Quick viewing(Text Mode)

Specificity in Mineralocorticoid Versus Glucocorticoid Action

Specificity in Mineralocorticoid Versus Glucocorticoid Action

Kidney International, Vol. 57 (2000), pp. 1256–1264

Specificity in versus action

PETER J. FULLER,SYLVIA S. LIM-TIO,1 and FRANCINE E. BRENNAN

Prince Henry’s Institute of Medical Research, Clayton, Victoria, Australia

Specificity in mineralocorticoid versus glucocorticoid action. extent and molecular basis of this overlap of mineralo- The mineralocorticoid (MR) and the glucocorticoid corticoid and glucocorticoid actions have been the sub- receptor (GR) share considerable structural and functional ject of both debate and speculation for many years [3–9]. homology. Overlapping effects on epithelial transport are observed in vivo; in vitro, both are able to bind and trans- The MR is expressed at the greatest abundance in activate through a common hormone response element. This the sodium-transporting epithelia of the distal colon and has led several investigators to suggest that specificity is con- distal nephron [3, 4], in certain regions of the central ferred primarily by prereceptor mechanisms, and we have ad- , particularly the hippocampus [10], and dressed this question using both in vitro and in vivo approaches. Although the MR has been regarded as less transcriptionally at lower abundance in a variety of other tissues. The active than the GR in vitro, significant differences are observed (GR) shows considerable structural when epithelial rather than fibroblast cell lines are used. These (Fig. 1) and functional homology with the MR [11, 12]. differences are mediated by the N-termini of the receptors. Acti- The GR, which is expressed relatively ubiquitously, me- vation of intracellular signaling pathways differentially modu- diates a diverse range of effects, including increased elec- lates MR- versus GR-mediated transactivation. Although these studies identify mechanisms by which specificity may be achieved, trolyte transport. The distinction mineralocorticoid:gluco- they do not prove that this occurs in vivo. Such studies have corticoid is confounded by the fact that, while been limited by an absence of MR-regulated . Known binds to the GR with an affinity several orders of magni- candidate aldosterone-responsive genes have been examined tude lower than that with which it binds to the MR, in the rat distal colon; the time course and the specificity of binds to the MR with a higher affinity than to the response to a single parenteral dose of has been characterized. The epithelial sodium channel ␤ and ␥ the GR [11, 12]. Given that cortisol circulates at much subunit genes are both up-regulated within 60 minutes by either higher concentrations than aldosterone, cortisol would MR or GR activation. Similar responses are observed for the be expected to fully occupy the MR under most condi- serum and glucocorticoid-regulated kinase and channel-induc- tions. It is now well established that specificity of receptor ing factor genes. All four genes show clear and rapid up-regula- occupancy is achieved at a prereceptor level by the juxta- tion of their mRNA levels by aldosterone, which is paralleled by GR-mediated up-regulation of expression. While they are position of the type II isoform of the enzyme 11␤-hydroxy- indeed aldosterone-responsive genes, genes that are uniquely dehydrogenase (11␤-HSD2) with the MR in aldosterone-regulated remain to be identified. epithelial cells [13, 14]. 11␤-HSD2 converts cortisol to (or to 11-dehydrocorticosterone in rodents). Cortisone is inactive with respect to both Aldosterone plays a central role in the control of the MR and the GR. pressure as a result of its effects on sodium homeostasis. As for the and receptors, the In addition, it has both direct and indirect effects on potas- MR can regulate expression, at least in an in vitro sium and hydrogen ion homeostasis. These mineralocorti- system, by binding to a glucocorticoid response element coid actions are mediated by the binding of aldosterone (GRE) in the promoter of a reporter gene (Fig. 2), such to the mineralocorticoid receptor (MR), a member of as the often used mouse mammary tumor virus (MMTV) the steroid/thyroid/retinoid superfamily of ligand-depen- promoter [11, 12]. This apparent lack of specificity with dent factors [1, 2]. The physiological gluco- respect to DNA binding has led various authors to argue corticoid cortisol also has effects on epithelial that mineralocorticoid versus glucocorticoid specificity transport and the maintenance of . The is entirely prereceptor [6, 7]. Under this scheme, physio- logical specificity relies on a combination of the patterns of and 11␤-HSD2 expression. In 1 Present address: Imperial Cancer Research Fund, PO Box 123, 44 the experimental situation, specificity can be expanded Lincoln’s Inn Fields, London WC2 A 3PX, United Kingdom. to include ligand specificity caused by the use of synthetic Key words: aldosterone, , cortisol, sodium transport. ligands, which putatively bind only MR or GR, be they  2000 by the International Society of Nephrology agonist or antagonist.

1256 Fuller et al: Specificity in MR versus GR action 1257

Fig. 1. Schematic representation of the struc- tures of the human mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) with the percentage amino acid identity between each of the domains shown (adapted with per- mission from Arriza et al in Science [11]).

This review seeks to address this issue from both a The time course of the response in any of these tissues conceptual or inferential perspective, and also with re- is important, particularly if one wishes to correlate physi- gard to studies of aldosterone-induced gene expression. ological and molecular responses. Verrey has recently The first section is an overview of the mineralocorticoid reviewed this issue in some detail [19]. In brief, aldoste- response in vivo as reported in studies in which the issue rone action mediated via the MR may be divided into of specificity has been specifically addressed. This is con- early effects with an onset of 30 to 60 minutes after trasted with studies in cell culture systems in which speci- aldosterone administration and late effects with a time ficity is not observed. Our own studies, as well as those of onset of three to six hours. The early response is of others, using in vitro systems are discussed with re- characterized by an increased amiloride-sensitive electro- spect to molecular mechanisms by which specificity may genic sodium flux through the sodium channels in the be achieved. Finally, we present recent data on the regu- apical membrane. During the later phase, there is accu- lation by of the expression of putative mulation of sodium channels and pumps [19]. Most stud- aldosterone-induced genes in vivo. ies of mineralocorticoid physiology commence at and often beyond three to six hours, so that back extrapola- tion to primary molecular events may be inappropriate. MINERALOCORTICOID PHYSIOLOGY Superficially, there are clear differences between GR In examining the studies of the physiology of aldoste- and MR activation seen most graphically when the clini- rone action, it is important to consider both the tissue cal syndromes of aldosterone and cortisol excess, Conn’s in which the response is being characterized and the and Cushing’s syndromes, respectively, are compared. time course of the response. Amphibian systems have Similarly, when receptor function is lost, as in the MR- provided very important insights into mineralocorticoid and GR-knockout mice, the phenotypes are distinctly physiology, particularly the toad bladder and more re- different [20, 21]. At least some of the differences seen cently the A6 renal epithelial cell line [15]; however, as in these in vivo situations may simply reflect differences discussed later in this article, there are certain caveats in the tissue distribution of the receptor. that must be placed on the interpretation of these studies. Turnamian and Binder have directly addressed the In mammalian systems, most studies are of either the question of MR versus GR action on electrolyte trans- kidney or the distal colon. The distal nephron of the port in rat distal colon [5]. They infused synthetic MR- kidney is a complex epithelium with clear partitioning or GR-specific for up to 11 days and character- of function, including mineralocorticoid responses. The ized the resulting changes in specific transport processes. distal colon is, in contrast, a relatively simple epithelium They found that, although the MR agonist induced elec- in which the actions of are well de- trogenic amiloride-sensitive sodium absorption with in- scribed [4]. There is an assumption that the mechanisms hibition of electroneutral Na-Cl absorption, the GR ago- of the colonic response are analogous to those of the nist induced only electroneutral Na-Cl absorption. In distal nephron, but this is by no means certain, and tissue- contrast to the synthetic mineralocorticoid, the glucocor- specific differences at both physiological and molecular ticoids also failed to increase active transport. levels have been identified [3, 4, 16–18]. Caution must These authors argued that previous studies using less be applied when drawing general conclusions about spec- receptor-selective agonists such as dexamethasone were ificity from studies in any one system. Our in vivo studies confounded by cross-over receptor activation, particu- have focused on the distal colon, and many of the physio- larly at higher concentrations. Bastl and Hayslett simi- logical studies that have sought to address MR versus larly concluded that acted primarily to GR specificity have used this system. increase electroneutral sodium transport in distal colon, 1258 Fuller et al: Specificity in MR versus GR action

Fig. 2. Schematic representation of the primary regulation of genes by the MR and the GR. Prereceptor specificity is provided by 11␤-hydroxysteroid dehydrogenase (11␤-HSD2). In most systems, both the MR and the GR can transactivate at a simple glucocorticoid receptor element (GRE), whereas specificity has been reported at a composite GRE through an interaction with the activator -1 (AP-1) complex [38]. An analogous mechanism that confers MR specificity remains to be identified.

with regulation of electrogenic transport being mediated salt replacement enables them to be rescued [27]. Al- by the MR [4]. Lomax and Sandle reported increased though this may reflect adverse effects of the high doses apical Naϩ conductance in the distal colon of adrenalec- of glucocorticoid, it may also imply that GR activation tomized rats after seven days of aldosterone or pure syn- cannot fully compensate for the lack of MR function [27]. thetic glucocorticoid treatment, whereas increased apical Kϩ conductance was only observed after aldosterone treatment [22]. These studies have conclusively shown STUDIES USING CELLS IN CULTURE that in the distal colon, MR but not GR mediates in- Studies in vitro using either cell lines or primary cul- creased Kϩ transport, whereas both MR and GR en- tures of isolated cells have supported the conclusion that hance Naϩ transport albeit through different pathways. the postreceptor events are indeed nonselective with re- The time courses of these studies, however, may have gard to MR versus GR. Husted, Laplace, and Stokes allowed secondary effects of the steroids [23] acting on reported that 24-hour exposure to both MR- and GR- other tissues or pathways to have contributed to the selective agonists could stimulate electrogenic sodium observed changes in electrolyte transport. transport in rat inner medullary collecting duct cells [28]. Fromm, Schulzke, and Hegel used isolated rat distal In a subsequent study, the same authors found differ- colon to examine Naϩ absorption over eight hours of ences in the time course of the response mediated by the steroid treatment [24]. An increase in electrogenic Naϩ two receptors, it being more prompt after glucocorticoid absorption was observed at two hours. In contrast to treatment [29]. On the basis of this and other observa- previous studies, this group reported increased electro- tions, the authors concluded that the MR and the GR genic absorption in response to the pure glucocorticoid activate different but overlapping cellular events leading RU28362 [9]. These authors argued against MR:GR to sodium transport [29]. Several of the cellular responses specificity and emphasized the possible role of GR:MR differed from those reported for cortical collecting duct heterodimerization in the response [25, 26]. Funder et cells, which suggests significant segmental differences in al observed a similar acute antinatriuretic response to responses within the nephron [29]. Na´ray-Fejes-To´ th and activation of either the GR or MR using a Kagawa bio- Fejes-To´ th used immunodissected rabbit cortical collect- assay, which led these authors to also conclude that MR ing duct cells to demonstrate that both transepithelial po- versus GR selectivity is entirely prereceptor [7]. tential difference and short-circuit current were increased Berger et al have recently reported the phenotype of by aldosterone and a selective glucocorticoid agonist [6]. transgenic mice in which a null mutation in the MR gene In this study, all of the cellular events measured exhibited has been created by homologous recombination [20]. a similar response to both classes of steroid. These mice show features of severe mineralocorticoid de- A6 cells, which are derived from Xenopus laevis renal ficiency, with marked salt wasting and dehydration. They tubules, have been used extensively for studies of miner- do not survive beyond eight days. Treatment with the alocorticoid responsivity. Their mineralocorticoid response synthetic glucocorticoid prolongs sur- to both aldosterone and glucocorticoids is mediated via vival, but the mice ultimately succumb, whereas careful the GR, as the endogenous MR is inactive [30, 31], which Fuller et al: Specificity in MR versus GR action 1259 suggests that some caution is required when extrapolat- [38]. The use of a DNA-binding mutation of the GR in ing studies from this amphibian cell line to the mamma- transgenic mice has further confirmed the importance of lian kidney. Nakhoul et al and Bens et al have recently protein:protein interactions independent of GR-DNA described the responses of two murine transformed renal binding in GR-mediated gene expression [35]. Identifi- cortical collecting duct cell lines to corticosteroids [32, 33]. cation of a MR-regulated gene or genes in vivo would In both lines, aldosterone induced amiloride-sensitive clearly facilitate the characterization of the molecular electrogenic sodium transport; however, in neither were basis of mineralocorticoid-specific regulation; however, highly selective GR agonists examined. Further studies MR-regulated genes have been identified only recently, of the response of one or both of these lines to selective and in all cases, these genes also appear to be GR- MR and GR agonists will be of considerable interest. regulated, as discussed later in this article. An alternate approach, in the absence of specifically MR-responsive promoters, is to identify the mechanisms by which MR- TRANSFECTION STUDIES specific regulation may be achieved, at least under in The observation in cotransfection experiments that vitro conditions. both the MR and the GR can transactivate at GRE In the classic cis-trans assay [11, 12] using the trans- sequences, particularly those of the MMTV promoter fected human GR or MR with MMTV luciferase (LUC) [11, 12], lent credence to the argument that the GR and or MMTV chloramphenicol acetyl transferase (CAT) as MR, once activated, exert equivalent effects (Fig. 2). The a reporter gene in CV-1 cells, the MR exhibits approxi- DNA-binding domains of the MR and the GR share mately 20% of the maximal response of the GR [39]. 94% identity [11], so this finding is not unexpected. MR- Creation of chimeric receptors between the MR and the mediated transactivation at other “physiologically rele- GR demonstrated that this difference was located in the vant” promoters has been reported. Zennaro, Le Men- N-terminal domains of the receptors [39], a somewhat uet, and Lombe`s reported that the more distal of the unsurprising finding given that (1) this region shows Ͻ15% two major promoters of the human mineralocorticoid identity between the receptors [11], and (2) that it con- receptor (hMR) gene was induced by both the MR and tains the major activation function (AF-1) for this system. the GR [34]; interestingly, the region of the promoter CV-1 cells are a fibroblast-derived cell line; when we ex- through which this effect was mediated does not contain amined the response in cell lines of either epithelial (LLC- an obvious GRE. This promoter is primarily used for PK1) or hippocampal (RN33B) origin (Fig. 3), the relative MR gene expression in the central nervous system, and levels of maximal MR-mediated transactivation doubled since corticosteroid regulation of receptor levels has [40]. Again using chimeric receptors, we were able to been clearly demonstrated in the central nervous system show that the effect was mediated by the N-terminal do- as opposed to peripheral tissues, the finding is therefore main. This suggested to us that cell-specific factors such potentially of physiological significance. It suggests that, as coactivators or corepressors are likely to interact very as with some actions of the GR [35], transactivation of the differently with the respective AF-1 regions of the two MR gene may be mediated via nonclassic albeit genomic receptors [41]. It follows that as for the GR [38], there mechanisms, and furthermore, as for classic GRE trans- is no reason a priori why MR-specific transactivation might activation, there may be an overlap of MR- and GR- not occur. We reasoned that not only might cell-specific mediated effects. Although the studies found similar re- profiles of transcription factors/coactivators be important sults in both CV-1 cells and a renal cortical cell line in defining GR versus MR specificity, but that, as with [34], it may be that a hippocampal cell line is needed to the GR and AP-1 interaction, activation of cell signaling explore specificity at this promoter fully. pathways may contribute to MR specificity. Using the Derfoul et al have reported MR-mediated transactiva- LLC-PK1 cells and the MMTV promoter, the relative tion at the human Na,K-ATPase ␤1 gene promoter [36]. transactivation by transfected MR and GR was examined A response element was identified within the promoter in the presence of various activators of cell surface recep- that responds both to activated MR and GR in CV-1 tors and/or signaling pathways [42]. Whereas treatment cells [36]. Although it is clear that this gene is regulated with 8 bromo-cAMP enhanced transcription by both re- under physiological conditions by glucocorticoids with a ceptors, as has been reported elsewhere [43], the addition relatively short time course, this has been demonstrated of the phosphatase inhibitor okadaic acid enhanced GR- not to occur with aldosterone in a mammalian system but not MR-mediated transcription. Dose-response stud- in vivo [37]. Thus, this natural, physiologically relevant ies with okadaic acid suggested that serine/threonine promoter did not exhibit receptor specificity under the protein phosphatase 1 is involved in GR- but not MR- conditions of the transfection. mediated transactivation [42]. For the GR, this is consis- Pearce and Yamamoto demonstrated in vitro that at tent with the effects of 8 bromo-cAMP alone, whereas least one action of the GR, interaction with the AP-1 in the case of the MR, phosphorylation/dephosphoryla- complex at a composite GRE, was GR-specific (Fig. 2) tion of another factor or of the MR itself must be invoked 1260 Fuller et al: Specificity in MR versus GR action

␣2-adrenergic agonist UK14304. Both of these inhibited MR-mediated transactivation but were without signifi- cant effect on the GR, although in both cases, there was a trend toward increased transactivation. The exact mechanism of the verapamil effect is unclear, as both a calcium ionophone and a calmodulin kinase inhibitor were without effect. UK14304 has effects on several pathways. Clonidine was without effect, although, unlike UK14304, it is only a partial ␣2-adrenergic agonist. The precise mechanism of these differential effects remains to be resolved; however, given the evidence suggesting a specific role for the MR in the central regulation of blood pressure, we speculated that the antihypertensive effect of these agonists might at least in part be mediated via inhibition of MR-specific transactivation [42]. The possibility of interactions between members of the gene superfamily of nuclear receptors was also examined [42]. Using the same system but with cotransfection of the thyroid (TR), activated TR was found to inhibit both MR- and GR-mediated transactiva- tion. However, in the absence of thyroid hormone, only MR-mediated transactivation was inhibited, albeit to a lesser extent. The reverse was also observed in that the MR without ligand inhibited TR-mediated transactiva- tion at a thyroid-response element. Whether this repre- sents a direct interaction between the MR and the TR or competition for a limiting cellular factor is not clear. The critical significance of these studies is the clear demonstration that under appropriate conditions both MR- and GR-specific mediation of corticosteroid-induced transactivation can occur, even at a relatively nonspecific promoter. From the studies of Pearce and Yamamoto, receptor-specific interactions with other cellular factors clearly can also be exploited at noncanonical hormone response elements [38]. In the absence of clearly defined aldosterone-responsive genes and/or uniquely aldosterone- responsive promoters, these concepts, however, remain just that. MR versus GR specificity is, in principle, an issue only where the MR and GR are coexpressed. Spe- cific effects must therefore be distinguished using recep- tor-specific ligands. Under physiological conditions, both receptors may be activated which may result in MR:GR Fig. 3. Cellular variations in relative GR and MR responses. The dose– heterodimers [25, 26, 36]. In CV-1 cells, this interaction response curves to cortisol for the GR (᭺) and the MR (᭹) in each of inhibits GR-mediated transactivation [25, 36], but in SK- three cell lines is shown (reprinted with permission from Lim-Tio, Keightley and Fuller, and [40]). N-MC cells using an MMTV promoter, an enhanced transcriptional response was observed [26]. Such interac- tions may clearly confound analysis of specificity.

[44]. Other agents were examined in the system including the phorbol ester, phorbol 12-myristate 13-acetate (PMA), SPECIFICITY OF REGULATION OF GENE which was without effect on transcription by either recep- EXPRESSION IN VIVO tor, and sodium nitroprusside, which enhanced GR- but The preceding discussion is clearly hypothetical in the not MR-mediated transactivation. Two agents, however, absence of clearly defined aldosterone-responsive genes. were found to exhibit MR-specific effects, the calcium Identification of genes that are regulated in vivo by aldo- channel blocker verapamil and the clonidine analog and sterone has proven elusive [19, 45]. Fuller et al: Specificity in MR versus GR action 1261

Fig. 4. Time course of the increase in mRNA levels in response to a single parenteral dose of aldosterone (A) or dexamethasone (B). The results of studies of the expression of the CHIF [60], sgk (Brennan and Fuller, unpub- lished data), and ENaC subunit (Fuller et al, unpublished data) genes have been plotted relative to control levels. The results are de- rived from Northern blot analysis of total co- lonic RNA taken at 0, 0.5, 1, 2, and 4 hours after a single injection of steroid (aldosterone 50 ␮g/kg, intraperitoneally, or dexamethasone 0.5 ng/kg, intramuscularly). The CHIF (᭜), sgk (ᮀ), ␣ENaC (᭿), ␤ENaC (᭹), and ␥ENaC (᭡) mRNA levels (as a ratio of GAPDH mRNA) are expressed as the mean Ϯ SEM.

Essential to the characterization of MR versus GR this central role of the ENaC in the mineralocorticoid specificity is the identification of genes that are regulated response, it follows that aldosterone may induce expres- by corticosteroids as part of the physiological mineralo- sion of the subunit genes or that modify some corticoid response. Such genes need to be primary re- property of existing channels to increase their open prob- sponse genes [23]. In addition, it follows that the re- ability [45, 47–49]. A number of studies have shown that sponse should occur in an acute time frame. Verrey has the ENaC subunit genes are regulated in a differential very clearly articulated the temporal basis of the - and tissue-specific manner [16–18]. Most of these studies ocorticoid response [19]. The earliest physiological re- have examined the response to either aldosterone or sponse is seen with a latency of 30 to 60 minutes during dexamethasone over several hours or days. In the rat which primary response genes are being induced. distal colon, these studies have consistently demonstrated We have sought to identify genes that are regulated elevated ␤ENaC and ␥ENaC but not ␣ENaC mRNA acutely by aldosterone in vivo in the distal colon. Our levels in response to corticosteroids or an elevation of attempts to identify such genes using differential hybrid- endogenous aldosterone levels by salt restriction [16–18]. ization, subtractive hybridization, and differential dis- These studies have not, however, defined the time course play have been unrewarding. In part, this may reflect of the response. We have examined the acute response the fact that an extremely small number of the total of the ENaC subunit genes in the rat distal colon to a genes expressed in distal colon are regulated, that the single parenteral dose of aldosterone, dexamethasone, degree of regulation is probably relatively modest 60 or the GR-specific ligand RU28362 (Fuller, Brennan, minutes after a single dose of aldosterone, and that the and Burgess, manuscript submitted). ␣ENaC mRNA use of an in vivo system still presents substantial difficul- levels were unaltered by either aldosterone or dexameth- ties in interpretation when compared with in vitro sys- asone treatment (Fig. 4). As in the previous longer term tems. In the last few years, several candidate genes have studies, both the ␤ENaC and ␥ENaC subunit genes re- emerged from studies by other groups, and we have sponded to aldosterone with a significant increase being characterized the acute responses of four of these genes seen by 30 minutes (Fig. 4A). Both subunits also re- to corticosteroids in the rat distal colon (Fig. 4). sponded to dexamethasone with the response being sig- The central role of the apical sodium channel in aldo- nificant at 60 minutes (Fig. 4B). RU28362 also induced sterone-mediated epithelial sodium transport is now both subunits, indicating that activation of the GR as clearly established through both electrophysiological and well as the MR can induce this response. When the molecular genetic studies [45–47]. The amiloride-sensi- baseline levels of ␤ENaC and ␥ENaC mRNA in the tive epithelial sodium channel subunit genes (␣, ␤, and distal colon of untreated adrenalectomized rats were ␥ENaC) were cloned from the rat distal colon [48, 49]. compared with the levels in intact rats, no significant The three homologous subunits are arranged in a hetero- difference was observed. It remains to be shown that tetrameric complex of two ␣ and one each of the ␤ and the observed responses are indeed primary responses; ␥ subunits [50]. Coexpression in Xenopus oocytes of however, they certainly occur within the appropriate all three subunits is required for maximal electrogenic time frame. There does not appear to be a clear differ- sodium flux [49], which is reflected in coexpression of ence between MR and GR activation, aside from a the three subunits in a variety of tissues [51]. Given slightly delayed response to dexamethasone relative to 1262 Fuller et al: Specificity in MR versus GR action aldosterone. This may reflect different modes of adminis- tration (intraperitoneally vs. intramuscularly) and/or the differing pharmakokinetics of the two steroids. A func- tional GRE has been reported in the human ␣ENaC promoter [52], which may mediate the response of this subunit to glucocorticoid in the lung and possibly in segments of the renal tubule. These same authors were, however, unable to identify a functional/structural ste- roid response element in either 450 or 800 bases of the 5Ј flanking regions of the hcENaC and rcENaC genes, respectively [53, 54]. Two other genes, recently identified by differential cloning strategies, are also regulated acutely by cortico- steroids in the distal colon. Chen et al applied a sup- pressive subtractive hybridization strategy to identify acutely regulated genes in amphibian A6 cells after a corticosteroid stimulus [55]. The cells were treated with dexamethasone or vehicle for one hour prior to RNA Fig. 5. Effect of cycloheximide on sgk mRNA expression in response to aldosterone. The results are derived from Northern blot analysis of extraction. One of the genes identified by subtraction of total colonic RNA taken at 30 (ᮀ) and 60 ( ) minutes after a single the RNA was found to be the Xenopus homologue of injection of aldosterone (Aldo). Cycloheximide (Cyclo) was adminis- the serum and glucocorticoid-regulated kinase (sgk), a tered 30 minutes prior to the steroid. mRNA levels are given relative to control levels, as a ratio of GAPDH mRNA, and are expressed as serine-threonine kinase identified previously by Webster mean Ϯ SEM (N ϭ 4). *P Ͻ 0.05. et al [56]. Chen et al provided convincing evidence that this is indeed a primary response gene in A6 cells [55], and Na´ray-Fejes-To´ th et al have reported similar find- ings in isolated cortical collecting duct cells [57]. Both previously in this article, this gene exhibited an acute groups provided evidence that sgk may directly regulate response to a single parenteral dose of either MR or GR ENaC activity. We have extended these studies to char- agonist in the distal colon (Fig. 4). The response was acterize the response in vivo. In this case, we saw a significant at 60 minutes for aldosterone and 2 hours response to adrenalectomy in both rat kidney and distal for dexamethasone, and induction was also seen with colon. Sgk mRNA levels responded to both aldosterone RU28362 [60]. The response to aldosterone was also not and dexamethasone (Fig. 4), with a significant increase inhibited by cycloheximide pretreatment of the rats [60]. seen by 30 minutes in the colon and slower increases in In distal colon after steroid treatment, Northern blot the kidney (Brennan and Fuller, manuscript submitted). analysis revealed multiple higher transcripts that hybrid- In A6 cells, the response was superinduced by cyclohexi- ized with an intron probe [61]. The increase in primary mide, suggesting that this is indeed a primary response and/or partially processed CHIF transcript levels after gene. We also observed an enhanced response to aldoste- steroid treatment argues in favor of a transcriptional rone in the colon when rats were pretreated with cyclo- response [61]. We also found that CHIF mRNA levels heximide (Fig. 5). sgk mRNA levels were also increased were decreased in the colon (but not the kidney) by by treatment of intact but not adrenalec- adrenalectomy and that the treatment of intact rats with tomized rats (Brennan and Fuller, manuscript submit- carbenoxolone sodium, an inhibitor of 11␤-HSD2, in- ted). sgk was also induced by dexamethasone in a range creased CHIF levels [60]. This latter response implies of other tissues in which the GR, but not the MR, is that corticosteroid-induced CHIF and also sgk expres- expressed. sion occurs in colonic epithelial cells that express 11␤- The fourth gene is the channel-inducing factor (CHIF) HSD2. In addition, this observation, coupled with the gene, which was identified by Attali et al [58]. This gene response to adrenalectomy, suggests that modulation of was differentially cloned from colonic RNA from rats CHIF mRNA levels in the distal colon occurs at circulat- treated with high doses of dexamethasone for two days. ing levels of corticosteroid. Although the exact function of this gene is not certain, Another gene putatively involved in colonic potassium it appears to be a transmembrane regulator or compo- transport, the ␣-subunit of the colonic Hϩ-KϩATPase, nent of potassium channels and/or pumps, and thus, its is regulated by two days of corticosteroid treatment [62]. role may be in the corticosteroid regulation of potassium However, using the experimental protocol described ear- transport independent of effects on sodium transport lier in this article, we have not observed acute regulation [59]. CHIF is expressed in great abundance in distal of this gene by either aldosterone or dexamethasone colon and kidney [59]. As with the other genes described (Verity and Fuller, unpublished observations). Fuller et al: Specificity in MR versus GR action 1263

CONCLUSIONS ACKNOWLEDGMENTS All four of the genes that we have shown to be acutely This work was supported by the National Health and Medical Re- regulated by aldosterone were also regulated through search Council of Australia through project grants (32994 and 35068), a Medical Postgraduate Scholarship to SSL-T, and a Principal Research GR-mediated pathways (Fig. 4), arguing strongly for a Fellowship to P.J.F. The authors wish to thank Mrs. Claudette Thiede- lack of specificity. However, it should be noted that un- man and Ms. Sue Panckridge for preparation of the manuscript. der the same conditions, the ␣ and ␤ subunits of Na,K- Reprint requests to Dr. Peter J. Fuller, Prince Henry’s Institute of ATPase were regulated by dexamethasone but not aldo- Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. sterone, indicating a GR-specific mechanism of activation E-mail: [email protected] [37]. The lack of obvious MR-mediated specific transacti- vation may reflect several factors. None of these genes REFERENCES were initially selected on the basis of a MR-specific 1. Evans RM: The steroid and superfamily. screen for induced genes, although the cloning of sgk by Science 240:889–895, 1988 2. Fuller PJ: The steroid receptor superfamily: Mechanisms of diver- Na´ray-Fejes-To´ th et al did use aldosterone treatment of sity. FASEB J 5:3092–3099, 1991 isolated cells from cortical collecting tubules [57]. Al- 3. Marver D: Evidence of corticosteroid action along the nephron. Am J Physiol 246:F111–F123, 1984 though most studies used a GR-specific agonist instead 4. Bastl CP, Hayslett JP: The cellular action of aldosterone in target of, or in addition to, dexamethasone, they also used epithelia. Kidney Int 42:250–264, 1992 aldosterone, which at high concentrations will cause 5. Turnamian SG, Binder HJ: Regulation of active sodium and potas- sium transport in the distal colon of the rat. J Clin Invest 84:1924– “cross-over” activation of the GR [12, 30, 31, 39]. In some 1929, 1989 cases, the aldosterone was administered in combination 6. Na´ ray-Fejes-To´ th A, Fejes-To´ th G: Glucocorticoid receptors me- diate mineralocorticoid-like effects in cultured collecting duct cells. with RU486 to prevent occupancy of the GR; however, Am J Physiol 259:F672–F678, 1990 under various circumstances, RU486 has been shown to 7. Funder JW, Pearce PT, Myles K, Roy LP: Apparent mineralo- clearly exhibit partial agonist activity [43], which may corticoid excess, pseudohypoaldosteronism, and urinary electro- lyte excretion: Toward a redefinition of mineralocorticoid action. confound the interpretation. FASEB J 4:3234–3238, 1990 The fact that only two of the genes unambiguously 8. Funder JW: Mineralocorticoids, glucocorticoids, receptors and re- respond to adrenalectomy is interesting in that at physio- sponse elements. Science 259:1132–1133, 1993 9. Grotjohann I, Schulzke J-D, Fromm M: Electrogenic Naϩ trans- logical concentrations, cortisol/corticosterone-mediated port in rat late distal colon by natural and synthetic glucocorticoste- actions of the GR are very modest, whereas occupancy roids. Am J Physiol 276:G491–G498, 1999 10. De Kloet ER, Vreugdenhil E, Oitzl MS, Joe¨ls M: Brain cortico- of the MR may be relatively more significant. In this steroid receptor balance in health and disease. Endocr Rev 19:269– context, the carbenoxolone studies are interesting in that 301, 1998 they expose the MR (and the GR [37]) to circulating 11. Arriza JL, Weinberger C, Cerelli G, Glaser TM, Handelin BL, Housman DE, Evans RM: Cloning of human mineralocorticoid levels of corticosterone. Subtle differences in the speed receptor complementary DNA: Structural and functional kinship of the response to aldosterone versus dexamethasone for with the glucocorticoid receptor. Science 237:268–275, 1987 each of the genes (Fig. 4) may reflect subtle differences in 12. Arriza JL: Aldosterone action: Perspectives from the cloning of the mineralocorticoid receptor, in Aldosterone: Fundamental As- the activation pathways involved. For CHIF expression pects, edited by Bonvalet JP, Farman N, Lombe`sM,Paris, Col- in the colon, where regulation appears to be more sensi- loque INSERM/John Libbey Eurotext, 1991, pp 13–21 13. White PC, Mune T, Agarwal AK: 11␤-Hydroxysteroid dehydro- tive, perhaps the ratio of MR to GR activation may be genase and the syndrome of apparent mineralocorticoid excess. a critical determinant of the specificity. Endocr Rev 18:135–156, 1997 Several studies, including our own, provide the basis 14. Krozowski Z: The 11␤-hydroxysteroid dehydrogenases: Functions and physiological effects. Mol Cell Endocrinol 151:121–127, 1999 for very plausible models in which MR versus GR speci- 15. Leaf A: From toad bladder to kidney. Am J Physiol 242:F103– ficity is observed, particularly through the AF-1 regions F111, 1982 where the amino acid sequence unambiguously differs 16. Escoubet B, Coureau C, Bonvalet J-P, Farman N: Noncoordi- nate regulation of epithelial Na channel and Na pump subunit between the two receptors. To apply these concepts, mRNAs in kidney and colon by aldosterone. Am J Physiol it is clearly necessary to identify uniquely aldosterone- 272:C1482–C1491, 1997 17. Renard S, Voilley N, Bassilana F, Lazdunski M, Barbry P: regulated genes (assuming these exist). Our focus has been Localization and regulation by steroids of the ␣, ␤ and ␥ subunits on the distal colon; it may be that this degree of specificity of the amiloride-sensitive Naϩ channel in colon, lung and kidney. is only observed in segments of the nephron in special- Pflu¨gers Arch 430:299–307, 1995 18. Stokes JB, Sigmund RD: Regulation of rENaC mRNA by dietary ized cells. Alternatively, it may be that in epithelial tissue, NaCl and steroids: Organ, tissue, and steroid heterogeneity. Am such genes do not exist and that, as has been suggested J Physiol 274:C1699–C1707, 1998 by others [8, 10], specificity only occurs in the central 19. Verrey F: Early aldosterone effects. Exp Nephrol 6:294–301, 1998 20. Berger S, Bleich M, Schmid W, Cole TJ, Peters J, Watanabe nervous system, where prereceptor specificity is not op- H, Kriz W, Warth R, Greger R, Schutz G: Mineralocorticoid erative. Before this can be concluded, a more rigorous receptor knockout mice: Pathophysiology of Naϩ . Proc Natl Acad Sci USA 95:9424–9429, 1998 screening approach that takes account of the above 21. Cole TJ, Blendy JA, Monaghan AP, Krieglstein K, Schmid W, points may be required. Aguzzi A, Fantuzzi G, Hummler E, Unsicker K, Schutz G: 1264 Fuller et al: Specificity in MR versus GR action

Targeted disruption of the glucocorticoid receptor gene blocks lators: Cellular and molecular biology. Endocrine Rev 20:321–344, adrenergic chromaffin cell development and severely retards lung 1999 maturation. Genes Dev 9:1608–1621, 1995 42. Lim-Tio SS, Fuller PJ: Intracellular signaling pathways confer 22. Lomax RB, Sandle GI: Comparison of aldosterone- and RU- specificity of transactivation by mineralocorticoid and glucocorti- 28362-induced apical Naϩ and Kϩ conductances in rat distal colon. coid receptors. Endocrinology 139:1653–1661, 1998 Am J Physiol 267:G485–G493, 1994 43. Keightley M-C, Fuller PJ: Cortisol resistance and the guinea- 23. Dean DM, Saunders MM: Ten years after: Teclassification of pig glucocorticoid receptor. Steroids 60:87–92, 1995 steroid-responsive genes. Mol Endocrinol 10:1489–1495, 1996 44. Galigniana MD: Native rat kidney mineralocorticoid receptor is 24. Fromm M, Schulzke JD, Hegel U: Control of electrogenic Naϩ a phosphoprotein whose transformation to a DNA-binding form absorption in rat late distal colon by nanomolar aldosterone added is induced by phosphatases. Biochem J 333:555–563, 1998 in vitro. Am J Physiol 264:E68–E73, 1993 45. Fuller PJ: Aldosterone effects and mechanism of action. Curr 25. Liu W, Wang J, Sauter NK, Pearce D: Steroid receptor hetero- Opin Endocrinol Diabetes 4:1383–1386, 1997 dimerization demonstrated in vitro and in vivo. Proc Natl Acad 46. Warnock DG: Liddle syndrome: An autosomal dominant form Sci USA 92:12480–12484, 1995 of human . Kidney Int 53:18–24, 1998 26. Trapp T, Rupprecht R, Castren M, Reul JMHM, Holsboer F: 47. Garty H, Palmer LG: Epithelial sodium channels: Function, struc- Heterodimerization between mineralocorticoid and glucocorticoid ture and regulation. Physiol Rev 77:359–396, 1997 receptor: A new principle of glucocorticoid action in the CNS. 48. Barbry P, Hofman P: Molecular biology of Naϩ absorption. Am Neuron 13:1457–1462, 1994 J Physiol 273:G571–G585, 1997 27. Bleich M, Warth R, Schmidt-Hieber M, Schulz-Baldes A, Has- 49. Gru¨ nder S, Rossier BC: A reappraisal of aldosterone effects on selblatt P, Fisch D, Berger S, Kunzelmann K, Kriz W, Schutz the kidney: New insights provided by epithelial sodium channel G, Greger R: Rescue of the mineralocorticoid receptor knock- cloning. Curr Opin Nephrol Hypertens 6:35–39, 1997 out mouse. Pflu¨gers Arch 438:245–254, 1999 50. Firsov D, Gautschi I, Merillat A-M, Rossier BC, Schild L: 28. Husted RF, Laplace JR, Stokes JB: Enhancement of electrogenic The heterotetrameric architecture of the epithelial sodium channel ϩ Na transport across rat inner medullary collecting duct by gluco- (ENaC). EMBO J 17:344–352, 1998 corticoid and by mineralocorticoid hormones. J Clin Invest 86:497– 51. Duc C, Farman N, Canessa CM, Bonvalet J-P, Rossier BC: Cell- 506, 1990 specific expression of epithelial sodium channel ␣, ␤ and ␥ subunits 29. Laplace JR, Husted RF, Stokes JB: Cellular responses to steroids ϩ in aldosterone-responsive epithelia from the rat: Localization by in the enhancement of Na transport by rat collecting duct cells in situ hybridization and immunocytochemistry. J Cell Biol 127: in culture. J Clin Invest 90:1370–1378, 1992 1907–1921, 1994 30. Schmidt TJ, Husted RF, Stokes JB: stimulation 52. Sayegh R, Auerbach SD, Li X, Loftus RW, Husted RF, Stokes of Naϩ transport in A6 cells is mediated via glucocorticoid recep- JB, Thomas CP: Glucocorticoid induction of epithelial sodium tors. Am J Physiol 264:C875–C884, 1993 channel expression in lung and renal epithelia occurs via trans- 31. Chen SY, Wang J, Liu W, Pearce D: Aldosterone responsiveness activation of a hormone response element in the 5Ј-flanking region of A6 cells is restored by cloned rat mineralocorticoid receptor. of the human epithelial sodium channel ␣ subunit gene. J Biol Am J Physiol 274:C39–C46, 1998 Chem 274:12431–12437, 1999 32. Nakhoul NL, Hering-Smith KS, Gambala CT, Hamm LL: Regula- 53. Thomas CP, Doggett NA, Fisher R, Stokes JB: Genomic organi- tion of sodium transport in M-1 cells. Am J Physiol 275:F998– Ј ␥ F1007, 1998 zation and the 5 flanking region of the -subunit of the human 33. Bens M, Vallet V, Cluzeaud F, Pascual-Letallec L, Kahn A, amiloride-sensitive epithelial sodium channel. J Biol Chem 271: Rafestin-Oblin ME, Rossier BC, Vandewalle A: Corticoste- 26062–26066, 1996 roid-dependent sodium transport in a novel immortalized mouse 54. Thomas CP, Auerbach SD, Zhang C, Stokes JB: The structure collecting duct principal cell line. J Am Soc Nephrol 10:923–934, of the rat amiloride-sensitive epithelial sodium channel gamma 1999 subunit gene and functional analysis of its promoter. Gene 228:111– 34. Zennaro M-C, Le Menuet D, Lombe`sM:Characterization of 122, 1999 the human mineralocorticoid receptor gene 5Ј-regulatory region: 55. Chen S-Y, Bhargava A, Mastroberardino L, Meijer OC, Wang Evidence for differential hormonal regulation of two alternative J, Buse P, Firestone GL, Verrey F, Pearce D: Epithelial sodium promoters via nonclassical mechanisms. Mol Endocrinol 10:1549– channel regulated by aldosterone-induced protein sgk. Proc Natl 1560, 1996 Acad Sci USA 96:2514–2519, 1999 35. Reichardt HM, Kaestner KH, Tuckermann J, Kretz O, Wessely 56. Webster MK, Goya L, Ge Y, Maiyar AC, Firestone GL: Charac- O, Bock R, Gass P, Schmid W, Herrlich P, Angel P, Schutz terization of sgk, a novel member of the serine/threonine protein G: DNA binding of the glucocorticoid receptor is not essential for kinase gene family which is transcriptionally induced by glucocorti- survival. Cell 93:531–542, 1998 coids and serum. Mol Cell Biol 13:2031–2040, 1993 36. Derfoul A, Robertson NM, Lingrel JB, Hall DJ, Litwack G: 57. Na´ ray-Fejes-To´ th A, Canessa C, Cleaveland ES, Aldrich G, Regulation of the human Na/K-ATPase ␤1 gene promoter by miner- Fejes-To´ th G: sgk is an aldosterone-induced kinase in the renal alocorticoid and glucocorticoid receptors. J Biol Chem 273:20702– collecting duct. J Biol Chem 274:16973–16978, 1999 20711, 1998 58. Attali B, Latter H, Rachamim N, Garty H: A corticosteroid- 37. Fuller PJ, Verity K: Colonic Na,K-ATPase subunit gene expres- induced gene expressing an “Isk-like” Kϩ channel activity in Xeno- sion: Ontogeny and regulation by adrenocortical steroids. Endocri- pus oocytes. Proc Natl Acad Sci USA 92:6092–6096, 1995 nology 127:32–38, 1990 59. Young DB: Quantitative analysis of aldosterone’s role in potas- 38. Pearce D, Yamamoto KR: Mineralocorticoid and glucocorticoid sium regulation. Am J Physiol 255:F811–F822, 1998 receptor activities distinguished by nonreceptor factors at a com- 60. Brennan FE, Fuller PJ: Acute regulation by corticosteroids of posite response element. Science 259:1161–1165, 1993 channel-inducing factor gene messenger ribonucleic acid in the 39. Rupprecht R, Arriza JL, Spengler D, Reul JMHM, Evans RM, distal colon. Endocrinology 140:1213–1218, 1999 Holsboer F, Damm KZ: Transactivation and synergistic properties 61. Brennan FE, Fuller PJ: Transcriptional control by corticosteroids of the mineralocorticoid receptor: Relationship to the glucocorti- of CHIF gene expression in the rat distal colon. Clin Exp Pharma- coid receptor. Mol Endocrinol 7:597–603, 1993 col Physiol 26:489–491, 1999 40. Lim-Tio SS, Keightley M-C, Fuller PJ: Determinants of specific- 62. Jaisser F, Escoubet B, Coutry N, Eugene E, Bonvalet J-P, ity of transactivation by the mineralocorticoid or glucocorticoid Farman N: Differential regulation of putative Kϩ-ATPase by low-Kϩ receptor. Endocrinology 138:2537–2543, 1997 diet and corticosteroids in rat distal colon and kidney. Am J Physiol 41. McKenna NJ, Lanz RB, O’Malley BW: coregu- 270:C679–C687, 1996

Top View