1 Physiologisches Institut der Universität München, D-80336 Münich, Germany; and 2 Department of Medicine, Johns Hopkins University, Baltimore, Maryland 21205
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ABSTRACT |
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In response to ambient hypertonicity, TonEBP (tonicity-responsive enhancer binding protein) stimulates certain genes including those encoding cytokines, transporters for organic solutes, and a molecular chaperone. TonEBP is regulated in a bidirectional manner, upregulated by an increase in ambient tonicity while downregulated by a decrease. To investigate the role of intracellular ionic strength in the activity of TonEBP, we subjected Madin-Darby canine kidney cells to a variety of conditions. Electron microprobe analysis was performed to measure intracellular electrolytes. Under conditions in which changes in cell volume were similar, TonEBP activity correlated with the intracellular ionic strength regardless of the external tonicity. On the other hand, inhibition of the Na+/K+-ATPase and high external K+ concentration led to a decreased activity of TonEBP despite a marked increase in the intracellular ionic strength. Because isotonic swelling is known to occur under these conditions, these data suggest that dilution of the cytoplasmic constituents inhibits the activity of TonEBP. We conclude that intracellular ionic strength and water content are major factors that determine the activity of TonEBP.
cell ionic strength; cell volume; organic osmolytes; heat shock protein 70; Madin-Darby canine kidney cells
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INTRODUCTION |
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TONICITY-RESPONSIVE
ENHANCER BINDING PROTEIN (TonEBP) is a transcriptional activator
of the Rel family that includes nuclear factor-B (NF-
B) and
nuclear factor of activated T cell (NFAT). This family is
defined by similarity in structure rather than by the amino acid
sequence of the DNA binding domain (24). TonEBP is
expressed in many tissues including brain, heart, kidney, and T cells
(14, 25). So far, the function of TonEBP has been elucidated only in activated T cells and the kidney. In T cells, TonEBP
is induced following activation of the T-cell receptors. TonEBP
stimulates transcription of cytokines, including TNF-
and
lymphotoxin-
, in a manner dependent on the ambient tonicity or
concentration of NaCl (11).
In the renal medulla, TonEBP plays a key role in protection of cells from the deleterious effects of hypertonicity (hyperosmotic concentration of salt) and high urea. In the rat kidney, medullary cells are bathed in interstitial fluid containing typically ~800 mosomol/kgH2O of salt and over 1,000 mosomol/kgH2O of urea. Hypertonicity causes an increase in the intracellular ionic strength and double-stranded DNA breaks (9), leading to cell cycle arrest or cell death depending on the intensity of hypertonicity (13). Cells in the renal medulla accumulate high concentrations of organic osmolytes, which allows intracellular ionic strength to be maintained at a "normal" level via osmotic replacement and, as a result, protects cells from the deleterious effects of hypertonicity (1). TonEBP stimulates transcription of genes encoding transporters and a synthetic enzyme for organic osmolytes: sodium/myo-inositol cotransporter (SMIT), the sodium-chloride-betaine cotransporter (BGT1), and aldose reductase (AR), which synthesizes sorbitol (28). TonEBP also stimulates transcription of a heat shock protein 70 (HSP70) gene (29) that protects cells from the potentially lethal effects of urea (19). In addition to these protective effects, TonEBP stimulates expression of a renal medullary-specific gene encoding the vasopressin-regulated urea transporters (UT-A) (18). It is interesting to note that TonEBP contributes to the accumulation of a high concentration of urea in the renal medulla by stimulating the UT-A gene but, at the same time, protects the cells against the effects of a high urea concentration by stimulating expression of a HSP70 gene. Thus TonEBP is an important regulator of many pathways in the hyperosmotic renal medulla.
In cultured kidney cells, activity of TonEBP is stimulated in response to hypertonicity via several pathways. In response to hypertonicity, phosphorylation and nuclear localization of TonEBP are increased in Madin-Darby canine kidney (MDCK) cells after 30 min (4). Activity of the transactivation domain of TonEBP is also stimulated by hypertonicity (6). TonEBP abundance is increased fourfold after 12 h of exposure to hypertonicity as a result of an increase in mRNA abundance and increased synthesis of TonEBP (27). Interestingly, the TonEBP response to ambient tonicity is bidirectional. When cells are exposed to hypotonicity (low osmolality), nuclear localization, activity of transactivation, and abundance of TonEBP all decrease, leading to reduced expression of TonEBP target genes, i.e., the exact opposite of what happens when cells are exposed to hypertonicity (6, 27).
TonEBP is expressed abundantly in the renal medulla, whereas its expression is much lower in the cortex. In response to water diuresis, the nuclear localization (or distribution) of TonEBP decreases markedly, leading to a reduced expression of the SMIT mRNA. In antidiuresis, the nuclear distribution of TonEBP increases, leading to higher expression of the SMIT mRNA (3). These data clearly establish that TonEBP is regulated in vivo in response to changes in water intake.
Despite the remarkable progress in understanding the function of TonEBP, just how TonEBP senses changes in ambient tonicity is not well understood. On the basis of inhibitor studies, a role for p38 MAP kinase has been proposed (17). Because these inhibitors do not affect phosphorylation of TonEBP (4), the role of p38 MAP kinase is not yet understood. Induction of AR activity in response to hypertonicity correlates best with the sum of Na+ and K+ concentrations, or ionic strength, inside the cell (26). It has therefore been proposed that intracellular ionic strength stimulates transcription of the AR gene, presumably via activation of TonEBP. In this study, we confirmed that the intracellular ionic strength correlates with activity of TonEBP under certain conditions. Unexpectedly, the data also suggest that TonEBP is inhibited by increased water content of the cell or dilution of the cytoplasmic constituents.
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MATERIALS AND METHODS |
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Cell culture. MDCK cells were cultured in a defined medium made by mixing equal parts of Dulbecco's modified Eagle's medium and Coon's modified Ham's F-12 medium as described previously (27). Cells were grown to confluence in isotonic medium to form monolayers before being treated with experimental media. Normal isotonic medium contained 110 mM NaCl. The concentration of NaCl was changed to 35 or 185 mM NaCl to make hypotonic or hypertonic medium, respectively. Where indicated, the medium was made hypertonic by addition of 75 mM KCl. Betaine (100 mM) was added to the hypotonic medium to make an isotonic medium labeled "200 + Bet" (see Figs. 4-6).
Preparation of freeze-dried cryosections and electron microprobe
analysis.
For electron microprobe analysis, about 300,000 cells were seeded on
collagen-coated, permeable, 12-mm filter membranes (Millicell-CM; Millipore, Bedford, MA). Cells were cultured for 3 days to form confluent monolayers, whereafter appropriate experimental media were
applied to both upper and lower sides of the filter. At the end of the
incubation period, the medium was removed rapidly from both sides of
the filter and a thin layer of the same medium containing 0.25 g/ml of
BSA (Behring, Marburg, Germany) was applied to both sides of the
filter. The filter with BSA solution was frozen rapidly in a 3:1
(vol/vol) mixture of propane and isopentane cooled in liquid nitrogen
(196°C). Cryosections (1 µm) were cut at
80°C, freeze-dried,
and analyzed in a scanning transmission electron microscope equipped
with an energy-dispersive X-ray detector system. The collected X-ray
spectra were quantified as described elsewhere (2).
Presentation of electrolyte data and statistical analysis. Intracellular electrolyte concentrations are given as means ± SE in mmol/kg wet wt. For each group, electron microprobe analyses were performed on at least four filters, with 7-10 cells analyzed per individual filter. The mean intracellular electrolyte concentrations obtained from one filter was taken as one data point. The significance of differences between two means was established with Student's t-test for unpaired samples, with P < 0.05 indicating significance. For more than two groups, one-way ANOVA was used (SPSS/PC; SPSS, Chicago, IL). If significant effects were detected, t-tests were used to determine the significance of differences between individual means, with the significance levels appropriately adjusted (22).
Northern blot analysis. RNA was isolated from MDCK cells grown on plastic dishes by using Trizol reagent (Invitrogen, Carlsbad, CA). RNA (5-8 µg) from each sample was separated on 1% agarose gel containing 2.2 M formaldehyde and transferred to a nitrocellulose membrane. Membranes were hybridized overnight with 32P-labeled human AR (GenBank accession no. J05474), canine BGT1 (M80403), human HSP70 (M11717), canine SMIT (M85068), or human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; X01677) cDNA. After washing under stringent conditions (60°C in 75 mM NaCl, 7.5 mM Na-citrate, and 0.1% SDS), radioactivity was visualized and quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). All experiments were performed in three different passages of cells (n = 3). Because one-way ANOVA detected significant differences in the data set shown in each of the graphs in Figs. 2B and 5B, paired t-tests were performed to compare two groups using Excel software (Microsoft, Redmond, WA), with P < 0.05 indicating significance.
Immunoblot analyses. Cells were lysed for 30 min at 4°C in lysis buffer (50 mM Tris-Cl, pH 7.6, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100) with freshly added protease inhibitors: 0.2 µg/ml aprotinin, 5 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 10 µM E64. After clearing by centrifugation, an aliquot containing 40 µg of protein from each sample was separated on a 7% SDS-polyacrylamide gel and blotted onto a nitrocellulose membrane. To detect TonEBP, we incubated the blots with a 1:4,000 dilution of the TonEBP antiserum (14) for 1 h in 20 mM Tris · HCl, pH 7.6, 150 mM NaCl, 0.1% Tween 20, and 5% nonfat milk. To detect HSC70, the constitutive isoform of HSP70, we used a commercial antibody from Stressgen (Victoria, BC, Canada) at a 1:400 dilution. The blots were then incubated with a secondary antibody conjugated with alkaline phosphatase and visualized with a commercial substrate for alkaline phosphatase (Sigma Chemical, St. Louis, MO).
Immunohistochemistry. Cells were grown to confluence on 18 × 18-mm glass coverslips before experimental treatments. Cells on coverslips were fixed for 15 min in 3% paraformaldehyde in PBS and permeabilized for 15 min in 0.5% Triton X-100 in Tris-buffered saline. The cells were then incubated for 30 min in a 1:400 dilution of TonEBP antiserum (14) in PBS at room temperature. TonEBP was visualized by incubation in a 1:400 dilution of Alexa 568-conjugated goat anti-rabbit antiserum (Molecular Probes, Eugene, OR) in PBS containing 3% BSA. The coverslips were mounted in Prolong antifade (Molecular Probes) for observation.
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RESULTS |
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The goal of this study was to test the hypothesis that
intracellular ionic strength signals to TonEBP. To achieve this goal, we manipulated intracellular ionic strength using a variety of methods
under different ambient tonicity conditions and investigated subsequent
changes in the activity of TonEBP. Concentrations of Na+,
K+, and Cl were measured by using electron
microprobe analysis (2), and their sum was taken as a
measure of intracellular ionic strength (16). TonEBP
activity on transcription was measured by Northern analyses of the
TonEBP target genes AR, BGT1, HSP70, and SMIT. The abundance and
nucleocytoplasmic distribution of TonEBP were determined by using
immunoblot and immunohistochemical analyses, respectively. Because
changes in TonEBP activity are maximal in the first 12 h of shift
in tonicity (14, 27), we examined TonEBP activity within
8 h of treatments to measure initial changes with greater sensitivity.
Effects of ouabain in isotonicity.
Under isotonic conditions, K+ was the predominant
electrolyte inside the cell (Fig. 1).
Ouabain treatment dramatically reduced the concentration of
K+ while increasing the concentrations of Na+
and Cl, indicating that the Na+,
K+, and Cl
are present almost exclusively in
ionic form. The sum of Na+, K+, and
Cl
concentrations, i.e., ionic strength, increased
significantly in ouabain-treated cells to a level comparable to that in
cells in medium made hypertonic by addition of 75 mM NaCl (Fig. 1). Despite the rise in intracellular ionic strength, the mRNA abundance of
TonEBP target genes in the ouabain-treated cells did not increase as in
those cells treated with NaCl hypertonicity, except for small but
significant increases in BGT1 mRNA at 8 h and in SMIT mRNA at
4 h (Fig. 2B). In fact,
the mRNA abundance of AR and HSP70 actually decreased significantly
following exposure to ouabain for 8 h. The abundance of TonEBP
protein decreased consistently in cells treated with ouabain for 4 and
8 h (Fig. 3, top). There were no obvious differences in nucleocytoplasmic distribution of TonEBP
between control and ouabain-treated cells (Fig. 3, bottom). Thus ouabain treatment resulted in lower abundance of TonEBP protein and its activity, despite an increase in the intracellular ionic strength.
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NaCl vs. KCl as hypertonic agent.
When the medium tonicity was raised by the addition of 75 mM NaCl or
KCl, the abundance and nuclear localization of TonEBP increased
similarly (Fig. 3). However, NaCl and KCl differed in their effects on
cell electrolytes and mRNA abundance of TonEBP target genes. The KCl
hypertonicity increased the cellular ionic strength significantly more
than the NaCl hypertonicity due to higher concentrations of
K+ and Cl (Fig. 1). Whereas the NaCl
hypertonicity clearly increased the mRNA abundance of all four genes
(P < 0.05), the KCl hypertonicity did so only modestly
(Fig. 2). In fact, after 8 h of exposure to KCl hypertonicity, the
increase in mRNA abundance was either at the borderline of significance
(P = 0.05 for BGT1 and SMIT) or not significant
(P > 0.05 for AR and HSP70). Although the
intracellular ionic strength was higher in cells treated with the KCl
hypertonicity than those cells treated with the NaCl hypertonicity,
transcriptional activity of TonEBP measured by expression of its target
genes was lower, despite comparable abundance of TonEBP.
Effects of ouabain in hypertonicity. In the NaCl or KCl hypertonic medium, ouabain treatment increased the intracellular ionic strength as in isotonic medium (Fig. 1). In cells switched to the NaCl hypertonicity, the abundance of TonEBP decreased following ouabain treatment without changes in nucleocytoplasmic distribution of TonEBP (Fig. 3). In cells switched to the KCl hypertonicity, however, TonEBP abundance was not affected by the ouabain treatment. The same trend was apparent for mRNA abundance: with the NaCl hypertonicity, ouabain treatment for 8 h significantly decreased mRNA abundance of AR, BGT1, and SMIT, whereas with the KCl hypertonicity, only the decrease in SMIT mRNA abundance was significant (Fig. 2). The abundance of HSP70 mRNA was not affected by ouabain in cells treated with either hypertonic medium, indicating that the regulation of the HSP70 promoter is more complex than that of other TonEBP target genes. Thus, under the NaCl hypertonicity, ouabain treatment resulted in lower abundance and activity of TonEBP despite increased intracellular ionic strength. With the KCl hypertonicity, however, ouabain decreased the activity of TonEBP without affecting the abundance of TonEBP protein.
Effects of hypotonicity and betaine loading.
The data in Figs. 1-3 demonstrate that ouabain treatment and KCl
hypertonicity decreased the activity of TonEBP despite an increase in
the intracellular ionic strength. To examine the effect of a decrease
in the intracellular ionic strength, we switched MDCK cells to a
hypotonic medium (200 mosmol/kgH2O) or additionally loaded
them with betaine by providing 100 mM betaine in culture medium
(200 + Bet). Cellular ionic strength decreased similarly and
significantly in each case (Fig. 4).
Likewise, the abundance of TonEBP (see Fig. 6) and the mRNA abundance
of its target genes (Fig. 5) decreased
similarly and significantly in each case. Nucleocytoplasmic distribution of TonEBP was not different between the cells in hypotonic
medium and the cells loaded with betaine (not shown). Thus a decrease
in cellular ionic strength led to a clear decrease in TonEBP activity
regardless of extracellular tonicity, 200 or 300 mosmol/kgH2O (200 + Bet).
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DISCUSSION |
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The data in Figs. 4-6 support the hypothesis that the
intracellular ionic strength signals to TonEBP. A decrease in the
intracellular ionic strength led to decreased abundance and activity of
TonEBP regardless of the extracellular tonicity, 200 or 300 mosmol/kgH2O. In addition, an increase in the intracellular
ionic strength led to increased abundance and activity of TonEBP in
different tonicity of the extracellular fluid, 300 or 450 mosmol/kg
water. Although these data eliminate extracellular tonicity as the
signal for TonEBP activation, the role of cell volume is not resolved.
As summarized in Table 1, cell volume
increases when cells are exposed to hypotonicity. It is hard to predict
changes in cell volume by betaine loading, but it is unlikely to
decrease. Likewise, cell volume must have decreased when cells were
switched from hypotonicity to isotonicity or when they were switched
from isotonicity (200 + Bet) to hypertonicity. We attempted to
manipulate the intracellular ionic strength and cell volume
independently by using the cationic ionophore nystatin and sucrose to
manipulate the extracellular oncotic pressure as others did
(8). However, even a maximal concentration of nystatin was
not an effective ionophore in MDCK cells in that the intracellular
concentrations of Na+ and K+ were only
moderately affected and the intracellular ionic strength did not
increase (not shown).
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Whereas the data discussed in Figs. 4-6 show a positive correlation between the activity of TonEBP and the intracellular ionic strength, other data in Figs. 1-3 display an inverse correlation, as summarized in Table 1. In all conditions where the activity of TonEBP decreased despite clear increase in the intracellular ionic strength, tremendous isotonic swelling is known to occur. For example, ouabain treatment swells the cortical collecting duct, leading to a doubling of its volume (23). Likewise, elevation of extracellular K+ concentration by replacing Na+ causes cell swelling due to influx of KCl (23). For technical reasons, we did not attempt to measure cell water directly. On the other hand, the intracellular concentration of phosphorus, an indirect measure of the concentration of organic molecules and, hence, of water content (12), decreased in the ouabain- or high K+-treated cells, although the decreases were not significant due to large variations (not shown). Assuming 80% water content and a constant amount of intracellular organic solute, calculations predict a >60% increase in cell water following the ouabain treatment shown in Fig. 1. Similar calculations predict a >50% increase in cell water following replacement of extracellular K+ in hypertonicity (+KCl vs. +NaCl). The increase in cell water content should cause a significant dilution of cytoplasmic constituents such as protein molecules. A change in the concentration of cytoplasmic constituents is thought to be a key factor determining the cellular response to a change in cell volume (21). We suggest that dilution of the cytoplasm is an important negative regulator of the TonEBP function that overrides the rise in the intracellular ionic strength. Inhibition by the dilution appears specific to TonEBP in that the abundance of HSC70 or G3PDH mRNA was not affected.
Whereas high extracellular K+ concentration and ouabain treatment inhibited TonEBP activity, they differed in their effects on the abundance of TonEBP. Ouabain treatment decreased the abundance of TonEBP, which must have contributed to the decreased activity of TonEBP as reflected in the abundance of mRNA for TonEBP target genes. On the other hand, high extracellular K+ concentration did not affect the abundance or nuclear localization of TonEBP. The activity of the transactivation domain at the COOH terminus of TonEBP is regulated by ambient tonicity in a bidirectional manner: increased by hypertonicity and decreased by hypotonicity (6). It is possible that the transactivation is inhibited by the high extracellular K+ concentration, perhaps due to factors other than the cytoplasmic dilution.
The abundance of HSP70 mRNA responded differently from that of other TonEBP target genes in response to K+ replacement and ouabain (Fig. 2). A high extracellular K+ concentration increases the expression of HSP70 (20), and this effect is not due to hypertonicity (5). Because HSP70 expression is affected by a number of factors, including heat and heat shock factors (15), pH, and a combination of low pH and urea (20), it is not surprising that ouabain did not reduce expression of HSP70 like other TonEBP target genes.
Uchida et al. (26) observed a decrease in the intracellular ionic strength in response to ouabain treatment rather than an increase, as reported in this study. It should be pointed out that in the former study, the exposure to hypertonicity and ouabain lasted 48 h, compared with the 4 or 8 h in the present study (Fig. 1). It is possible that cells adapt to the long-term exposure to ouabain by lowering the intracellular ionic strength. Alternatively, the differences may be due to the techniques used, electron microprobe analysis vs. conventional flame photometry combined with cell water measurement using [14C]urea. On the other hand, the activity of AR decreased by the ouabain treatment (26) in agreement with the decreased mRNA shown in Fig. 2.
The intracellular ionic strength is kept quite constant in the renal papilla in vivo even when the osmolality of the papilla changes dramatically (reviewed in Ref. 1). Changes in the osmolality in kidney medulla and in systemic body fluid due to changes in the diuretic state or other causes are likely to be gradual rather than abrupt. Because cell volume does not change appreciably, or changes much less when the ambient tonicity is changed slowly (10), changes in cell water content or the dilution/concentration of cellular constituents are probably rare. In view of the changes in TonEBP activity in the renal medulla in response to changes in the diuretic state (3), we speculate that intracellular ionic strength in the renal medulla is maintained at near-constant level because the ionic strength signals to TonEBP in a feedback manner, thereby regulating the cellular accumulation of organic osmolytes.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-42479 (to H. M. Kwon) and Deutsche Forschungsgemeinschaft Grants BE 963/10-1 and NE 839/1-1 (to F. X. Beck and W. Neuhofer). S. K. Woo was supported by a fellowship from the Juvenile Diabetes Foundation International. O. Nahm was supported by National Research Service Award DK-9960.
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. M. Kwon, Dept. of Medicine, Johns Hopkins Univ., 963 Ross Bldg., 720 Rutland Ave., Baltimore, MD 21205 (E-mail: mkwon{at}jhmi.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.
August 1, 2002;10.1152/ajpcell.00216.2002
Received 11 May 2002; accepted in final form 26 July 2002.
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