Glucocorticoid Receptor Dynamics Research Articles

Quantitation of Glucocorticoid Receptor DNA-Binding Dynamics by Single-Molecule Microscopy and FRAP

Recent advances in live cell imaging have provided a wealth of data on the dynamics of transcription factors. However, a consistent quantitative description of these dynamics, explaining how transcription factors find their target sequences in the vast amount of DNA inside the nucleus, is still lacking. In the present study, we have combined two quantitative imaging methods, single-molecule microscopy and fluorescence recovery after photobleaching, to determine the mobility pattern of the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR), two ligand-activated transcription factors. For dexamethasone-activated GR, both techniques showed that approximately half of the population is freely diffusing, while the remaining population is bound to DNA. Of this DNA-bound population about half the GRs appeared to be bound for short periods of time (∼0.7 s) and the other half for longer time periods (∼2.3 s). A similar pattern of mobility was seen for the MR activated by aldosterone. Inactive receptors (mutant or antagonist-bound receptors) show a decreased DNA binding frequency and duration, but also a higher mobility for the diffusing population. Likely, very brief (≤1 ms) interactions with DNA induced by the agonists underlie this difference in diffusion behavior. Surprisingly, different agonists also induce different mobilities of both receptors, presumably due to differences in ligand-induced conformational changes and receptor complex formation. In summary, our data provide a consistent quantitative model of the dynamics of GR and MR, indicating three types of interactions with DNA, which fit into a model in which frequent low-affinity DNA binding facilitates the search for high-affinity target sequences.

Localization and Dynamics of Glucocorticoid Receptor at the Plasma Membrane of Activated Mast Cells

In addition to their actions in the cell nucleus, glucocorticoids exhibit rapid non-nuclear responses that are mechanistically not well understood. To explain these effects, the localization of a glucocorticoid receptor (GR) expressed in mast cells as a GFP fusion was analyzed after activation of the cells on allergenic lipid arrays. These arrays were produced on glass slides by dip-pen nanolithography (DPN) and total internal reflection (TIRF) microscopy was used to visualize the GR. A rapid glucocorticoid-independent and -dependent recruitment of the GR-GFP to the plasma cell membrane was observed following contact of the cells with the allergenic array. In addition, the mobility of the GR at the membrane was monitored by fluorescence recovery after photobleaching (FRAP) and shown to follow binding kinetics demonstrating interactions of the receptor with membrane-bound factors. Furthermore the recruitment of the GR to the cell membrane was shown to result in a glucocorticoid-mediated increase in Erk phosphorylation. This is evidenced by findings that destruction of the membrane composition of the mast cells by cholesterol depletion impairs the membrane localization of the GR and subsequent glucocorticoid-mediated enhancement of Erk phosphorylation. These results demonstrate a membrane localization and function of the GR in mast cell signaling.

1SD-05 蛍光相関分光法を用いた細胞内グルココルチコイド受容体の動態解析(1SD 核内分子動態から観る遺伝子発現機構,シンポジウム,日本生物物理学会第50回年会(2012年度))

1SD-05 蛍光相関分光法を用いた細胞内グルココルチコイド受容体の動態解析(1SD 核内分子動態から観る遺伝子発現機構,シンポジウム,日本生物物理学会第50回年会(2012年度))

Glucocorticoid receptor dynamics and gene regulation

The glucocorticoid receptor regulates the expression of a large number of genes in mammalian cells. The interaction of this receptor with regulatory elements has been discovered to be highly dynamic, with occupancy states measured in seconds, rather than minutes or hours. This finding has led to a paradigm shift in our understanding of receptor function throughout the genome. The mechanisms involved in these rapid exchange events, as well as the implications for receptor function, are discussed.

Assessing the dynamics of nuclear glucocorticoid-receptor complex: adding flexibility to gene expression modeling.

A retrospective analysis was performed to modify our fourth-generation pharmacodynamic model for glucocorticoid receptor (GR) dynamics with incorporation of more physiological features. This modified model was developed by integrating previously reported free cytosolic GR and GR mRNA data following single (10, 50 mg/kg) and dual (50 mg/kg at 0 and 24 hr) intravenous doses of methylprednisolone (MPL) in adrenalectomized (ADX) male Wistar rats with several in vitro studies describing real-time kinetics of the transfer of rat steroid-receptor complex from the cell cytosol to the nucleus. Additionally, free hepatic cytosolic GR and its mRNA data from a chronic infusion dosing study of MPL (0.1 and 0.3 mg/kg/hr) in male ADX Wistar rats were used to verify the predictability of the model. Incorporation of information regarding in vitro receptor kinetics allowed us to describe the receptor-mediated pharmacogenomic effects of MPL for a larger variety of genes in rat liver from microarray studies. These included early responsive gene like CCAAT/enhancer binding protein-beta (CEBP-beta), a transcription factor, as well as the later responsive gene for tyrosine aminotransferase (TAT), a classical biomarker of glucocorticoid (GC) genomic effects. This more mechanistic model of GR dynamics can be applied to characterize profiles for a greater number of genes in liver.

Visualization of glucocorticoid receptor in the brain of green fluorescent protein–glucocorticoid receptor knockin mice

Glucocorticoids exert various neuroendocrinological effects, including stress response, in the central nervous system via glucocorticoid receptor (GR). GRs are transported from the cytoplasm to the nucleus upon ligand binding, and then exert the transcriptional activity. Although it is important for unraveling the actual property of the GR in vivo, subcellular dynamics of the GR are still unclear within the brain tissue in which the neuronal circuitry is maintained. To address this issue, we generated green fluorescent protein (GFP)–GR knockin mice, whose GR has been replaced by a GFP–GR fusion protein that is functionally indistinguishable from endogenous GR. In fixed brain sections of the GFP–GR knockin mice, the distribution of the green fluorescence was similar to that of GR immunoreactivity. By subtracting autofluorescence using fluorescent emission fingerprinting method with confocal laser scanning microscope, nuclear localization of GFP–GR was identifiable in the hippocampal CA3 subregion, where subcellular localization of the GR has been unsolved compared with other areas. To examine the subcellular trafficking of GFP–GR in vivo, we performed adrenalectomy on the GFP–GR knockin mice. GFP–GR was translocated from the nucleus to the cytoplasm and neurites two days after adrenalectomy. Furthermore, laser scanning cytometry by which fluorescence intensity in situ can be quantitatively measured revealed the entire GFP–GR expression level was increased. We then examined the dynamic changes in the subcellular localization of GFP–GR in living hippocampal neurons both in dissociated culture and in tissue slices. GFP–GR was localized in not only the perikarya but also neurites in the absence of ligand, and nuclear translocation following ligand treatment was observed. This is the first report visualizing subcellular trafficking of the GR in the mouse brain in more physiological condition. The present results propose new avenues for the research of the GR dynamics both in vitro and in vivo.

Dynamics of glucocorticoid receptor and induction of tyrosine aminotransferase in rat liver.

In order to better understand the role of glucocorticoid receptor in the hormonal action of glucocorticoid, depletion and replenishment of the cytoplasmic receptor and kinetics of induction of tyrosine aminotransferase (TAT) in rat liver were examined after administration of dexamethasone (Dex) and prednisolone. The extent of the receptor depletion and the duration of the depletion period were dose-dependent and were well correlated not only with the biological potency of the steroids administered but also with the kinetics of TAT induction. A linear relationship between the amount of depleted receptor and the kinetics of TAT induction was observed. Pretreatment of animals with a large dose of Dex reduced the response of TAT induction by Dex which was administered 24 h later. This seems to be attributable, at least in part, to the incomplete replenishment of the receptor and also to the increased rate of replenishment of the receptor. The reduced affinity of the partially replenished receptor to glucocorticoid might also participate in lowering the response to the second injection. In conclusion, depletion and replenishment of the cytoplasmic glucocorticoid receptor appeared to be directly responsible for the physiological action of glucocorticoid.