produces males that are sterile due to a complete cell-autonomous defect in spermatogenesis. The resulting male chimeras can be sterile but when fertile, they transmit the ES cell-derived genome to 100% of their offspring. The method was validated extensively and in two laboratories for gene-targeted ES clones that were derived from the commonly used parental ES cell lines Bruce4, E14, and JM8A3. The complete elimination of the collateral birth of undesired, non-ES cell-derived offspring in goGermline technology fulfills the reduction imperative of the 3R principle of humane experimental technique with animals. genesis 54:326-333, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.

Tsc22d3 promotes morphine tolerance in mice through the GPX4 ferroptosis pathway.

BACKGROUND: Morphine tolerance refers to gradual reduction in response to drug with continuous or repeated use of morphine, requiring higher doses to achieve same effect. METHODS: The morphine tolerance dataset GSE7762 profiles, obtained from gene expression omnibus (GEO) database, were used to identify differentially expressed genes (DEGs). Weighted Gene Co-expression Network Analysis (WGCNA) was applied to explore core modules of DEGs related to morphine tolerance. Core genes were input into Comparative Toxicogenomics Database (CTD). Animal experiments were performed to validate role of Tsc22d3 in morphine tolerance and its relationship with ferroptosis-related pathway. RESULTS: 500 DEGs were identified. DEGs were primarily enriched in negative regulation of brain development, neuronal apoptosis processes, and neurosystem development. Core gene was identified as Tsc22d3. Tsc22d3 gene-associated miRNAs were mmu-miR-196b-5p and mmu-miR-196a-5p. Compared to Non-morphine tolerant group, Tsc22d3 expression was significantly upregulated in Morphine tolerant group. Tsc22d3 expression was upregulated in Morphine tolerant+Tsc22d3_OE, expression of HIF-1alpha, GSH, GPX4 in GPX4 ferroptosis-related pathway showed a more pronounced decrease. As Tsc22d3 expression was downregulated in Morphine tolerant+Tsc22d3_KO, expression of HIF-1alpha, GSH, GPX4 in GPX4 ferroptosis-related pathway exhibited a more pronounced increase. Upregulation of Tsc22d3 in Morphine tolerant+Tsc22d3_OE led to a more pronounced increase in expression of apoptosis proteins (P53, Caspase-3, Bax, SMAC, FAS). The expression of inflammatory factors (IL6, TNF-alpha, CXCL1, CXCL2) showed a more pronounced increase with upregulated Tsc22d3 expression in Morphine tolerant+Tsc22d3_OE. CONCLUSIONS: Tsc22d3 is highly expressed in brain tissue of morphine-tolerant mice, activating ferroptosis pathway, enhancing apoptosis, promoting inflammatory responses in brain cells.

Elucidation of the mechanisms underlying tumor aggravation by the activation of stress-related neurons in the paraventricular nucleus of the hypothalamus.

A growing body of evidence suggests that excess stress could aggravate tumor progression. The paraventricular nucleus (PVN) of the hypothalamus plays an important role in the adaptation to stress because the hypothalamic-pituitary-adrenal (HPA) axis can be activated by inducing the release of corticotropin-releasing hormone (CRH) from the PVN. In this study, we used pharmacogenetic techniques to investigate whether concomitant activation of CRHPVN neurons could directly contribute to tumor progression. Tumor growth was significantly promoted by repeated activation of CRHPVN neurons, which was followed by an increase in the plasma levels of corticosterone. Consistent with these results, chronic administration of glucocorticoids induced tumor progression. Under the concomitant activation of CRHPVN neurons, the number of cytotoxic CD8+ T cells in the tumor microenvironment was dramatically decreased, and the mRNA expression levels of hypoxia inducible factor 1 subunit α (HIF1α), glucocorticoid receptor (GR) and Tsc22d3 were upregulated in inhibitory lymphocytes, tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). Furthermore, the mRNA levels of various kinds of driver molecules related to tumor progression and tumor metastasis were prominently elevated in cancer cells by concomitant activation of CRHPVN neurons. These findings suggest that repeated activation of the PVN-CRHergic system may aggravate tumor growth through a central-peripheral-associated tumor immune system.

Multi-Tissue Characterization of GILZ Expression in Dendritic Cell Subsets at Steady State and in Inflammatory Contexts.

Dendritic cells (DCs) are key players in the control of tolerance and immunity. Glucocorticoids (GCs) are known to regulate DC function by promoting their tolerogenic differentiation through the induction of inhibitory ligands, cytokines, and enzymes. The GC-induced effects in DCs were shown to critically depend on increased expression of the Glucocorticoid-Induced Leucine Zipper protein (GILZ). GILZ expression levels were further shown to control antigen-presenting cell function, as well as T-cell priming capacity of DCs. However, the pattern of GILZ expression in DC subsets across tissues remains poorly described, as well as the modulation of its expression levels in different pathological settings. To fill in this knowledge gap, we conducted an exhaustive analysis of GILZ relative expression levels in DC subsets from various tissues using multiparametric flow cytometry. This study was performed at steady state, in the context of acute as well as chronic skin inflammation, and in a model of cancer. Our results show the heterogeneity of GILZ expression among DC subsets as well as the complexity of its modulation, that varies in a cell subset- and context-specific manner. Considering the contribution of GILZ in the control of DC functions and its potential as an immune checkpoint in cancer settings, these results are of high relevance for optimal GILZ targeting in therapeutic strategies.

Endogenous and exogenous glucocorticoids abolish the efficacy of immune-dependent cancer therapies.

Glucocorticoids mediate potent anti-inflammatory and immunosuppressive effects. A chronic elevation of the endogenous glucocorticoid tonus subsequent to mental stress, as well as continuous treatment with exogenous glucocorticoids, activate an immunosuppressive transcription factor, TSC22D3, in dendritic cells, causing the subversion of cancer therapy-elicited antineoplastic immune responses and subsequent therapeutic failure.

Altered glucocorticoid metabolism represents a feature of macroph-aging.

The aging process is characterized by a chronic, low-grade inflammatory state, termed "inflammaging." It has been suggested that macrophage activation plays a key role in the induction and maintenance of this state. In the present study, we aimed to elucidate the mechanisms responsible for aging-associated changes in the myeloid compartment of mice. The aging phenotype, characterized by elevated cytokine production, was associated with a dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis and diminished serum corticosteroid levels. In particular, the concentration of corticosterone, the major active glucocorticoid in rodents, was decreased. This could be explained by an impaired expression and activity of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), an enzyme that determines the extent of cellular glucocorticoid responses by reducing the corticosteroids cortisone/11-dehydrocorticosterone to their active forms cortisol/corticosterone, in aged macrophages and peripheral leukocytes. These changes were accompanied by a downregulation of the glucocorticoid receptor target gene glucocorticoid-induced leucine zipper (GILZ) in vitro and in vivo. Since GILZ plays a central role in macrophage activation, we hypothesized that the loss of GILZ contributed to the process of macroph-aging. The phenotype of macrophages from aged mice was indeed mimicked in young GILZ knockout mice. In summary, the current study provides insight into the role of glucocorticoid metabolism and GILZ regulation during aging.

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: Fine-Tuning of Dendritic Cells Function.

Dendritic cells (DCs) are key antigen-presenting cells that control the induction of both tolerance and immunity. Understanding the molecular mechanisms regulating DCs commitment toward a regulatory- or effector-inducing profile is critical for better designing prophylactic and therapeutic approaches. Initially identified in dexamethasone-treated thymocytes, the glucocorticoid-induced leucine zipper (GILZ) protein has emerged as a critical factor mediating most, but not all, glucocorticoids effects in both non-immune and immune cells. This intracellular protein exerts pleiotropic effects through interactions with transcription factors and signaling proteins, thus modulating signal transduction and gene expression. GILZ has been reported to control the proliferation, survival, and differentiation of lymphocytes, while its expression confers anti-inflammatory phenotype to monocytes and macrophages. In the past twelve years, a growing set of data has also established that GILZ expression in DCs is a molecular switch controlling their T-cell-priming capacity. Here, after a brief presentation of GILZ isoforms and functions, we summarize current knowledge regarding GILZ expression and regulation in DCs, in both health and disease. We further present the functional consequences of GILZ expression on DCs capacity to prime effector or regulatory T-cell responses and highlight recent findings pointing to a broader role of GILZ in the fine tuning of antigen capture, processing, and presentation by DCs. Finally, we discuss future prospects regarding the possible roles for GILZ in the control of DCs function in the steady state and in the context of infections and chronic pathologies.

Regulation of SMAD transcription factors during freezing in the freeze tolerant wood frog, Rana sylvatica.

The wood frog, Rana sylvatica, survives sub-zero winter temperatures by undergoing full body freezing for weeks at a time, during which it displays no measurable brain activity, no breathing, and a flat-lined heart. Freezing is a hypometabolic state characterized by a global suppression of gene expression that is elicited in part by transcription factors that coordinate the activation of vital pro-survival pathways. Smad transcription factors respond to TGF-ß signalling and are involved in numerous cellular functions from development to stress. Given the identity of genes they regulate, we hypothesized that they may be involved in coordinating gene expression during freezing. Protein expression of Smad1/2/3/4/5 in response to freezing was examined in 24h frozen and 8h thawed wood frog tissues using western immunoblotting, with the determination of subcellular localization in muscle and liver tissues. Transcript levels of smad2, smad4 and downstream genes (serpine1, myostatin, and tsc22d3) were measured by RT-PCR. Tissue-specific responses were observed during freezing where brain, heart, and liver had elevated levels of pSmad3, and skeletal muscle and kidneys had increased levels of pSmad1/5 and pSmad2 during freeze/thaw cycle, while protein and transcript levels remained constant. There were increases in nuclear levels of pSmad2 in muscle and pSmad3 in liver. Transcript levels of serpine1 were induced in heart, muscle, and liver, myostatin in muscle, and tsc22d3 in heart, and liver during freezing. These results suggest a novel freeze-responsive activation of Smad proteins that may play an important role in coordinating pro-survival gene networks necessary for freeze tolerance.

Genotype-dependent consequences of traumatic stress in four inbred mouse strains.

Post-traumatic stress disorder (PTSD) is an anxiety disorder that develops in predisposed individuals following a terrifying event. Studies on isogenic animal populations might explain susceptibility to PTSD by revealing associations between the molecular and behavioural consequences of traumatic stress. Our study employed four inbred mouse strains to search for differences in post-stress response to a 1.5-mA electric foot shock. One day to 6 weeks after the foot shock anxiety, depression and addiction-like phenotypes were assessed. In addition, expression levels of selected stress-related genes were analysed in hippocampus and amygdala. C57BL/6J mice exhibited up-regulation in the expression of Tsc22d3, Nfkbia, Plat and Crhr1 genes in both brain regions. These alterations were associated with an increase of sensitized fear and depressive-like behaviour over time. Traumatic stress induced expression of Tsc22d3, Nfkbia, Plat and Fkbp5 genes and developed social withdrawal in DBA/2J mice. In 129P3/J strain, exposure to stress produced the up-regulation of Tsc22d3 and Nfkbia genes and enhanced sensitivity to the rewarding properties of morphine. Whereas, SWR/J mice displayed increase only in Pdyn expression in the amygdala and had the lowest conditioned fear. Our results reveal a complex genetic background of phenotypic variation in response to stress and indicate the SWR/J strain as a valuable model of stress resistance. We found potential links between the alterations in expression of Tsc22d3, Nfkbia and Pdyn, and different aspects of susceptibility to stress.