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Post-transcriptional and Nongenomic Effects of Glucocorticoids

Division of Allergy and Clinical Immunology, The Johns Hopkins University, Baltimore, Maryland

Correspondence and requests for reprints should be addressed to Cristiana Stellato, M.D., Ph.D., Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Room 1A.12A, Baltimore, MD 21224. E-mail: stellato{at}jhmi.edu

	ABSTRACT

TOP ABSTRACT POST-TRANSCRIPTIONAL GENE... NONGENOMIC ACTIONS OF STEROID... REFERENCES

 
The ability of glucocorticoids to interfere with post-transcriptional gene regulation has recently been recognized as a potentially important part of their anti-inflammatory property, and the mechanisms governing such activity are under active investigation. Several studies have shown that glucocorticoids can inhibit inflammatory signaling pathways known to control mRNA turnover and translation. Moreover, several glucocorticoid-sensitive determinants have been identified on mRNA molecules of inflammatory genes, and the RNA-binding factors interacting with them might constitute relevant glucocorticoid targets. Glucocorticoids also exert effects characterized as nongenomic, which occur within minutes of drug administration. The mechanisms of action of nongenomic glucocorticoid effects differ from the classical, transcription-dependent glucocorticoid action and involve the production of second-messenger molecules and activation of signal transduction pathways, either by the nuclear glucocorticoid receptor or by a membrane glucocorticoid receptor that has not yet been fully characterized. Ultimately, the discovery of novel pathways involved in mediating the actions of glucocorticoids should lead to improved targets for anti-inflammatory therapy.

Key Words: glucocorticoid receptor • mRNA stability • RNA-binding protein

The pathways that are targeted by glucocorticoids to yield their beneficial effects have not been fully identified. This research has become more important because of the quest for the "perfect" glucocorticoid—a molecule carrying the unequaled antiinflammatory activity of this class of compounds but devoid of the significant untoward effects on metabolism and growth. In the past decade, in parallel with seminal work unraveling the glucocorticoid mode of action on gene transcription, research has indicated that glucocorticoids broadly influence the complex network of pathways that regulate gene expression, from the signaling events that occur within minutes after cell activation to post-translational modifications that occur well after gene transcription. This article describes evidence that glucocorticoids have effects on post-transcriptional and nongenomic gene regulatory events that integrate with transcriptional regulation and are crucial in shaping gene expression during an inflammatory response. Some of the results of these studies have been previously reported in the form of an abstract (1).

	POST-TRANSCRIPTIONAL GENE REGULATION

TOP ABSTRACT POST-TRANSCRIPTIONAL GENE... NONGENOMIC ACTIONS OF STEROID... REFERENCES

 
Overview
Post-transcriptional gene regulation is increasingly recognized as a critical component in the upregulation of inflammatory genes. It seems to be important in keeping early response genes in a dynamic state, so that the protein output can be rapidly increased or terminated according to changes of transcription rate or changes in cell environment (2).

The stability of a mature cytoplasmic mRNA, as well as its translation, is regulated by complex interactions. These interactions are controlled by phosphorylation-mediated signaling pathways between cis-regulatory elements scattered throughout the RNA molecule and RNA-binding proteins of different natures and functional roles (3). The untranslated regions (UTRs) flanking the coding regions at the 5' and 3' ends of the mRNA molecule appear to be particularly important in conveying either pro- or antiinflammatory signals to relevant transcripts. For example, an element responsive to c-Jun N-terminal kinase (JNK) has been identified in the 5'-UTR of the interleukin (IL)-2 mRNA, which mediates activation-dependent mRNA stabilization in T lymphocytes (4). Conversely, the 5'-UTR of monocyte chemoattractant protein (MCP)-1 mRNA contains sequences mediating glucocorticoid-induced acceleration of mRNA decay (5). However, the majority of the phosphorylation-dependent post-transcriptional changes induced by proinflammatory stimuli, as well as many of the effects of glucocorticoids on these events, appear to be mediated by elements within the 3'-UTR of mRNAs, in particular by the adenylate/uridylate-rich elements (AREs). The AREs represent the most relevant and conserved group of sequences functionally associated with the regulation of mRNA stability and translation (6). The list of recognized ARE-bearing genes is growing rapidly (7): recently, an ARE-mRNA database has been established (ARED; http://rc.kfshrc.edu.sa/ared) (7), which shows the vast diversity of the pool of ARE-bearing mRNA encoding for proteins that belong to different biological processes and that participate in many disease states. Given the importance of the genes regulated post-transcriptionally through this pathway, AREs are now considered central cis-elements in gene regulation. Cloning of ARE-containing 3'-UTR sequences in reporter genes can alter the stability of the reporter mRNA (8). These sequences, which have been recently reclassified according to their number and configuration within the 3'-UTR (3) (Figure 1), are very heterogeneous, consisting of AUUUA pentamers and AT-rich stretches that can be found clustered in different combinations. In some cases, the AUUUA pentamer is not present at all. The sequences appear to be functionally heterogeneous as well, because they can mediate mRNA stability and translation through different pathways (9).

View larger version (33K): [in this window] [in a new window]   Figure 1. Cis-acting determinants of mRNA turnover for inflammatory genes. Sequences regulating mRNA turnover and translation are scattered throughout the mRNA molecule. However, the untranslated regions (UTRs) flanking the coding regions at the 5' and 3' ends of the mRNA molecule appear to be particularly important in regulation of inflammatory signals. JRE indicates a JNK-responsive element identified in the 5'-UTR of the IL-2 mRNA molecule; FGF, fibroblast growth factor; u-PA, urokinase plasminogen activator. The group table at the bottom of the figure is reprinted by permission from Wilusz and coworkers (3).

Several extracellular stimuli can trigger changes in mRNA stability, and the pathways regulating mRNA turnover, particularly AREs, appear to be an important crossroad for divergent regulatory signals (14). Studies conducted in our laboratory on the CC chemokine CCL11 (eotaxin) provide an example. CCL11 displays strong and selective chemoattractant and activating properties toward eosinophils (15). This molecule is considered relevant to allergic inflammation, (16) and it is known to be involved in other biological functions as well, such as angiogenesis (17). CCL11 is strongly upregulated in airway epithelial cells by IL-4, especially in combination with TNF- (18, 19), and its expression is profoundly inhibited by glucocorticoids (18). Both cytokine-induced upregulation and glucocorticoid-mediated inhibition of the CCL11 gene appear to be mediated only partially by transcriptional regulation (18, 20). Using the transcriptional inhibitor actinomycin D on epithelial cell cultures, we found that the combination of TNF- and IL-4, which yields a strong synergistic effect on CCL11 protein secretion, significantly increases the stability of CCL11 mRNA (1). Conversely, a potent topical glucocorticoid, budesonide, significantly decreases it (18). The 3'-UTR of CCL11 contains a tandem AUUUA sequence in a TA-rich stretch (21), indicating the potential for post-transcriptional regulation. Treatment with glucocorticoid significantly decreased the half-life of a heterologous reporter mRNA bearing the CCL11 3'-UTR, indicating that the effect of glucocorticoids was CCL11 3'-UTR–dependent, at least in part (1). Interestingly, the stabilizing effect of TNF- plus IL-4 on CCL11 mRNA was reproducible upon cytokine challenge of cells transfected with the reporter construct containing the CCL11 3'-UTR (1), which is further evidence that this region is a point of convergence of regulatory signals with either pro- or antiinflammatory effect.

Role in Glucocorticoid Antiinflammatory Action
Glucocorticoids influence gene expression by multiple mechanisms. The regulation of gene transcription by glucocorticoids is a key feature of the antiinflammatory activity of this class of drugs, and it is extensively reviewed elsewhere (22). Briefly, the glucocorticoid receptor, upon ligand binding, can influence transcription through DNA-dependent mechanisms by binding to a consensus sequence, the glucocorticoid response element (GRE), present within the 5' regulatory region of target genes. Such binding induces the transcription of potentially antiinflammatory genes. In rare instances, binding of the glucocorticoid receptor to a "negative" GRE can exert an inhibitory effect on gene transcription. However, the inhibitory effect of glucocorticoids on the transcription of proinflammatory genes is mostly due to DNA-independent mechanisms, mediated by protein–protein interactions between the ligand-activated glucocorticoid receptor and transcription factors, or cofactors (such as AP-1, members of the nuclear factor-B family, and others) that are crucial for the transcription of inflammatory genes. Through this interaction, glucocorticoids interfere with the binding of the trans-acting factor to its consensus sequence within the promoter of the targeted genes, thereby inhibiting the transcription of the genes under the transcription factor's control.

However, the mechanism of action of glucocorticoids is not limited to gene transcription. For an increasing number of genes, the inhibitory effect of glucocorticoids has been recognized to occur through alteration of mRNA turnover or translation (Table 1). A substantial number of these genes are involved in inflammation, such as IL-1, IL-6, CXCL8 (IL-8), IFN-ß, and granulocyte macrophage colony-stimulating factor (GM-CSF) (23–26). Among these, chemokine genes are being identified increasingly often (27), indicating the potential impact of this mode of action in the antiinflammatory activity of glucocorticoids. It appears that there are multiple molecular mechanisms by which glucocorticoids act on post-transcriptional events, and they are still far from being fully understood. Studies aimed at the identification of these mechanisms are reviewed below.

The following section briefly overviews the post-transcriptional activities of the major kinase pathways. It also evaluates the effect of glucocorticoids on each pathway, indicating, when available, studies that directly link the effects of glucocorticoids on mRNA turnover or translation in a gene target with their ability to inhibit kinase pathways involved in the post-transcriptional regulation of those genes (see Table 2). This review will focus primarily on data regarding the control of mRNA stability, although some of the effects on translational and post-translational control will be referenced as well.

Inhibition of the ERK pathway by glucocorticoids has been demonstrated in several in vitro studies using different models. The rapid induction of MAPK phosphatase 1 (MKP-1) appears to be responsible for glucocorticoid-induced inhibition of this pathway in mouse mast cells and human osteoblasts (32, 33). Similarly, the glucocorticoid-induced leucine zipper (GILZ) gene inhibited Raf-1 phosphorylation in T cells, leading to the inhibition of activation of the Raf-MEK-ERK pathway (where MEK designates MAPK/ERK kinase) (34). The inhibitory effect of glucocorticoid on this pathway appears to be cell type–depen-dent, because no effect on ERK activity by glucocorticoid was found in human airway smooth muscle cells (35) or in murine macrophages, where glucocorticoids selectively inhibited JNK/SAPK activity (36). However, no studies to date have directly addressed whether the known ability of glucocorticoid to inhibit some ERK-regulated genes, such as GM-CSF, through alteration of mRNA stability is due to the inhibition of ERK-dependent mRNA stabilization.

Activation of JNK/SAPK signaling induced ARE-dependent mRNA stabilization of IL-3 and vascular endothelial growth factor (VEGF) mRNAs (37, 38), whereas in activated T lymphocytes, stabilization of IL-2 mRNA was conferred by a JNK-responsive element present in the 5'-UTR (4). These data suggest that the function of JNK as an inducer of mRNA stabilization is mediated by multiple transcript-specific pathways. Another relevant post-transcriptional activity of JNK is the ARE-dependent translational regulation of TNF- expression, a function shared with the p38/SAPK2 pathway (36, 39).

Glucocorticoids inhibit the activity of JNK/SAPK in several cell types (36, 40). It has been postulated that they inhibit lipopolysaccharide-induced TNF- translation by blocking the activity of JNK/SAPK, as overexpression of SAPK-ß overrides the dexamethasone-induced inhibition of TNF- translation (36). Glucocorticoids increase mRNA turnover of the JNK-dependent VEGF gene in keratinocytes (41), and although direct supporting data are not available, inhibition of the JNK/SAPK pathway may mediate this effect.

The p38/SAPK2 pathway has been particularly linked to the promotion of increased mRNA stabilization and translation of numerous inflammatory genes. In different cell types, treatment with specific p38 inhibitors leads to a sharp decrease in the mRNA stability of several induced genes, as well as the stability of chimeric constructs carrying the ARE-bearing 3'-UTRs of these genes (reviewed in Reference 27). Translational regulation, as in the case of lipopolysaccharide-induced TNF-, has also been found to be dependent on p38 activation and on the presence of AREs (13, 28).

A number of genes that are post-transcriptionally repressed by glucocorticoid, such as IL-1, VEGF, TNF-, IL-6, IL-8, and cyclooxygenase-2 (COX-2), are also regulated by p38 (23, 24, 36, 40, 41). Consequently, studies that have investigated the links among the post-transcriptional effects of glucocorticoid, the signaling involved in mRNA stabilization, and the role of AREs in modulating these responses have focused on p38-regulated genes.

Lasa and colleagues investigated the mechanisms regulating COX-2 mRNA turnover in HeLa cells transiently transfected with a chimeric reporter construct including the full-length 3'-UTR of COX-2 (40, 42). The decay of the ß-globin reporter mRNA was examined in cells unstimulated or stimulated by a constitutively active form of MAPK kinase-6 (MKK-6), which activates the p38 kinase pathway and dramatically increases the stability of the reporter mRNA. Pretreatment of transfected cells with dexamethasone (40) or p38 antagonists (42) completely reversed MKK-6–induced stabilization of the reporter mRNA, an effect mediated in both cases by a small ARE cluster within the 3'-UTR of the COX-2 mRNA, which bears a large number of AREs. Cell treatment with glucocorticoids directly inhibited p38 phosphorylation, supporting the conclusion that the ability of glucocorticoid to accelerate COX-2 mRNA decay was due to the inhibition of the p38 MAPK, which was in turn responsible for COX-2 mRNA stabilization (40). Later, the same group established that glucocorticoids induced the expression of MKP-1 (43). Interestingly, glucocorticoid induction of MKP-1 was also responsible for inhibition of the ERK pathway in a mast cell line (32). As the effect of glucocorticoids on the mRNA stability of COX-2 and of other genes has been found to require gene expression (44), it is possible that glucocorticoid-induced genes such as MKP-1 play a significant role in post-transcriptional regulation of COX-2 by glucocorticoids.

Despite the evidence suggesting that ARE-dependent, kinase-mediated signaling pathways are targeted by glucocorticoids to alter mRNA turnover and translation, the data from different experimental models are somewhat contradictory and indicate that such a mechanism of action may be limited. These regulatory events occur in vitro in a cell type–specific fashion (40, 43) and possibly also in a transcript-specific fashion, because not all ARE-containing genes are p38-dependent (9, 45). Moreover, studies using synthetic inhibitors of signaling pathways on chimeric reporter constructs may not fully recapitulate the mechanisms regulating endogenous gene expression.

To better understand the physiologic role of AREs and the relevance of the p38 pathway in glucocorticoid-induced control of post-transcriptional regulation, it is useful to consider the results obtained in an in vivo model of targeted ARE deletion. Mice carrying a deletion of the ARE in the TNF- gene (ARE) displayed an increase in constitutive and inducible production of TNF- because of slower TNF- mRNA decay and loss of translational inhibition (39). The high circulating levels of TNF- were associated with early onset of inflammatory changes within the joints and the bowel, which closely resembled lesions present in human rheumatoid arthritis and Crohn's disease, respectively. The increase in TNF- protein secretion obtained in lipopolysaccharide-stimulated macrophages from the ARE mice was not responsive to cell treatment with a p38 inhibitor, showing that the ARE region is required for p38/JNK activation of TNF- translation. In contrast, TNF- secretion in macrophages from ARE mice was significantly inhibited by dexamethasone, although to a lesser extent in comparison with the effect observed in wild-type murine macrophages. The partial loss of inhibition by glucocorticoid in this model suggests that ARE-dependent inhibition of TNF-, likely mediated by inhibition of p38/JNK, is an important but not exclusive mechanism of inhibition of TNF- translation by glucocorticoids. It would be of interest to examine the effects of glucocorticoids on ARE-binding proteins acting as repressors of TNF- translation, such as T-cell intracellular antigen (TIA) and TIA-related protein (TIAR) (13). Glucocorticoids can influence translation by inhibiting translational initiation factors and ribosomal genes (46), and they can also target post-translational modifications. For example, glucocorticoids regulate the maturation of murine mammary tumor virus (MMTV) proteins in rat hepatoma cells by interfering with glycoprotein compartmentalization and processing, as well as protein phosphorylation (47). Similarly, glucocorticoids inhibit inducible nitric oxide synthase (iNOS) expression in rat glomerular mesangial cells by reducing iNOS mRNA translation and increasing degradation of iNOS protein, in parallel with actions on gene transcription and mRNA stability (48). Acceleration of protein degradation induced by glucocorticoids has also been reported for rat acetylcolinesterase (49) and GLUT2, a product of the rat pancreatic ß cells (50).

Heterogeneity of glucocorticoid-sensitive determinants of post-transcriptional regulation.
The pathways of post-transcriptional gene control may differ in their degrees of glucocorticoid sensitivity. Moreover, for a given target, glucocorticoids may modulate mRNA turnover in a cell type–specific fashion (27). This specificity may be due to differential expression of RNA-binding proteins among cell types, or it may arise from cell type–specific inhibition by glucocorticoids of signaling pathways controlling the target mRNA decay or translation. The RNA recognition motifs mediating the glucocorticoid action have been identified for only a few of the targeted genes (see Table 1). Most frequently, ARE dependence or at least involvement of the 3'-UTR has been documented (26, 51). As mentioned above, other sequences have been found to mediate the effect of glucocorticoids on mRNA turnover. Intronic sequences of prespliced nuclear fibronectin mRNA are thought to be responsible for the glucocorticoid-induced increase in mRNA decay (52). Acceleration of rat MCP-1 mRNA decay by glucocorticoids in smooth muscle cells was not dependent on the ARE-bearing 3'-UTR. Instead, it was mediated by a unique sequence in the 5'-UTR, which was also able to destabilize the mRNA of a reporter construct and constitutes, so far, the only glucocorticoid-sensitive region described in any 5'-UTR (5).

RNA-binding proteins, upon interaction with RNA recognition motifs, are ultimately responsible for the change in protein output that occurs as a result of post-transcriptional regulation (10). Despite the importance of these molecules, very little is known about the ability of glucocorticoids to alter their synthesis or activation. The phosphorylation status appears to be important for the activation and possibly the binding to RNA of some of these proteins, such as tristetraprolin (TTP) (53) or HuR (54). It can be hypothesized that inhibition of phosphorylation-mediated activation of RNA binding proteins, either directly or mediated by phosphatases, would be an efficient mechanism by which glucocorticoid could modulate mRNA stability or translation. In support of this hypothesis, the glucocorticoid effect on mRNA stability has often been found to be dependent on transcription or protein synthesis (see Table 1). It is tempting to speculate that glucocorticoid-induced phosphatases, such as MKP-1, mediate the effect of glucocorticoids on mRNA turnover. Acceleration of COX-2 mRNA decay by glucocorticoids involves primary loss of polyadenylated mRNA, an effect also dependent on ongoing transcription and protein synthesis (44). This process suggests that other classes of genes inducible by glucocorticoids could play a role in mediating the action of glucocorticoids on the deadenylation process (55).

There are also cases in which the action of glucocorticoids at the post-transcriptional level appears to be independent from de novo gene expression, as in the case of glucocorticoid-mediated acceleration of MCP-1 mRNA decay (5). These data suggest that glucocorticoids can act on constitutively expressed targets, possibly through phosphorylation-mediated changes or other post-translational modifications. Glucocorticoids might induce the activation or recruitment of RNAse complexes such as the exosome, a multi-subunit complex of RNAses that is critical in regulating the efficiency of ARE-dependent mRNA turnover (56). Alternatively, glucocorticoids might change the affinity of RNA-binding proteins for their recognition motifs, either favoring the displacement of a stabilizing protein, such as HuR, or promoting the activation and binding of a destabilizing protein, such as TTP (10). Very little is known about a direct effect of glucocorticoids on these regulatory RNA-binding proteins or on the activity of RNAse complexes.

The possibility cannot be excluded that the ligand-activated glucocorticoid receptor (GR) engages in protein–protein interactions with RNA-binding proteins, similar to its interactions with nuclear factor-B subunits and other transcription factors. The GR has been shown in vitro to bind at the nuclear level with nucleolin (57), a multifunctional protein that mediates, among other functions, increased IL-2 mRNA stability in T cells activated through the JNK pathway (12). The effect of this interaction on mRNA stability has not been evaluated. However, the formation of ligand-activated GR/RNA-binding complexes might mediate "post-transcriptional transrepression" by altering the composition and/or the configuration of the ribonucleoprotein complex, favoring mechanisms promoting mRNA decay.

In summary, AREs and other recognition motifs in the mRNA of inflammatory genes are indispensable binding sites for RNA-binding proteins (Figure 2). These proteins constitute the targets of key signaling pathways regulating gene expression upon cell activation. By increasing the mRNA stability and/or the translation rate, these pathways modulate the final protein output, thus providing an appropriate response to the specific activating stimulus received by the cells. Increasing evidence indicates that the inhibitory effect of glucocorticoids on these signaling pathways affects their post-transcriptional effects, leading to a glucocorticoid-mediated decrease in mRNA stability and/or translation. However, much remains to be learned about the full range of mRNA sequences involved in glucocorticoid responsiveness, the effects of glucocorticoids on other downstream effectors (such as RNA-binding factors and RNA-degrading enzymatic systems) that ultimately mediate mRNA turnover and translation, and how glucocorticoids achieve specificity in these actions.

View larger version (31K): [in this window] [in a new window]   Figure 2. Post-transcriptional gene regulation as a target of glucocorticoid (GC) action. Sequences contained in the mRNA of inflammatory genes function as binding sites for RNA-binding proteins, which are the targets of the key signaling pathways that regulate gene expression upon cell activation. These signaling pathways, in concert with transcriptional regulation, modulate the final protein output by increasing the mRNA stability and/or the translation rate, thus providing an appropriate response to the specific activating stimulus received by the cells. Inhibition of these signaling pathways by glucocorticoids interferes with their post-transcriptional effects, leading to a glucocorticoid-mediated decrease in mRNA stability and/or translation. Other, as-yet unidentified pathways (dotted lines) are likely to be involved in glucocorticoid responsiveness through multiple mRNA cis-elements, possibly through interference with downstream effector molecules, such as RNA-binding factors and RNA-degrading enzymatic systems, that ultimately mediate mRNA turnover and translation. JRE indicates a JNK-responsive element identified in the 5'-UTR of the mRNA molecule.

	NONGENOMIC ACTIONS OF STEROID HORMONES

TOP ABSTRACT POST-TRANSCRIPTIONAL GENE... NONGENOMIC ACTIONS OF STEROID... REFERENCES

 
Definition and Characteristics
From the early studies of the mechanism of action of glucocorticoids, it became apparent that all classes of steroid hormones can induce effects that occur in a very short time frame, within minutes or even seconds of their application. These changes have been well documented in vitro, on intracellular signaling pathways, and in vivo, in a wide array of human and animal models used to study biological functions such as oogenesis, vasoregulation, response to stress, and neurobehavioral changes (61). These rapid actions do not fit the classical "genomic" model of steroid action. Such a mechanism requires multiple receptor-mediated changes to occur upon translocation to the nucleus of the ligand-activated GR, followed by modulation of gene expression and possibly involving de novo production of genes. These actions require hours to be fully operative and are pharmacologically characterized by sensitivity to transcriptional and translational inhibitors such as actinomycin D or cycloheximide. Glucocorticoids' effects on mRNA stability can be also included in the classical mode of action, because they affect gene expression and may be sensitive to protein synthesis inhibitors. To distinguish between this well established mode of steroid action and the effects occurring acutely following steroid administration, the latter effects have been referred to as "nongenomic." It is important to understand, however, that these effects are not solely alternative to the genomic effects. Very useful in this matter is the definition of nongenomic effects recently given by Lösel and Wehling: "Any action that does not directly and initially influence gene expression, but rather drives more rapid effects such as the activation of signaling cascades" (62). This definition recognizes that the distinction between the two modes of action is not always clear-cut. The nongenomic effects of glucocorticoids do have some distinctive characteristics, however. In addition to the short time frame in which the effects occur, these characteristics include: (1) a different pharmacologic profile, because the effects are insensitive to transcriptional and protein synthesis inhibitors; (2) action on nonnucleated cells, such as platelets, erythrocytes, and spermatozoa; and (3) the ability of steroid analogs (such as bovine serum albumin-conjugated steroid molecules) that cannot access the intracellular compartment to elicit a response (57).

A classification of rapid steroid effects according to the very heterogeneous mechanisms involved has recently been proposed (63). The majority of these rapid effects have been documented at physiologic concentrations. The mechanisms of action, with different specificity according to the cell type and the steroid studied, are frequently mediated by the generation of a variety of second-messenger systems, by changes in ion fluxes, and by the activation of different kinase pathways (57). In several cases, the rapid effects are mediated by the classical GR, as proven by their sensitivity to nuclear receptor antagonists; however, some of the nongenomic effects appear to be nuclear antagonist–independent, suggesting that a different, membrane-bound GR mediates the nongenomic action. To include this alternative mode of action in the definition of nongenomic effects, the term "membrane-initiated steroid signaling" has been proposed (57). The identities of membrane-bound GRs have been elusive so far, although recently a seven-transmembrane progestin receptor was cloned and characterized (64).

Besides the homeostatic functions of glucocorticoids, these hormones are secreted by adrenal glands in stress-induced responses, which are rapid and therefore likely to be mediated by nongenomic mechanisms. Indeed, neurophysiologic and behavioral responses following glucocorticoid administration, as well as changes in a wide array of cellular responses, have been well characterized as nongenomic (56). The rest of this section provides examples of the nongenomic effects of glucocorticoids, divided according to some of the modes of action proposed in the recent classification (63).

Nongenomic Actions without Receptor Involvement
Some nongenomic effects of steroids seem to be mediated by alteration of the physicochemical properties of the cell membrane without the involvement of the GR. Steroids are highly lipophilic molecules and diffuse easily in lipid membranes, where they are thought to interfere, especially at high concentrations, with the function of membrane-bound molecules, such as ion channels or receptors. These effects have been observed in vitro in several cell types (61). They appear to be steroid-specific: micromolar concentrations of progesterone altered several physicochemical parameters of membrane vesicles from human spermatozoa, whereas none of the effects were produced by equimolar concentrations of testosterone or estrogen (65). In the majority of the studies the concentrations necessary to achieve these effects in vitro were above the physiologic and therapeutic range (> 10 µM) (66), and, therefore, their relevance in vivo is questionable. However, in a recent in vitro study using a more physiologic range of glucocorticoid concentrations (0.1–1 µM), a rapid (within 15 minutes) antisecretory response occurred in human primary bronchial epithelial cells following cell treatment with dexamethasone, an effect that was optimal at a low glucocorticoid concentration (1 nM) (67). There were rapid decreases, after dexamethasone treatment, in basal levels of intracellular Ca²⁺ ([Ca²⁺]_i) and in levels of [Ca²⁺]_i induced by adenosine triphosphate (ATP), which were insensitive to cycloheximide and were unaltered by the GR antagonist RU486. Treatment with dexamethasone also partially decreased Ca²⁺-dependent, ATP-induced epithelial Cl^– secretion. Experiments with specific antagonists showed that the effect of dexamethasone on [Ca²⁺]_i was dependent on stimulation of a Ca²⁺-ATPase via adenylate cyclase and protein kinase A signaling (67). As mucus hyperproduction in asthma is associated with upregulated expression of Ca²⁺-activated Cl^– channels (68), the antisecretory role of glucocorticoids may contribute to the beneficial effects of glucocorticoid therapy in asthma. However, in this study, the topical glucocorticoids hydrocortisone, triamcinolone, and budesonide did not reproduce the effect of dexamethasone on [Ca²⁺]_i (62). In fact, they were increasingly ineffective in the order listed here, even though their affinity for the GR increases in the same order. The authors speculate that since dexamethasone has the lowest lipophilicity among the glucocorticoids tested, it is best suited to interact directly with the cell membrane.

The issue of lipophilicity might be important in understanding the relevance of nongenomic mechanisms to the very rapid effects elicited by glucocorticoids in high-dose bolus therapy (> 250 mg prednisone-equivalent/day) for acute spinal cord trauma, multiple sclerosis, or anaphylaxis (69–71), such as prevention of early-onset edema. It has been proposed that nongenomic mechanisms may be clinically relevant over a limited period, "bridging the gap" until the long-term, genomic effects take place (72). Along these lines, high concentrations of dexamethasone stabilized lysosomal membranes, an effect thought to be involved in the antianaphylactic action of glucocorticoids, in a rapid (< 10 minutes) but sustained fashion (73). This effect was blocked by RU486 only at a later time point (24 hours), indicating a "dual action" mechanism of glucocorticoids that involves both nongenomic and genomic components. More controlled studies are needed, testing glucocorticoid concentrations obtainable in vivo during high-dose therapy, to verify the occurrence and the relevance of these nongenomic effects and to firmly establish whether the ability to elicit these responses should be considered, as recently suggested, in the choice of glucocorticoid for high-dose therapeutic regimens (74).

Nongenomic Actions via Classical Intracellular Receptors
Some effects of glucocorticoids are characterized pharmacologically by insensitivity to transcriptional or translational inhibitors, which indicates their nongenomic nature, coupled with susceptibility to antagonists of the nuclear receptor, which indicates the involvement of classical (or nuclear) receptor-mediated mechanisms. An example is the effect of glucocorticoids on the activation of endothelial nitric oxide synthase (eNOS) (75). Glucocorticoids have been shown to have some acute cardioprotective effects on cardiac ischemia (76), and nitric oxide is a major mediator of cardiovascular protection (77). Therapeutic concentrations of dexamethasone (100 nM) induced significant activation of eNOS in human endothelial cells within 10 minutes and up to 24 hours after stimulation (75). Glucocorticoid-induced early eNOS activity (detectable at 30 minutes), as well as nitric oxide production, were abolished by treatment with RU486 but were resistant to treatment with actinomycin D, indicating a GR-dependent and transcription-independent action. This response was significantly suppressed by specific inhibitors of phosphatidyl inositol 3-kinase (PI3K), as well as by a specific inhibitor of eNOS. In contrast, it took at least 4 hours for the same concentration of dexamethasone to activate the transcription of a reporter construct driven by multiple glucocorticoid response elements, an effect that was sensitive to RU486 and actinomycin D but insensitive to PI3K inhibitors. The authors concluded that eNOS activation by dexamethasone involves rapid, nontranscriptional mechanisms. This effect was found to be mediated by glucocorticoid-induced increased activity of PI3K, which, through an increase in phosphatidyl inositol 3,4,5-triphosphate, activated downstream pathways involving Akt, a kinase shown to activate eNOS via PI3K in response to VEGF (78). Importantly, in the same study the physiologic significance of the in vitro data was tested in an in vivo model of ischemia and reperfusion (I/R). Acute administration of high-dose dexamethasone (1,000 nM) blocked signs of I/R-induced vascular inflammation and decreased the extent of myocardial infarction. The latter effect was paralleled by a dexamethasone-induced increase in eNOS activity and was blocked by GR and eNOS antagonists. The nongenomic effect of glucocorticoids on eNOS was later found to be relevant as well in a model of ischemia in the brain (79).

Nongenomic Actions via Nonclassical Receptors
Two major candidates for membrane-associated glucocorticoid (GC) receptor have been so far studied: the first is a 63-kD acidic glycoprotein, which has been identified in neuronal plasma membranes of the amphibian Taricha granulosa. It has been functionally characterized as a putative membrane receptor for GC, with pharmacologic characteristics completely distinct from the known GR (80). Data from in vitro and in vivo models suggest a key role of this receptor in mediating at least some of the nongenomic neurophysiologic and behavioral effects of GC (reviewed in Ref. 61). The second candidate has been identified, in mammalian cells, as a modified form of the classical GR, which has been postulated to function as a membrane-bound glucocorticoid receptor (mGR) (81). Identification and subsequent data on this putative mGR have been gained mostly by epitope-recognition techniques using mouse lymphoma and human acute lymphoblastic leukemia cells (81). Several studies have characterized differences in cell localization, molecular size, and specificity to glucocorticoid between the GR and the mGR, but they have also pointed to several common aspects of the two receptors that support the identity of the mGR as a modified GR: shared epitope recognition for different antibodies directed against the GR, similar ability to bind to heat shock proteins or DNA, and similar phosphorylation patterns (77). Using cell populations with either high or low expression of the mGR, the presence of this receptor has been functionally linked to glucocorticoid-induced lysis of lymphoma cells, and it has been postulated to play a role in thymic involution and apoptosis (81, 82). Cloning of the putative mGR will be necessary to gain more definitive data on its nature and, most importantly, on the functions it mediates.

CONCLUSIONS
The antiinflammatory action of glucocorticoids appears to be mediated by a continuum of actions that interfere with a wide range of events regulating gene expression throughout the cell, from generation of early signaling events to post-translational modifications occurring well after transcription has occurred. The multiple effects of glucocorticoids appear to be highly integrated; for example, signaling molecules targeted nongenomically by glucocorticoid can indirectly modulate gene expression by activating transcription factors or kinase pathways crucial for transcriptional activation of gene expression (62). Furthermore, kinase pathways that are glucocorticoid-sensitive regulate gene expression at the transcriptional and post-transcriptional levels in a coordinated fashion.

The relevance of studies unraveling the cross-talk between the different mechanisms of glucocorticoid action lies in identifying novel, physiologically important signaling pathways by which steroids exert antiinflammatory effects that were previously overlooked because of the distance of the mechanisms involved from the transcription-dependent mode of glucocorticoid action. It will be important to discover how much of the anti-inflammatory activity of glucocorticoids lies in these newly recognized mechanisms of action and how these effects integrate with those exerted upon transcription.

	ACKNOWLEDGMENTS

 
C.S. received $150,000 in 2001 and 2002 from AstraZeneca UK Ltd. as research grant on "Preclinical studies on the function of CCR3 on epithelial cells".

The author is indebted to Drs. Ulus Atasoy, Vincenzo Casolaro, Steve Georas, and Robert Schleimer for discussions and review on the topic of the manuscript.

	FOOTNOTES

 
Dr. Stellato's work is supported by National Institutes of Health Grant AI 44242-0142.

(Received in original form February 18, 2004; accepted in final form May 10, 2004)

	REFERENCES

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