Cycloheximide increases glucocorticoid-stimulated -ENaCmRNA in collecting duct c - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:50 |只看该作者 |倒序浏览 |打印

作者：Omar A.Itani, Kristyn L.Cornish, Kang Z.Liu,  Christie P.Thomas,作者单位：1 Department of Internal Medicine, and Graduate Program in Molecular Biology, Universityof Iowa College of Medicine, and Veterans AffairsMedical Center, Iowa City, Iowa 52242 . p1 ~6 Q, m( n: w$ j

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      【摘要】* B- b$ k3 t* p: V" x7 u2 W( {
   Aldosteroneand glucocorticoids (GCs) stimulate Na reabsorption in thecollecting ducts by increasing the activity of the epithelialNa channel (ENaC). Our laboratory has usedMadin-Darby canine kidney-C7 cells to demonstrate that this effect isassociated with an increase in -ENaC gene transcription (Mick VE,Itani OA, Loftus RW, Husted RF, Schmidt TJ, and Thomas CP, MolEndocrinol 15: 575-588, 2001). Cycloheximide (CHX)superinduced the GC-stimulated -ENaC expression in a dose-dependentmanner, but had no effect on basal or aldosterone-stimulated -ENaCexpression, whereas anisomycin inhibited basal andcorticosteroid-stimulated -ENaC expression. The superinduction of -ENaC expression was also seen with hypotonicity, was blocked byRU-38486, and was independent of protein synthesis. CHX had no effecton -ENaC mRNA half-life, confirming that its effect was via anincrease in -ENaC transcription. The effect of CHX and hypotonicityon -ENaC expression was abolished by SB-202190, indicating an effect mediated via p38 MAPK. Consistent with this scheme, CHX increased pp38and MKK6, an upstream activator of p38, stimulated -ENaC promoteractivity. These data confirm a model in which CHX activates p38 inMadin-Darby canine kidney-C7 cells to increase -ENaC gene transcription in a GC-dependent manner. $ m5 D, m8 ]+ D0 o
      【关键词】 epithelial sodium channel aldosterone glucocorticoid generegulation" K+ @- m- i7 ]! x0 a. G, j# @
   INTRODUCTION3 q4 u4 a5 P+ j  B! U

SODIUM REABSORPTION IN THE cortical and medullary collecting duct istightly regulated in response to the perceived extracellular fluidvolume and to dietary Na intake. Sodium transport in thissegment of the nephron occurs via the epithelial Na channel (ENaC), and an important class of physiological regulators ofthis transport pathway is the corticosteroids, aldosterone, andglucocorticoids (GCs). GCs are also important regulators of ENaC-dependent Na transport in the lung, whereasaldosterone influences Na transport in othermineralocorticoid-responsive tissues, such as the distal colon, sweatducts, and salivary glands ( 21, 48 ). One importantmolecular target for aldosterone and GC action in the collecting ductis the -subunit of ENaC itself, which is transcriptionally regulatedvia a GC response element (GRE) in the 5'-flanking region of the gene( 27, 32, 38, 44 ).

In addition to corticosteroids, many other signaling pathways appear toregulate Na transport in the collecting duct, includingthose activated by AVP, prostaglandins, and EGF. The effects of AVP aresynergistic to the effects of corticosteroids and, in some cases, canbe mimicked by activators of adenylate cyclase and by membrane-permeantanalogs of cAMP ( 21, 45 ). Short- and long-term infusion of1-desamino-8- D -AVP to Brattleboro rats increases theabundance of each of the three ENaC subunit proteins, an effect thatmay be mediated by an increase in ENaC mRNA abundance( 15 ). In contrast to corticosteroids and AVP, EGFand PGE 2 inhibit collecting-duct Na transport( 53 ). Although the mechanism of inhibition ofNa transport in the collecting duct is unknown, growthfactors can modulate gene expression by activation of protein kinase Cand mitogen-activated protein (MAP) kinases. In parotid salivaryepithelial cells, protein kinase C activation leads to transcriptionaldownregulation of the -subunit of ENaC, an effect mediated by theERK ( 57 ). The ERK pathway antagonizes the GC-dependent trans -activation of the -ENaC subunit gene and providedthe first evidence for a direct cross talk between a nuclear hormonereceptor and a MAP kinase signaling pathway in the regulation of thisNa channel ( 27 ).; m0 ]5 U6 |4 I- v5 S* b
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In the course of our investigation of the mechanism of GC stimulationof ENaC gene expression, we found evidence that a second MAP kinase,p38 MAP kinase, stimulates -ENaC gene transcription in aGC-dependent manner in Madin-Darby canine kidney (MDCK)-C7 cells, acollecting duct cell line with regulated Na transport.3 @/ U: o, Y) P' n& D

METHODS
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Materials. Dexamethasone, aldosterone, cycloheximide, anisomycin, emetinehydrochloride, puromycin, and puromycin aminonucleoside were purchasedfrom Sigma (St. Louis, MO), and RU-28362, and RU-38486 were generousgifts from Roussel Uclaf (Romainville, France). SB-202190, SB-203580,and U-0126 were obtained from Calbiochem-Novabiochem (San Diego, CA),and actinomycin D was from Roche Molecular Biochemicals (Indianapolis,IN). Culture materials were from Life Technologies (Gaithersburg, MD),and [ - 32 P]UTP and [ 35 S]methionine werefrom NEN Life Science Products (Boston, MA). Stock solutions ofactinomycin D, cycloheximide, emetine, puromycin, U-0126,SB-202190 and SB-203580 were made in Me 2 SO, whereasdexamethasone, aldosterone, RU-28362, RU-38486, and anisomycin weremade in ethanol.1 [8 l% q' I$ d
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Tissue culture and RNA preparation. MDCK-C7 cells (gift from B. Blazer-Yost and H. Oberleithner) weremaintained in MEM with 10% fetal bovine serum ( 7 ). For RNA experiments, cells were grown in 100 mM petri dishes or six-well plates, switched to serum-free media, and incubated with 100 nM dexamethasone, aldosterone, or vehicle (ethanol) in the presence orabsence of other reagents for 24 h, except where indicated. Todetermine mRNA turnover, MDCK-C7 cells were stimulated for 12 hwith 100 nM dexamethasone alone or with cycloheximide, and thentreatment continued with these reagents for various times in thepresence of 1 µM actinomycin D. For tonicity experiments, MDCK-C7cells were exposed to an external osmolality of 250 or 200 mosmol/kgH 2 O by the addition of 0.2 or 0.5 vol ofH 2 O to the culture media for 4 h. As a controlfor dilution of media contents, isotonic conditions were maintained inother samples by the addition of 0.2 or 0.5 vol of 150 mM NaCl for4 h. RNA from cultured cells was prepared with the RNeasy minikit(Qiagen, Valencia, CA), following the manufacturer's recommendations.3 u- \  k) t2 s, s6 n$ l0 k0 j

RNAse protection assay. A template containing the cloned canine -ENaC and sgk1 cDNA and an18S rRNA cDNA fragment (pTR1 RNA 18S; Ambion, Austin, Tx) were used togenerate radiolabeled antisense cRNA probes, as previously described( 32 ). RNA samples were cohybridized overnight with -ENaC or sgk1 and 18S cRNAs and then digested with RNase A and T1and nuclease-protected fragments resolved by polyacrylamide gelelectrophoresis. To quantitate mRNA expression, autoradiograms werescanned, and the density of individual bands was measured by usingKodak Digital Science Image Analysis software (Rochester, NY). The -ENaC band was normalized for the density of the 18S rRNA band, andthe data from multiple experiments were pooled and analyzed by one-wayanalysis of variance and/or by Student's t -test. Forcalculation of mRNA half-life, data points were plotted on asemilogarithmic scale, and exponential regression lines were derivedfor both experimental conditions.
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Radiolabeling of MDCK-C7 proteins. For metabolic labeling of MDCK-C7 proteins, cells were seeded into24-well plates, grown until subconfluent, and switched to serum-freemedia for an additional 18-24 h. After they were washed with PBS,cells were incubated for 10 min with methionine-free RPMI 1640, andthen for 2 h with the same medium containing 15 µCi/ml of[ 35 S]methionine in the presence or absence ofcycloheximide or serum. The cells were then washed twice with ice-coldPBS, twice with 5% TCA, and solubilized in 0.5 ml of 0.25 N NaOH, andan aliquot was counted in a liquid scintillation counter.

Western blotting. MDCK-C7 cells were grown in six-well plates until subconfluent,switched to serum-free media for 24 h, and then treated with various reagents for the indicated times. Samples were then lysed in asolution containing 50 mM Tris, pH 7.4, 76 mM NaCl, 2 mM EGTA, 1%Nonidet P-40, 0.5% SDS, 10% glycerol, 0.4 mM PMSF, 2 µg/mlleupeptin, 2 µg/ml pepstatin, and 2 µg/ml aprotinin for 1 h at4°C and then homogenized by passing through an 18-gauge needleseveral times. Protein concentrations were determined by the Bradfordmethod, and 100 µg of each lysate were run on a 10% polyacrylamidegel at 30 V for 16 h. Resolved proteins were transferred ontonitrocellulose (Transblot, Bio-Rad, Hercules, CA) by using the OwlSeparation System (Midwest Scientific, Valley Park, MO) at 400 mA for45 min and blocked in 5% nonfat dry milk/0.05% TBS-Tween (TTBS) for1 h. Blots were incubated with 1:250 to 1:1,000 dilution of p38MAP kinase antibody, stress-activated protein kinase (SAPK)/c-Jun NH 2 -terminal kinase (JNK) (p54/46) antibody,phospho-SAPK/JNK (p54/46) antibody (all from Cell SignalingTechnologies, Beverly, MA), or phospho-p38 MAP kinase antibody (Sigma)in 3% nonfat dry milk/TTBS for 1 h. Blots were then washed oncein TTBS and incubated with 1:1,000 to 1:3,000 horseradishperoxidase-conjugated secondary IgG antibody for 1 h. After three10-min washes in TTBS, horseradish peroxidase detection was performedwith the SuperSignal chemiluminescence substrate (Pierce, Rockford, IL).

Transient transfection assays. An ~1,400-nt fragment of the h -ENaC 5'-flanking region( 1,388 55/ -ENaC-luc) cloned upstream of the firefly luciferasecoding region of pGL3basic (Promega, Madison, WI) was used fortransfection experiments ( 32 ). This construct wastransiently transfected into MDCK-C7 by using LipofectAMINE Plus (LifeTechnologies), along with a control plasmid, pRLSV40, which expressesthe sea pansy luciferase, to correct for differences in transfectionefficiency and in recovery of cytosolic extracts. The day aftertransfection, cells were treated for 24 h with 100 nMdexamethasone or aldosterone and 20 µM SB-202190 or its vehicle. Somewells were also treated with 1 µM cycloheximide for the last 6 h. The transfected cells were maintained in serum-free media from thetime of transcription until cells were lysed. Cell lysates wereprepared ~48 h after transfection, and reporter gene activity wasmeasured as previously described ( 44 ).( v, Y2 L1 u. E  A. s& Y/ N9 L* \

In another set of experiments, the plasmid, 1,388 55/ -ENaC-luc orTAT3-luc, a GC-responsive reporter plasmid (gift from David Pearce andKeith Yamamoto), was cotransfected into MDCK-C7 cells along withpRLSV40 and MKK6b, a constitutively active form of the upstream kinasefor p38 MAP kinase ( 39 ). Six hours after transfection, 100 nM dexamethasone or its vehicle was added, and 24 h later, lysateswere prepared and analyzed as described.5 f5 B! J8 {/ q; w/ a

RESULTS9 n) I5 d5 A' ]; s8 U1 h
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Our laboratory has previously reported that dexamethasone andaldosterone increase amiloride-sensitive Na transport inMDCK-C7 cells, an effect temporally associated with increasedexpression of -ENaC mRNA ( 32 ). The stimulation of -ENaC expression is mediated in both cases by GC receptor(GR)-mediated activation of a GRE in the 5'-flanking region of the -ENaC gene. To determine whether protein synthesis was required forthe corticosteroid effect on -ENaC gene transcription, we usedcycloheximide, a general protein synthesis inhibitor, simultaneouslywith dexamethasone, aldosterone, or vehicle for 24 h and measured -ENaC mRNA levels. Although cycloheximide had no effect on vehicleor aldosterone-stimulated -ENaC, a more than twofold increase in -ENaC mRNA levels was seen in the presence of dexamethasone (Fig. 1, A and B ). Theinitial interpretation of this finding was that cycloheximide reduced the synthesis of an intermediary protein that functioned to inhibit theGC-stimulated but not basal -ENaC gene transcription. It isimportant to note that MDCK-C7 cells lack the mineralocorticoid receptor (MR) and that aldosterone-stimulated -ENaC expression occurs through GR ( 32 ). The inability of cycloheximide tosuperinduce -ENaC mRNA levels in the presence of aldosterone was,therefore, surprising, because the effect of aldosterone and GC on -ENaC mRNA is mediated by GR. To determine whether the cycloheximide effect was dependent on activation of GR, we used a specific GR agonist, RU-28362, and a specific GR antagonist, RU-38486, and examined -ENaC mRNA levels. As expected, RU-28362 stimulated -ENaC mRNAlevels, and this was abolished by simultaneous treatment with RU-38486(Fig. 1, C and D ). Cycloheximide had additional stimulatory effects on RU-28362-treated MDCK-C7 cells, an effect thatwas completely abrogated by the GR antagonist RU-38486 (Fig. 1, C and D ).$ ?3 e: W, \* C! x7 B

Fig. 1. Effect of cycloheximide (chx) on -epithelialNa channel (ENaC) expression. Madin-Darby canine kidney(MDCK)-C7 cells were exposed to dexamethasone (dex) or aldosterone(aldo) in the presence or absence of 10 µM chx for 24 h, and -ENaC mRNA was measured by RNase protection assay (RPA) and comparedwith vehicle (ctrl). A : representative RPA is shown, and thelast lane indicates yeast (Y) control. B : RPA data arequantitated and pooled ( n = 3; means ± SE). Thedata are significantly different by one-way analysis of variance: dexand aldo increase -ENaC expression (* P -ENaCexpression ( # P C : the effect of chx on RU-28362-stimulated -ENaC expression. D : representative RPA and pooled dataare shown ( n = 3; means ± SE). The data aresignificantly different by one-way analysis of variance: RU-28362increases -ENaC gene expression (* P P -ENaC gene expression iscompletely inhibited by RU-38486.

To confirm the cycloheximide effect, we used another protein synthesisinhibitor, emetine, with vehicle and dexamethasone treatment. Bothemetine and cycloheximide dose dependently increased dexamethasone-stimulated -ENaC expression, with negligible effects in vehicle-treated cells (Fig. 2 A ). We also tested the effect of hypotonicity on dexamethasone-stimulated -ENaC expression, because, in an amphibian model of the collecting duct, hypotonicity stimulates Na transport and increases the expression ofanother GC-regulated gene, sgk1 ( 42, 55 ). WhenMDCK-C7 cells were switched to hypotonic media in the presence ofdexamethasone, an increase in -ENaC expression was seen that wasmore pronounced the greater the degree of hypotonicity (Fig. 2 B ). We then asked whether cycloheximide could have asimilar effect on other dexamethasone-regulated genes in MDCK-C7 cells.Sgk1 is a serine-threonine kinase that appears, at least in part, tomediate the corticosteroid effects on Na transport in thecollecting duct ( 5, 10, 18, 33 ). As our laboratory haspreviously reported, dexamethasone increases sgk1 expression in MDCK-C7cells ( 32 ). Simultaneous incubation with cycloheximidesubstantially increased dexamethasone-stimulated sgk1 expression (Fig. 2 C ).
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Fig. 2. Effect of chx, emetine, and hypotonicity on gene expression. A : MDCK-C7 cells were exposed to increasing concentrationsof emetine or chx in the presence of dex or its vehicle for 24 h,and -ENaC mRNA levels were measured. RPA is shown, and the last laneindicates yeast (Y) control. Emetine and chx increaseglucocorticoid-stimulated -ENaC expression in a dose-dependentmanner. B : MDCK-C7 exposed to dex under isotonic(300 mosmol/kgH 2 O) or hypotonic (250 and 200 mosmol/kgH 2 O) conditions for 4 h, and -ENaC mRNAlevels were measured. Hypotonicity increases -ENaC expression. C : MDCK-C7 cells treated with 100 nM dex for 1 or 2 hin the presence or absence of 10 mM chx, and sgk1 mRNA levels weremeasured. Chx increases glucocorticoid-stimulated sgk1 expression.

The results with cycloheximide and emetine raised the possibility thattheir effects on -ENaC expression may be mediated by inhibition ofprotein synthesis. To determine whether the effect of cycloheximidecorrelated with its effects on protein synthesis inhibition, wemeasured protein synthesis rates in MDCK-C7 cells labeled with[ 35 S]methionine. As expected, cycloheximide inhibitedprotein synthesis but profoundly only at 10 µM, while there was amodest but significant effect at 1 µM, and no effect at 0.1 µM,concentrations that were sufficient to superinduce -ENaC genetranscription (Fig. 3 ). These resultssuggested that the effects of cycloheximide were unlikely to besecondary to inhibition of protein synthesis.

Fig. 3. MDCK cell proteins were metabolically labeled with[ 35 S]methionine in the presence of complete medium(serum), various concentrations of chx (in µM), or its vehicle; andmethionine incorporation into cell lysates was measured. Values aremeans ± SE; n = 3. * P P

We then tested two other protein synthesis inhibitors and sawdramatically different effects on -ENaC mRNA expression. Puromycin and its inactive analog puromycin aminonucleoside had no effect on -ENaC mRNA levels, whereas anisomycin inhibited vehicle- and dexamethasone-treated -ENaC mRNA levels in a dose-dependent manner (Fig. 4, A and B ).These contrasting results clearly indicated that the effects of theseagents on -ENaC gene expression could not be explained by inhibitionof protein synthesis. We then evaluated the effect of anisomycin onaldosterone-stimulated -ENaC expression and confirmed thatanisomycin inhibited aldosterone-stimulated -ENAC expression in adose-dependent manner (Fig. 4 C ). These results demonstratethat, in contrast to the cycloheximide effect to superinduceGC-stimulated but not aldosterone-stimulated -ENaC expression,anisomycin was able to inhibit constitutive -ENAC expression, aswell as that stimulated by GC and aldosterone.
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Fig. 4. Effect of puromycin (Puro) and anisomycin on -ENaCexpression. A : MDCK-C7 cells were exposed toincreasing concentrations of puromycin or puromycin aminonucleoside inthe presence of dex or its vehicle for 24 h, and -ENaC mRNAlevels were measured. RPA is shown, and the last lane indicates yeast(Y) control. Neither puromycin nor its inactive analog altersglucocorticoid-stimulated -ENaC expression. B : MDCK-C7cells were exposed to increasing concentrations of anisomycin in thepresence of dex or its vehicle for 24 h, and -ENaC mRNA levelswere measured. RPA is shown, with the position of -ENaC mRNA and 18SrRNA indicated. The last lane indicates Y control. While chxsuperinduces dex-stimulated -ENaC expression, anisomycin inhibitsctrl and dex-stimulated -ENaC expression in a dose-dependent manner. C : MDCK-C7 cells were exposed to increasing concentrationsof anisomycin in the presence of aldo or its vehicle for 24 h, and -ENaC mRNA levels were measured. Similar to dex, the ctrl andaldo-stimulated -ENaC expression is inhibited by anisomycin./ k' P% {' u- x0 Y' x7 M7 O! I

To begin to understand the basis for the effect of cycloheximide,we examined the kinetics of the cycloheximide response on dexamethasone-stimulated -ENaC expression. Dexamethasone stimulated -ENaC expression, which was barely evident by 1 h, but in the presence of cycloheximide the transcript was clearly evident at 1 h, and with cycloheximide was more abundant at every time point tested(Fig. 5 A ). Because -ENaCmRNA appeared earlier, these results suggested that cycloheximideincreased the rate of transcription of -ENaC. To evaluate thisfurther, we measured -ENaC mRNA decay characteristics in thepresence of dexamethasone alone and in the presence of dexamethasoneand cycloheximide (Fig. 5 B ). The mRNA half-life under bothconditions was remarkably similar at ~9 h, confirming thatcycloheximide did not alter -ENaC mRNA stability. This findingfurther argues for an effect of cycloheximide on GC-stimulated -ENaCgene transcription.
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Fig. 5. Transcription and turnover of c -ENaC mRNA. A : effect of chx on the kinetics of -ENaC expressionafter stimulation for 24 h with dex. RPA is shown, with theposition of -ENaC mRNA and 18S rRNA indicated. Chx increases therate of appearance of -ENaC mRNA. B : decay of -ENaCmRNA with dex (D) or dex chx (D C) in the presence ofactinomycin D (A). The means ± SE of duplicate samples were usedto derive an exponential regression line for both sets. The chx-treatedsamples have more -ENaC mRNA and hence begin their decay at a higherlevel. The half-time for both sets is 9 h.
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A mechanism by which protein synthesis inhibitors may superinduce geneexpression is by activation of MAP kinases ( 3, 58 ). Weused SB-202190 or SB-203580 p38 MAP kinase inhibitors along withcycloheximide and dexamethasone and measured -ENaC mRNA levels. Thesuperinduction of dexamethasone-stimulated -ENaC expression bycycloheximide was abolished by simultaneous treatment with SB-202190and inhibited by SB-203580 (Fig. 5 A ). U-0126, an inhibitorof MEK, the upstream activator of ERK, had no effect oncycloheximide-stimulated -ENaC expression (Fig. 6 A ). These data suggested thatcycloheximide increased -ENaC expression by activation of p38 MAPkinase. The hypotonicity-mediated increase in GC-regulated -ENaCexpression was also blocked by SB-202190 and SB-203580, but not byU-0126 or PD-98059 (Fig. 6 B ). These results indicated thatthe effect of hypotonicity was also mediated by p38 MAP kinase." ]' ]; m" u3 A! t% A0 H

Fig. 6. Effect of mitogen-activated protein (MAP) kinaseinhibitors on -ENaC expression. A : effect of the specificp38 MAP kinase inhibitor SB-202190 on the chx stimulation of -ENaCexpression. MDCK-C7 cells were placed in serum-free media for 24 hand then treated with 100 nM dex for 4 h; 10 µM chx for 24 h; and/or 20 µM SB-202190, SB-203580, or U-0126 for 24 h.SB-202190 and SB-203580 inhibit the chx effect on -ENaC expression. A is representative of at least 2 experiments. B :effect of MAP kinase inhibitors on the stimulation of -ENaCexpression by hypotonicity. MDCK-C7 cells were placed in serum-freemedia for 24 h and then treated with 100 nM dex under hypotonicconditions (200 mosmol/kgH 2 O) for 4 h, in the presenceof 20 µM SB-202190, SB-203580, PD-98059, or 10 µM U-0126. SB-202190and SB-203580 inhibit the tonicity effect on -ENaC expression. B is representative of at least 2 experiments.% b; Z  S* T  F5 y- z# T9 a

To examine the effect of cycloheximide on MAP kinase activation, wemeasured total and phosphorylated p38 and JNK in MDCK-C7 lysates aftertreatment with either agent. Whereas total p38 did not vary betweenconditions, short-term treatment with cycloheximide or anisomycinincreased phospho-p38 abundance (Fig. 7 ).In addition to the principal band, a lower band was also seen,especially in cycloheximide-treated lanes, which may represent anotherisoform of p38 or a breakdown product. By contrast, anisomycin led to adramatic increase in phosphorylated JNKs, principally p46 (pJNK), whereas the corresponding treatment with cycloheximide showed a barelydetectable increase in pJNK, even when higherconcentrations of cycloheximide (25 µM) were used (Fig. 7 ). Therewas, however, no change in the total JNK between conditions. The datasuggest that cycloheximide principally activates p38 MAP kinase,whereas anisomycin is a potent activator of JNK and p38. Based on the data in Fig. 6 A, the activation of p38 MAP kinase bycycloheximide appears to increase transcription of the -ENaC gene.Despite activation of p38, anisomycin reduces -ENaC gene expression, presumably because activation of other pathways results in inhibition of -ENAC gene transcription.

Fig. 7. Immunoblotting for inactive and active forms of p38 andc-Jun NH 2 -terminal kinase (JNK). MDCK-C7 cells were placedin serum-free media for 24 h and then treated with variousreagents for the indicated times. Lysates were resolved by SDS-PAGE andthen immunoblotted with the indicated antibodies. Anisomycin (Aniso)and chx-activated p38 (pp38) were compared with vehicle-treated (ctrl)lane. Anisomycin increases phospho-JNK (pJNK), whereas vehicle and chxtreatments do not lead to a detectable change in pJNK.% ^1 i- A2 S3 w  m
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To confirm the effect of cycloheximide and SB-202190 on -ENaCgene transcription, we used the 1,388-bp human -ENaC promoter coupled to luciferase in transfection assays. This promoter contains the -ENaC GRE, and, as our laboratory has shown before( 32 ), is robustly stimulated by treatment with 100 nM dexamethasone for 24 h (Fig. 8 A ). Tominimize the effects of cycloheximide on translation of luciferase,this agent was used at a lower dose (1 µM) and for the last 6 honly. The results demonstrate that cycloheximide significantlyincreased -ENaC promoter-driven luciferase activity in the presenceof dexamethasone, indicating that the cycloheximide effect was at thelevel of gene transcription and that cis -elements within theincluded 5'-flanking sequence were sufficient to confer this effect.SB-202190 abolished the effect of cycloheximide (Fig. 8 A ),correlating with the inhibition of -ENaC gene expression seen inFig. 6 A. As in Fig. 6 A, SB-202190 appeared topartially inhibit the dexamethasone effect, suggesting that p38 MAPkinase activation may also support basal or dexamethasone-stimulated -ENaC gene expression. We next asked whether cycloheximide would have an effect on aldosterone-stimulated gene transcription. Consistent with the data seen in Fig. 1, the effect of cycloheximide to increase -ENaC gene transcription is seen only with dexamethasone and notwith aldosterone (Fig. 8 B )." V* \4 k% k$ O: u% E

Fig. 8. Transient transfection assays. A : MDCK-C7cells were transfected with 1,388 55/ -ENaC-luc and pRLSV40 andtreated with dex in the presence or absence of 20 µM SB-202190 (SB)for 24 h, with 1 µM chx added for the last 6 h. Values aremeans ± SE; n = 4. # P B :MDCK-C7 cells were transfected with 1,388 55/ -ENaC-luc and pRLSV40and treated with 100 nM dex or aldo for 24 h, with 1 µM chxadded for the last 6 h. Values are means ± SE; n = 4. * P P C : MDCK-C7 cells were transfected with 1,388 55/ -ENaC-luc (ENaC-luc) or TAT3-luc with pRLSV40 and MKK6band then treated for 24 h with 100 nM dex or vehicle. Values aremeans ± SE; n = 3. Results significantlydifferent by one-way ANOVA: * P P1 Q0 ?5 ^+ Y( a& x' T
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Finally, we asked whether activation of p38 MAP kinase was sufficientto stimulate -ENaC gene transcription. We used a plasmid thatoverexpressed a MAP kinase kinase, MKK6b, which constitutively activates p38 in transient transfection assays ( 1 ). Aftertransfection with the -ENaC promoter-luciferase construct, MDCK-C7cells were treated with 100 nM dexamethasone for 24 h, and ourresults demonstrate that cotransfection of MKK6b increases -ENaCgene transcription (Fig. 8 C ). Similar results were seen withTAT3-luc, a GC-responsive reporter plasmid in which three tandem copiesof the GRE in the rat tyrosine amino transferase gene are placedupstream of a TATA-driven firefly luciferase construct( 29 ). These results suggest that the effects of p38 MAPkinase on GC-dependent gene transcription may be seen with other genesthat are regulated by GREs.

DISCUSSION; u5 k, q" n* r6 B$ W) i6 x$ ?$ ^+ x

GC and mineralocorticoids are important physiological regulatorsof ENaC, and most, if not all, of their effects are mediated byincreases in transcription of target genes. In vivo, in kidney cortexand medulla and in the lung, -ENaC is itself a target of hormoneaction ( 2, 17, 49, 50 ). The increase in transcription ofthe -ENaC subunit is mediated by trans -activation of aGRE in the 5'-flanking region of the -ENaC gene by hormone-bound GRor MR ( 27, 32, 38, 44 ). However, the presence ofhormone-bound receptor is not sufficient for -ENaC gene activation,because, in colonic epithelia, treatment with GC or mineralocorticoids increases steady-state levels of - and -ENaC mRNA without any effect on -ENaC mRNA ( 28, 40, 49 ). One explanation for these results is that cell-specific coactivators present in the collecting duct or lung epithelia are required for the corticosteroid effect or that cell-specific repressors in colonic epithelia prevent the corticosteroid response.
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To begin to explore the mechanism of GC-stimulated -ENaC mRNAtranscription in the collecting duct, we used cycloheximide, a widelyused inhibitor of protein synthesis, with dexamethasone and aldosteroneand found that it specifically increased dexamethasone-stimulated, butnot aldosterone-stimulated or basal, -ENaC mRNA levels. The phenomenon of a further increase in mRNA levels with a protein synthesis inhibitor over that seen with an inducing agent such asdexamethasone is well known and is termed superinduction ( 12, 26 ). This phenomenon was first observed for labile immediate early-response genes such as c- fos, c- jun, and egr-1, which function as transcription factors, but morerecently has been seen with enzymes such as cyclooxygenase and proteinphosphatases and signaling molecules such as cytokines ( 14, 16, 31 ). Classically, these gene products are expressed rapidly andtransiently, and protein synthesis inhibitors typically induce basaland superinduce hormone or growth factor-stimulated gene expression.Our observations with cycloheximide and the -ENaC transcript wereintriguing in many ways. First, the -ENaC transcript is not animmediate early gene product, because, after stimulation by aldosteroneor dexamethasone, mRNA levels begin to increase by ~2 h and continueto increase for the next 24-48 h ( 24, 32 ). Second,this transcript encodes a channel protein rather than a transcriptionfactor, enzyme, or signaling molecule. Third, the superinduction isseen with dexamethasone and not with aldosterone, although bothagonists bind to the same transcription factor, GR, and activate -ENaC gene transcription via the same cis element inthese cells ( 32 ).
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Several mechanisms have been proposed to explain superinduction ofimmediate early genes by inhibitors of protein synthesis. These include 1 ) an increase in mRNA stability ( 37, 56 ); 2 ) increase in transcription, perhaps by inhibition oftranscriptional downregulation ( 23, 41 ); and 3 )stimulation of intracellular signaling pathways ( 30, 58 ).Increasingly, differential effects of various protein synthesisinhibitors have been reported, and it appears that inhibition ofprotein synthesis may not be required for some of these effects. In ourown studies, we show that, whereas emetine and cycloheximidesuperinduce -ENaC expression, puromycin has no effect and anisomycininhibits gene expression, suggesting that protein synthesis inhibitionis not sufficient for superinduction (Figs. 2 A and 4, A and B ). Our metabolic labeling studies also confirmed that inhibition of protein synthesis is not required for thecycloheximide effect (Fig. 3 ).* N+ ^0 n3 x0 Y' ~( q

Given the disparate effects between cycloheximide and anisomycin, webegan to wonder whether our results with -ENaC expression could beexplained by differential stimulation of intracellular signalingpathways. Recently, anisomycin has been shown to be a potent stimulantof JNK and is increasingly being used as a tool to examine the role ofJNK in subcellular biological phenomena ( 8, 9 ). It hasbecome clear, however, that, in some cell systems, anisomycin mayactivate each of the three known MAP kinase-signaling pathways,including JNK, p38, and ERK. In HeLa cells, for example, anisomycinactivates JNK and ERK and p38 ( 43, 58 ), whereas, in othercells such as NIH3T3 cells, ERK activation is not seen ( 11 ). Cycloheximide is a considerably weaker activator ofJNK ( 3, 58 ), and differing effects on MAP kinases couldexplain the contrasting effects of cycloheximide and anisomycin. When we used SB-202190, a specific p38 MAP kinase inhibitor, the effect ofcycloheximide was abolished in MDCK-C7 cells, confirming that cycloheximide stimulated -ENaC mRNA expression in a p38-dependent manner. Indeed, by immunoblot analysis, we demonstrated thatcycloheximide activated p38 alone, whereas anisomycin increasedphosphorylation of JNK and p38. Cycloheximide also enhanced GC- but notaldosterone-stimulated -ENaC promoter activity in luciferase assays,an activity that was blocked by SB-202190, confirming that its effectwas mediated by p38 MAP kinase. Finally, direct activation of p38 MAPkinase by overexpression of its upstream kinase MKK6 was also able to increase -ENaC gene transcription in a reporter gene assay. Taken together, the data strongly suggest that cycloheximide activates p38MAP kinase and that this activation increases -ENaC transcription ina GC-dependent manner in MDCK-C7 cells.$ P# T* l( c: G+ _' ?4 g* I8 _

What is the relevance of GC-stimulated transcription of genes that areinvolved in Na transport in the collecting duct? In vivo,under physiological conditions, aldosterone rather than cortisolengages the MR to increase gene transcription and stimulateNa transport ( 19, 20, 34 ). The selectivity ofMR for aldosterone comes from the actions of the enzyme11 -hydroxysteroid dehydrogenase type 2 (11 -OHSD2), whichinactivates cortisol to cortisone, a metabolite that no longer hasaffinity for MR or GR ( 19 ). Under pathophysiologicalconditions in which the activity of 11 -OHSD2 is diminished or whencirculating levels of cortisol are high enough to overwhelm thecapacity of this enzyme, endogenous GC can bind either the GR or MR tostimulate Na transport. The syndrome of apparentmineralocorticoid excess, which is due to mutations in 11 -OHSD2,exemplifies a situation in which cortisol functions as amineralocorticoid ( 13, 54 ). Additionally, severalsynthetic GCs in clinical use, such as dexamethasone, have very lowaffinity for MR and are not substrates for metabolism by 11 -OHSD2( 35 ). Thus the effects of these agents on -ENaC mRNAexpression and on amiloride-sensitive Na transportpathways in vivo and in cultured cells derived from the collecting ductmust be mediated virtually exclusively via activation of GR ( 2, 36, 46, 49, 51 ).

In contrast to the MR, GR binds with high affinity to cortisol anddexamethasone but with much lower affinity to aldosterone ( 19 ). With supraphysiological doses, aldosterone increases -ENaC mRNA expression and Na transport in somecollecting duct cell lines that lack functional MR ( 6, 32, 46, 52 ), suggesting that aldosterone may be able to exertmineralocorticoid effects through GR under certain circumstances. Underphysiological conditions, it is unlikely that aldosterone binds to GRin mineralocorticoid-responsive tissues, but, under conditions in whichcirculating levels of aldosterone are very high or where aldosterone iscompetitively displaced from MR (e.g., with the use of spironolactone),crossover binding to GR can occur. The identification of loss offunction mutations of MR in human disease and the creation oftransgenic mice with targeted deletion of MR have begun to provide uswith information on MR-independent pathways that contribute toregulation of ENaC and the reabsorption of Na in thecollecting duct ( 4, 22, 47 ). Understanding the downstreameffects of GR occupation when aldosterone, rather than a classic GC, isthe activating ligand has arguably become important. Our studiesdemonstrate that the p38 MAP kinase pathway has stimulatory effects onGC-mediated -ENaC gene expression. This effect is GR dependent andis only evident in the presence of its high-affinity ligand,dexamethasone or RU-28362, and not aldosterone. These results suggestone of two possibilities: a direct physical interaction between a p38MAP kinase-dependent coactivator and the GR-ligand complex, orcooperative interactions between p38 and GR-activated cis elements in the regulatory regions of the -ENaC gene. The dataobtained with TAT3-luc, in which the regulatory elements consist onlyof a minimal promoter and multiple GREs, would suggest that the formeris more likely. The data presented here thus fit a model in whichcycloheximide or hypotonicity activates p38 MAP kinase, which in turnsrecruits a transcriptional coactivator that recognizes GR bound to GCssuch as dexamethasone, but not aldosterone (Fig. 9 ). In fact, a nuclear receptorcoactivator, PGC-1, has recently been described that, on activation byp38 MAP kinase, enhances GR-dependent gene transcription, at least whenreconstituted in HeLa cells ( 25 ). Our studies point to mechanisms whereby ENaC gene transcription can be enhanced by activation of p38 MAP kinase following the engagement of a variety ofmembrane receptors or in response to adverse conditions such asischemia.
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Fig. 9. Model for stimulation of dex-regulated gene expression bychx and hypotonicity. p38 MAP kinase activation via its upstream kinaseMKK6 leads to the recruitment of a transcriptional coactivator thatenhances transcription from glucocorticoid response elements (GREs) inthe presence of GR bound to dex but not GR bound to aldo., R1 ]1 T3 K2 X" R

ACKNOWLEDGEMENTS
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The authors thank H. Oberleithner and B. Blazer-Yost for the giftof the MDCK-C7 cell line, Jiahuai Han for the gift of plasmid MKK6b,David Pearce and Keith Yamamoto for the gift of the plasmid TAT3-luc,and acknowledge the DNA synthesis and sequencing services provided bythe University of Iowa DNA core facility.
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