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1Department of Internal Medicine and the 2Graduate Program in Molecular Biology, University of Iowa College of Medicine
the 3Veterans Affairs Medical Center, Iowa City, Iowa
ABSTRACT
Medroxyprogesterone acetate (MPA), a widely used synthetic progestational contraceptive, occasionally leads to Cushingoid side effects such as hypertension, fluid retention, and centripetal obesity. We investigated the effect of MPA on classic mineralocorticoid target genes, -epithelial Na channel (ENaC) and sgk1, in the collecting duct. In adrenalectomized mice, aldosterone, dexamethasone, and MPA increased -ENaC mRNA levels in kidney cortex. MPA and dexamethasone, but not progesterone, dose dependently increased -ENaC and sgk1 mRNA in M-1 and in Madin-Darby canine kidney-C7 cells, both collecting duct cell lines. The stimulatory effect of MPA and dexamethasone on -ENaC expression was inhibited by RU-38486, a combined glucocorticoid receptor (GR) and progesterone receptor (PR) antagonist, but not by Org31710, a pure PR antagonist. MPA and dexamethasone dose dependently increased -ENaC promoter-driven luciferase activity in M-1 cells, which was not inhibited by Org31710, indicating that MPA regulates -ENaC in a PR-independent manner. When tested in HT29 cells, MPA could only stimulate -ENaC-driven reporter activity when GR was coexpressed, confirming the requirement for functional GR in the transcriptional effect of MPA. The activation of steroid receptors such as GR can explain the apparent glucocorticoid effects of MPA, independent of PR activation.
epithelial sodium channel; Na+ transport; aldosterone; glucocorticoid response element
MEDROXYPROGESTERONE ACETATE (MPA) is a potent synthetic progestin that has been in widespread use as an injectable long-active contraceptive (Depo-Provera) and is also used to treat endometriosis, as well as endometrial and breast cancer. MPA, like some of the other newly available progestins, may not only activate the progesterone receptor (PR) but in some cases may have stimulatory or inhibitory glucocorticoid, androgenic, or mineralocorticoid effects in vitro (27). Although there is no clear increase in weight, mood changes, hirsutism, or hypertension reported from the initial pivotal studies, occasional reports of Cushing's syndrome and hypertension in patients on MPA suggest that MPA may activate glucocorticoid receptor (GR) or mineralocorticoid receptor (MR) in susceptible individuals in vivo (3, 6, 14, 15, 18, 21, 24, 29, 30).
Aldosterone is one of the principal physiological regulators of epithelial sodium channel (ENaC) function in the connecting tubule (CNT) and the cortical collecting duct (CCD) of the kidney. The enzyme 11-hydroxysteroid dehydrogenase type 2 (11HSD2), which is expressed in the CNT, CCD, and other classic aldosterone-responsive tissues, metabolizes cortisol to the inactive cortisone, allowing aldosterone unrestricted access to its cognate receptor, the MR. When 11HSD2 is inactivated, as in the syndrome of apparent mineralocorticoid excess, or is overwhelmed by an excess of circulating glucocorticoids, as in Cushing's syndrome, cortisol binds to MR to activate a gene profile that results in the stimulation of benzamil-sensitive Na+ transport in the CNT and throughout the collecting duct (4, 20, 34).
Among the targets of aldosterone action in the distal nephron are the -subunit of ENaC and the serum and glucocorticoid-regulated kinase 1 (sgk1). Under Na+-loaded conditions with no circulating aldosterone, there is minimal ENaC activity at the apical membrane of the CNT and CCD and Na+ reabsorption is turned off allowing the excess Na+ to be excreted. In sodium-avid states when there is an increase in aldosterone levels, there is the rapid appearance of the -ENaC subunit and a shift in ENaC subunits from the cytosol to the apical membrane (32, 35). Sgk1 appears to play an important role in the redistribution of ENaC to the cell surface and like -ENaC is transcriptionally regulated by corticosteroids via a glucocorticoid response element (GRE) in the 5'-flanking regulatory region (9, 22).
In this study, we examined the effect of MPA on -ENaC and sgk1 expression in the mouse collecting duct. We demonstrate that MPA stimulates -ENaC in kidney cortex in vivo and in CCD cell lines and confirm that the increase in -ENaC is mediated via the GRE in its 5'-flanking region.
EXPERIMENTAL PROCEDURES
Materials. Dexamethasone, progesterone, and MPA were purchased from Sigma (St. Louis, MO). RU-38486 was a generous gift from Roussel Uclaf (Romainville, France), and Org31710 was a generous gift from Organon (Oss, Netherlands). Culture materials were from Life Technologies (Gaithersburg, MD), and all radionucleotides were from PerkinElmer Life Sciences (Boston, MA). Stock solutions of steroid compounds and receptor antagonists were made in ethanol.
Tissue culture and RNA extraction. The mouse renal CCD cell line M-1, the canine CCD cell line Madin-Darby canine kidney (MDCK)-C7, and the human colonic epithelial cell line HT29 were cultured as previously described (22, 26). To examine the effects of various steroids on gene expression, cell culture media were switched to serum-free media and then exposed to these agents or vehicle for various time periods. RU-38486 and Org31710 were used in some experiments and compared with control cultures in the presence of vehicle alone. Total RNA was prepared from cultured cells using TRI reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions.
Adrenalectomized mice. Adrenalectomized 30- to 32-day-old C57BL6 male mice were obtained from Charles River Laboratories (Wilmington, MA). Mice were provided 0.9% saline solution rather than drinking water and maintained on normal rat chow. Mice were injected intraperitoneally with aldosterone (1.5 mg/kg), dexamethasone (1 mg/kg), medroxyprogesterone (1 mg/kg), or vehicle (ethanol) 12 h apart for a total of three doses and then killed 2 h after the last injection. Total RNA was prepared from kidney cortex and medulla after they were homogenized in TRI reagent (Molecular Research Center). The research using animals was approved by the University of Iowa Institutional Animal Care and Use Committee.
Ribonuclease protection assay. A mouse sgk1 cDNA fragment was amplified by RT-PCR from mouse kidney using primers 5'-TGATCCCGAGTTTACCGAGG and 5'-TCAGAGGAAGGAATCCACAG. Mouse and canine -ENaC cDNAs and canine sgk1 cDNAs were cloned previously and have been described (22, 26). These cDNA products in pCRXl-topo were linearized and then used to synthesize radiolabeled antisense cRNA probes. RNA samples were cohybridized in solution with 18S rRNA (Ambion, Austin, TX) as a control for global changes in transcription and for RNA loading. Ribonuclease digestion and evaluation of protected fragments by PAGE were performed as previously described (26).
Transfection and functional analysis of 5'-flanking -ENaC DNA. Subconfluent M-1 and HT29 cells grown in 24-well plates were used for transfection assays using Lipofectamine Plus and Lipofectamine 2000, respectively (Invitrogen, Carlsbad, CA), as previously described (22, 26). The -ENaC promoter reporter plasmids contain the 5'-flanking region of the h-ENaC gene (1388 + 55 or 487+ 55), including the functional GRE cloned upstream of the firefly luciferase gene in the plasmid pGL3basic (Promega) and has also been previously described (22, 26). The plasmid -ENaC-mutGRE is a luciferase plasmid-containing sequence from 481 to +55 of the 5'-flanking region of -ENAC with the GRE mutated as has been previously described (8). The luciferase reporter plasmid TAT3-luc contains three tandem copies of the GRE of the rat tyrosine amino transferase gene and PRE-TATA-Luc contains two copies of the distal GRE of the MMTV promoter placed upstream of a TATA-driven firefly luciferase construct (16, 23). One microgram of the luicferase reporter constructs or the parent plasmid pGL3basic and 0.51 μg of a control plasmid, pRL-SV40 (Promega), where the Renilla luciferase gene is cloned downstream of the SV40 promoter, were cotransfected into each well. In some experiments, 0.5 μg of an expression vector for the glucocorticoid receptor, hGR, or progesterone receptor, PR-B (gift from S. Oate), or the empty plasmids, pCDNA3 (Invitrogen), or p-Len (gift from S. Oate) was cotransfected along with luciferase plasmids. The following day, dexamethasone, progesterone, or MPA was added to these wells and 24 h later cell lysates were obtained and dual luciferase activities were measured as previously described (26).
RESULTS
In the CNT and CCD of the kidney, aldosterone and dexamethasone increase the transcription of at least two genes, -ENaC and sgk1, that are thought to be important for the early and sustained stimulation of benzamil-sensitive Na+ transport (32, 35). We asked whether MPA stimulates -ENaC and sgk1 expression in kidneys of adrenalectomized mice. The effect of MPA was compared with that of aldosterone and dexamethasone each given as three doses over 36 h. MPA, like aldosterone and dexamethasone, significantly increased -ENaC expression in kidney cortex of adrenalectomized mice (Fig. 1, A and B). In kidney medulla, neither dexamethasone nor MPA had a statistically significant effect on -ENaC expression, although there was a clear trend toward an increase in -ENaC expression. In contrast to the effect on -ENaC, we were unable to see a significant effect of MPA on sgk1 in kidney cortex or medulla (data not shown).
To begin to examine the effect of MPA on -ENaC and sgk1 expression, we used two CCD cell lines, M-1 and MDCK-C7, where ENaC and sgk1 are expressed and where there is regulated benzamil-sensitive Na+ transport (22, 26). Dexamethasone and MPA, but not progesterone, were shown to increase -ENaC and sgk1 expression in M-1 cells (Fig. 2, A, B, C). There was no additive effect of MPA with dexamethasone on either gene suggesting that they may increase gene expression via a common pathway. Similar results were seen in MDCK-C7 cells where aldosterone and MPA increased -ENaC expression with no additive effect when both were combined (Fig. 2D). We then tested the effect of MPA on benzamil-sensitive Na+ transport in M-1 cells. Unlike dexamethasone which robustly stimulated Na+ transport, MPA had no effect on Na+ transport, even at 100 nM (data not shown).
A dose response for MPA, progesterone, and dexamethasone was then performed in M-1 cells. MPA and dexamethasone dose dependently increased -ENaC expression with the earliest effect seen at 10 nM for MPA and at 1 nM for dexamethasone (Fig. 3, A and B). There was no response to progesterone at all doses tested. To determine whether the effect of MPA on -ENaC gene expression was transcriptional, we compared the effect of each steroid on an -ENaC promoter reporter construct. This construct includes about 1,500 nt of the 5'-flanking region of -ENaC ligated upstream of the firefly luciferase reporter. MPA and to a smaller extent progesterone, at a dose of 1 μM, increased luciferase expression from the -ENaC promoter (Fig. 3C). This result suggested that the increase in steady-state expression of -ENaC mRNA was due to an increase in transcription of -ENaC. The stimulatory effect of MPA was also seen with TAT3-luc, where a trimerized GRE is coupled to a minimal promoter upstream of the luciferase coding region.
To examine the effect of MPA on -ENaC gene transcription in more detail, the dose-response characteristics on the -ENaC promoter were examined. As seen with -ENaC mRNA studies, dexamethasone robustly increased -ENaC promoter activity with the effect beginning after 1 nM (Fig. 3D). MPA increased -ENaC promoter activity beginning after 10 nM, while progesterone had a small stimulatory effect that was only seen at 1 μM, the highest dose used.
The experiments in Fig. 3, B and C, suggested that MPA may be mediating its effect via the GR. To evaluate this possibility, we tested the effect of MPA on -ENaC promoter-reporter activity in HT29 cells, a GR negative cell line (22). In these cells, MPA had no effect on -ENaC promoter activity unless GR was cotransfected in (Fig. 4A). These results indicate that GR is sufficient for the MPA effect and is consistent with activation of the GRE in the -ENaC promoter.
We then tested M-1 cells to see whether the effect of MPA on -ENaC expression was secondary to signaling via GR or PR. We reasoned that it was unlikely to be due to PR as progesterone had little or no effect on endogenous -ENaC or sgk1 gene expression and on -ENaC promoter activity (Figs. 2 and 3). To exclude the possibility that MPA was acting via PR, in M-1 cells, we tested the effect of MPA on PRE-TATA-luc, a reporter vector containing a weak hormone response element coupled to a minimal promoter. Neither progesterone nor MPA in two doses was able to stimulate reporter activity unless the PR type B receptor was cotransfected in (Fig. 4B). This is in contrast to the -ENaC promoter which is clearly stimulated by MPA in the absence of cotransfected PR (see Fig. 3, C and D). These results indicate that there is little if any functional PR present normally in M-1 cells and that the effect of MPA is thus likely to be independent of PR.
To further confirm that the effect of MPA on -ENaC mRNA was not via PR, we tested the effect of RU-38486, a widely used steroid receptor antagonist, and Org31710, a selective PR blocker, on -ENaC gene expression (12). RU-38486, but not Org31701, inhibited MPA-induced -ENaC expression (Fig. 5, A and B). RU-38486 is a potent PR blocker, but can also inhibit GR-dependent molecular events. The results are thus consistent with MPA acting via GR in M-1 cells. To confirm that Org31710 does not inhibit GR, we tested Org31710 on dexamethasone-stimulated -ENaC expression and compared it with RU-486. In contrast to 10 nM RU-38486, 10 nM Org31710 had little effect on -ENaC expression (Fig. 5, C and D).
We then tested the effect of Org31710 on -ENaC promoter constructs in transient transfection assays (Fig. 6A). Org31710 had no effect on MPA and dexamethasone-stimulated -ENaC promoter activity. These results are consistent with the idea that MPA and dexamethasone activate the -ENaC promoter via GR and not via PR. To demonstrate that Org31710, in the doses used, inhibits PR function, we tested the effect of Org31710 on PR-dependent trans-activation of PRE-TATA-luc by MPA (Fig. 6B). The data demonstrate that Org31710 at two concentrations inhibits MPA-stimulated PR activation. Finally, to determine whether the stimulation of -ENaC was mediated via the GRE in its 5'-flanking region, we tested the effect of MPA on the 485 +55 -ENaC promoter reporter construct where the GRE had been mutated. MPA and dexamethasone were unable to stimulate the -ENaC-mutGRE construct, indicating that the GRE is necessary for the effect of MPA on ENaC expression (Fig. 6C).
DISCUSSION
The natural progestin, progesterone, binds its cognate receptor in classic target tissues such as the uterine endometrium, where it is required for the normal menstrual cycle and for maintenance of pregnancy (27). Progesterone and its derivatives are used to treat amenorrhea, dysfunctional uterine bleeding, and infertility and are components of hormone replacement therapy. MPA is a synthetic 17-hydroxyprogesterone derivative that is a potent PR ligand that has been in use for over 40 years (37). MPA in a parenteral long-acting form is a popular long-term contraceptive because of its ability to inhibit ovulation and create a hostile environment for fertilization and implantation. Many synthetic progestins such as MPA may have pro- and antiglucocorticoid, promineralocorticoid, or androgenic effects because of crossover binding to these steroid receptors (27). Recently, the use of hormone replacement therapy with an estrogen-MPA combination pill in otherwise healthy postmenopausal women has been called into question because of unexpected adverse coronary events and breast cancer development (2, 19, 36). These side effects were not seen in women taking estrogen alone underscoring the notion that some progestins may cause unwanted side effects perhaps by activation of nonclassical pathways.
Although there is no evidence of widespread clinically significant MPA-mediated glucocorticoid or mineralocorticoid effects, there have been anecdotal reports of Cushing's syndrome and of a possible increase in the risk for diabetes, weight gain, and bone loss; side effects that are classic for the corticosteroids (3, 6, 14, 21, 30). Our studies on corticosteroid target genes in the CCD were prompted by the dramatic appearance of severe hypertension with weight gain in a young woman that coincided with the use of parenteral long-acting MPA and appeared to resolve completely within months of cessation of therapy. Our study demonstrates that MPA stimulates the expression of -ENaC transcripts in vivo and -ENaC and sgk1 in CCD cell lines. The peak plasma concentration of MPA following a standard parenteral dose of DepoProvera is 1 to 7 ng/ml (2.5 to 18 nM) (17). The circulating peak concentrations of MPA in vivo are clearly sufficient to increase -ENaC expression in culture (see Fig. 3A).
The effect of MPA on -ENaC expression was examined in some detail, and we confirmed that MPA, like the glucocorticoid, dexamethasone, increases the transcription of -ENaC by trans-activating an imperfect GRE in its 5'-flanking region. This increase in transcription accounts for the increase in -ENaC expression. It is interesting to note however that there is a discordance between the dose response for dexamethasone on -ENaC promoter-luciferase expression (Fig. 3D) with the dose response for endogenous -ENaC expression (Fig. 3, A and B). This difference may be primarily because the effect of dexamethasone to increase transcription is more marked when mediated through native 5'-flanking regulatory elements in the context of intact chromatin compared with the effect of dexamethasone on naked unwound DNA in plasmid constructs. It is also possible that there are additional cis-elements that regulate endogenous gene expression that are not present in the promoter-luciferase construct. Nevertheless, the increase in transcription is mediated by GR and may explain corticosteroid side effects that have been seen in some patients.
The effect of MPA on GR binding and on GR-dependent gene expression has been previously studied in different models with varying affinity for GR reported. For example, the relative binding affinity of MPA and cortisol for GR in human lymphocytes was 42 and 25%, respectively, of that seen with dexamethasone (11, 25, 28). In rat ovarian granulosa cells, the relative binding affinity of MPA was only 10% of dexamethasone (25). In displacement studies using labeled dexamethasone bound to canine liver cytosol, the Ki for MPA was 3.7 nM compared with 1.2 nM for cortisol and 0.8 nM for dexamethasone (28). These studies indicate that MPA, at least in some tissues, is a potent GR ligand.
On binding to GR, similar to the classic glucocorticoids, dexamethasone and cortisol, MPA increases the transcription of some target genes and represses others. Trans-activation has been reported with both native genes and with transfected reporter genes and appears to be mediated via a classical GRE (1, 7). The GR-mediated trans-repression of target genes is less well understood and may involve a negative GRE (nGRE); nevertheless, in some cell culture systems, MPA is as potent as dexamethasone in trans-repression (1, 13). Both trans-activation and trans-repression may be enhanced by increasing GR receptor density and may explain the variable glucocorticoid effects of MPA in different tissues (33, 39).
The early effect of corticosteroids to increase Na+ transport in the CNT and CCD has been thought to require the induction of sgk1 which then increases surface expression and function of Na+ channels, in part, by inactivation of Nedd42 (10, 31, 34). The sustained effect of corticosteroids to increase Na+ transport has been associated with the transcription of new -ENaC subunits in the CNT and CCD, although there has been no evidence that this increase in synthesis is required for the late effects of aldosterone. The role of sgk1 in renal Na+ handling was explored by creating sgk1 knockout mice (38). When sgk1 was ablated in these animals, there was no evidence of hypotension or salt wasting in sgk1/ mice under resting conditions. However, when mice were placed on a Na+-free diet, there was evidence of impaired Na+ conservation indicating that sgk1 is required under conditions where Na+ transport needs to be maximally stimulated.
MPA, like dexamethasone and aldosterone, increases the abundance of -ENaC1 and sgk1 in cultured CCD cell lines. Yet, this increase in gene expression is not sufficient to increase Na+ transport in these cells. There are at least two possible interpretations of these findings. The first is that the level of induction of sgk1 and -ENaC, which is less than that seen with dexamethasone, is too little to affect a downstream increase in Na+ transport. The second is that the increase in sgk1 and -ENaC1 is not sufficient by themselves to increase Na+ transport and that there must be other proteins or signaling pathways activated by dexamethasone and aldosterone that are required for the integrated corticosteroid effect on Na+ transport.
Although there are anecdotal reports of Cushing's syndrome with MPA, considering its widespread use, the incidence of glucocorticoid side effects appears to be very low. It is possible that the glucocorticoid effects of MPA are minimal in the doses used in vivo and that a glucocorticoid effect is manifest only in exceptional circumstances. This could come about because of a selective impairment in MPA metabolism or because of an increased affinity of GR variants for MPA or because of altered function of a corepressor or coactivator, which then results in amplification of the GC effect of MPA. In this regard, a rare mutation in MR converts it into a high affinity receptor for progesterone, which results in severe hypertension in pregnancy induced by the high circulating level of progesterone (5). It is possible that polymorphisms or mutations in GR may alter its affinity for progesterone, although this has not yet been described.
In summary, our studies demonstrate that MPA increases -ENaC1 and sgk1 in the CCD by binding to GR. We were unable to ascertain whether MPA could bind MR in CCD cells, as these cell lines do not express MR. Unlike dexamethasone and aldosterone which increase Na+ transport in CCD cells via GR and MR, respectively, we could not demonstrate a significant effect of MPA on Na+ transport. Nevertheless, our studies identify the collecting duct as a potential site for crossover effects of MPA on glucocorticoid target genes.
GRANTS
A Department of Veterans Affairs Merit Review Grant, United States Public Health Service Grants DK-54348 and HL-71664, and an Established Investigatorship of the American Heart Association supported this work.
ACKNOWLEDGMENTS
The authors thank H. Oberleithner and B. Blazer-Yost for the gift of the MDCK-C7 cell line, R. Evans for the hGR expression vector, D. Pearce for TAT3-luc, S. Oate for p-Len, PR-B, and PRE-TATA-Luc, Organon for Org31710, and J. Dillon and T. Schmidt for helpful discussions. We also thank the University of Iowa DNA core facility for DNA synthesis and sequencing services provided.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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