糖皮质激素非基因组效应的研究

在过去的几十年间,人们认为糖皮质激素（glucocorticoid,GC）仅仅是通过改变基因的表达来发挥其生理作用,这个过程需要几个小时来完成。然而,近年来越来越多的证据表明GC对激素分泌、神经元兴奋性、机体行为及细胞形态、糖类代谢等具备快速效应,这些过程往往在数秒钟或者分种内完成,这种作用机制被称为GC的非基因组作用机制。GC的非基因组作用主要可能通过两种不同的机制起作用：（1）通过细胞膜上或者细胞质内结构未知的糖皮质激素受体（glucocorticoid Recptor,GR）来发挥非基因组作用,即为特异性非基因组效应,（2）GC主要通过改变细胞膜理化作用来发挥效应。也称为非特异性非基因组效应（non-specific nongenomic effects,NSNE）。本文通过阐述近年来GC的非基因组的作用的最新研究进展并且讨论了这些非基因组作用临床治疗过程中的联系。对糖皮质激素基因组和非基因组作用机制的深入了解有助于指导我们在临床合理用药并减少其副作用。     

Rapid Elevation of Calcium Concentration in Cultured Dorsal Spinal Cord Astrocytes by Corticosterone

In addition to the classic genomic effects, increasing evidence suggests that GC can generate multiple rapid effects on many tissues and cells through nongenomic pathway. In the present study, the effects of corticosterone (CORT) on the intracellular calcium concentration ([Ca²⁺]i) in cultured dorsal spinal cord astrocytes were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator that could monitor real-time alterations of [Ca²⁺]i. CORT (0.01–10 μM) caused a rapid increase in [Ca²⁺]i with a dose-dependent manner in cultured dorsal spinal cord astrocytes. The action of CORT on astrocytic [Ca²⁺]i was blocked by pertussis toxin (a blocker of G protein activation, 100 ng/ml), but was unaffected by RU38486 (glucocorticoid receptor antagonist, 10 μM). In addition, cycloheximide (protein-synthesis inhibitor, 10 μg/ml) pretreatment could not impair the CORT-evoked [Ca²⁺]i elevation. Furthermore, Ca²⁺ mobilization induced by CORT was abolished by chelerythrine chloride (protein kinase C inhibitor, 10 μM), but was not impaired by H89 (protein kinase A inhibitor, 10 μM). These observations suggest that a nongenomic pathways might be involved in the effect of CORT on [Ca²⁺]i in cultured dorsal spinal cord astrocytes. In addition, our results also raise a possibility that a putative pertussis toxin-sensitive mGCR (G-protein-coupled membrane-bound glucocorticoid receptor) and the downstream activation of protein kinase C may be responsible for CORT-induced Ca²⁺ mobilization in cultured dorsal spinal cord astrocytes.     

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.     

A potential mechanism in medroxyprogesterone acetate teratogenesis.

MPA (medroxyprogeste)rone acetate) has been shown to be te)ratogenic in rabbits but not in rats or mice (Andrew and Staples 1977). Since normal steroid action appears to be mediated, in large part, through interaction with specific steroid receptors, it was postulated that the species difference in teratogenicity might be due to a difference in the interaction of MPA with target cells. A primary event in steroid-cell interaction is the binding of a steroid to intracellular receptors. Studies were initiated to measure the specific nature of MPA binding to glucocorticoid and progestin receptors in appropriate rat and rabbit target tissues. The competition of MPA with 3H-dexamethasone binding in liver cytosol (glucocorticoid receptor) and with 3H-progesterone binding in uterine cytosol (progesterone receptor) was determined. In rabbit liver cytosol, MPA was as effective at competing for specific dexamethasone binding as the natural glucocorticoids and considerably more effective than the nonspecific steroids. In rat liver cytosol MPA was only 10% as effective as the natural glucocorticoids and the competition could not be distinguished from that of nonspecific steroids. A similar species difference was not seen in uterine cytosol; MPA competed with progesterone in a similar fashion in both rat and rabbit. These data demonstrate a distinct species difference in the competitive nature of MPA for the glucocorticoid receptor but not for the progestin receptor. The results suggest that MPA, or possibly a metabolite, may be teratogenic in rabbits by binding with specific glucocorticoid receptors to inhibit or alter normal steroidal function in embryo-fetal development.     

Effects of Cortisol and Dexamethasone on Insulin Signalling Pathways in Skeletal Muscle of the Ovine Fetus during Late Gestation

Before birth, glucocorticoids retard growth, although the extent to which this is mediated by changes in insulin signalling pathways in the skeletal muscle of the fetus is unknown. The current study determined the effects of endogenous and synthetic glucocorticoid exposure on insulin signalling proteins in skeletal muscle of fetal sheep during late gestation. Experimental manipulation of fetal plasma glucocorticoid concentration was achieved by fetal cortisol infusion and maternal dexamethasone treatment. Cortisol infusion significantly increased muscle protein levels of Akt2 and phosphorylated Akt at Ser473, and decreased protein levels of phosphorylated forms of mTOR at Ser2448 and S6K at Thr389. Muscle GLUT4 protein expression was significantly higher in fetuses whose mothers were treated with dexamethasone compared to those treated with saline. There were no significant effects of glucocorticoid exposure on muscle protein abundance of IR-β, IGF-1R, PKCζ, Akt1, calpastatin or muscle glycogen content. The present study demonstrated that components of the insulin signalling pathway in skeletal muscle of the ovine fetus are influenced differentially by naturally occurring and synthetic glucocorticoids. These findings may provide a mechanism by which elevated concentrations of endogenous glucocorticoids retard fetal growth.     

Spatially regulated recruitment of clathrin to the plasma membrane during capping and cell translocation

Clathrin-coated vesicles bud from selected cellular membranes to traffic-specific intracellular proteins. To study the dynamic properties of clathrin-coated membranes, we expressed clathrin heavy chain tagged with green fluorescent protein (GFP) in Dictyostelium cells. GFP-clathrin was functional and retained the native properties of clathrin: the chimeric protein formed classic clathrin lattices on cellular membranes and also rescued phenotypic defects of clathrin null cells. GFP-clathrin distributed into punctate loci found throughout the cytoplasm, on the plasma membrane, and concentrated to a perinuclear location. These clathrin-coated structures were remarkably motile and capable of rapid and bidirectional transport across the cell. We identified two local domains of the plasma membrane as sites for clathrin recruitment in motile cells. First, as cells translocated or changed shape and retracted their tails, clathrin was transiently concentrated on the membrane at the back of the cell tail. Second, as cells capped their cell surface receptors, clathrin was recruited locally to the membrane under the tight cap of cross-linked receptors. This suggests that local sites for clathrin polymerization on specific domains of the plasma membrane undergo rapid and dynamic regulation in motile cells.     

The inhibition of commitment of mouse erythroleukemia cells by steroids involves a glucocorticoid-receptor mediated process(es) acting at the nuclear level

Dexamethasone has been shown to inhibit dimethylsulfoxide (DMSO)-induced differentiation of mouse erythroleukemia (or Friend) cells by blocking commitment to terminal erythroid maturation. In this study, we confirmed previous reports indicating the presence of glucocorticoid receptors in murine erythroleukemia cells and examined the mechanism(s) by which steroids block commitment. Untreated murine erythroleukemia cells contain dexamethasone receptors which decrease in number during DMSO-induced cell differentiation. When steroids of different classes (estrogenic, androgenic, glucocorticoid) were tested for inhibition of commitment and for displacement of [3H]dexamethasone from its receptors in DMSO-treated cells, we observed that the glucocorticoids dexamethasone, prednisolone and hydrocortisone, all blocked commitment and substantially displaced [3H]dexamethasone. In contrast, steroids other than glucocorticoids failed to inhibit commitment or displace [3H]dexamethasone. Analysis of kinetics of dexamethasone binding to chromatin revealed that dexamethasone binds to the nucleus via the receptor and preferentially interacts with active chromatin. Inhibition of commitment by dexamethasone persisted in cells released from this agent and reincubated with DMSO in the presence of another glucocorticoid of similar affinity to steroid receptors; inhibition of commitment, however, was not obtained when cells removed from dexamethasone were incubated in the presence of beta-estradiol, progesterone and testosterone. These data indicate that inhibition of commitment of mouse erythroleukemia cells by steroids is associated with binding to glucocorticoid receptors and may involve interactions of steroids and their receptors with regions of chromatin.     

Signal transduction in smooth muscle as studied by the subcellular membrane approach.

Many of the contractile regulatory events in smooth muscle reside in various cellular membrane components as functional membrane constituents that interact in a variably complex manner. The physiological handling of ionized calcium (Ca2+), which serves multiple roles as an extracellular signal, a second messenger, and an activator interacting directly with myofilaments to effectuate contractile responses, referred to as Ca2+ signalling processes, represents an integral part of a more complicated membrane transduction mechanism. The subcellular membrane approach toward the understanding of Ca2+ signalling as well as the transduction mechanisms involving membrane receptors, GTP binding proteins, ion channels, membrane-bound enzymes, and the production of intracellular second messengers has made a significant contribution in smooth muscle research for the past decade. This review summarizes the current state of knowledge about the multiplicity of interactions between Ca2+ and various membrane constituents in the surface membranes and sarcoplasmic reticulum, such as Ca2+ binding, Ca2+ ATPase pumps, Ca2+ channels, and Ca2+Na+ or related ion exchangers. A number of recent novel findings from this laboratory have also been discussed. First of all, the technical refinement of membrane separation and characterization, which permits better identification of neuronal membranes in highly innervated smooth muscle tissues, led to the distinction of prejunctional and postjunctional membrane receptors. Secondly, unlike the Ca(2+)-release channels labelled with [3H]inositol 1,4,5-trisphosphate, the other type of internal membrane Ca(2+)-release channels labelled by [3H]ryanodine has been identified only recently in smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Melatonin prevents glucocorticoid inhibition of cell proliferation and toxicity in hippocampal cells by reducing glucocorticoid receptor nuclear translocation

Glucocorticoids are the main product of the adrenal cortex and participate in multiple cell functions as immunosupressors and modulators of neural function. Within the brain, glucocorticoid activity is mediated by high-affinity mineralocorticoid and low-affinity glucocorticoid receptors. Among brain cells, hippocampal cells are rich in glucocorticoid receptors where they regulate excitability and morphology. Also, elevated glucocorticoid levels suppress hippocampal neurogenesis in adults. The pineal neuroindole, melatonin, reduces the affinity of glucocorticoid receptors in rat brain and prevents glucocorticoid-induced apoptosis. Here, the ability of melatonin to prevent glucocorticoid-induced cell death in hippocampal HT22 cells was investigated in the presence of neurotoxins. Results showed that glucocorticoids reduce cellular growth and also enhance sensitivity to neurotoxins. We found a G(1) cell cycle arrest mediated by an increase of cyclin/cyclin-dependent kinase inhibitor p21(WAF1/CIP1) protein after dexamethasone treatment and incremental change in amyloid beta protein and glutamate toxicity. Melatonin prevents glucocorticoids inhibition of cell proliferation and reduces the toxicity caused by glucocorticoids when cells were treated with dexamethasone in combination with neurotoxins. Although, melatonin does not reduce glucocorticoid receptor mRNA or protein levels, it decreases receptor translocation to nuclei in these cells.     

Lymphoid cell resistance to glucocorticoids in HIV infection

In humans infected with the HIV-1 virus there may be a disproportionate severity of signs and symptoms of illness compared to the fraction of CD4⁺ infected T-lymphoid cells. In part, this may be due to altered intercellular signalling systems and intracellular signal transduction. Glucocorticoids are well known for their effects on the vitality and function of lymphoid cells. Patients with HIV infections often show elevated circulating levels of cortisol, suggesting some misfunction in the regulatory systems that maintain the levels of this critical hormone. At the cellular level, it is known that both acute HIV infection and glucocorticoids can cause apoptotic cell death in thymic lymphocytes. However, chronically HIV-infected cells appear to be resistant to glucocorticoid-evoked cell death. Glucocorticoid receptor-ligand binding studies on patients' cells have shown reduced affinity between the receptor binding sites and test steroids. In vitro, chronically HIV-infected cells of the lymphoid CEM line displayed resistance to glucocorticoid-induced apoptosis. These cells showed reduced numbers of binding sites with little alteration in apparent affinity between ligand and receptor. Thus it appears that there may often be malfunction of the normal glucocorticoid response in HIV-infected cells probably due to altered interactions between the glucocorticoid receptor and its hormone. Such alterations may have clinical consequences, including the possibility of a relatively longer life span of infected CD4⁺ T-lymphocytes, as well as systemic effects of chronically elevated cortisol levels.     

Glucocorticoid binding during the differentiation of 3T3-F442A fibroblasts into adipocytes. A possible regulatory effect of insulin

Until recently, few studies had been carried out on receptors for glucocorticoids in adipocytes, although the role of these steroids is considerable. In the present studies, we chose the pre-adipocyte line 3T3-F442A, which constitutes an excellent model for investigating the differentiation and function of adipocytes. Using a whole cell assay system, we showed the existence of a homogenous class of sites with the characteristics of glucocorticoid receptors, that is, high-affinity binding which is reversible, specific and saturable. Whatever the state of cellular differentiation, the affinity of the receptor for dexamethasone did not vary, although we observed an increase in the number of sites during differentiation. When cells were differentiated in the presence of insulin, there was a further increase in the binding capacity; moreover, insulin deprivation of such adipocytes caused a decrease in the number of sites. Our results therefore suggest that factors other than the glucocorticoids themselves influence dexamethasone binding. It is suggested that insulin plays a role in the regulation of the number of glucocorticoid receptors.