Tonicity-responsive enhancer binding protein (TonEBP) regulates transcription of tonicity responsive genes such as the sodium-myo-inositol cotransporter (SMIT), the sodium-chloride-betaine cotransporter (BGT1), and aldose reductase (AR). To characterize signals that activate TonEBP in Madin-Darby canine kidney (MDCK) cells, the abundance and nuclear distribution of TonEBP were studied after the osmolality of the culture medium was changed. Hypertonicity but not hyperosmolality is effective in activation of TonEBP as expected. Surprisingly, exposure to hypotonic medium leads to a dramatic downregulation of TonEBP both in abundance and nuclear distribution, indicating that under isotonic conditions, TonEBP is at a low-level activated state and can respond to both increase and decrease in tonicity. Additional experiments suggest that cellular ionic strength is the signal that initiates regulation of TonEBP. The increase in abundance of TonEBP is mediated by an increase in mRNA abundance and a parallel increase in synthesis of TonEBP. The stability of TonEBP mRNA is not affected by hypertonicity indicating that transcription plays a major role in the induction of TonEBP by hypertonicity.
- hypertonicity-stimulated transcription
- sodium-myo-inositol cotransporter
- sodium-chloride-betaine cotransporter
- aldose reductase
the osmolality of the renal medulla varies widely in response to changes in water balance. When concentrated urine is produced in response to water deprivation, the osmolality of the medullary interstitial fluid is very high due to accumulation of urea and NaCl. Although urea equilibrates across the plasma membrane, NaCl is restricted to the interstitial fluid and the ionic strength inside medullary cells remains isotonic (3, 34). The osmotic gap across the plasma membrane is made up by the cellular accumulation of compatible osmolytes such as myo-inositol, betaine, sorbitol, taurine, and glycerophosphorylcholine (9). The accumulation of compatible osmolytes is a widespread and fundamental cellular response to hypertonicity because bacteria and yeast (45) and nonrenal tissues of mammals, for example, brain (12, 24), endothelia (40), and macrophages (6), also accumulate compatible osmolytes when they are exposed to hypertonic (hypernatremic) insterstitial fluid.
When accumulation of compatible osmolytes is blocked, cells in hypertonic culture media fail to grow (36, 44) and die (14). In macrophages cultured in hypertonic medium, phagocytic activity (38) and production of prostaglandin E2 (39) require, the accumulation of compatible osmolytes. Thus compatible osmolytes protect cellular function from the adversity of hypertonicity, although the underlying molecular mechanism is not clearly understood. High cellular ionic strength, which occurs when accumulation of compatible osmolytes is blocked, interferes with a variety of cellular processes (45).
There has been a great deal of progress in understanding the process of osmolyte accumulation. Enzymes and membrane transporters responsible for osmolyte accumulation have been identified. Interestingly, transcription plays a key role in the regulation of these proteins. When cells are exposed to a hypertonic medium, transcription of genes coding for sodium-myo-inositol cotransporter (SMIT) (43), sodium-chloride-betaine cotransporter (BGT1) (37), and aldose reductase (AR) (32), which catalyzes synthesis of sorbitol, is markedly stimulated, leading to increased activity of the proteins and accumulation of compatible osmolytes. A common genomiccis-element named tonicity-responsive enhancer (TonE) or osmotic response element is responsible for the transcriptional regulation of SMIT (30), BGT1 (25, 26), and AR (8, 15). A protein that specifically interacts with TonE was identified and named TonEBP (26). The activity/abundance of TonEBP in the nucleus increases in parallel with transcription of the BGT1 and SMIT genes when Madin-Darby canine kidney (MDCK) cells are switched to a hypertonic medium (26, 27). In vivo footprinting analysis reveals that the increase in nuclear TonEBP activity/abundance results in a parallel increase in TonEBP binding to the TonE sites upstream of the SMIT (30) and BGT1 (25, 26) genes. Thus the increase in nuclear TonEBP appears to be the key event in stimulation of the SMIT and BGT1 genes in response to hypertonicity.
Recent cloning of TonEBP revealed that it has a DNA binding domain that shares ∼45% amino acid identity with the DNA binding domain of the NFAT family of transcription factors (27). Outside the DNA binding domain, TonEBP does not resemble the NFATs. TonEBP is functionally different from the NFAT family as well in that it does not interact with AP1 and it is not regulated by calcineurin (22). On the other hand, hypertonicity dramatically increases the abundance of TonEBP in the nucleus via two pathways (27, 41). First, the abundance of TonEBP is increased at the whole cell level, induction. Second, the proportion of TonEBP in the nucleus is also increased, nuclear redistribution. Interestingly, inhibition of proteasome leads to decreased induction of SMIT and BGT1 mRNA in response to hypertonicity secondary to inhibition of the nuclear redistribution of TonEBP without affecting its abundance (41). Thus both induction and nuclear redistribution contribute to the activity of TonEBP in the nucleus. In this study, we provide evidence that TonEBP is regulated by changes in cellular ionic strength.
MATERIALS AND METHODS
MDCK cells were maintained in defined medium that was made by mixing equal parts of DMEM and Coon's modified Ham's F-12 medium as described previously (42). Where indicated, osmolality of the medium was increased by addition of NaCl, raffinose, or urea. In some experiments, NaCl was removed from the medium (Biofluids) to make a hypotonic medium of 135 mosmol/kgH20.
Western blot analysis and immunostaining.
For Western blot analysis of TonEBP, cells were lysed by rocking for 30 min at 4°C in a buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing protease inhibitors: 0.2 μg/ml aprotinin, 5 μM leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 10 μM E64. After being cleared by centrifugation, an aliquot containing 40 μg protein was taken from each sample and separated on a 6% SDS-polyacrylamide gel. The gel was blotted onto a nitrocellulose membrane. To detect TonEBP, blots were incubated with the TonEBP antiserum (27) at a 2,000-fold dilution for 1 h in 20 mM Tris ⋅ HCl, pH 7.6, 150 mM NaCl, 0.1% Tween 20, and 5% nonfat milk. The blots were then incubated with a secondary antibody conjugated with alkaline phosphatase that was visualized by using a commercial substrate (Sigma Chemical). Immunostaining of MDCK cells was performed as described (27) with the following modification: the TonEBP antiserum and rhodamine-conjugated secondary antibody were incubated at 1:400 dilution.
Northern blot analysis.
RNA was isolated by using Trizol reagent (GIBCO/BRL). Ten micrograms of RNA from each sample were separated on an 1% agarose gel containing 2.2 M formaldehyde and transferred onto a nitrocellulose membrane. Membranes were hybridized overnight with radiolabeled SMIT (21) or TonEBP cDNA. Canine TonEBP cDNA corresponding to nucleotides 730–2187 of the human TonEBP (27) was obtained by using RT-PCR. This canine cDNA shares 96% of nucleotides with the human cDNA. After washing under stringent conditions [65°C in 0.5× standard sodium citrate (SSC)-75 mM NaCl and 7.5 mM Na3 citrate with 0.1% SDS], radioactivity was visualized and quantified by using a Phosphorimager (Molecular Dynamics). To measure degradation of mRNA, cells were treated with 5 μg/ml of actinomycin D for up to 9 h before isolation of RNA. At this concentration, actinomycin D inhibits >90% of RNA synthesis as measured by trichloroacetic acid-precipitable incorporation of [3H]uridine (data not shown).
Pulse-chase of TonEBP.
Confluent MDCK cells were labeled for 2 h in methionine-free DMEM (Gibco/BRL) supplemented with 20 μCi/ml of [35S]-methionine (Amersham). After labeling, cells were washed and chased in defined medium containing 2 mM nonradioactive methionine. Where indicated, the defined medium or methionine-free DMEM was made hypertonic by adding 100 mM NaCl. Cell lysate was prepared as described above, and an aliquot of 250 μg of protein from each sample was taken for immunoprecipitation by using 5 μl of the TonEBP antiserum. After 1-h incubation with rocking at 4°C, 50 μl of a 50% slurry of protein A-agarose were added to each mixture and the incubation was continued for another hour. Reaction mixtures were centrifuged, and the pellets were washed three times with the lysis buffer. Western analysis of supernatants indicated that more than 95% of TonEBP was precipitated. Washed pellets were separated on a 6% SDS-acrylamide gel and blotted onto nitrocellulose membranes. The membranes were sequentially subjected to Western analysis to locate TonEBP bands and to autoradiography to visualize radioactivity.
TonEBP is stimulated by hypertonicity rather than hyperosmolarity. In this study, stimulation of TonEBP is defined as an increase in TonEBP abundance in the nucleus because the nuclear abundance of TonEBP correlates with binding of TonEBP to the TonE sites and increased transcription (see above). When MDCK cells are switched to a hypertonic medium containing 200 mM raffinose, stimulation of TonEBP occurs due to a combination of increased abundance (i.e., induction) of TonEBP and increased distribution into the nucleus from the cytoplasm (27, 41). To characterize the TonEBP response further, cells were cultured in media with various concentrations of raffinose. As shown in Fig.1 A, induction of TonEBP reaches a maximum when the concentration of raffinose exceeds 150 mM. Addition of 100 mM NaCl is as effective as 200 mM raffinose in inducing TonEBP. Addition of 200 mM urea to the isotonic medium or to hypertonic medium containing an extra 100 mM NaCl does not affect the abundance of TonEBP compared with the same solution without urea. Nuclear distribution is also increased by addition of NaCl but not urea (Fig. 1 B). These results demonstrate that hypertonicity rather than hyperosmolality per se is the effective signal for activation of TonEBP. Hypertonicity activates both pathways of TonEBP regulation, induction (increased abundance) and increased nuclear distribution.
Cellular accumulation of betaine and inositol reduces induction of TonEBP. One of the immediate consequences of exposing cells to a hypertonic fluid is an increase in cellular ionic strength due to osmotic water loss. Induction of AR in cells exposed to various hypertonic media correlates highly with the cellular ionic strength (35). Induction of SMIT (19) and BGT1 (7) mRNA also appears to correlate with the cellular ion concentration because inhibiting the accumulation of compatible osmolytes (which should increase cellular ionic strength) leads to higher abundance of those mRNAs. To examine the role of cellular ionic strength in regulation of TonEBP, excess (5 mM) betaine or inositol was added to the medium to accelerate accumulation of the compatible osmolyte, which should lead to a reduction in the cellular ionic strength. As shown in Fig.2, addition of betaine reduces the abundance of TonEBP and its mRNA by ∼50% in hypertonic condition. A slight decrease in TonEBP abundance is also observed in isotonic conditions, most likely because the cellular ionic strength is reduced as a result of excess betaine accumulation. The mRNA abundance of SMIT (Fig. 2) and BGT1 (not shown) was decreased far more than 50% by addition of betaine. Addition of inositol, on the other hand, results in moderate inhibition. This is expected because the normal medium contains 70 μM inositol and nominally no betaine. Thus accumulation of compatible osmolytes reduces the abundance and induction of TonEBP and its downstream genes, SMIT and BGT1.
Hypotonicity downregulates TonEBP. To explore the effects of reducing cellular ionic strength further, we grew MDCK cells in normal isotonic medium and then switched to a hypotonic medium of 135 mosmol made by removing NaCl (see materials and methods). As shown in Figure3 A, the abundance of TonEBP declines slowly to ∼50% 12 h after the switch. Nuclear distribution of TonEBP also decreases slowly, and by 18 h the intensity of TonEBP immuno staining in nuclei is equal to or less than that in cytoplasm (Fig. 3 B). On the other hand, the abundance of mRNA for TonEBP and SMIT drops rapidly with half-times <3 h. Because the half-life of TonEBP mRNA is ∼6 h in cells in isotonic and hypertonic medium (see below), it appears that switching cells to hypotonic medium accelerates turnover of this mRNA. Thus lowering the cellular ionic strength by exposing cells to hypotonic medium results in downregulation of TonEBP via dual pathways, abundance and nuclear distribution. Taken together, the data in Figs. 1-3 demonstrate that the activity of TonEBP is regulated by changes in tonicity or cellular ionic strength in both directions, up and down.
Induction of TonEBP involves an increase in mRNA.
To understand the mechanism of TonEBP induction, the time course of the abundance of TonEBP and its mRNA was measured for 18 h after medium tonicity was raised. As shown in Fig.4, the abundance of TonEBP rises slowly and reaches a plateau about fourfold the level in isotonic cells at 12 h. The abundance of TonEBP mRNA also increases and reaches a peak of threefold at 12 h. Next, we asked whether the increase in mRNA abundance leads to increased synthesis of TonEBP. To answer this, MDCK cells were pulse-labeled with [35S]-methionine after they were cultured for 12 h in hypertonic or isotonic medium. Radioactivity of TonEBP immunoprecipitated from the pulsed cells shows that the rate of TonEBP synthesis is about three times higher in cells cultured in hypertonic medium compared with those in isotonic medium (Fig. 5B), commensurate with the abundance of TonEBP mRNA (Fig. 4). On the other hand, chasing [35S]-methionine for the next 22 h shows that the half-life of TonEBP is ∼10 h and that there is no apparent difference between isotonic and hypertonic conditions (Fig.5 C). It is concluded that increased synthesis of TonEBP secondary to the increase in mRNA abundance is responsible for the induction of TonEBP in response to hypertonicity.
To investigate the mechanism of the increase in TonEBP mRNA abundance in response to hypertonicity, we have attempted to measure transcription of the TonEBP gene employing nuclear runoff assays. Unfortunately, the signal of runoff transcripts from the TonEBP gene was too low to be reliably quantified, even though the run-off transcripts of SMIT and BGT1 genes consistently yielded clear signals as reported previously (37, 43). We speculate that the low signal of the TonEBP transcript may be due to structural features of the gene. Work in progress (not shown) reveals that the TonEBP gene is large (>200 kbp) with numerous small (>200 bp) exons and large (typically several kilobase pairs) introns. Therefore, the primary transcript of the TonEBP gene is not expected to hybridize efficiently to the immobilized cDNA sequences in the nuclear run-off assay. On the other hand, the stability of TonEBP mRNA measured after treatment with actinomycin D is not affected by hypertonicity (Fig.6). It is likely that transcription plays a major role in the induction of the TonEBP mRNA by hypertonicity.
To explore the signaling pathways leading to TonEBP, we focused our efforts on early (up to 18 h) changes in TonEBP in response to perturbations in tonicity. The observation that hypertonicity rather than hyperosmolality is effective in stimulating TonEBP (Fig. 1) suggests two possible initial signals. First, shrinkage of cells may activate a sensor in the plasma membrane. Such a mechanism is well described in yeast. When exposed to hypertonicity, two membrane sensors of yeast, Sln1p and Sho1p, are activated fully within minutes and their signals converge to activate Hog1p, an isozyme of the mitogen-activated protein kinases (MAPKs) (23). Activated Hog1p is responsible for the early phase (up to 60 min) induction of theGPD1 mRNA but not the late phase of mRNA induction (29).GPD1 encodes glycerol-3-phosphate dehydrogenase that is a key enzyme in synthesis of glycerol, the major compatible osmolyte in yeast (1).
In mammalian cells, a tyrosine kinase (16) and isoforms of MAPKs (reviewed in Ref. 17) are also activated within minutes of exposure to hypertonicity. Because activity of these kinases returns to the basal level before a significant increase in TonEBP activity takes place (i.e., ∼4 h), they may be an upstream regulator in a cascade of signaling events. Inhibition of these kinases does not affect induction of downstream genes such as SMIT and BGT1 (2, 20) and TonE-mediated transcription (18). However, SB203580, which is an inhibitor of p38, and PD098059, which is an inhibitor of mitogen-activated extracellular regulated kinase (ERK) kinase (MEK-1, an upstream kinase for ERKs), blunt induction of SMIT and BGT1 (6, 31), and AR- and TonE-mediated transcription (28). For the studies using the inhibitors, there are two issues to be resolved. First, none of these studies documents inhibition of p38 and ERKs at early time points, i.e., before activation of TonEBP. Second, SB203580 and PD098059 are potent inhibitors of cyclooxygenase-1 and -2 and thromboxane synthase (5), and potentially other signaling pathways whose role in the response to hypertonicity remains to be evaluated. Thus it is not clear whether the effects of the inhibitors are directly due to inhibition of p38 and MEK-1. On the other hand, in those studies where inhibition of p38 and c-Jun NH2-terminal kinase (JNK) is demonstrated as a result of overexpressing dominant negative forms of the upstream kinases (18), TonE-mediated luciferase expression is not affected. In addition, hyperosmolar urea activates ERKs and p38 (46) and JNK (4) but not TonEBP (Fig. 1). At this time, there is no direct evidence that any of the hypertonicity or shrinkage activated protein kinases are involved in activation of TonEBP.
A second candidate for the initial signal to TonEBP is the rise in cellular ionic strength as a result of osmotic water loss. The best support for this concept is a study where it was shown that induction of AR correlates tightly with cellular ionic strength whereas there is no correlation with cell volume measured by water content (35). Lowering cellular ionic strength in hypertonic (Fig. 2) and hypotonic conditions (Fig. 3) leads to down regulation of TonEBP, providing further support that ionic strength is the signal. In animals switched abruptly to antidiuresis after a chronic diuresis with water loading, cellular ionic strength in the renal medulla initially rises but returns to isotonic level within 24 h as compatible osmolytes are accumulated (33, 34). It is likely that TonEBP serves to regulate cellular ion concentration to isotonic levels (via regulating accumulation of compatible osmolytes) when medullary cells are exposed to hypertonic interstitial fluid.
Downregulation of TonEBP abundance and nuclear distribution in hypotonic medium (Fig. 3), the opposite of upregulation in hypertonicity, demonstrates that TonEBP is not at an inactive state under isotonic conditions. In isotonicity, TonEBP is at a low-level activated state, positioned to respond to both an increase or decrease in tonicity. In hypotonicity, the downregulation of TonEBP should contribute to the downregulation of SMIT (Fig.3) and BGT1 mRNA (not shown). Related to this, when astrocytes are cultured in a hypotonic medium of 180 mosmol made by a partial removal of NaCl, the activity of SMIT diminishes by ∼90% in 24 h (13).
In primary cultures of canine lens epithelial cells, raising the concentration of glucose to 25 mM in the culture medium leads to a dramatic accumulation of sorbitol to over 100 mM in cell water in 24 h (11). The accumulation of sorbitol appears to be driven by AR activity because inhibitors of AR prevent the accumulation. Coincident with the sorbitol accumulation, the abundance of SMIT mRNA drops by 70% in 24 h. When sorbitol accumulation is blocked by using inhibitors of AR, the downregulation of the SMIT mRNA is prevented (11). It is most likely that a decrease in the cellular ionic strength as a direct result of sorbitol accumulation is responsible for the downregulation of the SMIT mRNA via down regulation of TonEBP. This mechanism may explain depletion of cellular myo-inositol in certain tissues during hyperglycemia and might contribute to the pathogenesis of diabetic complication (10).
Although direct evidence is not available (see Results ), it is likely that the induction of TonEBP by hypertonicity is mediated by an increase in transcription of the TonEBP gene. Induction of TonEBP mRNA peaks at 12 h after the switch to hypertonicity (Fig. 4), several hours faster than mRNA of its downstream genes such as SMIT, BGT1, and AR that peak at ∼18 h (32, 37, 43). Thus it appears that TonEBP is an early gene to SMIT, BGT1, and AR in the process leading to accumulation of compatible osmolytes in response to hypertonicity.
In summary, the regulation of TonEBP was characterized in response to various signals. The results suggest that cellular ionic strength rather than the cell volume is the signal for regulation of TonEBP. TonEBP is not inactive in isotonic conditions: it can be up regulated or down regulated depending on the changes in tonicity. The regulation of TonEBP involves two pathways: changes in abundance and distribution between the nucleus and cytoplasm.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-44484 and DK-42479.
Address for reprint requests and other correspondence: H. Moo Kwon, Div. of Nephrology, School of Medicine, The Johns Hopkins Univ., 963 Ross Bldg., 720 Rutland Ave., Baltimore, MD 21205 (E-mail:).
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- Copyright © 2000 the American Physiological Society