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作者：Zheng Zhang, Joan D. Ferraris, Heddwen L. Brooks, Ioana Brisc, Maurice B. Burg作者单位：Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung,and Blood Institute, Bethesda, Maryland 20892 p, J3 x, r( b! F- H* |! d
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【摘要】4 C. N; \% f4 t
TonEBP is a transcription factor that, when activated by hypertonicity, increases transcription of genes, including those involved in organic osmolyteaccumulation. Surprisingly, it is expressed in virtually all tissues,including many never normally exposed to hypertonicity. We measured TonEBPmRNA (real-time PCR) and protein (Western blot analysis) in tissues of control(plasma osmolality 294 ± 1 mosmol/kgH 2 O) and hyposmotic(dDAVP infusion plus water loading for 3 days, 241 ± 2mosmol/kgH 2 O) rats to test whether the ubiquitous expression ofTonEBP mRNA is osmotically regulated around the normal plasma osmolality. TonEBP protein is reduced by hyposmolality in thymus and liver, but not inbrain, and is not detected in heart and skeletal muscle. TonEBP mRNA decreasesin brain and liver but is unchanged in other tissues. There are no generalchanges in mRNA of TonEBP-mediated genes: aldose reductase (AR) does notchange in any tissue, betaine transporter (BGT1) decreases only in liver,taurine transporter (TauT) only in brain and thymus, and inositol transporter (SMIT) only in skeletal muscle and liver. Heat shock protein (Hsp)70-1and Hsp70-2 mRNA increase greatly in most tissues, which cannot beattributed to decreased TonEBP activity. The conclusions are as follows: 1 ) TonEBP protein or mRNA expression is reduced by hyposmolality inthymus, liver, and brain. 2 ) TonEBP protein and mRNA expression aredifferentially regulated in some tissues. 3 ) Although AR, SMIT, BGT1,and TauT are regulated by TonEBP in renal medullary cells, other sources ofregulation may predominate in other tissues. 4 ) TonEBP abundance andactivity are regulated by factors other than tonicity in some tissues. 9 e; D8 O: d7 F! R: ?$ m
【关键词】 aldose reductase transcription factor TonEBP' S" `1 U$ U+ U
TON EBP [also called OREBP( 7 ), NFAT5( 10 ), NFATz( 14 ), and NFATL1( 17 )] is a transcriptionfactor, originally identified from its role in adaptation of cells toincreases in tonicity ( 13 ).This role is clear in the renal inner medulla, where tonicity is higher thanin the rest of the body, and can vary greatly with urinary concentration( 1 ). In inner medullary cells,TonEBP regulates transcription of genes that protect against hypertonicity,including genes such as those for aldose reductase (AR) and betaine (BGT1),inositol (SMIT), and taurine (TauT) transporters( 2, 12, 13 ) that direct organicosmolyte accumulation and genes for heat shock proteins( 21 ). TonEBP is also expressedin cultured cells derived from tissues not normally exposed to hypertonicity,for example, HEP G2 cells from liver( 5 ), Chang liver cells( 7 ), HeLa cells( 7 ), embryonic stem (ES) cells( 11 ), and HEK 293 cells( 5 ). In these cells, as well asin kidney-derived cells such as Madin-Darby canine kidney (MDCK)( 12, 20 ), COS 7( 7, 9 ), and mIMCD3 cells ( 21 ), TonEBP is expressed andis transcriptionally active in the 300-mosmol/kgH 2 O media inwhich they are routinely grown. Furthermore, expression and transcriptionalactivity of TonEBP at 300 mosmol/kgH 2 O are bidirectionallyregulated by tonicity. These effects include that at 300mosmol/kgH 2 O 1 ) dominant negative TonEBP reducesexpression of TonE-mediated luciferase constructs( 13 ) and reduces the abundanceof SMIT and BGT1 mRNA; and 2 ) reducing tonicity decreases abundanceof TonEBP protein and mRNA and causes it to move out of the nucleus, opposite to the result of increasing tonicity( 20 ). Because TonEBP mRNA falls more rapidly than can be accounted for by its half-life, hypotonicityapparently accelerates its turnover; 3 ) reducing tonicity decreasesthe transactivating activity of TonEBP, opposite to the effect of increasingtonicity ( 5, 13 ). In short, in cellcultures, TonEBP expression and transcriptional activity are present at 300 mosmol/kgH 2 O and respond to decreases, as well asincreases, in tonicity.+ p. m+ N6 m/ c) B. N
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After its cloning, it became evident that TonEBP mRNA is constitutively expressed in virtually all tissues in vivo, including the majority that arenever normally exposed to hypertonicity( 10, 13, 17 ). Also, TonEBP protein wasdetected in adult murine thymus and testes( 17 ). TonEBP is expressed inES 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 thequestion 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 300mosmol/kgH 2 O, 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 isundefined.& z( k# B$ }& S9 e
; E: e9 q7 u: I. XIn the present studies, we tested two hypotheses: 1 ) thatpreviously undetected protein expression of TonEBP might occur in vivo in thetissues expressing its mRNA, and to test this we examined TonEBP proteinexpression in several tissues of rats; and 2 ) that TonEBP might servean osmoregulatory role in nonrenal tissues in vivo, responding to decreases,as well as increases, in tonicity of 300 mosmol/kgH 2 O. To testthis, we looked for downregulation of TonEBP mRNA and protein expression innonrenal rat tissues exposed to hypotonicity during hyposmolality and fordecreases in RNA expression of genes that are transcriptional targets ofTonEBP.) C6 E$ V; q8 E4 E1 ^ j
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MATERIALS AND METHODS
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4 @% `. C# u" @Experimental animals. Osmolality of the blood in rats was reduced by using the vasopressin escape protocol previously described ( 4 ). Briefly, under isofluoraneanesthesia, male Sprague-Dawley rats were subcutaneously implanted withosmotic minipumps (model 2002, Alzet, Palo Alto, CA), which delivered 20 ngdDAVP/h (Peninsula Laboratories, Belmont, CA) for 4 days. All rats received pellets of rat chow (Formula 53140000, Ziegler Brothers, Gardner, PA) and adlibitum water during this period. After 4 days, the rats were divided intocontrol (3 rats for measurement of specific proteins and 3 rats formeasurement of specific mRNAs) and experimental (hyposmolality, similarly 6rats) groups. Vasopressin escape rats were given a daily water load via a gelled-agar diet (71% water, 28% finely ground rat chow, 1% agar) and had freeaccess to water. They received 55-65 g of the gel diet per 250 g body wtper day. Control rats continued to receive pellets of rat chow and ad libitumwater. After 3 days of treatment, the rats were killed and tissues were harvested. All experiments were carried out according to the guidelines laiddown by the National Institutes of Health Animal Care and Use Committee.5 ]5 w9 w9 Q' K. o$ C' z7 G3 k
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Sample preparation, SDS-PAGE electrophoresis, and immunoblotting formeasurement of TonEBP protein abundance. Tissue samples of brain, thymus,skeletal muscle, liver, and heart from three control and three hyposmotic ratswere homogenized in 10 ml of ice-cold isolation solution (250 mM sucrose, 10mM triethanolamine, pH 7.6, containing 1 µg/ml leupeptin and 0.1 mg/mlphenylmethylsulfonyl fluoride), using a tissue homogenizer (Omni 1000, with amicro saw tooth generator) at maximum speed for three 20-s intervals. Totalprotein concentrations were measured (BCA kit, Pierce Chemical), and thesamples 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 theproteins of interest. Equal loading of the gels with the same amount of totalprotein from each sample was tested using preliminary 12% polyacrylamide gelsthat were stained with Coomassie blue. Densitometry (Personal Densitometer SI,Molecular Dynamics, San Jose, CA) was performed on representative bands, andloading was adjusted so that protein loaded in each lane would not differ bymore than 5% of the mean. Proteins were then separated on 7.5% polyacrylamidegels by SDS-PAGE and were transferred to nitrocellulose membraneselectrophoretically (Bio-Rad Mini Trans-Blot Cell). Membranes were blocked for1 h at room temperature with 5% nonfat dried milk and probed overnight at4°C with rabbit polyclonal anti-NFAT5 COOH-terminal diluted antibodies (Affinity BioReagents, no. PA1-023; diluted to 1:1,000). Membranes werewashed and exposed to secondary antibody (goat anti-rabbit IgG conjugated tohorseradish peroxidase, Pierce no. 31463; diluted to 1:5,000) for 1 h at roomtemperature. After being washed, bands were visualized using a luminol-based enhanced chemiluminescence substrate (LumiGLO, Kirkegaard and PerryLaboratories, Gaithersburg, MD). Band densities were determined by laserdensitometry (Personal Densitometer SI).
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Sample preparation, RNA isolation, cDNA preparation, and real-time PCRfor measurement of abundance of specific RNAs. Total RNA was isolatedfrom brain, thymus, skeletal muscle, liver, and heart of three control andthree hyposmotic rats, using an RNeasy kit (Qiagen). This procedure includestreatment with DNase to minimize contamination by genomic DNA. cDNA wasprepared with Taq Man reverse transcription reagents, using randomhexamers, according to the manufacturer's instructions (Applied Biosystems). Real-time PCR was performed in triplicate on both 8- and 80-ng aliquots ofeach cDNA sample using Taq Man universal PCR master mix in a totalvolume of 20 µl (ABI PRISM 7900HT Sequence Detection System, AppliedBiosystems). In this system, the accumulation of the PCR product is monitoredin real time by a fluorogenic 5'-nuclease assay, using probes specificfor each cDNA being tested. Primers and probes were designed from rat cDNAsequences. The PCR primers were designed to span an intron of genes thatcontain introns, namely TonEBP, AR, BGT1, TauT, and -actin. This was notpossible for SMIT and heat shock protein (HSP)70 genes, which contain nointrons. The sequences of the primers and probes are shown in Table 1. We sequenced the PCRproducts produced by each primer set and found that all sequences matchedthose of the intended targets. Significant contamination by genomic DNA wasexcluded by failure to generate a product in PCR reactions on total RNA thatwas not reverse transcribed, using the SMIT- and HSP70-specific primers and probes.
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9 ?' [0 N2 j, _! TTable 1. Primers and probes designed for TonEBP and downstream genes* p z4 O; H/ P& }. M* u
5 [: ]" q" ~# T* V0 `. sCalculation of relative tissue mRNA abundance from the real-time PCRdata. The detection system records the number of PCR cycles (Ct) requiredto produce an amount of product equal to a threshold value, which is aconstant. From the Ct values, we calculated means ± SE of tissue mRNAabundance in each tissue from experimental animals relative to the mean ofcontrol animals (taken as 100%), using the following principles. 1 )By definition, the number of specific cDNA molecules at the threshold( N Ct ) is constant for a given cDNA, independent of thenumber of cycles that it takes to reach it. 2 ) For a specific cDNA,the ratio N (exp) i / N (cont) i isindependent of i, assuming only that the efficiency (E) of PCR for aspecific template is constant and the same for samples from experimental andcontrol animals, where i is the cycle number, and N ( X ) i is the number of specific cDNAmolecules in a sample ( X = control or experimental) at cycle i.3 ) The ratio of the number of specific cDNA molecules at a cycle, Ct, tothe number at another cycle, i, is N i / N Ct =1/E (Ct- i ).
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5 [' S/ S: J7 ^, }6 U5 ~8 i% u @/ zTo normalize the comparison between control and experimental results, wecompared all results to the number of specific molecules at an arbitrarycycle, I, chosen for convenience to be the largest whole number thatis less than any of the experimental values of Ct. Then, we calculated N ( X ) I / N Ct for eachsample. From those results, i.e.,avg[ N (cont) I / N Ct ], or theaverage of N (cont) I / N Ct wasobtained. Each result for a given tissue from an experimental animal wasnormalized to a mean control value of 100% by dividing each value of N ( X ) I / N Ct (controland experimental) byavg[ N (cont) I / N Ct ] andmultiplying by 100. Then, means ± SE were calculated for control andexperimental samples.
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3 I z# K+ q; ]$ ]Statistics. Statistical significance was calculated using the one-tail t -test to evaluate for each tissue the differences betweencontrol and experimental conditions (GraphPad Instat 3.0). Results areexpressed as means ± SE. Differences were considered significant for P
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Serum osmolality differs significantly ( P rats in the control group (294 ± 1 mosmol/kgH 2 O, n = 6) and the hyposmotic group (241 ± 2 mosmol/kgH 2 O) after the latter are subjected to 3 days of water loading in the presence of dDAVP.The decrease is similar to that observed in earlier studies, using a similarprotocol ( 4 ), in whichdifferences in concentration of sodium and its associated anions accounted formost of the decrease in osmolality. Thus the lower plasma osmolality reflectshyponatremia and hypotonicity.
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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, butthe abundance varies greatly from tissue to tissue. The lowest level is inskeletal muscle (mean Ct = 31.59 with 80 ng of cDNA reverse transcribed fromtotal 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, andliver 40.$ L& D8 z! A# h9 N
% p0 C" x' d; F; bBrains and livers from the hyposmotic rats contain less TonEBP mRNA thanthose from the control rats, but there is no significant difference in thymus,heart, or skeletal muscle ( Fig.1 A ). Representative amplification curves of TonEBP mRNAfrom brains of control and hyposmotic rats are shown in Fig. 2. Simultaneously measured18s RNA does not differ significantly with serum osmolality in any tissue( Fig. 1 B ), confirmingthat reverse transcription, cDNA loading, and PCR are equivalent. Also,expression of -actin does not differ( Fig. 1 C ), confirmingthat the changes in TonEBP in brain and liver do not reflect a general changein mRNA level in those organs.
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* m: R( O7 C6 mFig. 1. Expression of specific RNAs in tissues from control and hyposmotic rats.Results are expressed relative to a mean control value of 100% ( n =3, * P A : TonEBP RNA expression. B : 18sRNA expression. C : -actin RNA expression.% w/ Y0 I O( m, j9 x2 z- h; g
( C+ E2 x( k( Z$ ?$ u. Q; ~Fig. 2. Representative real-time PCR plot by the ABI Prism 7900HT SequenceDetection System. Points are mean of TonEBP result from brains of 3 controland 3 hyposmotic rats. Representative amplification curve showing cycle numberof control and hyposmotic rats (broken line showing position of ampliconthreshold).3 O9 U. h3 F- K8 D M
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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 notdetected in heart and skeletal muscle, even loading 80 µg of protein( Fig. 3 ). Hypotonicitysignificantly lowers TonEBP protein expression in thymus and liver but not inbrain ( Fig. 3 ).9 p; E- X& A4 A! \
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Fig. 3. TonEBP protein expression in tissues from control and hyposmotic rats. The5 tissues shown were collected from each rat. Protein loading was 40µg/lane from brain, liver, and thymus; 80 µg/lane from heart andskeletal muscle. In all blots, the left -most 3 lanes were from 3individual control rats and the right -most 3 lanes were from 3individual hyposmotic rats.- W ^- H% Z' f: R) g
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Effect of hypotonicity on expression of other mRNAs. We also measured the mRNA abundance of several genes that are known transcriptionaltargets of TonEBP, namely AR, BGT1, SMIT, and TauT( Table 2 )( 2, 12, 13 ). AR mRNA does not differsignificantly between osmotic conditions in any tissue. BGT1 decreasessignificantly with osmolality only in liver. SMIT decreases significantly withosmolality in liver and muscle. TauT decreases significantly with osmolalityin brain and thymus.! J1 [8 q! O+ P$ m& ?- s
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Table 2. Effect of hyposmolality on RNA expression (% of control by real-timePCR) of genes regulated by TonEBP and of Hsp70-2" ~4 v) [, n% }
* H. T, Z. ?; F& S- Q2 E1 U- DHSP70 expression is known to be affected by hypertonicity. Hypertonicity increases HSP70 mRNA and protein expression in mIMCD3 cells ( 15, 21 ). Dominant negative TonEBPreduces HSP70 mRNA expression in MDCK cells under isotonic and hypertonicconditions, consistent with regulation of transcription of HSP70 by TonEBP( 21 ). Examination of HSP70mRNA expression is complicated because two different HSP70 genes expressvirtually identical proteins( 18, 21 ), and the names given inrat, 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 byspecies and the name of the gene in that species, according to GenBank. The5'-flanking region of the human HSP70-2 gene contains TonEs( 21 ), as it does also in thehomologous 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 aluciferase reporter construct containing 4 kb of the 5'-flankingregion of mouse hsp70.1 gene is increased by hypertonicity( 21 ). Given this evidence thatTonEBP regulates tonicity-dependent transcription of mouse hsp70.1, but notmouse hsp70A1, we measured the effect of hypotonicity on the homologous ratHsp70-1 and rat Hsp70-2 genes. Surprisingly, hypotonicity causes a largeincrease 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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Table 3. Comparison of nomenclature (from GenBank) of rat, mouse, and humanHSP70 genes ( 18 )) a0 n. L. |2 ^
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The expression and transcriptional activity of TonEBP in immortalized celllines at 300 mosmol/kgH 2 O are bidirectionally regulated bytonicity, suggesting that they might also be bidirectionally regulated bytonicity at 295 mosmol/kgH 2 O in the multiple nonrenal tissuesin which they are expressed in vivo.' h* z, Z% v# _: W+ Z" J" k' [
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We used the rat "vasopressin escape" model( 4 ) of hyposmolality to testthis hypothesis. By infusing rats with dDAVP while maintaining a high waterintake, we lowered their plasma osmolality from the normal 294 to 241mosmol/kgH 2 O. Then, we measured in brain, liver, thymus, skeletalmuscle, 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 todecreases, as well as increases, in tonicity around the normal plasmaosmolality in vivo, which would be the case if there was a generalosmoregulatory role for its ubiquitous expression in nonrenal tissues. Inimmortalized cells, hypotonicity decreases TonEBP mRNA and protein( 20 ). In contrast, in vivo liver is the only organ in which plasma hypotonicity results in decreases inboth TonEBP mRNA and protein. In brain, TonEBP mRNA decreases, but TonEBPprotein does not change. In thymus, TonEBP protein decreases, but mRNA doesnot change. In heart and skeletal muscle, TonEBP mRNA does not change andTonEBP protein is immeasurably low.2 }: P7 T$ x' A$ h7 [' N0 Z% H
& K% \3 c3 L8 y# J$ s' PIn immortalized cells in tissue culture, hypotonicity decreases AR, BGT1mRNA abundance ( 5 ), and SMITmRNA abundance ( 20 ). Incontrast, in vivo hypotonicity ( Table2 ) does not change AR mRNA in any of the five tissues that weretested. It decreases BGT1, SMIT, and TauT mRNA in liver (although the latterreduction is not statistically significant), SMIT mRNA in muscle, and TauTmRNA in brain and thymus. Thus we find that hypotonicity decreases mRNA ofsome transcriptional targets of TonEBP in some nonrenal tissues in vivo, asmight be expected if expression of these genes were under bidirectionalconstitutive osmotic regulation by TonEBP, but not all targets and not in alltissues. Liver is the only organ in which we found that hypotonicity consistently reduces not only TonEBP mRNA and protein but also BGT1, SMIT, andTauT mRNA. The latter results are reminiscent of a previous study of primaryculture of rat liver sinusoidal endothelial cells( 19 ) in which hypotonicitydecreases BGT1, SMIT, and TauT mRNA and reduces uptake of betaine, inositol, and taurine. We infer that, although tonicity may bidirectionally controlTonEBP activity in liver, other sources of regulation predominate in the othertissues in which it is constitutively expressed.
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Thus TonEBP apparently plays a nonosmotic role in most nonrenal tissues inwhich it is constitutively expressed in vivo. Some nonosmotic roles of TonEBPare already known, namely in lymphocytes proinflammatory stimuli, as well ashypertonicity, increase TonEBP transcriptional activity, suggesting a role insignaling inflammation ( 17 ),and TonEBP is involved in promotion of carcinoma invasion downstream ofintegrin, suggesting a role in tumor metastasis( 6 ). It seems likely that othernonosmotic roles of TonEBP remain to be discovered.0 [* J4 S9 F6 _" W
" E% m }3 U1 v+ o' pLiver is the only organ in which hypotonicity-induced decreases of TonEBPmRNA and protein expression correlate. In brain, TonEBP mRNA decreases but notprotein; in thymus, TonEBP protein decreases but not mRNA. This lack ofcorrelation is not surprising, however, because protein abundance is alsoregulated by translation and degradation, which may or may not follow mRNAabundance.
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$ s- G* y& Q k0 t5 dHypertonicity increases HSP70 expression( 3 ) because of TonEBP-mediated increase in transcription( 21 ). Therefore, we expectedthat hypotonicity might decrease HSP70 in the nonrenal tissues that we studiedin vivo. Surprisingly, we found that hypotonicity greatly increases Hsp70-1and Hsp70-2 mRNA in brain, thymus, skeletal muscle, and heart. Greaterinduction of HSP70 mRNA and protein by heat in primary cultures of rathepatocytes was observed at 205 than at 305 mosmol/kgH 2 O, but theresult of hypotonicity alone was not reported( 8 ). We are unaware of anyother previous evidence that hypotonicity induces HSP70. A specificTonEBP-mediated response to hypotonicity per se seems unlikely, if onlybecause Hsp70-2 is not mediated by TonEBP( 21 ). Instead, we suppose thathypotonicity might trigger a general stress response akin to heat shock butunrelated to TonEBP.3 C3 E* H s0 M. d1 ~( ], D5 U2 x! M4 ^
$ N# F" |) H: \# OACKNOWLEDGMENTS
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Present address of H. L. Brooks: Dept. of Physiology, Univ. of Arizona,Tucson, AZ 85724.& S7 K" x1 \ g
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发表于 2009-4-21 13:46
