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(Hypertension. 1997;30:191-198.)
© 1997 American Heart Association, Inc.

Articles

Characterization of a Sodium-Response Transcriptional Mechanism

Nelson Ruiz-Opazo; Jean-François Cloix; Maria-Grazia Melis; Xie Hou Xiang; ; Victoria L. M. Herrera

From the Section of Molecular Genetics, Whitaker Cardiovascular Institute, and Section of Cardiology (V.L.M.H.), Evans Department of Medicine, Boston (Mass) University School of Medicine.

Correspondence to Victoria L.M. Herrera, MD, Section of Molecular Genetics, W-609, Whitaker Cardiovascular Institute, Boston University School of Medicine, 80 E Concord St, Boston, MA 02118.

	Abstract

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Abstract On the basis of paradigms in development wherein discrete transcriptional events are pivotal regulatory steps, we tested the hypothesis that transcriptional sodium (Na⁺)–response mechanisms are involved in in vivo Na⁺-induced responses relevant to normal (homeostatic) and pathophysiological (salt-sensitive hypertension) conditions. We used Na,K-ATPase -subunit genes as molecular probes and the Na⁺ ionophore monensin to induce a dose-specific incremental increase in [Na⁺]_i in rat A10 embryonic aortic smooth muscle cells. RNA blot analysis of rat A10 cells revealed a dose-specific (0.022 to 30 µmol/L monensin) upregulation of ₁-, ₂-, and ß₁-subunit Na,K-ATPase RNA levels. Control ß-actin and -tropomyosin RNA levels did not change. With the use of chloramphenicol acetyltransferase (CAT) as reporter gene, CAT assays of rat ₁[-1288]CAT and human ₂[-798]CAT promoter constructs exhibited induction of CAT activity in monensin (10 µmol/L)–treated A10 cells compared with untreated A10 cells. Promoter deletion constructs for rat ₁[-1288]CAT defined a positive Na⁺-response regulatory region within -358 to -169 that is distinct from the basal transcriptional activation region of -155 to -49 previously defined. Similarly, a positive Na⁺-response regulatory region is delimited to within -301 in the human ₂ Na,K-ATPase 5' flanking region. Analysis of transgenic TgH₂[-798]CAT rats demonstrated sodium activation of human ₂[-798]CAT transgene expression in aorta parallel to observations made in rat A10 aortic tissue culture cells. Southwestern blot analysis of nuclear extracts from monensin (10 µmol/L)–treated and control untreated A10 cells revealed a nuclear DNA binding protein (approximately 95 kD) that is upregulated by increased [Na⁺]_i. These data provide initial characterization of a transcriptional Na⁺-response mechanism delimiting a positive Na⁺-response regulatory region in two target genes (₁ and ₂ Na,K-ATPase) as well as detection of a Na⁺-response nuclear DNA binding protein. The in vitro data are corroborated by in vivo experimental and transgenic promoter expression studies, thus validating the biological relevance of the observations.

Key Words: monensin • gene regulation • muscle, smooth • rats, transgenic

	Introduction

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High salt intake as a risk factor for hypertension has been delineated in humans^{1 2 3 4} as well as in critical animal models, such as primates⁵ and inbred rats,⁶ elucidating the phenomenon of salt sensitivity. Salt sensitivity has been associated with increased renal disease and cardiac hypertrophy in hypertensive patients.^{7 8} It has also been associated with vascular abnormalities, stroke, and cardiac hypertrophy in the stroke-prone spontaneously hypertensive rat (SHRSP) model.^{9 10} These observations indicate that the pathogenic events initiated by high salt intake are putative major determinants of the course and/or outcome of hypertension. The molecular basis of these observations has not been elucidated.

In response to a given stimulus, short-term cellular changes that are immediate, classically involving changes in enzymatic activities and cell surface receptor activation, as well as longer term changes that involve transcription and translation could be expected. As seen in development, transcriptional events alter set points that contribute to paradigms of ordered and coordinate events relevant to organogenesis and patterning, to name a few.¹¹ This transcriptional regulation-based paradigm forms the theoretical basis for the hypothesis investigated in this study, that is, that transcriptional sodium-response (Na⁺-response) mechanisms exist that effectively modulate salt-induced homeostatic changes as well as play pivotal roles in the pathogenesis of salt-induced hypertension.

The current experimental evidence that supports the hypothesis of putative transcriptional Na⁺-response mechanisms derives from in vivo rat studies and in vitro tissue culture studies. In vivo rat studies have identified a panel of candidate Na⁺-response genes, such as the induction of gene expression of the ₁-subunit Na,K-ATPase gene in prehypertensive salt-loaded Dahl salt-sensitive rat kidney¹² and the endothelin-1 gene in salt-loaded SHRSP hearts.⁹ Salt loading in a normotensive Wistar rat also induced ₁ Na,K-ATPase mRNA levels in aorta,¹³ indicating that putative Na⁺-response mechanisms are most likely adaptive to restore Na⁺ homeostasis when challenged. However, when coupled with dysfunctional hypertension gene products, Na⁺-response mechanisms putatively go awry or amplify the primary dysfunction of said hypertension genes and contribute to the pathogenesis of salt-sensitive hypertension and/or its target-organ complications. It is also evident that the Na⁺ induction of gene expression is targeted to specific genes, distinguishing it from a generalized, nonspecific cellular response.

The upregulation of ₁ Na,K-ATPase gene expression by high [Na⁺]_i has been observed in tissue culture systems regardless of the mode of induction of increased [Na⁺]_i, that is, by veratridine,¹⁴ incubation in low-K⁺ medium,^{15 16} or serum.¹⁷ Given that other cellular changes also occur that are distinct for veratridine, low-K⁺ medium, and serum, the commonality of both the increase in [Na⁺]_i and induction of ₁ Na,K-ATPase gene expression in these tissue culture systems^{14 15 16 17} suggests that the ₁ Na,K-ATPase gene is most likely a target gene of a putative Na⁺-response transcriptional mechanism.

We investigated these issues in an integrated, stepwise manner and present data that show dose-responsive increases in ₁, ₂, and ß₁ Na,K-ATPase RNA levels in rat A10 cells with increased [Na⁺]_i via monensin treatment (0.022 mol/L to 30 µmol/L); that define transcriptional activation of rat (R) ₁ Na,K-ATPase and human (H) ₂ Na,K-ATPase promoter–chloramphenicol acetyltransferase (CAT) constructs in rat A10 cells treated with 10 µmol/L monensin; that delimit a Na⁺-response regulatory region in both R₁ and H₂ Na,K-ATPase genes via promoter deletion analysis of reporter gene activity; that document the Na⁺ activation of H₂ Na,K-ATPase in a transgenic rat model; and that detect a Na⁺-response nuclear DNA binding protein upregulated in rat A10 cells with increased [Na⁺]_i via treatment with 10 µmol/L monensin.

	Methods

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Culture and Monensin Treatment of Rat A10 Cells
Rat A10 cells (American Type Culture Collection [ATCC]) were grown in 20% fetal calf serum (GIBCO-BRL) and Dulbecco's modified Eagle's medium. All experiments were conducted with cells from the same passage. Transfection efficiency decreased with high passage number. Monensin (Calbiochem) was dissolved in 95% ethanol and kept as a 1 mmol/L stock, which was used as stock for all dilutions used in the experiment. Serial dilutions from the same stock were made for the dose-response curve analysis.

RNA Blot Analysis
RNA blot analysis of total cellular RNA was done essentially as described previously,¹³ with the following specifications. Total cellular RNAs were isolated by the CsCl gradient method using guanidinium thiocyanate as homogenizing buffer. Concentrations of RNA were obtained by UV spectroscopy, and equal amounts of RNA were loaded in formaldehyde agarose gels. Nondegraded RNA was ascertained by ethidium bromide staining of ribosomal RNA. New RNA blots were used in the analysis of ₁, ₂, and ß₁ Na,K-ATPase RNA levels. RNA blots were then stripped and probed with the control probes -tropomyosin and ß-actin. The specificity of all probes had been previously ascertained.¹³ RNA levels were measured by densitometry. Analyses of ₁, ₂, and ß₁ RNA levels were corrected for variation in loading on the basis of control -tropomyosin RNA levels. The degree of induction (-fold increase) for ₁, ₂, and ß₁ Na,K-ATPase gene expression was assessed by the ratio of corrected RNA levels with monensin over corrected baseline RNA level (no monensin treatment).

Promoter Deletion Constructs
Deletion constructs were made in the pSP73 vector (Promega) using the Erase-a-Base System kit (Promega) according to the manufacturer's specifications. All deletion constructs were identified by nucleotide sequencing and subcloned into a promoterless CAT vector. Identification numbers were assigned on the basis of the nucleotide position from the first transcription start site identified respectively for the R₁ and H₂ Na,K-ATPase genes.^{18 19} The pCAT-basic vector (Promega) was used for R₁ promoter deletion constructs; a pSP73-CAT[SV40 polyA] vector was used for H₂ promoter deletion constructs. We modified this vector so as to add more cloning sites for promoter insertion into a CAT vector. All promoter deletion CAT constructs were resequenced to ascertain deletion identity and correct orientation with respect to the CAT coding region. Large-scale DNA preparations were made by double CsCl gradients or Qiagen columns per the manufacturer's specifications.

Transfection and CAT Assays of Rat A10 Cells
Transfection was done with Lipofectin (GIBCO-BRL) following the manufacturer's specifications. To ascertain equivalent transfection of control and monensin-treated A10 cells, a 2x Lipofectin-DNA cocktail was made and divided for two culture plates that were then assigned as test and cognate control plates. Equimolar amounts of promoter deletion CAT constructs were used for transfection to validate comparative analysis along with 5 µg of pSV–ß-galactosidase (Promega) as internal control. DNA concentrations were determined by UV spectroscopy and confirmed on agarose gels. Identical passage number of A10 cells was maintained for the experiments. Transfection was done 24 hours after plating at identical densities based on cell count. Transfected cells were maintained in growing medium for 24 hours. Then test A10 cells were placed in growing medium containing 10 µmol/L monensin, and control cells were placed in fresh growing medium containing an equivalent amount (5 µL) of vehicle alone. Incubation in 10 µmol/L monensin or vehicle alone (control) was carried out for 16 hours as determined by time course studies (data not shown). These experimental conditions were maintained throughout the study.

CAT assays were done essentially as described²⁰ with the following specifications: protein concentrations were determined with the BCA (Pierce) assay per the manufacturer's instructions; equal amounts of protein extracts were used for all CAT assays to validate visual comparative analysis when transfection efficiencies varied less than 10% as determined by ß-galactosidase assays; and equal amounts of [¹⁴C]chloramphenicol (New England Nuclear) were used for all experimental points. Quantitative analysis was done by autoradiographic densitometry of acetylated [¹⁴C]chloramphenicol and nonacetylated substrate. CAT activity was determined as the percent conversion of acetylated [¹⁴C]chloramphenicol over total substrate used. Quantitative CAT activities were corrected for transfection efficiencies as determined by ß-galactosidase activity using the ß-galactosidase assay kit (Promega) per the manufacturer's instructions. The percent increase or decrease of CAT activity was determined from the ratio of CAT activity in monensin-treated A10 cells over untreated control cells per promoter deletion construct. This assessment is limited per promoter deletion construct because of varying levels of basal transcriptional activation with each promoter deletion construct. The percent increase or decrease reflects the capacity for each promoter construct for Na⁺ activation of transcription.

CAT Assays of TgH₂-CAT Transgenic Rats
We used the TgH₂[-798]CAT transgenic rat model to validate the in vivo Na⁺ response of the H₂ Na,K-ATPase -798 5' flanking regulatory region.²¹ As described previously, this model reveals that the development and tissue-specific expression patterns of H₂ Na,K-ATPase gene expression, as reflected in the CAT activity of the -798 5' flanking region, are similar to R₂ Na,K-ATPase gene expression.²¹ The TgH₂[-798]CAT₈₈ line, Tg88, was chosen because of optimal expression in the aorta.²¹ Test Tg88 male rats (n=4) were given a high salt (8% NaCl) diet for 2 weeks. Littermate control Tg88 males (n=4) were maintained on normal rat chow. After 2 weeks of high salt challenge, protein extracts were obtained from aorta, brain, and heart. Aortic samples were pooled (two rats per sample) because of tissue size. All other tissues were analyzed individually.

All procedures were carried out in accordance with our institutional guidelines.

CAT assays were done on the cardiac left ventricle, brain, and aorta essentially as described.²² Protein concentrations were measured with the BCA assay (Pierce), and equal amounts of protein extract were used for CAT assays. CAT assays were set up essentially as described.²⁰

Southwestern Blot Analysis
Cell nuclear extracts were prepared essentially as described.²⁰ Southwestern blot analysis was done as described,²⁰ with the following specifications: Equal amounts of nuclear extracts were used for A10 cells and human glioblastoma (HTB-17 [ATCC]) cell line; denaturation and renaturation were done at 4°C. Monensin treatment was done as described above for RNA analysis and CAT assays. Random-primed labeling was used to make the ³²P-labeled double-stranded DNA probe. The standard protocol for Coomassie staining of protein gels was used. Competition was done with 10-fold and 100-fold molar excesses of unlabeled double-stranded DNA probe. The double-stranded DNA probe spans -798 to +67 of the H₂ Na,K-ATPase gene.

	Results

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Upregulation of ₁, ₂, and ß₁ Na,K-ATPase Gene Expression by Na⁺
Although an imperfect experimental system, a monensin-induced increase in [Na⁺]_i in rat A10 cells provides a strategic experimental model that, when coupled with complementary transgenic experiments, provides an integrated approach to the analysis of molecular mechanisms underlying salt-induced homeostatic and pathological changes. The rat A10 cell line (ATCC) is a clonal cell line derived from embryonic rat thoracic aorta that proliferates as myoblasts and develops into cells that phenotypically resemble smooth muscle cells,²³ albeit imperfectly. As an established clonal cell line, the variability seen in aortic smooth muscle primary cells is removed as an uncontrolled experimental variable. Additionally, consistency in transfection experiments testing for the functionality of different lengths of the 5' flanking regulatory region is maximized since the cell line is clonally derived and easily propagated.

Monensin is an antibiotic that mediates electroneutral exchange of external Na⁺ for internal H⁺, resulting in a rise in [Na⁺]_i and fall in [H⁺]_i.²⁴ In rat parotid acinar cells, monensin increased [Na⁺]_i in a concentration-dependent manner (0.01 to 100 µmol/L).²⁵ Stable incremental elevations of [Na⁺]_i by graded concentrations of monensin associated with increased Na,K-ATPase activity have also been described in adult rat liver (ARL) cells.²⁶ In cultured rat neurons and C6 glioma cells, 10 µmol/L monensin induced a twofold rise in [Na⁺]_i and stimulated Na,K-ATPase activity.²⁷ This was associated with an increase in pH_i by 0.4 to 0.5 unit and only a transient decline in membrane potential.²⁷ High doses (100 µmol/L) of monensin induce other non–Na⁺-related cellular effects. By ascertaining the experimental doses of monensin usage at 10 µmol/L, a true Na⁺ response will be identified while maximizing the measurement of differential responses of transcriptional activation by increased [Na⁺]_i.

RNA blot analysis of total cellular RNA of A10 cells treated with increasing doses of monensin (22 nmol/L to 100 µmol/L) showed an increase in RNA levels of ₁, ₂, and ß₁ Na,K-ATPase genes over baseline levels (Fig 1), in contrast with ß-actin and -tropomyosin genes, which remained unchanged up to 10 µmol/L monensin; these were then used as internal controls. At 100 µmol/L monensin, -tropomyosin decreased, indicating the specificity of the increase of ₁, ₂, and ß₁ Na,K-ATPase RNA levels. Quantitative analysis of RNA levels by densitometry corroborated the observed dose-specific upregulation of ₁, ₂, and ß₁ Na,K-ATPase RNA levels (Fig 1).

View larger version (25K): [in this window] [in a new window]   Figure 1. Quantitation of dose-response increase of steady-state RNA levels of 1, 2, and ß1 Na,K-ATPase in monensin-treated rat A10 cells. RNA blot analysis revealed increased 1, 2, and ß1 Na,K-ATPase levels in monensin-treated A10 cells in contrast to -tropomyosin RNA and ß-actin levels, which did not change (data not shown). Densitometric quantification of autoradiographic signals were analyzed using -tropomyosin and rRNA as controls. Compared with baseline control levels, 1, 2, and ß1 increased 1.8- to 3.4-fold at 0.022 µmol/L monensin and increased correspondingly with increasing monensin doses up to 30 µmol/L. At 9 µmol/L, 1 increased 6.5-fold, 2 increased 5-fold, and ß1 increased 3-fold. The 1 isoform increased markedly (12-fold) at 30 µmol/L, with 2 increasing 6.7-fold and ß1 5.3-fold.

Transcriptional Upregulation of ₁ and ₂ Na,K-ATPase Genes
The comparative advantage inherent in the analysis of the 5' flanking region of two -subunit isoform genes, ₁ and ₂ Na,K-ATPase genes, and of two species, rat and human, directed the design of the current study. Genomic clones of the 5' flanking regulatory region of both R₁[-1288] Na,K-ATPase and H₂[-798] Na,K-ATPase were isolated and characterized by sequence analysis, confirming identities with previously published sequences^{18 19} (data not shown). Promoter-reporter gene (CAT) expression minigenes were then constructed for R₁[-1288] and H₂[-798] and tested for functionality by CAT assays. Promoter-reporter gene expression experiments documented basal activity of both R₁[-1288]CAT and H₂[-798]CAT in rat A10 (ATCC) cells and induction by 10 and 100 µmol/L monensin treatment for 16 hours (data not shown). A panel of promoter deletion constructs was then made for both R₁[-1288] and H₂[-798] and tested together using identical experimental conditions and equimolar amounts of promoter deletion constructs (Figs 2 and 3). The ß-galactosidase (pSVß) plasmid was cotransfected as internal control for transfection efficiency. Autoradiographic signals were quantitated by densitometry and ß-galactosidase activity was corrected for each point. The percent increase or decrease in CAT activity with monensin treatment compared with baseline control conditions was taken as the Na⁺-induced activation/deactivation of transcription. Because basal activity is affected by promoter deletions,²⁸ the Na⁺ response was defined by the ratio of monensin-treated CAT activity over baseline per promoter deletion construct.

View larger version (45K): [in this window] [in a new window]   Figure 2. A, Quantitative analysis of promoter deletion studies of rat 1 Na,K-ATPase gene expression in rat A10 cells treated with 10 µmol/L monensin. Per promoter deletion construct, the percent increase in chloramphenicol acetyltransferase (CAT) activity in monensin-treated cells compared with baseline control cells is depicted alongside the respective promoter construct. All points were corrected for ß-galactosidase enzyme activity. Equimolar amounts of promoter deletion constructs were used. The promoter deletion studies indicate putative positive Na+-response element (Na-RE) in the region between -358 and -169 in rat 1 Na,K-ATPase as well as putative negative and other positive modulatory elements between -1288 and -375. B, Representative CAT assay experiments of rat 1 Na,K-ATPase deletion constructs show increased acetylated [14C]chloramphenicol products in 10 µmol/L monensin-treated (m) cells compared with control (c) baseline cells. Deletion constructs for rat 1 Na,K-ATPase (-375, -358, and -169) are presented. Equal amounts of cell extracts were used; transfection efficiency varied less than 10% for all points, validating visual comparative analysis. Because of changing levels of basal transcriptional activation with each promoter deletion construct, assessment of percent Na+ activation of transcription is valid per promoter construct only comparing CAT activity in monensin-treated vs control baseline conditions.

View larger version (57K): [in this window] [in a new window]   Figure 3. A, Quantitative analysis of promoter deletion studies of human 2 Na,K-ATPase gene expression in rat A10 cells treated with monensin. Percent increase in chloramphenicol acetyltransferase (CAT) activity in monensin-treated cells compared with baseline control cells is depicted alongside the respective promoter construct. Experimental specifications are identical to those described in Fig 2 legend for 1 Na,K-ATPase gene analysis. Promoter deletion constructs indicate putative positive Na+-response element (Na-RE) in the region between -301 and -1 in human 2 Na,K-ATPase as well as putative negative and other positive modulatory elements between -798 and -570. B, Representative CAT assay experiments of human 2 Na,K-ATPase deletion constructs show increased acetylated [14C]chloramphenicol products in monensin-treated (m) cells compared with control (c) baseline cells. Deletion constructs for human 2 Na,K-ATPase (-642, -570, and -301) are presented. The pCAT-basic plasmid used as negative control (-) and pCAT-enhancer plasmid used as positive control (+) do not show induction of gene expression in identical experimental conditions, indicating the specificity of Na+ response of the human 2 Na,K-ATPase gene constructs as well as of the rat 1 Na,K-ATPase gene (Fig 2). Experimental specifications were as described in Fig 2B legend.

As seen in Figs 2A and 3A, a spectrum of monensin-induced increased CAT activity was detected. The highest levels of expression were detected with the R₁[-375]CAT construct, at an 80% increase, and the R₁[-358]CAT construct, at a 110% increase (Fig 2A). A lower induction level was noted with the R₁[-169]CAT construct. These data delineate a putative Na⁺-response element (Na⁺-RE) between -358 and -169 in the R₁ Na,K-ATPase gene (Fig 2A and 2B) and indicate that other modifying elements, both positive and negative, are most likely present farther 5' between -1288 and -358. Likewise, for H₂ Na,K-ATPase deletion regulatory CAT constructs, peak induction of CAT activity was detected with the -301 CAT construct (Fig 3A and 3B), followed by 240% induction at -570 and 50% induction at the -798 and -1576 CAT constructs. These data delineate a putative major Na⁺-RE within 301 bp of the transcription start site of the H₂ Na,K-ATPase 5' flanking region but indicate that positive and negative cis-acting regulatory elements modulating the Na⁺ response also exist farther 5' between -1576 and -301.

As seen in representative CAT assays wherein transfection variation was less than 10% for valid visual comparative analysis (Figs 2B and 3B), both R₁ and H₂ promoter deletion constructs exhibited upregulation with monensin treatment compared with baseline conditions; this upregulation varied per promoter deletion construct (Figs 2A and 3A). Additionally, the baseline level of R₁ Na,K-ATPase promoter-CAT construct expression was greater than H₂ Na,K-ATPase promoter-CAT construct expression, consistent with the relative ratios of endogenous levels of ₁ to ₂ Na,K-ATPase RNA in rat A10 cells (Fig 1).

Sodium Activation of H₂[-798]CAT Transgene
To establish in vivo validation of the previously undescribed induction of H₂[-798]CAT in rat A10 cells by monensin-induced increased [Na⁺]_i, we analyzed salt-loaded (8% NaCl diet challenge) transgenic Sprague-Dawley rats homozygous for the H₂[-798]CAT gene (TgH₂[-798]CAT₈₈) compared with non–salt-loaded (regular diet) TgH₂[-798]CAT rats. The 8% NaCl diet is the established high salt diet used to elicit salt-sensitive hypertension in the Dahl salt-sensitive rat model.⁶ From four TgH₂[-798]CAT rat lines with parallel tissue-specific expression of the transgene, one line, Tg88, was chosen because it exhibited optimal H₂[-798]CAT transgene expression in aorta.²¹ Since the Sprague-Dawley rat strain is outbred, varying responses to salt loading are expected. This is an experimentally proven observation given that both the Dahl salt-sensitive and Dahl salt-resistant rat lines were derived from Sprague-Dawley rats. A representative experiment of salt-responding TgH₂[-798]CAT₈₈ rats is shown in Fig 4. In contrast to control transgene rat littermates kept on a regular salt (0.4% NaCl) diet, four Na⁺-loaded transgenic rats were found to exhibit Na⁺-response activation of H₂[-798]CAT transgene transcription. As seen in Fig 4, CAT assays of equal amounts of extracts from pooled aortas (two rats per sample) of Na⁺-loaded TgH₂[-798]CAT rats (lanes 1 through 4) on high salt rat chow demonstrated upregulation of H₂[-798]CAT activity compared with aortic extracts from control non–Na⁺-loaded TgH₂[-798]CAT rats (lanes 5 through 8) on normal rat chow. Tissue extracts from total heart (data not shown) and total brain (Fig 4) did not show Na⁺-induced changes in salt-loaded transgenic rats (lanes 1 through 4) compared with control transgenic rats (single lanes 5 through 8), indicating specificity of Na⁺ activation of H₂-CAT in aortic tissues. These data corroborate the in vitro data detecting upregulation of H₂ Na,K-ATPase promoter-CAT constructs in monensin-treated rat A10 cells (Fig 1B). These data mirror the induction of ₁ Na,K-ATPase gene expression in vitro in monensin-treated rat aortic A10 cell experiments as well as in vivo in aortas from salt-loaded rats¹³ and in transgenic rats bearing the ₁[-1288] promoter–directed minigene (V.L.M.H. et al, unpublished data, 1997).

View larger version (48K): [in this window] [in a new window]   Figure 4. Analysis of human (H) 2[-798]CAT gene expression in transgenic rats. Induction of chloramphenicol acetyltransferase (CAT) activity was observed in aortas of H2[-798]CAT transgenic Sprague-Dawley rats given a high salt (8% NaCl) diet (S, lanes 1 through 4) compared with aortas from H2[-798]CAT transgenic rats maintained on a regular rat diet (C, lanes 5 through 8). CAT assays of salt-loaded H2[-798]CAT transgenic rats demonstrate upregulation of transgene expression in aorta (S, pooled lane 1,2 and pooled lane 3,4) compared with control rat aortas (C, pooled lane 5,6 and pooled lane 7,8). This upregulation was not observed in extracts prepared from total brain homogenates from salt-loaded rats (S, lanes 1 through 4) compared with brain extracts from control rats (C, lanes 5 through 6). These data parallel previous observations of induction of rat 1 Na,K-ATPase mRNA levels detected in aorta13 in salt-loaded Sprague-Dawley rats. Results depict a representative experiment from salt-sensitive Sprague-Dawley rats. Being members of an outbred strain, not all Sprague-Dawley rats are salt sensitive.

Na⁺-Response Nuclear DNA Binding Protein
To investigate putative Na⁺-response trans-acting factors, we performed Southwestern blot analysis and gained insight into the size, amount, and minimal number of nuclear DNA binding proteins interacting with Na⁺-response regions in rat A10 cells treated with 10 µmol/L monensin to increase [Na⁺]_i. At the outset, the simple definition of a putative Na⁺-response DNA binding protein would be induction or unique presence in monensin-treated rat A10 cell nuclear extracts compared with control rat A10 cells. The H₂ Na,K-ATPase -798 5' flanking region was used as probe.

As seen in Fig 5, Southwestern blot analysis of rat A10 nuclear proteins revealed binding of the H₂[-798] probe to three nuclear DNA binding proteins with molecular masses of approximately 41, 85, and 95 kD (Fig 5A, lanes 3 and 4; Fig 5B, lanes 1 through 6). Interestingly, the 95-kD protein was increased in the monensin-treated rat A10 cells (Fig 5A, lane 4; Fig 5B, lanes 2, 4, and 6) compared with control untreated cells (Fig 5A, lane 3; Fig 5B, lanes 1, 3, and 5). As seen in Fig 5A, lanes 7 and 8, Southwestern blot analysis of human glioblastoma (HTB-17) cell nuclear extracts did not detect any specific binding of nuclear DNA binding proteins. Coomassie-stained lanes of both A10 (Fig 5A, lanes 1 and 2) and HTB-17 (Fig 5A, lanes 5 and 6) cells documented the equivalent amounts of nuclear extract protein loaded, supporting the specificity of nuclear DNA binding proteins detected. Furthermore, to show specificity by competition, we performed Southwestern blot analysis using identical experimental conditions with the addition of unlabeled -798 probe in 10-fold and 100-fold molar excesses (Fig 5B). "Cold" unlabeled probe (lanes 3 through 6) successfully diminished the amount of binding of the approximately 41-, 85-, and 95-kD nuclear DNA binding proteins compared with baseline conditions (lanes 1 and 2) in both untreated (Fig 5B, lanes 3 and 5) and monensin-treated (Fig 5B, lanes 4 and 6) A10 cells. This supports the specificity of the binding of the H₂[-798] probe to the approximately 41-, 85-, and 95-kD proteins. The upregulation of the 95-kD protein by monensin treatment suggests that this protein most likely represents a putative Na⁺-response transcription factor and provides a glimpse into the trans-acting component of the Na⁺-response transcriptional machinery. We note that the rat ₂ Na,K-ATPase probe gave identical results on Southwestern blot analysis (data not shown).

View larger version (65K): [in this window] [in a new window]   Figure 5. Southwestern blot analysis of Na+-response DNA binding proteins. A, Southwestern blot analysis of rat A10 nuclear extract (lanes 1 through 4) reveals binding of the human (H) 2[-798] 5' flanking probe to approximately 41-, 85-, and 95-kD nuclear DNA binding proteins (lanes 3 and 4). Interestingly, the 95-kD protein is increased in monensin-treated rat A10 cells (lane 4) compared with control untreated cells (lane 3). Coomassie-stained lanes document the quality and equal amount of nuclear extract protein loaded for control (lane 1) and monensin-treated (lane 2) cells. In contrast, analysis of equal amounts of human glioblastoma (HTB-17) nuclear extracts, control cells (lanes 5 and 7), and monensin-treated cells (lanes 6 and 8) did not detect any nuclear DNA binding by H2[-798] probe in identical Southwestern blotting experimental conditions (lanes 7 and 8). This supports the specificity of the nuclear DNA binding observed in rat A10 nuclear extracts (lanes 3 and 4). Coomassie-stained lanes reveal equivalent amounts of nuclear protein extract from HTB-17 cells in control (lane 5) and monensin-treated (lane 6) cells. Molecular weight markers (Bio-Rad) are noted on the left; from top to bottom: 130 000, 75 000, 50 000, 39 000, 27 000, and 17 000. B, Competition Southwestern blot analysis using the identical experimental conditions as described above but adding unlabeled -798 probe in molar excesses of 10-fold to 100-fold to show specificity by competition. Shown are baseline condition (lanes 1 and 2), 10-fold molar excess unlabeled probe (lanes 3 and 4), and 100-fold molar excess unlabeled probe (lanes 5 and 6). "Cold" unlabeled probe increasingly diminished the amount of binding of the approximately 41-, 85-, and 95-kD nuclear DNA binding proteins in both control A10 cells (lanes 1, 3, and 5) and monensin-treated cells (lanes 2, 4, and 6).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Perspective on Mechanisms
This study documents the involvement of transcriptional events in salt-induced response mechanisms. The data include in vitro evidence delineating the components of a Na⁺-response transcriptional machinery using the ₁ and ₂ Na,K-ATPase genes as target genes dually investigating both cis-acting and trans-acting aspects. Data from the analyses of ₁[-1288] and ₂[-798] Na,K-ATPase promoter regions identify a positive Na⁺-response regulatory region to within -358 bp from the transcription start site in both 5' flanking regions. When our data delimiting the Na⁺-response region to -358 to -169 in the ₁ Na,K-ATPase gene are correlated with previous observations by Suzuki-Yagawa et al²⁸ defining a positive basal regulatory region for ₁ Na,K-ATPase gene expression in five different cell lines (MDCK canine kidney, HepG2 human liver, L6 rat skeletal myoblast, B103 rat neuroblastoma, and 3Y1 rat embryo fibroblast) to be between -155 and -49, it becomes apparent that the ₁ Na,K-ATPase gene Na⁺-response regulatory region is outside the basal activation regulatory region. These data suggest that the region from -358 to -169 in the ₁ Na,K-ATPase gene most likely contains a positive Na⁺-RE inducing basal activation in the presence of increased intracellular Na⁺. A parallel situation is predicted in the H₂ Na,K-ATPase gene because the Na⁺-response region is delineated within -301 bp of the 5' flanking sequences (Figs 2 and 3) and a basal activation region has been delineated within -175 to -108 of the 5' flanking sequences in the R₂ Na,K-ATPase gene.²⁹ The identification of the core consensus sequences of the putative Na⁺-RE remains to be determined, but the current data localize the putative Na⁺-RE to within an approximately 200-bp region. Sequence homology between R₁ and H₂ Na,K-ATPase in the Na⁺-response region would facilitate further testing for putative core consensus sequences of Na⁺-RE(s). Determining whether there is a conserved Na⁺-RE between the ₁ and ₂ Na,K-ATPase genes or whether their respective Na⁺-REs are distinct would also be highly informative. Distinct Na⁺-RE consensus sequences would indicate a far more complex molecular mechanism underlying salt-load–induced responses.

The observation of a greater Na⁺ response by the promoter deletion constructs R₁[-358]CAT and H₂[-301]CAT compared with longer-length promoter constructs reveals the likely presence of negative and other positive regulatory sites 5' to these proximal regions. Intuitively, the use of multiple positive and negative elements could contribute to mechanisms of specificity and versatility of Na⁺-response gene regulation in different pathophysiological states and remains to be elucidated.

This study also provides insight into trans-acting components of the hypothesized Na⁺-response transcriptional mechanism. The coordinate upregulation of a nuclear DNA binding protein (an approximately 95-kD protein) and the induction of gene expression of its target gene (₂ Na,K-ATPase gene) suggest a positive regulatory mechanism of gene regulation. A priori, a positive feedback mechanism would ensure the continuing upregulation with persistent increased [Na⁺]_i. Intuitively, the negative regulatory elements deduced from the promoter deletion expression analysis (Fig 3) would be involved in the turning off of the Na⁺ response when [Na⁺]_i is normalized or presumably when a new "steady state" is reached.

The concordance of our experimental in vitro tissue culture data and in vivo experimental rat and transgenic rat data strengthen the deduced implications as bona fide insights into a Na⁺-response mechanism. More specifically, these parallel molecular events that we have documented are as follows: (1) R₁-CAT activity is greater than H₂-CAT activity in rat aortic A10 cells, just as ₁ Na,K-ATPase RNA levels are greater than ₂ Na,K-ATPase RNA levels in rat aorta¹³ and rat A10 cells (Fig 1); (2) both ₁ and ß₁ Na,K-ATPase mRNA levels increase on increased [Na⁺]_i by monensin in in vitro rat A10 cells (Fig 1) as well as by high salt intake in in vivo rat studies¹³ ; (3) H₂-CAT gene expression is induced by increased Na⁺ load in rat A10 cell expression experiments (Figs 2 and 3) as well as in transgenic TgH₂-CAT rats given a high salt diet (Fig 4); and (4) the R₁[-1288] promoter exhibits Na⁺ load–induced activation in transgenic Dahl salt-sensitive rats (unpublished data, 1997). These data suggest that the Na⁺-RE core sequences to be identified using R₁ and H₂ Na,K-ATPase as target genes in the rat A10 monensin expression system will be biologically relevant.

The Na⁺-response regulatory region delineated in the monensin–rat A10 aortic smooth muscle cell experimental system described here is most likely distinct from the Na⁺-response region proposed by Yamamoto et al,³⁰ which was delineated experimentally in rat primary cardiomyocytes treated with 1 mmol/L ouabain. Ouabain induced increases in the gene expression of all three Na,K-ATPase -subunit isoforms, which peaked at 60 minutes in isolated rat heart cardiocytes and declined significantly thereafter.³⁰ The authors attributed this transient induction of gene expression to measured increases in [Na⁺]_i on incubation with 1 mmol/L ouabain³⁰ ; however, they did not explain the deinduction of ₁-, ₂-, and ₃-subunit RNA levels after 60 minutes despite the continuing incubation in ouabain and the ensuing increase in [Na⁺]_i.³⁰ Their observations of a transient (60-minute) Na⁺ response³⁰ are in contrast to our in vitro data showing the persistence of Na⁺ induction at 16 to 24 hours in rat A10 cells treated with monensin (Fig 1) and to previous in vivo data showing induction by high salt intake of ₁ Na,K-ATPase gene expression in kidney after 3 days,¹² in aorta after 2 and 4 weeks,¹³ and in kidney after 8 weeks (V.L.M.H. et al, unpublished results, 1997) of high salt intake.

Perspective on Pathophysiology
The parallel study of both the ₁ and ₂ Na,K-ATPase genes is relevant to the study of salt-sensitive hypertension since ₁ Na,K-ATPase is the sole -subunit isoform in renal epithelial cells,^{31 32} whereas ₁ and ₂ Na,K-ATPases are both present in vascular media.¹³ Delineation of a Na⁺-response mechanism involving ₁ and ₂ Na,K-ATPase genes would contribute to the hierarchical dissection of salt-induced molecular events that are pertinent to hypertensive renal and vascular pathology. With the delineation of ₁ and ₂ Na,K-ATPase genes as Na⁺-response target genes, it could be hypothesized that the effects of their individual (renal) or combinatorial (vascular) modulation could affect integrated tissue responses to multiple blood pressure regulatory systems. This deduction is based on data implicating Na,K-ATPases to be involved in respective physiological effects of various blood pressure regulatory hormones, such as norepinephrine,^{33 34} dopamine,^{33 34} angiotensin,^{34 35} and atrial natriuretic peptide.³⁴ Given a normal genetic background, these Na⁺-induced responses could be expected to be homeostatic. Given a pathological genetic background, however, these Na⁺-induced responses could contribute to salt-induced hypertension, as seen in the Dahl salt-sensitive inbred rat,⁶ and in salt-induced stroke and salt-induced increase in cardiac hypertrophy, as seen in the SHRSP model.^{9 10} Additionally, Na⁺-induced upregulation of ₁ Na,K-ATPase gene expression could affect cell growth characteristics of vascular smooth muscle cells since cell proliferation is associated with higher levels of ₁ Na,K-ATPase gene expression and since ₁ Na,K-ATPase activity has been implicated as a general mechanism by which basal transcriptional activity of c-fos is modulated.³⁶ This association might also account for the increase in cardiac hypertrophy observed in salt-loaded SHRSP compared with non–salt-loaded SHRSP despite equivalent blood pressure levels.⁹ Their respective roles in Na⁺-induced homeostatic and pathophysiological responses remain to be explored and defined.

Because Na⁺-induced changes and pressure-overload changes are equally cogent to the pathogenesis of hypertension, it is interesting to note that ₂ Na,K-ATPase is induced by Na⁺ load but deinduced by pressure overload.¹³ In fact, this has been experimentally documented in the same transgenic rat model.²¹ These observations suggest a logical mechanistic hypothesis that pressure-overload deinduction of ₂ Na,K-ATPase overrides the initial Na⁺-response induction of ₂ Na,K-ATPase in aorta. It is significant to note that our experimental data indicate that this hypothesis most likely applies to human pathogenic mechanisms as well. This hypothesis remains to be tested in other target gene systems.

The observations of parallel events in both R₂ and H₂ Na,K-ATPase gene regulation in basal tissue-specific expression as well as in responses to increased pressure overload²¹ and increased Na⁺ load (the present study) imply that the Na⁺ activation of H₂ Na,K-ATPase observed here is a bona fide pattern of modulation of the human gene. The concordance of tissue culture and transgenic observations attests to the validation of the observation on H₂ Na,K-ATPase regulation, albeit in rat A10 cells and in transgenic rats. These experimental approximations provide a valid extension of derived hypotheses into human pathogenic mechanisms.

	Acknowledgments

 
This work was supported by National Institutes of Health grant HL-48903. We acknowledge Fang Qing, Lyle V. Lopez, and Alec Goodman for excellent technical assistance and Ari Tsikoudakis for preparation of the manuscript and graphics.

Received September 6, 1996; first decision October 10, 1996; accepted January 15, 1997.

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