The glucocorticoid receptor is increased in Atm-/- thymocytes and in Atm-/- thymic lymphoma cells, and its nuclear translocation counteracts c-myc expression

We have previously demonstrated that spontaneous DNA synthesis in immature thymocytes of Atm-/- mice is elevated, and that treatment with the glucocorticoid dexamethasone (Dex) attenuates this increased DNA synthesis and prevents the development of thymic lymphomas. Deregulation of c-myc may drive the uncontrolled proliferation of Atm-/- thymocytes, since upregulation of c-myc parallels the elevated DNA synthesis in the cells. In this study, we show that the glucocorticoid receptor (GR) is expressed at high levels in Atm-/- thymocytes and in Atm-/- thymic lymphoma cells, although serum glucocorticoid (GC) levels in Atm-/- mice are similar to those in Atm+/+ mice. In cultured Atm-/- thymic lymphoma cells treated with Dex, GR nuclear translocation occurs, resulting in suppression of DNA synthesis and c-myc expression at both the mRNA and protein levels. Interestingly, the GR antagonist RU486 also causes GR nuclear translocation, but does not affect DNA synthesis and c-myc expression in Atm-/- thymic lymphoma cells. As expected, RU486 reverses the suppressive effects of Dex on DNA synthesis and c-myc expression. Administration of Dex to Atm-/- mice decreases the elevated c-Myc protein levels in their thymocytes. These findings suggest that GC/GR signaling plays an important role in regulating c-myc expression in Atm-/- thymocytes and thymic lymphoma cells.     

Identification of the functional domain of glucocorticoid receptor involved in RU486 antagonism

Mifepristone, also known as RU486, is a potent glucocorticoid receptor (GR) antagonist that inhibits GR-mediated transactivation. As an alternative to existing antidepressants, RU486 has been shown to rapidly reverse psychotic depression, most likely by blocking GR. Although a number of studies have demonstrated RU486-induced GR antagonism, the precise mechanism of action still remains unclear. To identify the GR domain involved in RU486-induced suppression, GR transactivation and nuclear translocation were examined using cells transfected with human GR (hGR), Guyanese squirrel monkey GR (gsmGR), and GR chimeras into COS-1 cells. RU486 showed a much more potent suppressive effect in gsmGR-expressing cells versus hGR-expressing cells, without significant cortisol- or RU486-induced changes in nuclear translocation. A GR chimera containing the gsmGR AF1 domain (amino acids 132–428) showed a marked decrease in luciferase activity, suggesting that this domain plays an important role in RU486-induced GR antagonism. Furthermore, fluorescence recovery after photobleaching (FRAP) analysis indicated that, in the presence of RU486, gsmGR AF1 domain contributes to GR mobility in living COS-1 cells. Taken together, these results demonstrate, for the first time, that the antagonistic effects of RU486 on GR transactivation involve a specific GR domain.     

大鼠海马神经元内11β-HSD1和GR的共存及其意义

本研究旨在探讨糖皮质激素代谢酶-11β-羟基类固醇脱氢酶Ⅰ型（11β-HSD1）和糖皮质激素受体（GR）在大鼠海马神经元内的共同分布及其意义。用免疫细胞化学方法研究显示,海马神经元内不仅存在11β-HSD1免疫反应物质,还存在GR免疫反应物质,而且11β-HSD1与GR共存于同一个海马神经元内,用Western印迹杂交和薄层层析（TLC）方法研究表明,地塞米松（DEX）可以促进11β-HSD1与GR共存于同一个海马神经元内,用Western印迹杂交和薄层层析（TLG）方法研究表明,地塞米松（DEX）可以促进11β-HSD1蛋白表达及其酶的活性,利用11β-HSD1基因启动子区序列构建的以CAT酶为报告基因的pBLCAT6质粒转染PC12细胞,证实DEX能够促进CAT酶的表达。以上糖皮质激素的作用均可为GR受体阻断剂RU38486所阻断,结果提示;糖皮质激素（GC）与GR结合后,可以作用于与其共存的11β-HSD1基因启动子区,使11β-HSD1表达增加,从而使更多的GC代谢产物转化为有活性的GC,此机制可能与保证GC在海马神经元内与亲和力较低的GR结合有关。     

Suppressive effect of dexamethasone on TIMP-1 production involves murine osteoblastic MC3T3-E1 cell apoptosis

High dose glucocorticoid (GC) treatment induces osteoporosis partly via increasing osteoblast apoptosis. However, the mechanism of GC-induced apoptosis has not been fully elucidated. Osteoblast-derived tissue inhibitor of metalloproteinase-1 (TIMP-1) was recently reported to be involved in bone metabolism. Our previous study demonstrated that TIMP-1 suppressed apoptosis of the mouse bone marrow stromal cell line MBA-1 (pre-osteoblast) induced by serum deprivation. Therefore, we tested the effect of the GC dexamethasone (Dex) on TIMP-1 production in murine osteoblastic MC3T3-E1 cells and further determined whether this action is associated with Dex-induced osteoblast apoptosis. Dex decreased TIMP-1 production in MC3T3-E1 cells, and this effect was blocked by the glucocorticoid receptor (GR) antagonists, RU486 and RU40555. Recombinant TIMP-1 protein reduced caspase-3 activation and apoptosis induced by Dex in MC3T3-E1 cells. In addition, the pro-apoptotic effect of the Dex was augmented by suppression of TIMP-1 with siRNA. Furthermore, mutant TIMP-1, which has no inhibitory effects on MMPs, yet protects MC3T3-E1 cells against Dex-induced apoptosis. Our study demonstrates that Dex suppresses TIMP-1 production in osteoblasts through GR, and this effect is associated with its induction of osteoblast apoptosis. The anti-apoptotic action of TIMP-1 is independent of its inhibitory effects on MMPs activities. The decrease in TIMP-1 production caused by Dex may contribute to the mechanisms of Dex-induced bone loss.     

Recycling and desensitization of glucocorticoid receptors in v-mos transformed cells depend on the ability of nuclear receptors to modulate gene expression

In v-mos transformed cells, glucocorticoid receptor (GR) proteins that bind hormone agonist are not efficiently retained within nuclei and redistribute to the cytoplasmic compartment. These cytoplasmic desensitized receptors cannot be reutilized and may represent trapped intermediates derived from GR recycling. We have used the glucocorticoid antagonist RU486 to examine whether v-mos effects can be exerted on any ligand-bound GR. In the rat 6m2 cell line that expresses a temperature-sensitive p85gag-mos oncoprotein, RU486 is a complete antagonist and suppresses dexamethasone induction of metallothionein-1 mRNA at equimolar concentrations. Using indirect immunofluorescence, we observe efficient nuclear translocation of GR in response to RU486 treatment in either the presence or absence of v-mos oncoproteins. However, in contrast to the redistribution of agonist-bound nuclear receptors to the cytoplasm of v-mos-transformed cells, RU486-bound GRs are efficiently retained within nuclei. Interestingly, withdrawal of RU486 does not lead to efficient depletion of nuclear GR in either nontransformed or v-mos transformed cells. It is only after the addition of hormone agonist to RU486 withdrawn v-mos-transformed cells that GRs are depleted from nuclei and subsequently redistributed to the cytoplasm. Thus, only nuclear GRs that are agonist-bound and capable of modulating gene activity can be subsequently processed and recycled into the cytoplasm.     

Age-related synthesis of glucocorticoids in thymocytes

Glucocorticoids (GCs) are primarily synthesized in the adrenal glands but an ectopic production has also been reported in the brain, the gastrointestinal tract and in thymic epithelial cells (TEC). Here we show that thymocytes express genes encoding for all enzymes required for de novo GC synthesis and produce the hormone as demonstrated by both a GC specific reporter assay and a corticosterone specific ELISA assay. Interestingly, GC synthesis is detectable in cells from young mice (4 weeks) and thereafter increases during aging (14-22 weeks) together with an increased gene expression of the rate-limiting enzymes StAR and CYP11A1. Hormone production occurred at a thymocyte differentiation stage characterized by being double positive for the CD4 and CD8 surface markers but was found to be unrelated to CD69 expression, a marker for thymocytes undergoing positive selection. No GC synthesis was found in resting or anti-CD3 activated CD4 and CD8 positive T cells isolated from the spleen. Thymocyte-derived GC had an anti-proliferative effect on a GR-transfected cell line and induced apoptosis in thymocytes. The age- and differentiation stage-related GC synthesis in thymocytes may play a role in the involution process that the thymus gland undergoes.     

The glucocorticoid receptor mediates the thymic epithelial cell-induced apoptosis of CD4+8+ thymic lymphoma cells

"Negative selection" and "death by neglect" are governed by apoptotic processes occurring in the thymus that shape the repertoire of maturing T cells. We have previously developed an in vitro model that recapitulates "death by neglect": Co-cultivation of double positive (DP) thymocytes or thymic lymphoma cells (PD1.6) with thymic epithelial cells (TEC) caused TcR-independent apoptosis of the former. We further demonstrated that this apoptosis could be attenuated by aminoglutethimide, an inhibitor of steroid synthesis, suggesting a role of TEC-derived glucocorticoids (GC) in this death process. We have now substantiated the role of the GC-glucocorticoid receptor (GR) axis by using a GC-resistant subline (PD1.6Dex(-)) obtained from the GC-sensitive PD1.6 cells by repeated exposures to increasing doses of dexamethasone (Dex). The PD1.6Dex(-) cells barely express GR and are much less sensitive to TEC-induced apoptosis. Re-expression of GR in PD1.6Dex(-) cells restored their sensitivity to both Dex and TEC, highlighting the central role of GR in these apoptotic processes. Likewise, repeated exposures of PD1.6 cells to TEC led to the selection of TEC-resistant cells (PD1.6TEC(-)) that are insensitive to corticosterone and less sensitive to Dex, though their GR level was only moderately reduced. This is in line with the low levels of corticosterone secreted by TEC. Altogether, our data show that TEC eliminates DP thymic lymphoma cells in a GR-dependent manner and modulates the GC sensitivity of the surviving cells.     

11-Beta hydroxysteroid dehydrogenase type 2 expression in white adipose tissue is strongly correlated with adiposity

Glucocorticoid action within the cells is regulated by the levels of glucocorticoid receptor (GR) expression and two enzymes, 11-beta hydroxysteroid dehydrogenase type 1 (11betaHSD1), which converts inactive to active glucocorticoids, and 11-beta hydroxysteroid dehydrogenase type 2 (11betaHSD2), which regulates the access of active glucocorticoids to the receptor by converting cortisol/corticosterone to the glucocorticoid-inactive form cortisone/dehydrocorticosterone. Male Wistar rats developed obesity by being fed a high-fat diet for 56 days, and GR, 11betaHSD1 and 11betaHSD2 gene expression were compared with control-diet fed animals. Gene expression analysis of 11betaHSD1, 11betaHSD2 and GR were performed by RT-PCR in subcutaneous and retroperitoneal adipose tissue. High-fat fed animals overexpressed 11betaHSD2 in subcutaneous but not in retroperitoneal fat. Interestingly, mRNA levels strongly correlated in both tissues with different parameters related to obesity, such as body weight, adiposity and insulin resistance, suggesting that this gene is a reliable marker of adiposity in this rat model of obesity. Thus, 11betaHSD2 is expressed in adipose tissue by both adipocytes and stromal-vascular cells, which suggests that this enzyme may play an important role in preventing fat accumulation in adipose tissue.     

Glucocorticoid receptor antagonist and siRNA prevent senescence of human bone marrow mesenchymal stromal cells in vitro

We investigate the effects mediated by glucocorticoid (GC) receptor (GR) blockage by using RU486, a GR antagonist and GR short interfering RNA (GR siRNA) on the proliferative and differentiation capabilities of human bone marrow mesenchymal stromal/stem cells (MSCs) and on their senescence and antioxidant levels during extended in vitro culture. Treatment with either RU486 or GR siRNA for a 7-day period significantly increased the proliferation of MSCs and their osteogenic capabilities, as reflected by an increase in alkaline phosphatase (ALP) levels after differentiation. Following 4 weeks of treatment, MSCs improved or maintained their proliferation rates, whereas control MSCs exhibited decreased proliferation. Although all MSCs exhibited reduced osteogenic potential after 4 weeks of in vitro culture, the MSCs treated with GR inhibitors showed higher ALP levels than untreated MSCs on being subjected to osteogenic differentiation. Such treatment also significantly down-regulated the adipogenic capabilities of MSCs. Telomere lengths and the activities of telomerase and superoxide dismutase of MSCs treated with either RU486 or GR siRNA appeared to be higher than those detected in controls. These results demonstrate that the blockage of effects mediated by the GCs normally found in fetal bovine serum might postpone senescence of these cells by up-regulating their antioxidant levels. Our data suggest that the blocking of the effects mediated by GCs might extend the lifespan of endogenous MSCs in patients who have elevated GC levels as a consequence of advancing age or estrogen depletion.     

Collaborated regulation of female-specific murine Cyp3a41 gene expression by growth and glucocorticoid hormones

CYP3A41 is a female-specific major CYP3A in mouse livers. Adrenalectomy decreased expression of CYP3A41 as well as CYP3A11, another major CYP3A, and dexamethasone (DEX) restored the decreased expression. Hypophysectomy completely abolished CYP3A41 expression and growth hormone (GH) replacement only slightly restored the expression. Treatment with DEX alone did not induce expression of either CYP3A41 or CYP3A11 in hypophysectomized mice. However, combined treatment with GH and DEX strongly induced expression of CYP3A41 but not CYP3A11. In primary cultured mouse hepatocytes, DEX induced expression of both CYP3A41 and CYP3A11, and DEX-inducible expression of CYP3A41 was suppressed by RU486, a potent antiglucocorticoid. In contrast, RU486 by itself enhanced basal expression of CYP3A11 mRNA, while it showed no inhibitory effect on DEX-inducible expression. These observations indicate that glucocorticoids may participate in the GH-dependent control of the Cyp3a41 gene expression, probably mediated via the glucocorticoid receptor, which may be different from that of the Cyp3a11 gene expression.     

Glucocorticoid receptors take part in the apoptotic process of human lens epithelial cells, but the glucocorticoid receptor antagonist RU486 does not rescue the cells fully

To identify an agent with specific activity against human lens epithelial cells (HLECs), we confirmed the presence of glucocorticoid receptors (GRs) and GR-α genes and evaluated whether GRs have a relationship with the apoptotic process in cultured HLECs. We also determined whether the inhibitor RU486 could rescue the cells from apoptosis when the HLECs were exposed to dexamethasone (Dex), a steroid, in 4 concentrations for 4 periods, or were co-treated with the antagonist RU486. We found that Dex, which has been used as a medical agent for a long time, resulted in increased expression of GRE-luciferase, the GR-α gene and GR-protein and, in contrast, decreased the viability of HLECs. The expression of Bax protein was increased in an earlier stage in contrast to the expression of Bcl-2 protein, which was increased in a later stage. Caspase-3 activity was significantly increased under lower concentrations of Dex in the last stage. The nuclear morphology of HLECs showed an obvious apoptotic phenomenon under greater concentrations of Dex in the last stage. However, RU486, a GR antagonist, could partially inhibit GR and Bax expressions and the expression of caspase-3 was increased so that there was not a decrease in the ratio of apoptotic cells and an increase in the viability of HLECs. Our data showed that GRs had a partial relationship to the apoptotic process of HLECs when exposed to Dex and RU486 did not rescue the cells fully. Because of its toxicity, RU486 did not provide a therapeutic benefit in a glucocorticoid induced cataract (GIC) for the in vitro model, however, its activity and pathway targeting should still be studied further with appropriate drug combinations.     

大鼠胸腺细胞糖皮质激素受体结合与糖皮质激素效应的定量关系

本文通过糖皮质激素拮抗剂 RU486阻断不同分数的糖皮质激素受体(GR)后,GR 结合与糖皮质激素(GC)效应的比较,研究于两者的量的关系。在地塞米松(Dex)10_(-9)—10_(-5)mol/L浓度范围内,Dex 和大鼠胸腺细胞 GR 的特异结合与 Dex 抑制细胞尿嘧啶核苷(Urd)参入效应呈正向平行趋势。用 RU486阻断5×10_(-8)mol/L 浓度的 Dex与 GR 结合的不同分数,比较 Dex 特异结合与抑制效应的关系,发现两者表现为线性正相关(P<0.01)。将 Dex 结合-效应直线外推,结果提示 GR 有约20%的占领阈但几乎没有备用受体。     

Rapid mechanisms of glucocorticoid signaling in the Leydig cell

Stress-mediated elevations in circulating glucocorticoid levels lead to corresponding rapid declines in testosterone production by Leydig cells in the testis. In previous studies we have established that glucocorticoids act on Leydig cells directly, through the classic glucocorticoid receptor (GR), and that access to the GR is controlled prior to the GR by a metabolizing pathway mediated by the type 1 isoform of 11beta-hydroxysteroid dehydrogenase (11betaHSD1). This enzyme is bidirectional (with both oxidase and reductase activities) and in the rat testis is exclusively localized in Leydig cells where it is abundantly expressed and may catalyze the oxidative inactivation of glucocorticoids. The predominant reductase direction of 11betaHSD1 activity in liver cells is determined by an enzyme, hexose-6-phosphate dehydrogenase (H6PDH), on the luminal side of the smooth endoplasmic reticulum (SER). Generation of the pyridine nucleotide cofactor NADPH by H6PDH stimulates the reductase direction of 11betaHSD1 resulting in increased levels of active glucocorticoids in liver cells. Unlike liver cells, steroidogenic enzymes including 17beta-hydroxysteroid dehydrogenase 3 (17betaHSD3) forms the coupling with 11betaHSD1. Thus the physiological concentrations of androstenedione serve as a substrate for 17betaHSD3 utilizing NADPH to generate NADP+, which drives 11betaHSD1 in Leydig cells primarily as an oxidase; thus eliminating the adverse effects of glucocorticoids on testosterone production. At the same time 11betaHSD1 generates NADPH which promotes testosterone biosynthesis by stimulating 17betaHSD3 in a cooperative cycle. This enzymatic coupling constitutes a rapid mechanism for modulating glucocorticoid control of testosterone biosynthesis. Under stress conditions, glucocorticoids also have rapid actions to suppress cAMP formation thus to lower testosterone production.     

Expression of the glucocorticoid receptor from the 1A promoter correlates with T lymphocyte sensitivity to glucocorticoid-induced cell death

Glucocorticoid (GC) hormones cause pronounced T cell apoptosis, particularly in immature thymic T cells. This is possibly due to tissue-specific regulation of the glucocorticoid receptor (GR) gene. In mice the GR gene is transcribed from five separate promoters designated: 1A, 1B, 1C, 1D, and 1E. Nearly all cells express GR from promoters 1B-1E, but the activity of the 1A promoter has only been reported in the whole thymus or lymphocyte cell lines. To directly assess the role of GR promoter use in sensitivity to glucocorticoid-induced cell death, we have compared the activity of the GR 1A promoter with GC sensitivity in different mouse lymphocyte populations. We report that GR 1A promoter activity is restricted to thymocyte and peripheral lymphocyte populations and the cortex of the brain. The relative level of expression of the 1A promoter to the 1B-1E promoters within a lymphocyte population was found to directly correlate with susceptibility to GC-induced cell death, with the extremely GC-sensitive CD4+CD8+ thymocytes having the highest levels of GR 1A promoter activity, and the relatively GC-resistant alphabetaTCR+CD24(int/low) thymocytes and peripheral T cells having the lowest levels. DNA sequencing of the mouse GR 1A promoter revealed a putative glucocorticoid-response element. Furthermore, GR 1A promoter use and GR protein levels were increased by GC treatment in thymocytes, but not in splenocytes. These data suggest that tissue-specific differences in GR promoter use determine T cell sensitivity to glucocorticoid-induced cell death.     

Identification of cis elements necessary for glucocorticoid induction of growth hormone gene expression in chicken embryonic pituitary cells

Glucocorticoid (GC) treatment of rat or chicken embryonic pituitary (CEP) cells induces premature production of growth hormone (GH). GC induction of the GH gene requires ongoing protein synthesis, and the GH genes lack a canonical GC response element (GRE). To characterize cis-acting elements and identify trans-acting proteins involved in this process, we characterized the regulation of a luciferase reporter containing a fragment of the chicken GH gene (-1727/+48) in embryonic day 11 CEP cells. Corticosterone (Cort) increased luciferase activity and mRNA expression, and mRNA induction was blocked by protein synthesis inhibition. Through deletion analysis, we identified a GC-responsive region (GCRR) at -1045 to -954. The GCRR includes an ETS-1 binding site and a degenerate GRE (dGRE) half site. Nuclear proteins, including ETS-1, bound to a GCRR probe in electrophoretic mobility shift assays, and Cort regulated protein binding. Using chromatin immunoprecipitation, we found that ETS-1 and GC receptor (GR) were associated with the GCRR in CEP cells, and Cort increased GR recruitment to the GCRR. Mutation of the ETS-1 site or dGRE site in the -1045/+48 GH reporter abolished Cort responsiveness. We conclude that GC regulation of the GH gene during development requires cis-acting elements in the GCRR and involves ETS-1 and GR binding to these elements. Similar ETS-1 elements/dGREs are located in the 5'-flanking regions of GH genes in mammals, including rodents and humans. This is the first study to demonstrate involvement of ETS-1 in GC regulation of the GH gene during embryonic development in any species, enhancing our understanding of GH regulation in vertebrates.