Transport Of Glucocorticoid Receptor Research Articles

The transportosome system as a model for the retrotransport of soluble proteins

The classic model of action of the glucocorticoid receptor (GR) sustains that its associated heat-shock protein of 90-kDa (HSP90) favours the cytoplasmic retention of the unliganded GR, whereas the binding of steroid triggers the dissociation of HSP90 allowing the passive nuclear accumulation of GR. In recent years, it was described a molecular machinery called transportosome that is responsible for the active retrograde transport of GR. The transportosome heterocomplex includes a dimer of HSP90, the stabilizer co-chaperone p23, and FKBP52 (FK506-binding protein of 52-kDa), an immunophilin that binds dynein/dynactin motor proteins. The model shows that upon steroid binding, FKBP52 is recruited to the GR allowing its active retrograde transport on cytoskeletal tracks. Then, the entire GR heterocomplex translocates through the nuclear pore complex. The HSP90-based heterocomplex is released in the nucleoplasm followed by receptor dimerization. Subsequent findings demonstrated that the transportosome is also responsible for the retrotransport of other soluble proteins. Importantly, the disruption of this molecular oligomer leads to several diseases. In this article, we discuss the relevance of this transport machinery in health and disease.

Differential regulation of the glucocorticoid receptor nucleocytoplasmic shuttling by TPR-domain proteins

A dimer of the heat-shock protein of 90-kDa (Hsp90) represents the critical core of the chaperone complex associated to the glucocorticoid receptor (GR) oligomer. The C-terminal end of the Hsp90 dimer shapes a functional acceptor site for co-chaperones carrying tetratricopeptide repeat (TPR) domains, where they bind in a mutually exclusive and competitive manner. They impact on the biological properties of the GR•Hsp90 complex and are major players of the GR transport machinery. Recently, we showed that the overexpression of a chimeric TPR peptide influences the subcellular distribution of GR. In this study, the functional role of endogenous proteins carrying TPR or TPR-like sequences on GR subcellular distribution was characterized. It is demonstrated that, contrarily to the positive influence of FKBP52 on GR nuclear accumulation, FKBP51 and 14-3-3 impaired this property. While SGT1α showed no significant effect, the overexpression of the Ser/Thr phosphatase PP5 resulted in a nearly equal nuclear-cytoplasmic redistribution of GR rather than its typical cytoplasmic localization in the absence of steroid. This observation led to analyse the influence of the phosphorylation status of GR, which resulted not linked to its nucleo-cytoplasmic shuttling mechanism. Nonetheless, it was evidenced that both PP5 and FKBP52 are related to the anchorage of the GR to nucleoskeleton structures. The influence of these TPR domain proteins on the steroid-dependent transcriptional activity of GR was also characterized. It is postulated that the pleiotropic actions of the GR in different cell types may be the consequence of the relative abundance of different TPR-domain interacting co-chaperones.

Effects of Maternal Chewing on Prenatal Stress-Induced Cognitive Impairments in the Offspring via Multiple Molecular Pathways.

We aimed to investigate the effects of maternal chewing on prenatal stress-induced cognitive impairments in the offspring and to explore the molecular pathways of maternal chewing in a mice model. Maternal chewing ameliorated spatial learning impairments in the offspring in a Morris water maze test. Immunohistochemistry and Western blot findings revealed that maternal chewing alleviated hippocampal neurogenesis impairment and increased the expression of hippocampal brain-derived neurotrophic factor in the offspring. In addition, maternal chewing increased the expression of glucocorticoid receptor (GR) and 11β-hydroxysteroid dehydrogenase isozyme 2 (11β-HSD2) and decreased the expression of 11β-HSD1 in the placenta, thereby attenuating the increase of glucocorticoid in the offspring. Furthermore, maternal chewing increased the expression of 11β-HSD2, FK506-binding protein 51 (FKBP51) and FKBP52 and decreased the expression of 11β-HSD1, thereby increasing hippocampal nuclear GR level. In addition, maternal chewing attenuated the increase in expression of DNMT1 and DNMT3a and the decrease in expression of histone H3 methylation at lysine 4, 9, 27 and histone H3 acetylation at lysine 9 induced by prenatal stress in the offspring. Our findings suggest that maternal chewing could ameliorate prenatal stress-induced cognitive impairments in the offspring at least in part by protecting placenta barrier function, alleviating hippocampal nuclear GR transport impairment and increasing the hippocampal brain-derived neurotrophic factor (BDNF) level.

Prenatal alcohol exposure modifies glucocorticoid receptor subcellular distribution in the medial prefrontal cortex and impairs frontal cortex-dependent learning.

Prenatal alcohol exposure (PAE) has been shown to impair learning, memory and executive functioning in children. Perseveration, or the failure to respond adaptively to changing contingencies, is a hallmark on neurobehavioral assessment tasks for human fetal alcohol spectrum disorder (FASD). Adaptive responding is predominantly a product of the medial prefrontal cortex (mPFC) and is regulated by corticosteroids. In our mouse model of PAE we recently reported deficits in hippocampal formation-dependent learning and memory and a dysregulation of hippocampal formation glucocorticoid receptor (GR) subcellular distribution. Here, we examined the effect of PAE on frontal cortical-dependent behavior, as well as mPFC GR subcellular distribution and the levels of regulators of intracellular GR transport. PAE mice displayed significantly reduced response flexibility in a Y-maze reversal learning task. While the levels of total nuclear GR were reduced in PAE mPFC, levels of GR phosphorylated at serines 203, 211 and 226 were not significantly changed. Cytosolic, but not nuclear, MR levels were elevated in the PAE mPFC. The levels of critical GR trafficking proteins, FKBP51, Hsp90, cyclophilin 40, dynamitin and dynein intermediate chain, were altered in PAE mice, in favor of the exclusion of GR from the nucleus, indicating dysregulation of GR trafficking. Our findings suggest that there may be a link between a deficit in GR nuclear localization and frontal cortical learning deficits in prenatal alcohol-exposed mice.

Reduced Glucocorticoid Receptor Protein Expression in Children with Critical Illness

Background/Aims: The diagnostic criteria for critical illness-related corticoid insufficiency (CIRCI) are not well established, particularly for children. In addition to alterations in adrenal function, cellular resistance to glucocorticoid action could contribute to CIRCI due to alterations in the functioning of the intracellular receptor protein for corticosteroids, the glucocorticoid receptor (GR). Methods: We have therefore undertaken a pilot, prospective study to assess whether cellular GR activity can be measured in peripheral blood mononuclear cells (PBMCs) from critically ill children. Results: Total and cytoplasmic, but not nuclear GR levels were significantly lower in PBMCs from critically ill children (i.e. sepsis/septic shock and traumatic brain injury) compared to healthy controls. While total cortisol concentrations did not differ between test groups, salivary and serum-free cortisol concentrations were significantly greater in both groups of children with critical illness. Cortisol-binding globulin levels were significantly lower in patients with sepsis/septic shock. Conclusions: The lower total and cytoplasmic receptor levels in critically ill children suggest that the GR-mediated response to exogenous glucocorticoid therapy may be limited. However, the nuclear transport of GR in critically ill patients suggests that residual receptors in these patients retain functionality and may be accessible to therapeutic treatments that maximize their activity.

On the Trail of the Glucocorticoid Receptor: Into the Nucleus and Back

The glucocorticoid receptor (GR) belongs to the superfamily of steroid receptors and is an important regulator of physiological and metabolic processes. In its inactive state, GR is unbound by ligand and resides in the cytoplasm in a chaperone complex. When it binds glucocorticoids, it is activated and translocates to the nucleus, where it functions as a transcription factor. However, the subcellular localization of GR is determined by the balance between its rates of nuclear import and export. The mechanism of GR nuclear transport has been extensively studied. Originally, it was believed that nuclear import of GR is initiated by dissociation of the chaperone complex in the cytoplasm. However, several studies show that the chaperone machinery is required for nuclear transport of GR. In this review, we summarize the contribution of various chaperone components involved in the nuclear transport of GR and propose an updated model of its nuclear import and export. Moreover, we review the importance of ligand-independent nuclear transport and compare the nuclear transport of GR with that of other steroid receptors.

Detection of Dynamic Spatiotemporal Response to Periodic Chemical Stimulation in a Xenopus Embryonic Tissue

Embryonic development is guided by a complex and integrated set of stimuli that results in collective system-wide organization that is both time and space regulated. These regulatory interactions result in the emergence of highly functional units, which are correlated to frequency-modulated stimulation profiles. We have determined the dynamic response of vertebrate embryonic tissues to highly controlled, time-varying localized chemical stimulation using a microfluidic system with feedback control. Our approach has enabled localized spatiotemporal manipulation of the steroid hormone dexamethasone (DEX) in Animal Cap (AC) tissues isolated from gastrulating Xenopus embryos. Using this approach we investigated cell-scale responses to precisely controlled stimulation by tracking the redistribution of a GFP-tagged DEX-reporter constructed from the human glucocorticoid receptor (GR). We exposed defined regions of a single AC explant to different stimulation conditions—continuous stimulation, periodic stimulation, and no stimulation. We observed collective behavior of the GR transport into the nucleus was first-order. Furthermore, the dynamic response was well-modeled by a first-order differential equation with a single time derivative. The model predicted that responses to periodic stimulations closely matched the results of the frequency-based experiments. We find that stimulation with localized bursts versus continuous stimulation can result in highly distinct responses. This finding is critical as controlled space and time exposure to growth factors is a hallmark of complex processes in embryonic development. These complex responses to cellular signaling and transport machinery were similar to emergent behaviors in other complex systems, suggesting that even within a complex embryonic tissue, the overall system can converge toward a predictive first-order response.

Reduced Viability of Mice with Lung Epithelial-Specific Knockout of Glucocorticoid Receptor

Glucocorticoid (GC)-responsive epithelial-mesenchymal interactions regulate lung development. The GC receptor (GR) mediates GC signaling. Mice lacking GR in all tissues die at birth of respiratory failure. To determine the specific need for epithelial GR in lung development, we bred triple transgenic mice that carry SPC/rtTA, tet-O-Cre, and floxed, but not wild-type, GR genes. When exposed to doxycycline in utero, triple transgenic (GRepi⁻) mice exhibit a Cre-mediated recombination event that inactivates the floxed GR gene in airway epithelial cells. Immunofluorescence confirmed the elimination of GR in Cre-positive airway epithelial cells of late gestation GRepi⁻ mice. Embryonic Day 18.5 pups had a relatively immature appearance with increased lung cellularity and increased pools of glycogen in the epithelium. Postnatal Day 0.5 pups had decreased viability. We used quantitative RT-PCR to demonstrate that specific elimination of epithelial immunoreactive GR in GRepi⁻ mice is associated with reduced mRNA expression for surfactant proteins (SPs) A, B, C, and D; β- and γ-ENaC; T1α; the 10-kD Clara cell protein (CCSP); and aquaporin 5 (AQP5). Western blots confirmed reduced levels of AQP5 protein. No reduction in the levels of the GR transport protein importin (IPO)-13 was observed. Our findings demonstrate a requirement for lung epithelial cell GR in normal lung development. We speculate that impaired epithelial differentiation, leading to decreased SPs, transepithelial Na, and liquid absorption at birth, may contribute to the reduced survival of newborn mice with suppressed lung epithelial GR.

The microtubule-associated protein doublecortin-like regulates the transport of the glucocorticoid receptor in neuronal progenitor cells.

In neuronal cells, activated glucocorticoid receptor (GR) translocates to the nucleus guided by the cytoskeleton. However, the detailed mechanisms underlying GR translocation remain unclear. Using gain and loss of function studies, we report here for the first time that the microtubule-associated protein doublecortin-like (DCL) controls GR translocation to the nucleus. DCL overexpression in COS-1 cells, neuroblastoma cells, and rat hippocampus organotypic slice cultures impaired GR translocation and decreased GR-dependent transcriptional activity, measured by a specific reporter gene assay, in COS-1 cells. Moreover, DCL and GR directly interact on microtubule bundles formed by DCL overexpression. A C-terminal truncated DCL with conserved microtubule-bundling activity did not influence GR translocation. In N1E-115 mouse neuroblastoma cells and neuronal progenitor cells in rat hippocampus organotypic slice cultures, laser-scanning confocal microscopy showed colabeling of endogenously expressed DCL and GR. In these systems, RNA-interference-mediated DCL knockdown hampered GR translocation. Thus, we conclude that DCL expression is tightly regulated to adequately control GR transport. Because DCL is primarily expressed in neuronal progenitor cells, our results introduce this microtubule-associated protein as a new modulator of GR signaling in this cell type and suggest the existence of cell-specific mechanisms regulating GR translocation to the nucleus.

Importin 13 Regulates Nuclear Import of the Glucocorticoid Receptor in Airway Epithelial Cells

Antiinflammatory effects of glucocorticoids are critical to treatment of airway inflammation in such common disorders as asthma. There is considerable variation in responsiveness to glucocorticoid, and prolonged exposure can result in glucocorticoid resistance. We cloned LGL2, a glucocorticoid-inducible gene in fetal rat lung. We described the characterization of lgl2 as a nuclear transport protein, classified as importin 13 (IPO13), and demonstrated developmental regulation of IPO13 nucleocytoplasmic shuttling. We now report on the identification of the glucocorticoid receptor (GR) as a cargo substrate for IPO13. Binding of GR and IPO13 was demonstrated by GR-GST pulldown and coimmunoprecipitation. To investigate the role of IPO13 in modulating GR signaling in the lung, we studied IPO13-regulated GR transport in airway epithelial cells. Small interfering RNAs that inhibited IPO13 synthesis prevented nuclear translocation of GR. Silencing of IPO13 also abrogated the ability of cortisol to inhibit synthesis of the inflammatory cytokine IL-8 after stimulation with TNF-alpha. Our findings support a role for IPO13 in promoting nuclear occupancy of GR in a way that strongly potentiates the antiinflammatory effects of glucocorticoids. We speculate that variation in cellular levels of IPO13 and intracellular IPO13 shuttling rates may contribute to glucocorticoid resistance.