Inhibition of the production of mediators of inflammation by corticosteroids is a glucocorticoid receptor-mediated process

In order to find an explanation for corticosteroid resistance we assessed whether inhibition by dexamethasone (DEX) of the stimulated production of TNF- proportional, variant, IL-6, PGE(2) and LTB(4) by peripheral blood mononuclear cells (MNC) depends on binding to the glucocorticoid receptor (GR), and whether it is determined by the number or the affinity of the GR of these cells. GR number and affinity of MNC were determined by means of a whole cell DEX binding assay. MNC were incubated with DEX and LPS or A23187 in the absence or presence of RU486, a potent steroid antagonist. DEX caused a concentration dependent inhibition of TNF- proportional, variant, IL-6 and PGE(2) production but had no effect on LTB(4) production. RU486 significantly blocked the effect of DEX, but no correlations were found between the inhibition of mediator release and the K(d) or receptor number.     

Characterization of glucocorticoid inhibition of antigen-induced inositolphosphate formation by rat basophilic leukemia cells: possible involvement of phosphatases.

The suppressive effect of glucocorticoids (GC) upon antigen-induced phosphatidylinositol phospholipase C (PI-PLC) activity and inositol phosphate formation by rat basophilic leukemia cells (RBL-2H3) has been characterized. Addition of antigen for a period of 1-30 min enhanced production of [3H]inositol monophosphate (IP1), inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3) by about 5-10-fold. Pretreatment with hydrocortisone (HC) reduced formation of the various inositol phosphates (IPs) and degradation of phosphatidylinositol 4,5-bisphosphate (PIP2) by an average of 50%. Maximal inhibition of hydrolysis of PIP2 and reduction in stimulation of IP3 formation was reached after 4 h of preincubation with 2.10(-6) M of HC. Cycloheximide and RU486, a GC receptor antagonist, completely prevented the inhibitory effect of HC on IP formation. Other GC, dexamethasone (DEX) and triamcinolone (each at 2.10(-7) M) markedly suppressed antigen induced IP3 production, while aldosterone and sex steroids such as estradiol and progesterone (each at 2.10(-6) M) were virtually inactive. Antigen-stimulated phosphorylation of a 18 kDa and other proteins was inhibited by about 60% following pretreatment with the GC. This inhibition was in turn prevented by cycloheximide. DEX also doubled the activity of cellular acid phosphatase activity. The results suggest that the inhibitory effect of GC is specific, receptor-mediated, dependent on protein synthesis and possibly mediated by protein phosphatase activity.     

RU486逆转糖皮质激素对淋巴细胞达白细胞介素—2受体的抑…

本文采用荧光标记ＣＤ２５单抗和放射性配基结合分析实验,观察了ＲＵ４８６地地塞公抑制淋巴细胞表达高、低亲和力ＩＬ－２受本的影响。结果显示,与地塞米松共同培养４８小时的大脾淋细胞,高、低亲和力ＩＬ－２受体的表达明显降低;在含有地塞米松的淋巴细胞培养体系中加入ＲＵ４８６后,表达ＣＤ２５（低亲和力ＩＬ－２受体）的阳性细胞率显著升高,淋巴细胞表面的高亲和力ＩＬ－２受体数量明显增加,基本恢复至正常水平,以上结     

A Novel Inhibitory Role for Glucocorticoids in the Secretion of Angiotensinogen by C6 Glioma Cells

Abstract: Astrocytes have been identified as the primary source of brain angiotensinogen (Ao), but the regulation of the secretion of this protein from astrocytes is poorly defined. In this study, the rat C6 glioma cell line was used as an astrocyte model to investigate the regulation of Ao secretion. C6 cultures secreted Ao at a rate of 4.05 ± 1.52 (mean ± SD) ng of Ao/10⁶ cells/24 h as determined by a direct radioimmunoassay. This rate was not significantly altered by the hormones thyroxine, estradiol, angiotensin II, growth hormone, and prostaglandins or by increased levels of intracellular cyclic AMP. Treatment with the synthetic glucocorticoid dexamethasone (DEX; 10–⁶M) reduced the rate of Ao secretion to 1.82 ± 0.28 ng of Ao/10⁸ cells/24 h. By comparison, the basal secretion rate for rat H4 hepatoma cells was 142.4 ± 10.0 ng of Ao/10⁶ cells/24 h, and this increased fourfold (572.4 ± 173.1 ng/10⁶ cells/ 24 h) in the presence of 10–⁶M DEX. Both these inhibitory (C6) and stimulatory (H4) actions of DEX were dose related. The inhibition observed in C6 cells was mimicked by RU28362, a pure glucocorticoid agonist, and reversed by the antagonist RU486, demonstrating that DEX was functioning as a true glucocorticoid. The action of DEX was also antagonized by the cyclic AMP analogue N⁶,2′-O- dibutyryladenosine 3′:5′-cyclic monophosphate (dBcAMP) (control, DEX, and DEX + dBcAMP, 3.58 ± 0.73, 1.69 ± 0.82, and 4.93 ± 1.88 ng of Ao/10⁶ cells/24 h, respectively, and by the β-adrenergic agonist isoprenaline, which stimulates cyclic AMP production. It was concluded that glucocorticoids inhibit Ao secretion, possibly by interacting with a cyclic AMP-responsive pathway. The inhibition of Ao production by DEX is a novel observation supporting the view that regulation of Ao is tissue specific.     

Effects of immune activation and glucocorticoid administration on feather growth in greenfinches

Elevation of glucocorticoid (GC) hormone levels is an integral part of stress response (as well as its termination) and immunomodulation. These hormones are also responsible for mobilizing energy stores by stimulation of gluconeogenesis and inhibition of protein synthesis. Elevation of GCs is thus incompatible with other protein-demanding processes, such as moult. Previous studies have shown that chronic elevation of GC hormones suppresses feather growth. Here, we asked whether similar effect would also occur in the case of acute GC elevation and induction of an inflammatory response by foreign antigen. We performed an experiment on captive wild-caught greenfinches (Carduelis chloris) injecting birds with phytohaemagglutinin (PHA) and dexamethasone (DEX) in a factorial design. To assess the possible somatic impacts of these manipulations, we removed one of the outermost tail feathers before the experiment and measured mass and rachis diameter and length of the replacement feathers grown in captivity. Immunostimulation by PHA reduced rachis length, but did not affect feather mass or rachis diameter. Single injection of a synthetic GC hormone DEX significantly reduced all three parameters of feather size. Altogether, these findings demonstrate the sensitivity of feather growth to manipulation of immune and adrenal functions. Our results corroborate the somatic costs of immune activation and suggest that even a short-term elevation of GC hormones may induce long-term somatic costs with a potential impact on fitness. Our findings also imply that a single injection of DEX, frequently used as a diagnostic tool, can have lasting effects and researchers must consider this when designing experiments.     

SERGE, the subcellular site of initial hepatic glycogen deposition in the rat: a radioautographic and cytochemical study

Hormonal control of hepatic glycogen and blood glucose levels is one of the major homeostatic mechanisms in mammals: glycogen is synthesized when portal glucose concentration is sufficiently elevated and degraded when glucose levels are low. We have studied initial events of hepatic glycogen synthesis by injecting the synthetic glucocorticoid dexamethasone (DEX) into adrenalectomized rats fasted overnight. Hepatic glycogen levels are very low in adrenalectomized rats, and DEX causes rapid deposition of the complex carbohydrate. Investigation of the process of glycogen deposition was performed by light and electron microscopic (EM) radioautography using [3H]galactose as a glycogen precursor. Rats injected with DEX for 2-3 h and [3H]galactose one hour before being killed displayed an increasing number of intensely labeled hepatocytes. EM radioautography revealed silver grains over small (+/- 1 micron) ovoid or round areas of the cytosome that were rich in smooth endoplasmic reticulum (SER) and contained a high concentration of small dense particles. These distinct areas or foci of SER and presumptive glycogen (SERGE) were most numerous during initial periods of glycogen synthesis. After longer exposure to DEX (4-5 h) more typical deposits of cytoplasmic glycogen were evident in the SERGE regions. Several criteria indicated that the SERGE foci contained glycogen or presumptive glycogen: resemblance of the largest dense particles to beta-glycogen particles in EM; association of 3H-carbohydrate with the foci; removal of particles and label with alpha-amylase; and positive reaction with periodic acid-chromic acid-silver methenamine. The concentration of SER in the small foci and the association of newly formed glycogen particles with elements of SER suggest a role for this organelle in the initial synthesis of glycogen.     

Glucocorticoid prevents brain-derived neurotrophic factor-mediated maturation of synaptic function in developing hippocampal neurons through reduction in the activity of mitogen-activated protein kinase

An increased level of glucocorticoid may be related to the pathophysiology of depressive disorder. The involvement of brain-derived neurotrophic factor (BDNF) in the antidepressive effect has also been suggested; however, the possible influence of glucocorticoid on the action of BDNF in the developing central nervous system has not been elucidated. In this study, we investigated the effect of glucocorticoid (dexamethasone, DEX) on synaptic maturation and function enhanced by BDNF in early developing hippocampal neurons. In the immature stage, BDNF increased the outgrowth of dendrites and the expression of synaptic proteins including glutamate receptors and presynaptic proteins. Pretreatment with DEX significantly inhibited the BDNF-dependent up-regulation of both dendritic outgrowth and synaptic proteins. In the more mature stage, the BDNF-reinforced postsynaptic Ca(2+) influx was decreased by DEX. BDNF-enhanced presynaptic glutamate release was also suppressed. RU486, a glucocorticoid receptor antagonist, canceled the DEX-dependent blocking effect on the action of BDNF. After down-regulation of glucocorticoid receptor by small interfering RNA application, no inhibitory effect of DEX on the BDNF-increased synaptic proteins was observed. Interestingly, the BDNF-activated MAPK/ERK pathway, which is an essential intracellular signaling pathway for the BDNF-increased synaptic proteins, was reduced by DEX. These results suggest that BDNF-mediated synaptic maturation is disturbed after neurons are exposed to high-level glucocorticoid in their development stage.     

Expression and induction of CYP3As in human fetal hepatocytes

CYP3A4 and CYP3A7 mRNA expression levels were markedly up-regulated by dexamethasone (DEX), but not by rifampicin (RIF). CYP3A5 mRNA level was not increased significantly by DEX, RIF, or phenobarbital. Testosterone 6beta-hydroxylase activity was induced to about 2-fold of control by DEX. However, concomitant treatment with RIF did not alter DEX-mediated induction of CYP3A mRNA expression and testosterone 6beta-hydroxylase activity. DEX-mediated induction of CYP3A mRNA was suppressed in a dose-dependent manner by RU486, a glucocorticoid receptor (GR) antagonist. At 5microM RU486, DEX-mediated induction of CYP3A4, CYP3A5, and CYP3A7 mRNA expression was inhibited almost completely. These results suggest that, in human fetal hepatocytes, PXR is not involved in DEX-mediated induction of CYP3A4 and CYP3A7, and that the induction is mediated directly by GR.     

Dexamethasone increases αvβ3 integrin expression and affinity through a calcineurin/NFAT pathway

The purpose of this study was to determine how dexamethasone (DEX) regulates the expression and activity of αvβ3 integrin. FACS analysis showed that DEX treatment induced expression of an activated αvβ3 integrin. Its expression remained high as long as DEX was present and continued following DEX removal. FACS analysis showed that the upregulation of αvβ3 integrin was the result of an increase in the expression of the β3 integrin subunit. By real time qPCR, DEX treatment induced a 6.2-fold increase (p < 0.04) in β3 integrin mRNA by day 2 compared to control and remained elevated for 6 days of treatment and then an additional 10 days once the DEX was removed. The increase in β3 integrin mRNA levels required only 1 day of DEX treatment to increase levels for 4 days in the absence of DEX. In contrast, DEX did not alter β1 integrin mRNA or protein levels. The DEX-induced upregulation of β3 integrin mRNA was partly due to an increase in its half-life to 60.7 h from 22.5 h in control cultures (p < 0.05) and could be inhibited by RU486 and cycloheximide, suggesting that DEX-induced de novo protein synthesis of an activation factor was needed. The calcineurin inhibitors cyclosporin A (CsA) and FK506 inhibited the DEX induced increase in β3 integrin mRNA. In summary, the DEX-induced increase in β3 integrin is a secondary glucocorticoid response that results in prolonged expression of αvβ3 integrin and the upregulation of the β3 integrin subunit through the calcineurin/NFAT pathway.     

Lobular and cellular patterns of early hepatic glycogen deposition in the rat as observed by light and electron microscopic radioautography after injection of 3H-galactose

Very low hepatic glycogen levels are achieved by overnight fasting of adrenalectomized (ADX) rats. Subsequent injection of dexamethasone (DEX), a synthetic glucocorticoid, stimulates marked increases in glycogen synthesis. Using this system and injecting 3H-galactose as a glycogen precursor 1 hr prior to sacrifice, the intralobular and intracellular patterns of labeled glycogen deposition were studied by light (LM) and electron (EM) microscopic radioautography. LM radioautography revealed that 1 hr after DEX treatment, labeling patterns for both periportal and centrilobular hepatocytes resembled those in rats with no DEX treatment: 18% of the hepatocytes were unlabeled, and 82% showed light labeling. Two hours after treatment with DEX, 14% of the hepatocytes remained unlabeled, and 78% were lightly labeled; however, 8% of the cells, located randomly throughout the lobule, were intensely labeled. An increased number of heavily labeled cells (26%) appeared 3 hr after DEX treatment; and by 5 hr 91% of the hepatocytes were intensely labeled. Label over the periportal cells at this time was aggregated, whereas centrilobular cells displayed dispersed label. EM radioautographs showed that 2 to 3 hr after DEX injection initial labeling of hepatocytes, regardless of their intralobular location, occurred over foci of smooth endoplasmic reticulum (SER) and small electron-dense particles of presumptive glycogen, and in areas of SER and distinct glycogen particles. After 5 hrs of treatment with DEX, the intracellular distribution of label reflected the glycogen patterns characteristic of periportal or centrilobular regions.     

Differential control of eosinophil survival by glucocorticoids

Glucocorticoids are effective drugs for eosinophil-related disorders, such as asthma and allergy. Previous studies have demonstrated that glucocorticoids increase eosinophil apoptosis and block the survival effect of submaximal concentrations of interleukin-5 (IL-5). We investigated the effect of glucocorticoids on eosinophil survival in the presence of a higher concentration of IL-5 (1 ng/ml), comparable to IL-5 levels in bronchoalveolar lavage and sputum specimens from patients with asthma. In contrast to incubation in the presence of submaximal concentrations of IL-5, the addition of dexamethasone (DEX) to media containing 1 ng/ml IL-5 led to a significant increase in eosinophil cell viability from 58 ± 6.9% to 87 ± 2.4% (p < 0.005) after 72 hours in culture. We found that RU486 blocked the DEX effect on cell viability confirming that glucocorticoid receptor functions are required. We investigated the possibility that the glucocorticoid enhancement of eosinophil survival may be due to an effect on IL-5 receptor expression. Our results show that the IL-5 associated decrease in IL-5 receptor -subunit expression was blocked significantly after 24 hrs in culture with media containing IL-5 plus DEX compared to IL-5 alone. It is tempting to speculate that the observed glucocorticoid enhancement of eosinophil survival in the presence of elevated concentrations of IL-5 could be a mechanism that contributes to glucocorticoid resistance in asthma.     

糖皮质激素对肝细胞内游离钙的作用

应用钙离子荧光指示剂ｆｕｒａ－２,对糖皮质激素（Ｇｌｕｃｏｃｏｒｔｉｃｏｉｄ,ＧＣ）是否影响肝细胞内游离钙（Ｉｎｔｒａｃｅｌｌｕｌａｒ　ｆｒｅｅ　ｃａｌｃｉｕｍ,［Ｃａ２＋］ｉ作了初步探讨。结果发现,ＧＣ在短期内能升高肝细胞［Ｃａ２＋］ｉ,水平,并具有明显的量效关系。以１．０μｍｏｌ／Ｌ的Ｃｏｒｔｉｓｏｌ和１０．０μｍｏｌ／Ｌ的Ｄｅｘａｍｅｔｈａｓｏｎｅ效果最好。加入１．０μｍｏｌ／Ｌ的Ｃｏｒｔｉｓｏｌ０．２５ｍｉｎ即可引起肝细胞［Ｃａ２＋］ｉ的明显升高,到１０分钟时效应达高峰。此时与静息状态的肝细胞［Ｃａ２＋］ｉ水平相比,胞浆内游离钙升高了近３倍;与相应对照组比较,胞浆内游离钙升高具有明显的统计学意义,Ｐ＜０．０１。ＲＵ４８６为一种人工合成的糖皮质激素受体（Ｇｌｕｃｏｃｏｒｔｉｃｏｉｄ　ｒｅｃｅｐｔｏｒ,ＧＲ）的拮抗剂,它可以取消ＧＣ升高肝细胞［Ｃａ２＋］ｉ的效应,提示ＧＣ升高肝细胞内［Ｃａ２＋］ｉ可能与ＧＲ介导有一定关系。鉴于ＧＣ升高［Ｃａ２＋］ｉ时间较短,推测与肝细胞膜ＧＲ的非基因快速调节作用影响钙离子通道有关。     

高脂饮食和地塞米松联合诱导胰岛素抵抗大鼠模型

目的用高脂饲料＋地塞米松（dexamethasone,DEX）隔日腹腔注射建立实验性胰岛素抵抗大鼠模型,研究该模型糖代谢、脂代谢和激素水平等方面的变化。方法采用Wistar雄性大鼠,分为正常对照组、高脂组、DEX组（1mg/kg,i.p.）和高脂＋DEX组（1mg/kg,i.p.）,连续观察8周,每周测定大鼠空腹血糖,分别于造模第2周和第8周测糖耐量,8周后处死大鼠,测定胸腺、脾脏、肝脏等脏器重量。结果高脂饲料能加重腹腔注射DEX造成的空腹血糖升高,造模第8周空腹血糖（7.7±0.7）较空白组（6.5±0.6）显著升高。使模型动物糖耐量明显异常,肝糖原、肌糖原含量显著增加,血浆胰岛素及游离脂肪酸水平显著升高,各脏器指数明显增加。结论高脂＋DEX隔日腹腔注射能成功诱导胰岛素抵抗大鼠模型,这种造模方法较单纯注射DEX或单纯高脂饲养成模率高,造模周期短。     

MicroRNA‐20a‐5p contributes to hepatic glycogen synthesis through targeting p63 to regulate p53 and PTEN expression

Recently, it is implicated that aberrant expression of microRNAs (miRs) is associated with insulin resistance. However, the role of miR‐17 family in hepatic insulin resistance and its underlying mechanisms remain unknown. In this study, we provided mechanistic insight into the effects of miR‐20a‐5p, a member of miR‐17 family, on the regulation of AKT/GSK pathway and glycogenesis in hepatocytes. MiR‐20a‐5p was down‐regulated in the liver of db/db mice, and NCTC1469 cells and Hep1‐6 cells treated with high glucose, accompanied by reduced glycogen content and impaired insulin signalling. Notably, inhibition of miR‐20a‐5p significantly reduced glycogen synthesis and AKT/GSK activation, whereas overexpression of miR‐20a‐5p led to elevated glycogenesis and activated AKT/GSK signalling pathway. In addition, miR‐20a‐5p mimic could reverse high glucose‐induced impaired glycogenesis and AKT/GSK activation in NCTC1469 and Hep1‐6 cells. P63 was identified as a target of miR‐20a‐5p by bioinformatics analysis and luciferase reporter assay. Knockdown of p63 in the NCTC1469 cells and the Hep1‐6 cells by transfecting with siRNA targeting p63 could increase glycogen content and reverse miR‐20a‐5p inhibition‐induced reduced glycogenesis and activation of AKT and GSK, suggesting that p63 participated in miR‐20a‐5p‐mediated glycogenesis in hepatocytes. Moreover, our results indicate that p63 might directly bind to p53, thereby regulating PTEN expression and in turn participating in glycogenesis. In conclusion, we found novel evidence suggesting that as a member of miR‐17 family, miR‐20a‐5p contributes to hepatic glycogen synthesis through targeting p63 to regulate p53 and PTEN expression.