Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892
Submitted 21 January 2003 ; accepted in final form 20 June 2003
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ABSTRACT |
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aldose reductase; transcription factor; TonEBP
After its cloning, it became evident that TonEBP mRNA is constitutively
expressed in virtually all tissues in vivo, including the majority that are
never normally exposed to hypertonicity
(10,
13,
17). Also, TonEBP protein was
detected in adult murine thymus and testes
(17). TonEBP is expressed in
ES cells and throughout the stages of fetal development
(11). Immunostaining shows
expression of TonEBP in almost all developing tissues, including the brain,
colon, heart, muscle, and eyes
(11). These findings raise the
question of what the role of TonEBP expression might be in nonrenal tissues.
One possibility is that it serves an osmoregulatory role in these tissues,
responding to decreases and increases in tonicity at 300
mosmol/kgH2O, as discusssed above for tissue culture cells.
However, other roles for TonEBP have also been identified. In lymphocytes,
hypertonicity increases TonEBP transcriptional activity
(16,
17), but so also do
proinflammatory stimuli (6).
However, the role of constitutive expression of TonEBP mRNA in most tissues is
undefined.
In the present studies, we tested two hypotheses: 1) that
previously undetected protein expression of TonEBP might occur in vivo in the
tissues expressing its mRNA, and to test this we examined TonEBP protein
expression in several tissues of rats; and 2) that TonEBP might serve
an osmoregulatory role in nonrenal tissues in vivo, responding to decreases,
as well as increases, in tonicity of 300 mosmol/kgH2O. To test
this, we looked for downregulation of TonEBP mRNA and protein expression in
nonrenal rat tissues exposed to hypotonicity during hyposmolality and for
decreases in RNA expression of genes that are transcriptional targets of
TonEBP.
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MATERIALS AND METHODS |
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Sample preparation, SDS-PAGE electrophoresis, and immunoblotting for measurement of TonEBP protein abundance. Tissue samples of brain, thymus, skeletal muscle, liver, and heart from three control and three hyposmotic rats were homogenized in 10 ml of ice-cold isolation solution (250 mM sucrose, 10 mM triethanolamine, pH 7.6, containing 1 µg/ml leupeptin and 0.1 mg/ml phenylmethylsulfonyl fluoride), using a tissue homogenizer (Omni 1000, with a micro saw tooth generator) at maximum speed for three 20-s intervals. Total protein concentrations were measured (BCA kit, Pierce Chemical), and the samples were solubilized in Laemmli sample buffer at 60°C for 15 min. Semiquantitative immunoblotting was carried out as previously described (Terris J, unpublished observations) to assess the relative abundances of the proteins of interest. Equal loading of the gels with the same amount of total protein from each sample was tested using preliminary 12% polyacrylamide gels that were stained with Coomassie blue. Densitometry (Personal Densitometer SI, Molecular Dynamics, San Jose, CA) was performed on representative bands, and loading was adjusted so that protein loaded in each lane would not differ by more than 5% of the mean. Proteins were then separated on 7.5% polyacrylamide gels by SDS-PAGE and were transferred to nitrocellulose membranes electrophoretically (Bio-Rad Mini Trans-Blot Cell). Membranes were blocked for 1 h at room temperature with 5% nonfat dried milk and probed overnight at 4°C with rabbit polyclonal anti-NFAT5 COOH-terminal diluted antibodies (Affinity BioReagents, no. PA1023; diluted to 1:1,000). Membranes were washed and exposed to secondary antibody (goat anti-rabbit IgG conjugated to horseradish peroxidase, Pierce no. 31463; diluted to 1:5,000) for 1 h at room temperature. After being washed, bands were visualized using a luminol-based enhanced chemiluminescence substrate (LumiGLO, Kirkegaard and Perry Laboratories, Gaithersburg, MD). Band densities were determined by laser densitometry (Personal Densitometer SI).
Sample preparation, RNA isolation, cDNA preparation, and real-time PCR
for measurement of abundance of specific RNAs. Total RNA was isolated
from brain, thymus, skeletal muscle, liver, and heart of three control and
three hyposmotic rats, using an RNeasy kit (Qiagen). This procedure includes
treatment with DNase to minimize contamination by genomic DNA. cDNA was
prepared with TaqMan reverse transcription reagents, using random
hexamers, according to the manufacturer's instructions (Applied Biosystems).
Real-time PCR was performed in triplicate on both 8- and 80-ng aliquots of
each cDNA sample using TaqMan universal PCR master mix in a total
volume of 20 µl (ABI PRISM 7900HT Sequence Detection System, Applied
Biosystems). In this system, the accumulation of the PCR product is monitored
in real time by a fluorogenic 5'-nuclease assay, using probes specific
for each cDNA being tested. Primers and probes were designed from rat cDNA
sequences. The PCR primers were designed to span an intron of genes that
contain introns, namely TonEBP, AR, BGT1, TauT, and -actin. This was not
possible for SMIT and heat shock protein (HSP)70 genes, which contain no
introns. The sequences of the primers and probes are shown in
Table 1. We sequenced the PCR
products produced by each primer set and found that all sequences matched
those of the intended targets. Significant contamination by genomic DNA was
excluded by failure to generate a product in PCR reactions on total RNA that
was not reverse transcribed, using the SMIT- and HSP70-specific primers and
probes.
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Calculation of relative tissue mRNA abundance from the real-time PCR data. The detection system records the number of PCR cycles (Ct) required to produce an amount of product equal to a threshold value, which is a constant. From the Ct values, we calculated means ± SE of tissue mRNA abundance in each tissue from experimental animals relative to the mean of control animals (taken as 100%), using the following principles. 1) By definition, the number of specific cDNA molecules at the threshold (NCt) is constant for a given cDNA, independent of the number of cycles that it takes to reach it. 2) For a specific cDNA, the ratio N(exp)i/N(cont)i is independent of i, assuming only that the efficiency (E) of PCR for a specific template is constant and the same for samples from experimental and control animals, where i is the cycle number, and N(X)i is the number of specific cDNA molecules in a sample (X = control or experimental) at cycle i. 3) The ratio of the number of specific cDNA molecules at a cycle, Ct, to the number at another cycle, i, is Ni/NCt = 1/E(Cti).
To normalize the comparison between control and experimental results, we compared all results to the number of specific molecules at an arbitrary cycle, I, chosen for convenience to be the largest whole number that is less than any of the experimental values of Ct. Then, we calculated N(X)I/NCt for each sample. From those results, i.e., avg[N(cont)I/NCt], or the average of N(cont)I/NCt was obtained. Each result for a given tissue from an experimental animal was normalized to a mean control value of 100% by dividing each value of N(X)I/NCt (control and experimental) by avg[N(cont)I/NCt] and multiplying by 100. Then, means ± SE were calculated for control and experimental samples.
Statistics. Statistical significance was calculated using the one-tail t-test to evaluate for each tissue the differences between control and experimental conditions (GraphPad Instat 3.0). Results are expressed as means ± SE. Differences were considered significant for P < 0.05.
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RESULTS |
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Effect of hypotonicity on TonEBP mRNA expression. Detectable levels of TonEBP mRNA are present in all tissues tested from control rats, but the abundance varies greatly from tissue to tissue. The lowest level is in skeletal muscle (mean Ct = 31.59 with 80 ng of cDNA reverse transcribed from total RNA). The levels in the other tissues are higher than in skeletal muscle: brain 209 times as high as skeletal muscle, thymus 251, heart 81, and liver 40.
Brains and livers from the hyposmotic rats contain less TonEBP mRNA than
those from the control rats, but there is no significant difference in thymus,
heart, or skeletal muscle (Fig.
1A). Representative amplification curves of TonEBP mRNA
from brains of control and hyposmotic rats are shown in
Fig. 2. Simultaneously measured
18s RNA does not differ significantly with serum osmolality in any tissue
(Fig. 1B), confirming
that reverse transcription, cDNA loading, and PCR are equivalent. Also,
expression of -actin does not differ
(Fig. 1C), confirming
that the changes in TonEBP in brain and liver do not reflect a general change
in mRNA level in those organs.
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Effect of hypotonicity on TonEBP protein expression. Although TonEBP mRNA is detected in all tissues examined (Fig. 1), TonEBP protein is not detected in heart and skeletal muscle, even loading 80 µg of protein (Fig. 3). Hypotonicity significantly lowers TonEBP protein expression in thymus and liver but not in brain (Fig. 3).
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Effect of hypotonicity on expression of other mRNAs. We also measured the mRNA abundance of several genes that are known transcriptional targets of TonEBP, namely AR, BGT1, SMIT, and TauT (Table 2) (2, 12, 13). AR mRNA does not differ significantly between osmotic conditions in any tissue. BGT1 decreases significantly with osmolality only in liver. SMIT decreases significantly with osmolality in liver and muscle. TauT decreases significantly with osmolality in brain and thymus.
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HSP70 expression is known to be affected by hypertonicity. Hypertonicity
increases HSP70 mRNA and protein expression in mIMCD3 cells
(15,
21). Dominant negative TonEBP
reduces HSP70 mRNA expression in MDCK cells under isotonic and hypertonic
conditions, consistent with regulation of transcription of HSP70 by TonEBP
(21). Examination of HSP70
mRNA expression is complicated because two different HSP70 genes express
virtually identical proteins
(18,
21), and the names given in
rat, human, and mouse for the homologous genes differ in a confusing fashion
(Table 3) and are used
inconsistently in the literature. In what follows a gene is designated by
species and the name of the gene in that species, according to GenBank. The
5'-flanking region of the human HSP70-2 gene contains TonEs
(21), as it does also in the
homologous rat Hsp70-1 and mouse hsp70.1 genes. Hypertonicity increases mouse
(mIMCD3 cells) hsp70.1 mRNA expression but not hsp70A1 expression
(21). Also, transcription of a
luciferase reporter construct containing 4 kb of the 5'-flanking
region of mouse hsp70.1 gene is increased by hypertonicity
(21). Given this evidence that
TonEBP regulates tonicity-dependent transcription of mouse hsp70.1, but not
mouse hsp70A1, we measured the effect of hypotonicity on the homologous rat
Hsp70-1 and rat Hsp70-2 genes. Surprisingly, hypotonicity causes a large
increase of rat Hsp70-1 and Hsp70-2 mRNA in brain, thymus, heart, and skeletal
muscle, without significant change in liver
(Table 2).
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DISCUSSION |
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We used the rat "vasopressin escape" model (4) of hyposmolality to test this hypothesis. By infusing rats with dDAVP while maintaining a high water intake, we lowered their plasma osmolality from the normal 294 to 241 mosmol/kgH2O. Then, we measured in brain, liver, thymus, skeletal muscle, and heart TonEBP mRNA and protein and mRNA expression of AR, BGT1, SMIT, TauT, and HSP70 genes that are osmotically regulated by TonEBP. The results do not support the hypothesis that TonEBP generally responds to decreases, as well as increases, in tonicity around the normal plasma osmolality in vivo, which would be the case if there was a general osmoregulatory role for its ubiquitous expression in nonrenal tissues. In immortalized cells, hypotonicity decreases TonEBP mRNA and protein (20). In contrast, in vivo liver is the only organ in which plasma hypotonicity results in decreases in both TonEBP mRNA and protein. In brain, TonEBP mRNA decreases, but TonEBP protein does not change. In thymus, TonEBP protein decreases, but mRNA does not change. In heart and skeletal muscle, TonEBP mRNA does not change and TonEBP protein is immeasurably low.
In immortalized cells in tissue culture, hypotonicity decreases AR, BGT1 mRNA abundance (5), and SMIT mRNA abundance (20). In contrast, in vivo hypotonicity (Table 2) does not change AR mRNA in any of the five tissues that were tested. It decreases BGT1, SMIT, and TauT mRNA in liver (although the latter reduction is not statistically significant), SMIT mRNA in muscle, and TauT mRNA in brain and thymus. Thus we find that hypotonicity decreases mRNA of some transcriptional targets of TonEBP in some nonrenal tissues in vivo, as might be expected if expression of these genes were under bidirectional constitutive osmotic regulation by TonEBP, but not all targets and not in all tissues. Liver is the only organ in which we found that hypotonicity consistently reduces not only TonEBP mRNA and protein but also BGT1, SMIT, and TauT mRNA. The latter results are reminiscent of a previous study of primary culture of rat liver sinusoidal endothelial cells (19) in which hypotonicity decreases BGT1, SMIT, and TauT mRNA and reduces uptake of betaine, inositol, and taurine. We infer that, although tonicity may bidirectionally control TonEBP activity in liver, other sources of regulation predominate in the other tissues in which it is constitutively expressed.
Thus TonEBP apparently plays a nonosmotic role in most nonrenal tissues in which it is constitutively expressed in vivo. Some nonosmotic roles of TonEBP are already known, namely in lymphocytes proinflammatory stimuli, as well as hypertonicity, increase TonEBP transcriptional activity, suggesting a role in signaling inflammation (17), and TonEBP is involved in promotion of carcinoma invasion downstream of integrin, suggesting a role in tumor metastasis (6). It seems likely that other nonosmotic roles of TonEBP remain to be discovered.
Liver is the only organ in which hypotonicity-induced decreases of TonEBP mRNA and protein expression correlate. In brain, TonEBP mRNA decreases but not protein; in thymus, TonEBP protein decreases but not mRNA. This lack of correlation is not surprising, however, because protein abundance is also regulated by translation and degradation, which may or may not follow mRNA abundance.
Hypertonicity increases HSP70 expression (3) because of TonEBP-mediated increase in transcription (21). Therefore, we expected that hypotonicity might decrease HSP70 in the nonrenal tissues that we studied in vivo. Surprisingly, we found that hypotonicity greatly increases Hsp70-1 and Hsp70-2 mRNA in brain, thymus, skeletal muscle, and heart. Greater induction of HSP70 mRNA and protein by heat in primary cultures of rat hepatocytes was observed at 205 than at 305 mosmol/kgH2O, but the result of hypotonicity alone was not reported (8). We are unaware of any other previous evidence that hypotonicity induces HSP70. A specific TonEBP-mediated response to hypotonicity per se seems unlikely, if only because Hsp70-2 is not mediated by TonEBP (21). Instead, we suppose that hypotonicity might trigger a general stress response akin to heat shock but unrelated to TonEBP.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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