Hippocampal glucocorticoid receptor expression in the tree shrew: Regulation by psychosocial conflict

Summary 1. This study was conducted to determine whether chronic psychosocial conflict alters the expression of glucocorticoid receptor (GR) mRNA in the hippocampus of male tree shrews (Tupaia belangeri).2. To generate probes for thein situ hybridization, the tree shrew GR gene was partly cloned. There was a 90% homology between the deduced amino acid sequence of the cloned tree shrew GR and that of the corresponding human GR sequence.³⁵S-Labeled riboprobes which had been transcribed from the tree shrew GR clone hybridized to pyramidal neurons in all subregions of the tree shrew hippocampal formation and to granule neurons in the dentate gyrus.3. Afterin situ hybridization, the expression of GR mRNA was semiquantitatively determined by counting silver grains over single neurons of the hippocampal formation of psychosocially stressed tree shrews and control animals. After 12 days of social conflict, the number of silver grains in the CA1 and CA3 pyramidal neurons of stressed animals was significantly lower than in controls. No statistically significant differences in mRNA expression were observed in the pyramidal neurons of the subiculum and in the granule neurons of the dentate gyrus.4. The present results suggest that psychosocial stress leads to a site-specific down-regulation of hippocampal GR via modification of mRNA expression.     

Effects of maternal methyl-supplement diet on hippocampal glucocorticoid receptor mRNA expression in rats selected for behavior

In the present work, we study glucocorticoid receptor (GR) gene expression in gray rats selected for aggressive and domestic behavior, as well as in offspring of mothers with methyl-supplemented diet during pregnancy. Tame selection is associated with increased GR mRNA expression as compared with aggressive rats, whereas maternal methyl-supplemented diet inhibits GR activity in tame rats. GR gene promoter methylation is an important way of altering GR expression.     

Mammary glucocorticoid receptor of mice in pregnancy and in lactation.

Activity of glucocorticoid receptor in mouse mammary cytosol changes during lactogenesis. The highest receptor activity is observed in the second half of pregnancy. The receptor from mammary glands from lactating and pregnant mice exhibits the same sedimentation pattern, as well as the same specificity and affinity for binding of steroid hormones.     

Molybdate-stabilized glucocorticoid receptor: evidence for a receptor heteromer

The composition of the molybdate-stabilized glucocorticoid receptor (GR) complex has been investigated with a monoclonal antibody against the steroid-binding Mr 94 000 (94K) GR protein. It was concluded that one antibody molecule binds one 94K GR molecule. This finding constituted the basis for calculating the number of antibodies bound to the molybdate-stabilized nonactivated GR complex, which has an Mr of 302 000 (302K). Gel filtration on Sephacryl S-400 and density gradient centrifugation showed that only one antibody molecule bound to the molybdate-stabilized GR complex (calculated relative molecular mass for the antibody--molybdate-stabilized GR complex, 456 000; relative molecular mass for one antibody molecule, 157 000). Furthermore, experiments performed with a second antibody immunoprecipitation assay in the presence of an excess of both antibody and GR confirmed the above results. The possibility of steric hindrance not allowing more than one antibody molecule to bind to the molybdate-stabilized GR complex could be excluded. These results suggest that the molybdate-stabilized GR complex with an Mr of 302K only contains one steroid-binding 94K GR molecule and therefore represents a heteromeric complex.     

糖皮质激素受体及其选择性调节剂研究进展

糖皮质激素（glucocorticoids,GCs）是临床上广泛使用的一类抗炎药物,在体内主要通过糖皮质激素受体（glucocorticoid receptor,GR）发挥生理和药理作用。GR是核受体超家族的成员之一,为配体激活的转录因子,在机体的多种生理和病理活动中扮演重要的角色。随着对GR信号通路的深入研究,寻找针对糖皮质激素受体的新型调节剂,以期将抗炎作用和现有糖皮质激素的副作用相分离,已经成为新药发现的研究热点。本文对近年来GR的分子结构、生物学作用及其选择性调节剂的研究进展作一简要的介绍。     

Evidence of a gene-dosage effect for the glucocorticoid receptor in trisomy 18 mice

The amount of cytosolic glucocorticoid receptor in liver of Ts18, Ts16, and Ts19 vs euploid mouse fetuses was studied after incubation of [3H]dexamethasone with cytosol followed by isoelectric focusing on polyacrylamide gels. In addition, corticosterone concentrations and enzyme activities of alanine aminotransferase and tyrosine aminotransferase were measured in the cytosol of the livers. The amount of glucocorticoid receptor in the cytosol fractions of the livers was always higher in the Ts18 than in the euploid fetuses of the same litter. It was also significantly (P less than 0.0005) higher if pooled data from different litters were analyzed. The ratio of the glucocorticoid receptor in Ts18 vs euploid mice varied between 1.3 and 4.7, with a mean of 2.1. In contrast, the glucocorticoid receptor levels in Ts16 and Ts19 fetuses were not different from the corresponding euploid controls. Comparing the corticosterone levels of the three trisomies tested with the corresponding euploid fetuses, no significant differences were found, indicating that the markedly elevated cytosolic glucocorticoid receptor concentrations in Ts18 were not due to different corticosterone levels. This finding is consistent with the assignment of the glucocorticoid receptor gene to chromosome 18 in the mouse. There was no correlation between glucocorticoid receptor levels and the activity of the two glucocorticoid inducible enzymes tested in the liver of mouse fetuses.     

Tumor necrosis factor inhibits glucocorticoid receptor function in mice: a strong signal toward lethal shock

As glucocorticoid resistance (GCR) and the concomitant burden pose a worldwide problem, there is an urgent need for a more effective glucocorticoid therapy, for which insights into the molecular mechanisms of GCR are essential. In this study, we addressed the hypothesis that TNFα, a strong pro-inflammatory mediator in numerous inflammatory diseases, compromises the protective function of the glucocorticoid receptor (GR) against TNFα-induced lethal inflammation. Indeed, protection of mice by dexamethasone against TNFα lethality was completely abolished when it was administered after TNFα stimulation, indicating compromised GR function upon TNFα challenge. TNFα-induced GCR was further demonstrated by impaired GR-dependent gene expression in the liver. Furthermore, TNFα down-regulates the levels of both GR mRNA and protein. However, this down-regulation seems to occur independently of GC production, as TNFα also resulted in down-regulation of GR levels in adrenalectomized mice. These findings suggest that the decreased amount of GR determines the GR response and outcome of TNFα-induced shock, as supported by our studies with GR heterozygous mice. We propose that by inducing GCR, TNFα inhibits a major brake on inflammation and thereby amplifies the pro-inflammatory response. Our findings might prove helpful in understanding GCR in inflammatory diseases in which TNFα is intimately involved.     

Hippocampal CA3 calcineurin activity participates in depressive-like behavior in rats

The reactive oxygen species (ROS) superoxide has been recognized as a critical signal triggering retinal ganglion cell (RGC) death after axonal injury. Although the downstream targets of superoxide are unknown, chemical reduction of oxidized sulfhydryls has been shown to be neuroprotective for injured RGCs. On the basis of this, we developed novel phosphine-borane complex compounds that are cell permeable and highly stable. Here, we report that our lead compound, bis (3-propionic acid methyl ester) phenylphosphine borane complex 1 (PB1) promotes RGC survival in rat models of optic nerve axotomy and in experimental glaucoma. PB1-mediated RGC neuroprotection did not correlate with inhibition of stress-activated protein kinase signaling, including apoptosis stimulating kinase 1 (ASK1), c-jun NH2-terminal kinase (JNK) or p38. Instead, PB1 led to a striking increase in retinal BDNF levels and downstream activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway. Pharmacological inhibition of ERK1/2 entirely blocked RGC neuroprotection induced by PB1. We conclude that PB1 protects damaged RGCs through activation of pro-survival signals. These data support a potential cross-talk between redox homeostasis and neurotrophin-related pathways leading to RGC survival after axonal injury.     

The dioxin receptor: a comparison with the glucocorticoid receptor

The physico-chemical properties of the dioxin and glucocorticoid receptors from rat liver and wild-type and mutant cell lines were investigated and compared. In rat liver, the receptors are virtually indistinguishable. Both are highly asymmetrical proteins with axial ratios of 12-15, have Stokes radii of 6 nm and sedimentation coefficients of approximately 4 S. This results in a calculated apparent mol. wt of approximately 100,000. The dioxin receptor from the mouse hepatoma cell line Hepa 1c1c7 represents an atypical form of the dioxin receptor with a pronounced tendency to aggregate to form Mr approximately equal to 300,000 complexes in high ionic strength and in the absence of sodium molybdate. In the presence of sulphydryl reducing agents, however, the Hepa 1c1c7 dioxin receptor dissociates to an Mr approximately 100,000 species. In analogy to the nt- mutant glucocorticoid receptor in mouse lymphoma cells, there is no gross change in the structure of the nt- dioxin mutant in mouse hepatoma cells compared with the wild-type receptor. The nt- dioxin receptor does, however, have a reduced affinity for DNA.     

Precocious induction of tryptophan dioxygenase by glucocorticoid in suckling rats and correlation with change in glucocorticoid receptor

When young rats (less than 14 days old) were treated once a day for 2 days with 100 micrograms/100 g body weight of dexamethasone, their liver cytosol showed a sharp new peak of glucocorticoid binding protein (peak C) eluted with 0.14 M NaCl on DEAE-cellulose chromatography. When the young rats were given a single injection of the hormone, the chromatogram showed a dominant peak of binding protein (peak B), eluted with 0.07 M NaCl, which was similar to that in untreated rats. The appearance of peak C on two treatments of young rats with hormone was confirmed by both in vivo and in vitro labeling and also studies on the nuclear fraction. Peaks B and C were specific hormone-binding proteins as shown with excess unlabeled hormone. The appearance of peak C was concomitant with the precocious induction of tryptophan dioxygenase in the liver of the young rats, and pretreatment with two injections of dexamethasone were necessary for maximal enzyme induction. On the other hand, in adult rats a single injection of dexamethasone (of 20 micrograms/100 g body weight or more) was enough to cause the appearance of peak C and induce tryptophan dioxygenase activity maximally; an additional injection of the hormone did not change the chromatographic pattern of the specific binding or the enzyme activity. For this effect in young rats, dexamethasone could not be replaced by other hormones such as glucagon, growth hormone, thyroid hormones, insulin, sex steroids or short-acting glucocorticoid.     

CRF-induced excessive grooming behavior in rats and mice

We studied the grooming response to lateral ventricle injection of CRF in both rats and mice under similar conditions. One microgram of CRF ICV induced a pronounced increase (3- to 4-fold) in the frequency of self-grooming in rats, but only a much smaller (less than 20%) increase in mice. The minimum effective dose of CRF in rats was 300 ng. Although ACTH1-24 induced less grooming in mice than in rats, the difference in potency did not appear to be sufficient to explain the differences between the effectiveness of CRF in the two species. Whereas ACTH increased all types of grooming scored. CRF increased all forms of grooming except flank scratching with the hind limb. The major effect of CRF was to increase the number of episodes of grooming, whereas ACTH1-24 tended to prolong the length of individual episodes. The excessive grooming induced by ICV CRF was not affected by prior treatment with dexamethasone, suggesting that the increased grooming was not due to secondary release of ACTH from the pituitary. Nevertheless, ICV CRF might induce grooming by releasing MSH/ACTH from cerebral storage sites. CRF-induced grooming, like ACTH-induced grooming, was inhibited by naloxone pretreatment. Despite the small qualitative differences, CRF-induced grooming could be due to secondary release of ACTH.     

Hippocampal representation of touch-guided behavior in rats: persistent and independent traces of stimulus and reward location

Understanding the mechanisms by which sensory experiences are stored remains a compelling challenge for neuroscience. Previous work has described how the activity of neurons in the sensory cortex allows rats to discriminate the physical features of an object contacted with their whiskers. But to date there is no evidence about how neurons represent the behavioural significance of tactile stimuli, or how they are encoded in memory. To investigate these issues, we recorded single-unit firing and local field potentials from the CA1 region of hippocampus while rats performed a task in which tactile stimuli specified reward location. On each trial the rat touched a textured plate with its whiskers, and then turned towards the Left or Right water spout. Two textures were associated with each reward location. To determine the influence of the rat's position on sensory coding, we placed it on a second platform in the same room where it performed the identical texture discrimination task. Over 25 percent of the sampled neurons encoded texture identity--their firing differed for two stimuli associated with the same reward location--and over 50 percent of neurons encoded the reward location with which the stimuli were associated. The neuronal population carried texture and reward location signals continuously, from the moment of stimulus contact until the end of reward collection. The set of neurons discriminating between one texture pair was found to be independent of, and partially overlapping, the set of neurons encoding the discrimination between a different texture pair. In a given neuron, the presence of a tactile signal was uncorrelated with the presence, magnitude, or timing of reward location signals. These experiments indicate that neurons in CA1 form a texture representation independently of the action the stimulus is associated with and retain the stimulus representation through reward collection.     

Peroxisome proliferator-activated receptor alpha agonist-induced down-regulation of hepatic glucocorticoid receptor expression in SD rats

It was reported that glucocorticoid production was inhibited by fenofibrate through suppression of type-1 11β-hydroxysteroid dehydrogenase gene expression in liver. The inhibition might be a negative-feedback regulation of glucocorticoid receptor (GR) activity by peroxisome proliferator-activated receptor alpha (PPARα), which is quickly induced by glucocorticoid in the liver. However, it is not clear if GR expression is changed by fenofibrate-induced PPARα activation. In this study, we tested this possibility in the liver of Sprague-Dawley rats. GR expression was reduced by fenofibrate in a time- and does-dependent manner. The inhibition was observed in liver, but not in fat and muscle. The corticosterone level in the blood was increased significantly by fenofibrate. These effects of fenofibrate were abolished by PPARα inhibitor MK886, suggesting that fenofibrate activated through PPARα. In conclusion, inhibition of GR expression may represent a new molecular mechanism for the negative feedback regulation of GR activity by PPARα.     

Myocardial citrullination in rheumatoid arthritis: a correlative histopathologic study

Introduction

The aim of this study was to explore the presence and localization of myocardial citrullination in samples from rheumatoid arthritis (RA) patients compared to rheumatic and non-rheumatic disease control groups.

Methods

Archived myocardial samples obtained during autopsy from 1995 to 2009 were assembled into four groups: RA; scleroderma; fatal myocarditis; and non-rheumatic disease controls. Samples were examined by immunohistochemistry (IHC) for the presence and localization of citrullination and peptidyl arginine deiminase enzymes (PADs) by a single cardiovascular pathologist blinded to disease group and clinical characteristics.

Results

Myocardial samples from seventeen RA patients were compared with those from fourteen controls, five fatal myocarditis patients, and ten scleroderma patients. Strong citrullination staining was detected exclusively in the myocardial interstitium in each of the groups. However, average and peak anti-citrulline staining was 59% and 44% higher, respectively, for the RA group compared to the combined non-RA groups (P < 0.05 for both comparisons). Myocardial fibrosis did not differ between the groups. In contrast to citrullination, PADs 1 to 3 and 6 were detected in cardiomyocytes (primarily PADs 1 and 3), resident inflammatory cells (primarily PADs 2 and 4), and, to a smaller extent, in endothelial cells and vascular smooth muscle cells. PAD staining did not co-localize with anti-citrulline staining in the interstitium and did not vary by disease state.

Conclusions

Staining for citrullination was higher in the myocardial interstitium of RA compared to other disease states, a finding that could link autoimmunity to the known increase in myocardial dysfunction and heart failure in RA.