Translational regulation of Na-K-ATPase subunit mRNAs by glucocorticoids

Prasad Devarajan1 and Edward J. Benz Jr.2

1 Pediatric Nephrology, Yale University School of Medicine, New Haven 06520, and Albert Einstein College of Medicine, Bronx, New York 10467; and 2 Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06520; and Johns Hopkins School of Medicine, Baltimore, Maryland 21205


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Glucocorticoids (GC) regulate Na-K-ATPase-subunit mRNA transcription. However, GC-induced increases in Na-K-ATPase activity are not always paralleled by changes in subunit mRNA abundance. We therefore examined posttranscriptional mechanisms of subunit gene regulation by GC. cDNA-derived mRNAs encoding alpha 1-, alpha 3-, and beta 1-subunits were tested for stability and translation efficiency in a cell-free lysate, in the presence of hydrocortisone (HC) or dexamethasone (Dex). No effect of HC on subunit mRNA stability was noted. Translation efficiency of alpha 1- and alpha 3-mRNAs, but not of beta 1-mRNA, was significantly increased by HC and Dex. Deletion of the 5'untranslated region (5'UT) of alpha 1-mRNA abolished this effect. Translation of a chimeric beta 1-mRNA, constructed by transposing the 5'UT of alpha 1 onto the coding region of beta 1, was enhanced by HC. Transposition of a putative steroid-modulatory element conserved in the 5'UT of all alpha  isoforms (ACAGGACCC) onto the coding region of beta 1-mRNA rendered it responsive to HC. A synthetic primer containing the ACAGGACCC sequence abolished the effect of HC on alpha 1- and chimeric beta 1-mRNAs. Our results indicate that GC can directly enhance Na-K-ATPase translation in vitro in a subunit-specific manner, via a putative GC-modulatory element situated in a predicted loop structure within the 5'UT of alpha -mRNAs.

mRNA stability; 5'untranslated region; hormonal regulation of sodium-potassium-adenosinetriphosphatase; glucocorticoid modulatory element; mRNA secondary structure.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE NA-K-ATPASE IS AN INTEGRAL membrane protein present in all eukaryotic cells. It is responsible for the ATP-dependent transport of sodium and potassium across the cell membrane. The ionic gradients thus created are essential for cell volume regulation, movement of ions and nutrients, and electrical activity of excitable tissues (26, 31). It is a heterodimer of two subunits in equal molar amounts. The alpha -subunit (Mr = 112,000) is catalytically active, whereas the beta -subunit (Mr = 35,000) facilitates the functional maturation and membrane insertion of the alpha beta -heterodimer (26). Molecular cloning of the cDNAs encoding the subunits has revealed that at least three isoforms of the alpha -subunit (termed alpha 1, alpha 2, and alpha 3) and two of the beta -subunit (beta 1 and beta 2) exist, which exhibit complex tissue-specific and developmental patterns of expression (19, 26, 31, 38, 44).

Regulation of Na-K-ATPase is especially critical in the kidney, where the alpha 1beta 1 unit is a major determinant of transepithelial sodium transport across the tubular epithelium in all nephron segments (15, 25). Mechanisms have evolved for short-term adaptation of the activity of existing pumps, and for long-term changes in pump biosynthesis (3, 10, 15, 26, 31). Acute changes in renal Na-K-ATPase biosynthesis can occur at the transcriptional (32, 39) and translational (13, 21) levels, and may be driven by monovalent ions (5) or hormones such as corticosteroids (4, 7-9, 17, 27, 42) and thyroid hormone (23).

Glucocorticoids (GC) increase renal Na-K-ATPase activity (16). This increase occurs in isolated perfused tubules and in cultured renal epithelial cells, suggesting that it is independent of changes in sodium load and hemodynamics. GC also increase Na-K-ATPase-subunit protein abundance (2, 27), and transcriptional regulatory mechanisms underlying these biosynthetic changes are being actively investigated (7-9). However, GC-induced increases in sodium pump activity are not always paralleled by changes in subunit mRNA abundance. For instance, during normal postnatal maturation, the upsurge in serum GC concentration is associated with a doubling of Na-K-ATPase activity in the renal cortex, but only a small increase in alpha 1-mRNA levels (7). In addition, the GC-induced increase in renal Na-K-ATPase activity in adrenalectomized rats is prevented by inhibition of protein synthesis, implicating translational control (2, 14). Furthermore, in alveolar epithelial cells, dexamethasone (Dex) upregulates Na-K-ATPase activity and alpha 1-subunit protein abundance with no changes in the alpha 1-mRNA abundance (2). Such observations suggest that glucocorticoids may additionally regulate Na-K-ATPase-subunit expression via posttranscriptional and/or translational mechanisms. However, these downstream effects have hitherto been difficult to assess in heterogeneous animal and cell culture systems that are typically under the influence of multiple interdependent regulatory mechanisms.

In this study, we examined the effect of GC on Na-K-ATPase-subunit mRNA stability and translation efficiency in a cell-free system. Our findings indicate that GC can directly enhance Na-K-ATPase translation in vitro in a subunit-specific manner, via a putative GC-modulatory element situated in a predicted loop structure within the 5'untranslated region (5'UT) of alpha -mRNAs.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

All restriction enzymes were from New England Biolabs. Kits for in vitro transcription and translation were from Promega. L-[35S]methionine and [32P]dCTP were from Amersham. Enlightening was from New England Nuclear. PCR kit was from Perkin-Elmer Cetus. Cap analog and random-primed DNA labeling kit were from Boehringer Mannheim. Hydrocortisone (HC), Dex, and aldosterone were from Sigma.

Na-K-ATPase clones. The Na-K-ATPase cDNA clones encoding full-length rat alpha 1-, alpha 3-, and beta 1-mRNAs have been previously described (13). The construction of a variant alpha 1-cDNA (termed alpha 1-5'UTdel) devoid of its 5'UT, and of a chimeric beta 1-cDNA (termed beta 1-chimera) with the 5'UT of alpha 1 transposed onto the coding region of beta 1 have also been published (13). A new variant beta 1-clone used in this study (termed beta 1/GME) was constructed as follows. Comparison of nucleotide sequences revealed a 9-bp segment (ACAGGACCC) in the 5'UT of Na-K-ATPase alpha 1-subunit (at nucleotide position -27 to -35), that is conserved among all alpha -subunit isoforms across several species (see Fig. 4A). This sequence is highly homologous to a steroid-modulatory element identified in the 5'UT of myelin basic protein mRNA (position -29 to -37), the translation efficiency of which has previously been shown to be GC responsive (41). A sense primer containing this segment, the Kozak consensus sequence, and the first five residues of beta 1-mRNA were utilized to amplify the entire coding region of beta 1 by standard PCR technique (13). The PCR product was ligated into pGEM vector (Promega), sequenced to confirm its identity, and termed beta 1/GME.

In vitro transcription. All cDNA clones were linearized with Sma I, and capped mRNAs were transcribed in vitro as described by the manufacturer (Promega). Transcription products were visualized by electrophoresis in 1%-formaldehyde agarose gels and quantitated by spectrophotometry. Comparable amounts of mRNA were obtained from each cDNA template. All in vitro transcribed mRNAs used in this study contain the entire coding regions and part of the untranslated regions, but are devoid of Poly(A) tails.

mRNA stability in reticulocyte lysate. Equal amounts (200 ng) of capped mRNA transcripts derived from full-length alpha 1- and beta 1-cDNAs were incubated in 100 µl of rabbit reticulocyte lysate supplemented with [35S]methionine at 30°C for 60 min, with or without HC (10-8 M, a physiologically relevant concentration). The mRNAs were extracted with phenolchloroform, ethanol precipitated, and analyzed by formaldehyde agarose gel electrophoresis, Northern blotting, and probing with random primer-labeled full-length cDNAs for alpha 1- or beta 1-subunit as previously described (13).

In vitro translation. Equal amounts (200 ng) of each capped mRNA species were heated to 67°C for 10 min and translated at 30°C for 60 min in a 25-µl reaction mixture containing 15 µl of nuclease-treated rabbit reticulocyte lysate, 40 units of RNasin, 20 µM amino acids minus methionine, [35S]methionine (20 µCi per reaction), and varying concentrations of HC, Dex, or aldosterone as described in previous studies (2, 41). For some experiments, the reaction mixture was supplemented with canine pancreatic microsomes. Translation products were analyzed by SDS-PAGE followed by fixation and fluorography of the dried gel with Enlightening. We have previously shown that all Na-K-ATPase mRNAs are most efficiently translated at a concentration of 200 ng per reaction, and extending the reaction time or altering magnesium concentrations was devoid of any effect (13). Also, we have demonstrated that the addition of microsomes to the lysate did not alter the translation efficiency of Na-K-ATPase subunits (13). For the oligonucleotide competition experiments, a synthetic primer, ACAGGACCC (termed GME oligo, representing the putative GME), was added to the translation mixture (10-fold molar excess of primer to mRNA) at the start of the reaction. To assess the role of specific bases within the GME oligo, substitutions were incorporated based on conserved sequences (Fig. 4, A and B). The synthetic primer GCGGCACCC (termed mutant oligo) was tested in the translation mixture at a 10-fold molar excess of primer to mRNA.

Analysis of mRNA secondary structure. The 5'UT sequence of alpha 1-mRNA was subjected to secondary structure analysis by using the FOLD program (13, 45), which predicts the most stable secondary structure for the mRNA under study, by using thermodynamics and auxiliary information.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Na-K-ATPase subunit mRNAs are stable in HC. The capped mRNAs transcribed from full-length cDNAs encoding alpha 1- and beta 1-Na-K-ATPase subunits were tested for stability in a translationally active reticulocyte lysate mixture, in the presence of physiological concentrations of HC. The alpha 1-Na-K-ATPase cDNA yielded a 3.7-kb mRNA, and the beta 1-cDNA a predominant 2.3-kb mRNA species, along with less abundant 1.7- and 1.4-kb messages (Fig. 1). This is consistent with the size of native Na-K-ATPase subunit mRNAs in vivo, namely, 3.7 kb for alpha 1 and multiple species of 2.7, 2.3, 1.7, and 1.4 (ending at different polyadenylation sites) for beta 1-mRNA (31). It should be noted that the largest mRNA transcribed by our beta 1-cDNA is ~400 bp smaller than the largest beta 1-mRNA transcribed in vivo, because part of the 3'untranslated region is missing. However, we and others have previously shown that, in spite of the varying sizes of the beta 1-mRNAs transcribed, they are very efficiently and rapidly translated in reticulocyte lysate to a single polypeptide of the predicted size and immunoreactivity (13). We have also previously shown that these mRNAs are stable in the lysate in the absence of HC (13). No significant effect of HC on the stability of either mRNA species was detected (Fig. 1). Therefore, the reticulocyte lysate represented an ideal system to explore the effects of GC on Na-K-ATPase subunit mRNA translatability alone, independent of transcriptional and mRNA stability considerations.


View larger version (75K):
[in this window]
[in a new window]
 
Fig. 1.   Na-K-ATPase mRNAs are stable in hydrocortisone (HC). Equal amounts (200 ng) of capped alpha 1- or beta 1-mRNA transcripts were incubated in a translationally active lysate with or without HC as indicated, and subjected to Northern analysis with probes encoding the respective cDNAs. Both mRNAs were stable in lysate with respect to time or the presence of HC.

Glucocorticoids enhance translation efficiency of alpha - mRNAs. We have previously demonstrated that cDNA-derived mRNAs encoding alpha 1-, alpha 3-, and beta 1-Na-K-ATPase subunits are reliably translated in reticulocyte lysate into polypeptides of the predicted size and immunoreactivity (13). In this study, we tested the translation efficiency of alpha 1-, alpha 3-, and beta 1-Na-K-ATPase subunits in the presence of various concentrations of HC, Dex, or aldosterone. The concentrations of hormones tested were chosen on the basis of known physiological plasma levels (7, 41) as well as previous in vitro studies (2, 41). At the physiological concentrations examined, both HC (Fig. 2A) and Dex (not shown) selectively enhanced translation efficiency of alpha 1- and alpha 3-mRNAs but not of beta 1-mRNA. Densitometric analysis of three separate experiments revealed an ~150% increase in alpha 1- and alpha 3-translation products in the presence of 10-6 and 10-10 M HC, and a 300% increase in 10-8 M HC (Fig. 3). The addition of microsomes did not alter these results for alpha 1- or beta 1-mRNA quantitatively, although the expected acquisition of core glycosylated sugars was apparent for the beta 1-subunit (Fig. 2B).. In contrast, addition of aldosterone at physiological or supraphysiological doses was devoid of any effect on alpha 1-mRNA translation (Fig. 2B), suggesting that the effect was specific to GC, and was unlikely to be GC-receptor mediated.


View larger version (44K):
[in this window]
[in a new window]
 
Fig. 2.   Glucocorticoids (GC) enhance translation efficiency of Na-K-ATPase alpha -mRNAs (M). Equal amounts (200 ng) of capped alpha 1-, alpha 3-, or beta 1-mRNA transcripts were incubated in a translationally-active lysate with varying concentrations of HC or aldosterone (Aldo) as indicated. HC selectively enhanced translation efficiency of alpha 1- and alpha 3-, but not beta 1-mRNA (A). Addition of microsomes (Mic) resulted in the expected appearance of larger, glycosylated forms of beta 1-protein, but did not alter the translational response of either alpha 1- or beta 1-mRNA to HC (B). Aldo was devoid of any effect on alpha 1-mRNA translation (C).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of physiological concentration of GC on translation of various Na-K-ATPase mRNAs. Densitometric abundance of the translation product of each mRNA designated on the horizontal axis is shown as a percent increase in 10-8 M HC compared with control values (same mRNA without HC). Values represent means ± SE from 3 separate experiments for each mRNA species.

The 5'UT of alpha 1-mRNA contains a transferable GC-modulatory element. To explore the potential role of 5'UT sequences, we examined the effect of HC on translation efficiency of variant clones. In previous studies, we have shown that translation of alpha 1-mRNA devoid of the 5'UT (alpha 1-5'UTdel), proceeds efficiently in reticulocyte lysate (13). The translation enhancing effect of HC on alpha 1-mRNA was abolished by removal of the 5'UT (Fig. 4), suggesting the existence of a GC-modulatory element within this sequence. Indeed, transposition of the 5'UT of alpha 1-mRNA onto the coding sequences of the previously unresponsive beta 1-mRNA rendered the translation efficiency of the chimeric beta 1-construct sensitive to physiological concentrations of HC (Fig. 4). By densitometry, the abundance of chimeric beta 1-translation product was increased by ~200% in 10-6 and 10-10 M HC, and ~350% in the presence of 10-8 M HC (Fig. 3). These results indicate that the GC-modulatory element within the 5'UT of alpha 1-mRNA can be transferred to a normally unresponsive message.


View larger version (59K):
[in this window]
[in a new window]
 
Fig. 4.   The 5' untranslated region (5'UT) of alpha 1-mRNA contains a transferable GC-modulatory element (GME). The variant alpha 1-mRNA devoid of its 5'UT (alpha 1-5'UTdel) and chimeric beta 1-mRNA with the 5'UT of alpha 1 transposed onto the coding region of beta 1 (beta 1-chimera) were translated in the presence of varying concentrations of HC as shown. The translation enhancing effect of HC on alpha 1-mRNA was abolished by removal of the 5'UT. Transposition of the 5'UT onto the previously unresponsive beta 1-mRNA increased the translation efficiency of the chimeric beta 1-construct in the presence of physiological concentrations of HC.

The sequence ACAGGACCC is a GC-modulatory element in alpha 1-mRNA. Comparison of nucleotide sequences revealed a 9-bp segment (ACAGGACCC) at position -27 to -35 for the alpha 1-isoform that is conserved in the 5'UT of all Na-K-ATPase alpha -subunit isoforms in several species (Fig. 5A). This sequence is absent from the normally unresponsive beta -subunits, but is highly homologous to a steroid-modulatory element identified in the 5'UT of myelin basic protein mRNA, the translation efficiency of which has previously been shown to be GC sensitive (41). We therefore tested the possibility that this segment may represent a GC-modulatory element, initially by transposing it onto the coding region of beta 1-mRNA. Indeed, the variant clone beta 1/GME produced a translation product of the appropriate size, the abundance of which was increased by ~250% in the presence of 10-8 M HC (Figs. 5B and 3).


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 5.   The sequence ACAGGACCC is a GME in alpha 1 mRNA. A: a comparison of nucleotide sequences within the 5'UT of Na-K-ATPase alpha -subunit mRNAs and the myelin basic protein (MBP). The segment ACAGGACCC (position -27 to -35 for alpha 1-mRNA) is highly conserved among several alpha -Na-K-ATPases across species and has been shown to represent a steroid modulatory element in MBP (41). *, Residues that are completely conserved. B: the sequences of the GME oligo (top) and the mutant oligo (bottom). The mutant oligo contains substitutions of the conserved residues. C (top 2 sequences): the translation products of the variant clone beta 1/GME, in the presence of varying concentrations of HC. This clone contains the above nucleotide segment of alpha 1-Na-K-ATPase transposed onto the coding region of beta 1-mRNA, and its translation is enhanced by physiological concentrations of HC, suggesting that the segment represents a GME. C (middle): the translation products of beta 1/GME and alpha 1-wild-type (WT) mRNAs, in the presence of the GME oligo, which abolished the enhancing effect of HC on translation of both of these mRNAs. C (bottom): the translation of alpha 1-mRNA in the presence of HC and mutant oligo. The mutant oligo was ineffective in blocking the effect of HC on alpha 1-mRNA translation.

We then tested the ability of a synthetic oligonucleotide, ACAGGACCC, to block the HC-mediated enhanced translation of wild-type alpha 1 and of the variant clone beta 1/GME. The primer (termed GME oligo, Fig. 5B) abolished the effect of HC on translation of both these mRNAs, when incubated in the mixture at a 10-fold molar excess of primer to mRNA (Fig. 5C). Taken together, these results identified the sequence ACAGGACCC as a transferable glucocorticoid-modulatory element within the 5'UT of alpha 1-Na-K-ATPase mRNA.

To confirm the role and specificity of the GME oligonucleotide, substitutions were incorporated in the primer based on conserved sequences (Fig. 5A). A synthetic primer GCGGCACCC (termed mutant oligo) did not have any effect on wild-type alpha 1-mRNA translation in the presence of HC, at a 10-fold molar excess of primer to mRNA (Fig. 5C). Thus substitutions of the conserved bases abolished the inhibitory effect seen with the GME oligonucleotide.

The alpha 1-GC-modulatory element is situated in a loop structure. Secondary structure examination of the 5'UT of alpha 1-mRNA by the FOLD program revealed that five of the nine residues representing the GC-modulatory element are situated in a loop structure (Fig. 6), within the first of four such loops previously described (13). Such loops are predicted to be accessible to interacting proteins.


View larger version (9K):
[in this window]
[in a new window]
 
Fig. 6.   The alpha 1-GME is situated in a loop structure. The 5'UT sequence of alpha 1-mRNA was subjected to secondary structure analysis by using the FOLD program. The first of four stem-loop structures previously identified (13) is shown. Numbers represent nucleotide positions, with the initiator methionine representing positions 1-3. The majority of residues representing the GME ACAGGACCC are in a loop region that is predicted to be accessible to interacting proteins.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we tested the hypothesis that GC influence Na-K-ATPase-subunit mRNA expression via posttranscriptional mechanisms. The results demonstrate for the first time that physiological concentrations of GC enhance the translation efficiency of catalytic subunit mRNAs in vitro. This effect is specific to the alpha -subunits, because beta 1-mRNA translation was unaltered by GC. In addition, this property is restricted to the glucocorticoids, because aldosterone did not influence alpha 1-mRNA translation even at supraphysiological doses. By using deletion mutants and chimeric clones, we established that the 5'UT of alpha 1-mRNA contains a modulatory element that confers GC responsiveness to the downstream coding sequences. By comparison of nucleotide sequences, we identified a 9-bp segment, ACAGGACCC, that is conserved across species within the 5'UT of all alpha -Na-K-ATPase mRNAs but is absent from the beta -subunit. This sequence is highly homologous to the steroid modulatory element previously identified in myelin basic protein mRNA, the translation of which is similarly steroid sensitive. By creation of chimeric mutants as well as oligonucleotide competition experiments, we have established that the sequence ACAGGACCC represents a transferable GC-modulatory element within the 5'UT of alpha 1-Na-K-ATPase mRNA, which is required to mediate the enhanced translational response to GC. Analysis of predicted mRNA secondary structure revealed that the GC-modulatory element is ideally situated in a loop structure that is predicted to be accessible to interacting proteins.

Glucocorticoids mediate profound physiological and developmental effects in eukaryotes, by regulating the expression of several target genes (6). For the majority of proteins, this is achieved via a well-studied pathway involving specific intracellular glucocorticoid receptors and a cognate glucocorticoid response element in the promoter region of target genes. Several lines of evidence have endorsed the importance of this transcriptional mechanism in the regulation of Na-K-ATPase-subunit gene expression by GC (4, 7-9, 17, 27, 42, 43). However, additional posttranscriptional mechanisms must exist, because GC-induced increases in sodium pump activity are not always paralleled by changes in subunit mRNA abundance. These mechanisms have hitherto been difficult to ascertain in animal and cell culture systems where transcriptional regulation may predominate. In this study, a cell-free translation system was used to determine the direct effects of GC on translation efficiency of Na-K-ATPase subunit mRNAs synthesized in vitro, thereby circumventing any effects on transcription. In addition, the absence of sequences homologous to the cognate-glucocorticoid response element in the Na-K-ATPase mRNAs tested attests to the lack of transcriptional influences. Furthermore, the inability of aldosterone to mimic the effect of GC, even at supraphysiological doses, suggests that the enhanced Na-K-ATPase translation noted in this study is not receptor mediated.

The posttranscriptional regulation of gene expression by GC may occur either through mRNA stabilization or via direct effects on translation. For example, Dex enhances Bcl-x gene expression by significantly extending mRNA stability (11). Although we have not performed a detailed kinetic assay of Na-K-ATPase mRNA stability, our results clearly indicate that both subunit mRNAs were equally stable in the lysate during the translationally active period, irrespective of the presence or absence of GC. Thus we were able to exclude any effects of GC on mRNA stability as a potential explanation for the enhanced translation of Na-K-ATPase alpha -mRNAs that we have observed. However, these in vitro studies do not rule out the potential for additional effects of GC on mRNA stability in nucleated cells. Indeed, the mRNAs used in this in vitro study were devoid of Poly(A) tails. In vivo, it is possible that the stability of native mRNAs, with their complete Poly(A) tails and all polyadenylation signals, may be significantly influenced by physiological concentrations of GC. This may be especially relevant to Na-K-ATPase-beta 1 mRNA translation in vivo, because the largest mRNA transcribed in vitro lacked ~400 bp of 3'untranslated sequences.

Several recent reports have indicated that GC can directly influence mRNA translation, either positively or negatively. For instance, Dex inhibits translation of inducible nitric oxide synthase (29), cyclooxygenase-2 (34), cytokines such as leukemia-inhibitory factor (22), and tumor necrosis factor-alpha (18). On the other hand, Dex has been shown to play a role in the enhanced translation efficiency of a variety of polypeptides, including myelin basic protein (41), myelin proteolipid protein (41), vasopressin receptor (12), glutamine synthetase (37), beta 2-adrenergic receptor (33) and, most recently, the proximal tubule Na/H antiporter (1). Perhaps the best-studied example is that of myelin basic protein, the translation efficiency of which was enhanced two- to threefold in a cell-free system in the presence of physiological concentrations of HC or Dex (41). This effect was mediated by a steroid- modulatory element situated in a predicted loop structure at position -29 to -37 within the 5'UT of myelin basic protein mRNA. It is interesting to note the several similarities between that study and ours. In the present report, the GC-induced threefold increase in translation efficiency of alpha 1-Na-K-ATPase mRNA was also mediated by a highly homologous GC-modulatory element in a predicted loop at position -27 to -35 within its 5'UT. Furthermore, a comparison of putative glucocorticoid modulatory elements within the 5'UTs of alpha -Na-K-ATPases across isoforms and species reveals the striking and complete conservation of the very same nucleotides that have been shown by site-directed mutational analysis to be critical for GC responsiveness in myelin basic protein (highlighted by asterisks in Fig. 4A). Indeed, the present study has demonstrated, by oligonucleotide-competition experiments, that the same nucleotides are also necessary for the translational control of alpha -Na-K-ATPase by GC.

The concentrations of GC that were found to be effective in this study are within the physiological range (41) and have been used in previous studies. For instance, during postnatal maturation of the kidney, a significant increase in Na-K-ATPase activity (paralleled by only a small increase in catalytic-subunit mRNA level) is associated with a measured plasma-glucocorticoid level in the 10-8 M range (7). In addition, the Dex-induced upregulation of Na-K-ATPase protein abundance and activity (with no change in alpha 1-mRNA level) in alveolar epithelial cells has been demonstrated to occur at GC concentrations of 10-7 and 10-8 M (2). In our study, the maximal stimulatory effect on alpha 1-mRNA translation was noted at GC concentrations of 10-8 M (3-fold increase); concentrations of 10-6 or 10-10 M yielded only modest increases (1.5-fold). Our results suggest that, in addition to the well-documented receptor-mediated transcriptional regulation of Na-K-ATPase by GC, direct translational control may represent an adjunctive, but physiologically relevant, mechanism for the basal, constitutive expression of Na-K-ATPase.

Regulation of gene expression at the level of translation is now widely appreciated as an important modulator of protein synthesis (24, 35). Elucidation of the ribosome-scanning model has shown that the peptide chain initiation phase is the rate-limiting step in protein translation (28, 36). Efficiency of translation initiation is heavily influenced by mRNA secondary structure of the 5'UT (28), and by the interactions between a variety of proteins that bind either to the 5'UT of mRNAs (20, 40) or directly to components of the translational machinery (30). Pertinent to this study, the proximity of the putative GC-modulatory element to the initiator methionine, combined with the fact that most of the known physiological effects on translation are exerted at the level of polypeptide chain initiation (35), suggests that GC increase translation of alpha 1-Na-K-ATPase by enhancing the formation of the initiation complex.

Can enhanced translation of Na-K-ATPase alpha -mRNAs (but not beta -mRNA) lead to increased activity of the alpha beta heterodimer? We have previously shown that the translation efficiency of beta -mRNAs is markedly greater than that of alpha -mRNAs, both in vitro and in vivo (13). It is therefore reasonable to speculate that newly synthesized beta -subunit protein is normally present in enough excess to incorporate all the alpha -protein made, including the enhanced amounts of alpha -protein induced by glucocorticoid surges, thereby resulting in increased pump activity. Furthermore, our study does not exclude any additional effects of GC on Na-K-ATPase activity from enhanced trafficking of the heterodimer, as has been suggested for the Na+/H+ exchanger (1).

In summary, we have shown that GC can directly enhance Na-K-ATPase translation in vitro in a subunit-specific manner, via a putative GC-modulatory element in a loop structure within the 5'UT of alpha -mRNAs. Several speculations regarding the physiological significance of these findings can be offered. First, translational control may represent an adjunctive mechanism for the constitutive, basal regulation of Na-K-ATPase function by GC. Second, such a mechanism could account for instances where GC-induced increases in sodium pump activity are not accompanied by changes in subunit mRNA abundance. Third, it may provide for a mechanism by which GC can regulate Na-K-ATPase-gene expression during the physiological GC surge characteristic of early postnatal development of several organ systems. Fourth, it may represent a general mechanism by which growth-related proteins (such as Na-K-ATPase and myelin basic protein) could rapidly overcome a state of repressed translation. We have previously shown that translation efficiency of the Na-K-ATPase alpha -subunit is chronically inhibited by its complex 5'UT secondary structure (13), reminiscent of the constitutionally repressed translation of growth-related proteins. It is therefore possible that the appearance of growth stimuli such as steroids could counteract the repressive influences on the 5'UT, perhaps by altering the phosphorylation state of translation regulatory molecules, thereby allowing for rapid translation of the proteins at a crucial period during altered cellular growth and development. It will be important in future experiments to document a direct mRNA-protein interaction between steroids and the GC-modulatory element, and to identify the critical residues within this element that mediate this interaction. It will also be of value to test the behavior of variant Na-K-ATPase clones in transfected cells, to search for similar translational effects of GC in vivo.


    ACKNOWLEDGEMENTS

This work was supported by grants from the National Institutes of Health to P. Devarajan (DK-47072, DK-53289) and E. J. Benz, Jr. (HL-24385, HL-44985, HL-23076).


    FOOTNOTES

Address for reprint requests and other correspondence: P. Devarajan, Children's Hospital at Montefiore, Albert Einstein College of Medicine, 111 East 210th St., Bronx, NY 10467 (E-mail: pdevaraj{at}aecom.yu.edu).

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.

Received 3 February 2000; accepted in final form 11 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ambühl, PM, Yang X, Peng Y, Preisig PA, Moe OW, and Alpern RJ. Glucocorticoids enhance acid activation of the Na+/H+ exchanger 3 (NHE3). J Clin Invest 103: 429-435, 1999[Abstract/Free Full Text].

2.   Barquin, N, Ciccolella DE, Ridge KM, and Sznajder JI. Dexamethasone upregulates the Na-K-ATPase in rat alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 273: L825-L830, 1997[ISI][Medline].

3.   Bertorello, AM, and Katz AI. Short-term regulation of renal Na-K-ATPase activity: physiological relevance and cellular mechanisms. Am J Physiol Renal Fluid Electrolyte Physiol 265: F743-F755, 1993[Abstract/Free Full Text].

4.   Bonvalet, JP. Regulation of sodium transport by steroid hormones. Kidney Int 53: S49-S56, 1998.

5.   Bowen, WJ, and McDonough AA. Pretranslational regulation of Na-K-ATPase in cultured canine kidney cells by low K. Am J Physiol 52: C179-C189, 1987.

6.   Burnstein, KL, and Cidlowski JA. Regulation of gene expression by glucocorticoids. Annu Rev Physiol 51: 683-699, 1989[ISI][Medline].

7.   Celsi, G, Nishi A, Akusjärvi G, and Aperia A. Abundance of Na+-K+-ATPase mRNA is regulated by glucocorticoid hormones in infant rat kidneys. Am J Physiol Renal Fluid Electrolyte Physiol 260: F192-F197, 1991[Abstract/Free Full Text].

8.   Celsi, G, Stålh J, Wang ZM, and Nishi A. Adrenocorticoid regulation of Na+,K+-ATPase in adult rat kidney: effects on post-translational processing and mRNA abundance. Acta Physiol Scand 145: 85-91, 1992[ISI][Medline].

9.   Celsi, G, Wang ZM, Akusjärvi G, and Aperia A. Sensitive periods for glucocorticoids' regulation of Na+-K+-ATPase mRNA in the developing lung and kidney. Pediatr Res 33: 5-9, 1992[Abstract].

10.   Celsi, G, and Wang ZM. Regulation of Na+-K+-ATPase gene expression: a model to study terminal differentiation. Pediatr Nephrol 7: 630-634, 1993[ISI][Medline].

11.   Chang, TC, Hung MW, Jiang SY, Chu JT, and Tsai LC. Dexamethasone suppresses apoptosis in a human gastric cancer cell line through modulation of bcl-x gene expression. FEBS Lett 415: 11-15, 1997[ISI][Medline].

12.   Colson, P, Ibarondo J, Devilliers G, Balestre MN, Duvoid A, and Guillon G. Upregulation of Via vasopressin receptors by glucocorticoids. Am J Physiol Endocrinol Metab 263: E1054-E1062, 1992[ISI][Medline].

13.   Devarajan, P, Gilmore-Hebert M, and Benz EJ, Jr. Differential translation of the Na,K-ATPase subunit mRNAs. J Biol Chem 267: 22435-22439, 1992[Abstract/Free Full Text].

14.   Doucet, A. Function and control of Na-K-ATPase in single nephron segments of the mammalian kidney. Kidney Int 34: 749-760, 1988[ISI][Medline].

15.   Doucet, A. Na,K-ATPase in the kidney tubule in relation to natriuresis. Kidney Int 41: S118-S124, 1992[ISI].

16.   Ellis, D, Sothi TD, and Avner ED. Glucocorticoids modulate renal glucocorticoid receptor and Na,K-ATPase activity. Kidney Int 32: 464-471, 1987[ISI][Medline].

17.   Ewart, HS, and Klip A. Hormonal regulation of Na(+)-K(+)-ATPase: mechanisms underlying rapid and sustained changes in pump activity. Am J Physiol Cell Physiol 269: C295-C311, 1995[Abstract/Free Full Text].

18.   Gewert, K, Svensson U, Andersson K, Holst E, and Sundler R. Dexamethasone differentially regulates cytokine transcription and translation in macrophages responding to bacteria or okadaic acid. Cell Signal 11: 665-670, 1999[ISI][Medline].

19.   Gloor, SH, Antonicek H, Sweadner KJ, Pagliussi S, Frank R, Moos M, and Schanchner M. The adhesion molecule on glia (AMOG) is a homologue of the beta -subunit of Na,K-ATPase. J Cell Biol 110: 165-174, 1990[Abstract].

20.   Gray, NK, and Wickens M. Control of translation initiation in animals. Annu Rev Cell Dev Biol 14: 399-458, 1998[ISI][Medline].

21.   Grindstaff, KK, Blanco G, and Mercer RW. Translational regulation of Na,K-ATPase alpha1 and beta1 polypeptide expression in epithelial cells. J Biol Chem 271: 23211-23221, 1996[Abstract/Free Full Text].

22.   Grosset, C, Taupin JL, Lemercier C, Moreau JF, Reiffers J, and Ripoche J. Leukaemia inhibitory factor expression is inhibited by glucocorticoids through post transcriptional mechanisms. Cytokine 11: 29-36, 1999[ISI][Medline].

23.   Horowitz, B, Hensley CB, Quintero M, Azuma KK, Putnam D, and McDonough AA. Differential regulation of Na,K-ATPase alpha1, alpha2 and beta subunit mRNA and protein levels by thyroid hormone. J Biol Chem 265: 14308-14314, 1990[Abstract/Free Full Text].

24.   Jansen, M, deMoor CH, Sussenbach JS, and van den Brande JL. Translational control of gene expression. Pediatr Res 37: 681-686, 1995[Abstract].

25.   Jorgensen, PL. Structure, function and regulation of Na,K-ATPase in the kidney. Kidney Int 29: 10-20, 1986[ISI][Medline].

26.   Jorgensen, PL, Meng LM, and Pedersen PA. Structure and regulation of Na,K-ATPase in the kidney. In: Molecular Nephrology: Kidney Function in Health and Disease, edited by Schlöndorff D., and Bonventre JV.. New York: Dekker, 1995, p. 349-368.

27.   Klein, LE, and Lo CS. Regulation of rat renal (Na+ + K+)-adenosine triphosphatase mRNA levels by corticosterone. Experientia 48: 768-773, 1992[ISI][Medline].

28.   Kozak, M. The scanning model for translation: an update. J Cell Biol 108: 229-241, 1989[Abstract].

29.   Kunz, D, Walker G, Eberhardt W, and Pfeilschifter J. Molecular mechanisms of dexamethasone inhibition of nitric oxide synthase expression in interleukin 1 beta stimulated mesangial cells: evidence for the involvement of transcriptional and posttranscriptional regulation. Proc Natl Acad Sci USA 93: 255-259, 1996[Abstract/Free Full Text].

30.   Labate, ME, Whelly SM, and Barker KL. Ribosome-associated estradiol-binding components in the uterus and their relationship to the translational capacity of uterine ribosomes. Endocrinology 119: 140-151, 1986[Abstract].

31.   Lingrel, JB, Orlowski J, Shull MM, and Price EM. Molecular genetics of Na,K-ATPase. Prog Nucleic Acid Res Mol Biol 38: 37-89, 1990[ISI][Medline].

32.   Liu, B, and Gick G. Characterization of the 5' flanking region of the rat Na+/K+-ATPase beta 1 subunit gene. Biochim Biophys Acta 1130: 336-338, 1992[ISI][Medline].

33.   McGraw, DW, Chai SE, Hiller FC, and Cornett LE. Regulation of the beta 2-adrenergic receptor and its mRNA in the rat lung by dexamethasone. Exp Lung Res 21: 535-546, 1995[ISI][Medline].

34.   Newton, R, Seybold J, Kuitert LM, Bergmann M, and Barnes PJ. Repression of cyclooxygenase-2 and prostaglandin E2 release by dexamethasone occurs by transcriptional and post-transcriptional mechanisms involving loss of polyadenylated mRNA. J Biol Chem 273: 32312-32321, 1998[Abstract/Free Full Text].

35.   Pain, VM. Initiation of protein synthesis in eukaryotic cells. Eur J Biochem 236: 747-771, 1996[Abstract].

36.   Sachs, AB, Sarnow P, and Hentze MW. Starting at the beginning, middle, and end: translation initiation in eukaryotes. Cell 89: 831-838, 1997[ISI][Medline].

37.   Sarantos, P, Chakrabarti R, Copeland EM, and Souba WW. Dexamethasone increases jejunal glutamine synthetase expression via translational regulation. Am J Surg 167: 8-13, 1994[ISI][Medline].

38.   Shull, GE, Greeb J, and Lingrel JB. Molecular cloning of three distinct forms of Na,K-ATPase alpha -subunit from rat brain. Biochemistry 25: 8125-8134, 1986[ISI][Medline].

39.   Shull, GE, Pugh DG, and Lingrel JB. The human Na,K-ATPase alpha 1 gene: characterization of the 5' flanking region and identification of a restriction fragment length polymorphism. Genomics 6: 451-460, 1990[ISI][Medline].

40.   Stripecke, R, Oliveira CC, McCarthy JE, and Hentze MW. Proteins binding to 5' untranslated region sites: a general mechanism for translational regulation of mRNAs in human and yeast cells. Mol Cell Biol 14: 5898-5909, 1994[Abstract].

41.   Verdi, JM, and Campagnoni AT. Translation regulation by steroids. Identification of a steroid modulatory element in the 5'-untranslated region of the myelin basic protein messenger RNA. J Biol Chem 265: 20314-20320, 1990[Abstract/Free Full Text].

42.   Verrey, F, Beron J, and Spindler B. Corticosteroid regulation of renal Na,K-ATPase. Miner Electrolyte Metab 22: 279-292, 1996[ISI][Medline].

43.   Welling, PA, Caplan M, Sutters M, and Giebisch G. Aldosterone mediated Na/K-ATPase expression is alpha(1) isoform specific in the renal cortical collecting duct. J Biol Chem 268: 23469-23476, 1993[Abstract/Free Full Text].

44.   Young, RM, Shull GE, and Lingrel JB. Multiple mRNAs from rat kidney and brain encode a single Na,K-ATPase beta subunit protein. J Biol Chem 262: 4905-4910, 1987[Abstract/Free Full Text].

45.   Zuker, M, and Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res 9: 133-148, 1981[Abstract].


Am J Physiol Renal Fluid Electrolyte Physiol 279(6):F1132-F1138
0363-6127/00 $5.00 Copyright © 2000 the American Physiological Society