Hormone-regulated transepithelial Na transportin mammalian CCD cells requires S - 《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：My N.Helms, GézaFejes-Tóth,  AnikóNáray-Fejes-Tóth作者单位：Department of Physiology, Dartmouth Medical School,Lebanon, New Hampshire 03756-0001 / ~1 I+ K3 h1 l7 M" T3 ~" i1 a
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      【摘要】
   To study the roleof serum and glucocorticoid-inducible kinase-1 (SGK1) in mammaliancells, we compared Na transport rates in wild-type (WT) M1cortical collecting duct cells with M1 populations stably expressinghuman full-length SGK1, NH 2 -terminal truncated ( N-60)SGK1, "kinase-dead" (K127M) SGK1, and cells that have downregulatedlevels of SGK1 mRNA (antisense SGK1). Basal rates of transepithelialNa transport were highest in full-length SGK1 populations,compared among the above populations. Dexamethasone treatment increased Na transport in WT and full-length SGK1 cells 2.7- and2-fold, respectively. Modest stimulation of Na absorptionwas detected after dexamethasone treatment in N-60 SGK1 populations.However, N-60 SGK1 transport rates remained substantially lower thanWT values. Importantly, a combination of high insulin, dexamethasone,and serum failed to significantly stimulate Na transportin antisense or K127M SGK1 cells. Additionally, expression of antisenseSGK1 significantly decreased transepithelial resistance values.Overall, we concluded that SGK1 is a critical component incorticosteroid-regulated Na transport in mammaliancortical collecting duct cells. Furthermore, our data suggest that theNH 2 terminus of SGK1 may contain a Phox homology-likedomain that may be necessary for effective Na transport.
      【关键词】 aldosterone epithelial sodium channel M cell line serum andglucocorticoidinducible kinase cortical collecting duct
   INTRODUCTION( K" E* J& ^- x6 F
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SERUM AND GLUCOCORTICOID-INDUCIBLE KINASE-1 (SGK1) is a novel member of theserine/threonine family of kinases and is induced in response to avariety of extracellular stimuli (listed in Ref. 18 ).Implicit in its name, SGK1 was originally identified as a serum andglucocorticoid-induced gene in rat mammary tumor cells by differentialdisplay ( 37 ). Since then, posttranslational regulation ofSGK1 has also been described. For example, components of thephosphoinositide 3-kinase signaling pathway are necessary for thephosphorylation of SGK1, which leads to its activation. Amino acidresidues Thr256 (located in the activation loop of SGK1) and Ser422(located in the COOH-terminal domain of SGK1) must both bephosphorylated by 3-phosphoinositide-dependent protein kinases-1 and -2 (PDK1 and PDK2) for complete activation of SGK1 ( 16, 26 ).Recently, our laboratory and others have demonstrated that the level ofSGK1 transcript is rapidly upregulated in response tomineralocorticoids, independently of de novo protein synthesis, intarget epithelial cells ( 2, 6, 23, 25, 30 ).
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Mineralocorticoids are the key regulators of transepithelialNa transport. In the absence of 11 -hydroxysteroiddehydrogenase-2 activity, glucocorticoids can also inducemineralocorticoid-like effects ( 11, 17, 19, 24 ).Mineralocorticoids and glucocorticoids are both effective inupregulating SGK1 transcript levels in epithelial cells, which impliesa relationship between SGK1 and epithelial Na channel(ENaC) activity in collecting duct cells. This notion is stronglysupported by several heterologous SGK1 expression studies. Forinstance, oocytes coexpressing SGK1 and ENaC subunits exhibited higherNa current and more channels localized to the plasmamembrane compared with oocytes expressing ENaC alone ( 1, 6, 23, 30 ). Additionally, ectopic expression of SGK1 in Xenopuslaevis A6 cells displayed exceptionally high levels ofNa transport under basal conditions, which were sustainedwith aldosterone treatment ( 9 ). Moreover, inhibition ofthe phosphoinositide 3-kinase signaling pathway with LY-294002 wasassociated with a substantial decrease in the rate of Na transport in A6 cells ( 28, 36 ). Together, the abovestudies strongly implicate SGK1 as a mediator of aldosterone-stimulated transcellular Na reabsorption./ _% n, U+ \  m8 j. @) g( U

Despite these recent advances, however, the physiological role of SGK1in mammalian kidney cells has not been definitively established. Thegoal of our study was to generate stable mouse cortical collecting duct(CCD) cells that either overexpress or downregulate SGK1 activity.Here, we report for the first time that SGK1 is a critical regulator ofENaC activity in mammalian CCD cells.& D7 ^9 f* o; `

MATERIALS AND METHODS& o% C( _: h+ @
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Generation of an antisense SGK1 construct. The antisense SGK1 construct was created by ligating a 708-bp fragmentof cDNA located in the 3'-untranslated region of mouse SGK1 into themultiple cloning site of TRE vector (Clontech, Palo Alto, CA) in thereverse (antisense) orientation. Specifically, the DNA fragment islocated in the mouse SGK1 between the Bam HI and the Hin dIII restriction sites and includes the poly-A tail (Fig. 1 A ). The newly constructedantisense TRE construct carries both ampicillin and hygromycinresistance genes for selection in Escherichia coli and inmammalian cells, respectively. The subsequent transcription ofantisense RNA downregulates the endogenous expression of the complementtranscript ( 20, 27, 39 ).

Fig. 1. Effect of antisense serum and glucocorticoid-induciblekinase-1 (SGK1) construct expression on the level of SGK1transcript in M1 cortical collecting duct cells. A :3'-untranslated region fragment (UTR; 709 bp) from the mouse SGK1genome cloned in the reverse (antisense) orientation into pTRE vector(Clontech, Palo Alto, CA). B : representative RT-PCR of SGK1and -actin from control and antisense M1 populations grown ininsulin- and dexamethasone-containing medium. Serial dilutions of cDNA(10-0.15 ng) were used as templates. Antisense SGK1 constructexpression downregulated endogenous levels of SGK1 transcript. C : -actin-normalized SGK1 transcript levels were 4 timeslower in the antisense cells ( n = 4) vs. wild-type (WT; n = 5) M1 cells ( P t -test (1-tailed; 2-sample equal variance). Valuesare averages ± SE.

Retroviral cDNA constructs of human full-length SGK1,NH 2 -terminal truncated ( N-60) SGK1, anddominant-negative K127M SGK1 tagged with hemaglutinnin (HA) epitopes atthe NH 2 terminus were generously provided by Dr. SuzanneConzen (University of Chicago). Retroviral vectors were produced bytransient LipofectAMINE (Invitrogen, Carlsbad, CA) transfection ofHA-tagged constructs into host amphotropic Pheonix cells (American TypeCulture Collection, Rockville, MD) according to the manufacturer'sprotocol and as previously described ( 22 ).
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Cell culture and construction of M1 CCD cell lines, whichoverexpress or downregulate SGK1 activity. M1 cells ( 33 ) were propagated in PC-1 growth medium(BioWhittaker, Walkersville, MD) containing an abundance of growthsupplements. The supplementary growth factors include 15 µg/mlinsulin, 5 µM dexamethasone, 5% FBS, 2 mM glutamine, and antibiotics(75 µg/ml penicillin, 100 µg/ml streptomycin, and 12.5 µg/mltylosin). This medium is hereafter referred to as insulin- anddexamethasone-containing (IDC) medium.
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Subconfluent M1 cells were cotransfected with antisense TRE vectorconcurrently with a Tet-On construct. The Tet-On construct carriesneomycin resistance and was designed to facilitate TRE vectorexpression in the presence of tetracycline. However, it was ourexperience that Tet-On vector provided strong, constitutive cytomegalovirus promoter activity in trans, which did notrequire additional tetracycline stimuli. The cotransfections werecarried out by using a modified LipofectAMINE protocol( 15 ). M1 cells stably expressing antisense SGK1 cDNA werethen selected by using 200 µg/ml hygromycin and 250 µg/ml neomycin.3 F( o4 Z- ?6 E7 B% a4 Z& u: P

Supernatant from amphotropic Phoenix cells producing retrovirusespackaging truncated ( N-60), full-length, and dominant-negative (K127M) SGK1 were collected and passed through a 0.45-µm filter toremove cellular debris. Twenty-four hours before infection, 6 × 10 5 M1 cells were seeded onto a 60-mm plastic dish in 2 mlIDC medium. M1 cells were inoculated with 2 ml filtered amphotropicsupernatant and allowed to incubate for an additional 48 h. Stable N-60, full-length, and K127M SGK1 expression in M1 cells wereselected with 700 ng/ml puromycin.
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All clones surviving antibiotic selection were expanded without theprocess of clonal selection on the basis of SGK1 vector expressionlevels. Cell populations will be referred to by the expression vectortransformed into parent M1 cells (full-length, N-60, K127M, orantisense SGK1).; P2 W5 F, A! w( A1 j5 C
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Quantitative RT-PCR. To compare the relative abundance of SGK1 mRNA between wild-type (WT)M1 cells and populations of M1 cells stably expressing antisense SGK1construct, we used quantitative RT-PCR methods, previously described in( 23 ). Briefly, total RNA was extracted from WT andantisense SGK1 M1 cells by using TRI Reagent per the manufacturer'sprotocol (Molecular Research Center, Cincinnati, OH). Then, 2 µg ofisolated total RNA was reverse transcribed by extension of randomprimers. SGK1 upper (5'-CTC AGT CTC TTT TGG GCT CTTT-3') and lower(5'-TTT CTT CTT CAG GAT GGC TTTC -3') PCR primers generated a 450-bpproduct. Reactions were performed with Ampli Taq DNApolymerase (Roche, Indianapolis, IN) under standard conditions withvarying amounts of template (10, 2.5, 0.62, and 0.15 ng of cDNA)originating from WT or antisense cells cultured in IDC medium. Thermalcycling conditions included a 2-min denaturing step at 96°C, followedby 25 cycles of 95°C for 1 min for denaturing, 57°C for 1 min toreanneal, and 72°C for 1 min for elongation of template, and then afinal extension of 72°C for 8 min. The abundance of -actin mRNAfor each sample was also determined by using primers and PCR conditionsoptimized for -actin, as previously described ( 34 ).Amplified SGK1 PCR products were separated on a 5% polyacrylamide gel,stained with ethidium bromide, quantified by using a FluoroImager andImageQUANT software (Molecular Dynamics, Sunnyvale, CA), and thennormalized for -actin mRNA levels.; d3 ?8 r5 B: n7 |5 D

Electrophysiological measurements. Cells were seeded onto Millicell permeable membranes (Millipore,Bedford, MA) at a density of 4 × 10 5 cells/chamber.After seeding, cells were bathed in IDC medium on the basolateral andapical sides until they formed confluent monolayers (~3-7 days).After the cells reached confluency, the bathing medium was changed toan insulin and steroid hormone-free (I-SterF) medium composed ofDMEM/F-12 (Mediatech, Herndon, VA) supplemented with 5% FBS strippedtwice with charcoal, 15 mM HEPES, 2 mM glutamine, and antibiotics.Cells were cultured in I-SterF medium for 48 h to establish basallevels of Na transport. Cells were then treated with 1 µM dexamethasone (in I-SterF medium) for a 24-h period to study theeffects of corticosteroids. The transepithelial voltage andtransepithelial resistance ( R TE ) values of eachpopulation of cells were regularly determined by using an EpithelialVoltohmmeter (World Precision Instruments, Sarasota, FL), and theequivalent short-circuit current ( I sc ) wascalculated. Our laboratory has previously demonstrated that I sc mainly represents transepithelialNa current in this model system ( 7 ). Cellswere regarded as confluent monolayers when voltage values remainedsteady for two independent readings over a 48-h time period and R TE values reached threshold levels of 900 · cm 2. All membrane resistancevalues reported represent the difference between measured R TE and porous membrane R TE (~125 · cm 2 ) in which the cells were grown.
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Western blot analysis. Confluent N-60 SGK1, full-length SGK1, K127M SGK1, and WT M1 cellswere rinsed twice with ice-cold PBS before lysing with 500 µl of SDSsolubilization buffer [48.2 mM 2-( N -hexylamino) ethanesulfonic acid, 1% SDS, 10% glycerol, and 1% protease and phosphatase inhibitor cocktail (Sigma, Palo Alto, CA)]. Theprotein concentration of cellular lysate was determined by using BCAProtein Assay Reagent (Pierce Chemical, Rockford, IL). Then, 7 µg oftotal protein lysate or immunoprecipitated product concentrated from a60-mm dish was electrophoresed on a 10% acrylamide gel under denaturing conditions. The proteins were then transferred toImmobilon-P membrane (Millipore) and blocked in buffer consisting of5% dry milk, 10 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween, 124 µMthimerosal, and 1% Mega Block (PGC Scientifics, Frederick, MD) for1 h. Next, the membrane was incubated in 4 µg of rabbitpolyclonal anti-HA antibody (Upstate Biotechnology, Cleveland, OH)diluted in 4 ml of blocking buffer at room temperature for 1 h.After extensive washes, anti-rabbit IgG-horseradish peroxidase(HRP)-labeled antibody (Cell Signaling Technology, Beverly, MA) wasadded at a concentration of 1 µg/ml and incubated for another hour atroom temperature. After thorough washes, HRP signal was detected byusing the ECL substrate (Amersham Pharmacia Biotech, Piscataway, NJ) orSuper Signal West Dura Substrate (Pierce Chemical). The membrane was finally exposed to Kodak X-OMAT AR scientific imaging film (Kodak, Rochester, NY).
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Immunoprecipitation. Confluent cells grown on a 60-mm plate were washed with ice-cold PBSand then lysed with buffer consisting of 20 mM Tris, pH 7.5, 150 mMNaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, and 1% protease andphosphatase inhibitor cocktail, further complemented with 1 mM -glycerol phosphate, 2.5 mM sodium pyrophosphate, 1 mMNa 3 VO 4, and 1 µg/ml leupeptin. Lysates werethen sonicated for 5-10 s. To immunoprecipitate SGK1 protein, 500 µl of supernatant from hybridomas-producing SGK1 antibody or 7 µganti-HA antibody diluted in lysis buffer were added to the cell lysate.SGK1 monoclonal antibodies were generated in mice immunized with mouseSGK1 amino acids 147-437, by using standard techniques previouslydescribed ( 10 ). Then, the antibody containing lysates wasincubated overnight at 4°C with gentle rocking. The followingmorning, 20 µl of protein A-Sepharose beads (Sigma) were added to thesample and incubated for an additional 2 h at 4°C with gentlerocking. The sample was then briefly centrifuged at 14,000 g. The precipitated pellet was washed in lysis buffer andthen resuspended in 25 µl SDS solubilization buffer. Theimmunoprecipitated complex was analyzed by Western blot technique, asdescribed above.

RESULTS

Stable integration of SGK1 constructs in the M1 CCD cell line. To study the role of SGK1 on transepithelial Na transportin mammalian cells, we generated M1 CCD cells stably expressing thehuman full-length SGK1, N-60 SGK1, and dominant-negative K127M SGK,as well as antisense SGK1, which downregulates the endogenous level ofSGK1 transcript.
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The abundance of SGK1 transcript in WT and antisense M1 cells grown inIDC medium was determined by using quantitative PCR techniques. TheSGK1 primer pair selected amplifies a region located within theendogenous mouse SGK1 transcript. Expression of antisense SGK1effectively downregulated the endogenous level of SGK1 transcript anddid not affect the expression levels of -actin (Fig. 1, B and C ). Normalized mouse SGK1 transcript levels were ~4times lower in the antisense cell populations compared with WT M1 cells ( P N-60, and K127M SGK1 wereconstructs incorporated into the M1 genome and were expressed, wedetermined HA-protein expression by using Western blot analysis. N-60 SGK1 signal was robust from 7 µg total protein lysate (Fig. 2, lane 4 ). Similarly, aspreviously reported for human embryonic kidney 293 and mammaryepithelial MCF10A cells ( 16, 22 ), we found thatfull-length SGK1 proteins are expressed at levels much lower thantruncated SGK1. Therefore, it was necessary to immunoprecipitatefull-length and K127M SGK1 protein by using an SGK1 antibody to enrichfor the signal in Western blot analysis. Western blot analysis ofimmunoprecipitated protein using SGK1 antibody from M1 cells expressingfull-length or K127M SGK1 displayed the appropriate size bands whenprobed with the HA antibody (Fig. 2, lanes 2 and 5 ). As a positive control, we used protein from COS-7 cellstransiently transfected with full-length HA-tagged SGK1 (Fig. 2, lane 3 ). We are confident of the specificity of the HAantibody, because immunoprecipitated protein from WT M1 cells subjectedto Western blot analysis was negative for the HRP signal (Fig. 2, lane 6 ). Furthermore, the immunoprecipitation was performedwith a mouse antibody, which does not cross-react with the HRP-labeledanti-rabbit antibody used in Western blot analysis. This is importantbecause immunoprecipitation of SGK1 using HA antibody raised in rabbitgave an immensely strong signal in Western blot (Fig. 2, lane1 ). The saturated signal in lane 1 most likelycorresponds to the secondary antibody bound to heavy-chain rabbitimmunoglobulins carried over from the immunoprecipitation. These datademonstrate that the stable M1 cells we generated effectively expressthe truncated, full-length, and dominant-negative SGK1 proteins.6 F) N, ?/ p2 |7 y2 o2 ~& B! Y
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Fig. 2. Western blot analysis confirms stable expression ofhemaglutinnin (HA) SGK1 proteins in M1 cortical collecting duct cells.Protein was separated on a polyacrylamide gel and with an anti-HAantibody as described in MATERIALS AND METHODS. Lane1 : protein immunoprecipitated (IP) with HA-antibody from M1 cellsexpressing K127M SGK1. Lane 2 : protein immunoprecipitatedwith SGK1 antibody from M1 cells expressing K127M SGK1. Lane3 : positive control ( ctr); protein immunoprecipitated with SGK1antibody from COS-7 cells transfected with 1 µg full-length HA SGK1construct. Lane 4 : 7 µg total protein from M1 cellsexpressing NH 2 -terminal truncated ( N-60) SGK1. Lane 5 : protein immunoprecipitated with SGK1 antibody fromM1 cells expressing full-length SGK1. Lane 6 : negativecontrol; protein immunoprecipitated with SGK1 antibody from WT cells.Western blot analysis and immunoprecipitation procedures (see MATERIALS AND METHODS ) were performed twice independently( n = 2). Signal was detected by using ECL substrate in lanes 1-4 and West Dura substrate in lanes 5 and 6.9 d* x- E0 H3 Q6 V" ?

Effect of changes in SGK1 expression on transepithelialNa transport in M1 cells. To observe the effect of expressing full-length, N-60, antisense,and K127M SGK1 on transepithelial Na transport, each cellline was cultured on permeable filters concurrently with WT M1 cells.Fig. 3 A, illustrates thesteady-state I sc of each cell line grown in IDCmedium. Noticeably, I sc in cells expressing full-length SGK1 was significantly higher than that in WT M1 cells ( P I sc values of N60 SGK1 populations were comparable to WT values.Importantly, the expression of antisense and K127M SGK1 in M1 cellsdecreased I sc values markedly, to ~20% of I sc in WT cells ( P medium that includes serum and growthfactors.
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Fig. 3. Transepithelial Na transport levels[short-circuit current ( I sc )] in WT M1 cellsand cells stably expressing full-length, N-60, antisense, and K127MSGK1. A : cells were maintained in complete insulin- anddexamethasone-containing growth medium until confluent monolayersdeveloped. I sc values were determined 48 hafter development of confluency. B : growth medium was thenreplaced with insulin and steroid hormone-free (I-SterF) medium for48 h to establish basal levels of Na transport. InI-SterF medium, basal rates of Na absorption weresignificantly higher in cells expressing full-length SGK1 compared withother cell populations. C : 1 µM dexamethasone was added tothe I-SterF medium and incubation continued for 24 h.Dexamethasone significantly increased Na current in WT andfull-length SGK1 cells but failed to substantially stimulate it inantisense and K127M SGK1 cells. The I sc valuesrepresent average values ± SE from independent Millicellcultures. Each experiment was repeated from 2 created progenitorpopulations. Statistical comparison between each respective cellpopulation and WT within each experimental parameter was made by usingStudent's paired t -test (1-tailed, 2-sample equalvariance); *** P P P left to right : WT, full-length, N-60, antisense, and K127M.
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In I-SterF, I sc values typically declined tolevels below 10 µA/cm 2 (see Fig. 3 B ).However, a noticeable difference was observed in the cell linesoverexpressing full-length SGK1, for which I sc remained high (16.18 µA/cm 2 ) even in the absence of serumand hormone stimulation. To demonstrate that these changes were notsimply due to a "time effect," we kept separate cultures incomplete growth medium for an additional 11 days. Within this timeframe, cells maintained steady-state levels of I sc and R TE (data not shown).
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After basal levels of transepithelial I sc wereestablished, cells were treated with 1 µM dexamethasone in I-SterFmedium for 24 h (Fig. 3 C ). Dexamethasone, as expected,increased I sc values (2.7-fold) in WT M1 cells.Similarly, I sc values in cells expressing full-length SGK1 increased twofold after dexamethasone treatment. Thesecells reached rates of Na transport that weresignificantly higher than those observed in WT cells ( P I sc values in the N-60populations increased approximately twofold with corticosteroidstimulation, the resulting I sc was stillsignificantly lower than that in WT levels ( P absorption in cells expressing antisense SGK1 and did not substantially increase Na transport above basal levels in cellsexpressing the dominant-negative K127M SGK1. I sc values for antisense and K127M SGK1 cell populations remainedsignificantly lower than WT M1 cells in the presence of 1 µMdexamethasone ( P P respectively).

Effect of changes in SGK1 expression on R TE. Generally, high- R TE values characterize celltypes with very tight junctions, whereas low R TE values are associated with leaky epithelia or multiple open ionchannels. In the present study, as well as in previouslypublished reports ( 33 ), M1 cells predominantly develop R TE 900 · cm 2. However, antisensepopulations incubated in I-SterF medium and 1 µMdexamethasone-containing medium displayed exceptionallylow- R TE values of 421.2 ± 71.8 and569.8 ± 87.4 · cm 2,respectively (Fig. 4, B and C ). The apical to basolateralpermeability of Millicell cultures were inspected to determine theintegrity, or "leakiness," of the monolayer. Four hundredmicroliters of tissue culture medium were applied to the apicalcompartment of the Millicell chamber, and 600 µl of the same mediumwere applied to the basolateral compartment. During regular changes ofthe culture medium, potential leakage through the monolayer wasdetermined by measuring fluid volume from each compartment. Under eachexperimental growth parameter, there was no significant change in fluidvolumes from each compartment. Additionally, [ 3 H]mannitolparacellular permeability assays (described in Ref. 41 )were performed to further verify that the monolayer was not leaky inantisense cell populations cultured on permeable supports. The percentleakage of radioactively labeled mannitol measured from WT cellscultured in IDC medium did not differ significantly compared withantisense cells incubated in I-SterF- and dexamethasone-containing medium (data not shown). Together, these observations are highly suggestive that downregulation of SGK1 alters R TE values. The resulting epithelial monolayeris not leaky and therefore is suitable for the determination oftransepithelial Na transport.$ h) f4 e, V8 L* q' O: Z

Fig. 4. Transepithelial resistance ( R TE ) in WT M1cells and cells stably expressing full-length, N-60, antisense, orK127M SGK1. The cell populations were maintained as described for Fig. 3. R TE values were measured with an epithelialvoltohmmeter. On the whole, R TE values in cellsexpressing different SGK1 constructs did not substantially deviate fromWT M1 values. However, cells expressing antisense SGK1 displayedsignificantly lower R TE values. Values areaverages ± SE. Statistical comparison among WT and each cellpopulation is made by using Student's paired t -test(1-tailed; 2-sample equal variance); *** P P P left to right : WT, full-length, N-60,antisense, and K127M.

Figure 4 A also demonstrates that cells overexpressingfull-length SGK1, grown in IDC medium, exhibited lower R TE values compared with control( P R TE values are a result of veryhigh-ENaC activity, maximally stimulated by high concentrations ofhormone and serum.

DISCUSSION: C7 R5 O) w: b/ q; _) H0 |

Since the recent identification of SGK1 as an earlyaldosterone-induced gene, it has been a priority to characterize thephysiological role of SGK1 in epithelial Na transport.Prior studies utilizing X. laevis oocytes and A6 cell linesare demonstrative of the ability of SGK1 to mediate ENaC activity.However, the role of SGK1 in effecting mineralocorticoid- andcorticosteroid-induced Na transport in mammalian CCD cellshad not been established before this study.

This study contributes to establishing SGK1 as acorticosteroid-regulated mediator of Na reabsorption inmammalian collecting duct cells. The overexpression of SGK1 in M1 celllines increased transepithelial I sc significantly above control levels under basal growth conditions andafter dexamethasone treatment. Furthermore, the downregulation of SGK1transcript using an antisense SGK1 construct and the expression of adominant-negative SGK1 decreased transepithelial I sc. Because we performed these studies by usinga mixed population of cells stably expressing SGK1 constructs, theresults cannot be attributed to mere clonal variations. Furthermore,studies published by Faletti et al. ( 9 ) similarlyascertain that SGK1 is a necessary component of ENaC-mediated Na transport in X. laevis A6 cells. Faletti etal. included overexpression of full-length human and dominant-negativeD222A mutant SGK1, which is unresponsive to phosphorylation by PDK2.Basal rates of Na transport in A6 cells overexpressinghuman SGK1 were 3.5 times higher than untransfected control.Conversely, D222A SGK1 clones did not respond to hormone stimulation,which is characteristic of A6 model epithelial cells.0 O& B0 ~; K' ^9 ~, C4 D# ~& L

However, our studies also examined populations of cells thatoverexpressed truncated SGK1. These cells did not respond to corticosteroid treatment to the extent observed in WT and full-length SGK1 populations. Because N-60 SGK1 is expressed at very high levelsin both M1 cells and other cell types ( 16, 22 ) and maintains its phosphorylation (activation) sites, the reason for thisobserved effect on ENaC function is not obvious. We speculate that thefirst 60 amino acids in SGK1 are necessary for stimulation of ENaCactivity. It is reasonable to propose this, because research within thepast decade has identified several proteins that contain NH 2 -terminal Phox homology domains, which specifically bindto phosphoinositides to target to their subcellular site ofactivation ( 5, 8, 14, 32, 40 ). Interestingly, themouse homolog of human SGK3, cytokine-independent survival kinase(CISK), contains a PX domain in the NH 2 -terminal region( 21 ). Mutation of this region inhibited CISKlocalization to endosomal compartments and therefore inhibited CISKfunction ( 38 ). The highly conserved PX sequence motifidentified in CISK [R(R/K)xxLxx(Y/F)] is also conserved within theNH 2 -terminal region of SGK1 that was truncated in our N-60 cell lines. Perhaps, in a manner similar to the Pleckstrin homology domain of PKB and the PX domain of CISK, SGK1 possesses atargeting motif necessary for PI-3 kinase-dependent activation at themembrane. We speculate that this may in part explain the lack of effecton Na transport in N-60 SGK1 cells when they aretreated with corticosteroid.% B6 w0 h# p# m* N
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Additionally, the NH 2 terminus of SGK1 may be responsiblefor targeting it to the appropriate ENaC-activating substrate. In thisinstance, the overexpression of truncated SGK1 in our studies couldpossibly be acting as a dominant-negative and thus inhibits full ENaCactivity in M1 cells. Recent evidence from Brickley et al.( 3 ) supports our contention that theNH 2 -terminal region targets SGK1 to the site ofNa transport action. Their studies demonstrated adifference between cellular localization of N-60 SGK1 greenfluorescent protein (GFP) and SGK1 GFP ( 3 ). Althoughfull-length SGK1 GFP is predominantly located in the cytoplasm andplasma membrane of HEK293T, Cos, SK-BR-3, and Madin-Darby canine kidneycells, truncated SGK1 GFP is homogenously distributed and fails tolocalize to the membrane ( 3, 29 ). This group haspreviously demonstrated that expression of truncated and WT SGK1similarly inhibit apoptosis in MCF10A-Myc cell lines. For thisreason, N-60 SGK1 does not behave as a dominant-negative kinase inrelation to apoptosis ( 22 ). We therefore suggestthat the putative SGK1-targeting motif may specifically regulate SGK1 activity in ENaC-mediated Na transport.
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Analysis of the R TE values obtained in thisstudy implicates SGK1 as a key component in the maintenance ofepithelial tight junctions, in addition to its role in mediatingcorticosteroid-induced Na reabsorption. Populations of M1cells stably expressing antisense SGK1 exhibited significantly lower R TE values compared with WT cells in eachexperimental medium. We demonstrated that the substantial decrease in R TE values observed did not result in a leakyepithelium by using apical-to-basolateral permeability assays.Additionally, the cells maintained viability, as suggested by a modestrise in R TE values when treated with corticosteroid.6 a) l2 ~* F+ f# s) ^1 j- s
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Of particular interest and relevance to the present study, severalinvestigators have reported that steroid hormones positively modulate R TE in other epithelial cells. In these studies,the R TE of rabbit distal colon ( 12, 13, 35 ) and mammary epithelial cells ( 4, 31, 41 )revealed a significant increase in R TE valuesafter aldosterone and dexamethasone treatment, respectively. Althoughthe precise molecular mechanism of steroid-regulated tight junctionformation is not clearly established, Singer et al. ( 31 )demonstrated that specific serine/threonine kinase inhibitors, such asH7, moderately reduced R TE values in mouse mammary epithelial cells after dexamethasone treatment. Accordingly, the effect of downregulation of SGK1 on R TE values in M1 cells in the present study suggests that SGK1 may be animportant regulator of tight junction formation. Given that CCD cells,in vivo, necessarily develop very tight junctions to minimize back-fluxof ions, SGK might have dual physiological roles in renal cells.Perhaps aldosterone concurrently modulates R TE and ENaC activity, by way of SGK1, to efficiently reabsorbNa  .. y1 p( T% H, }, E
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In summary, our data affirms that SGK1 is a critical player in themolecular pathway of ENaC activation. In our mammalian collecting ductcell lines, SGK1 activated transepithelial Na transportand ostensibly affected tight junction formation. Our study alsosuggests that the NH 2 terminus of SGK1 is necessary formodulating ENaC activity, possibly through a putative PX domain.# n( ]: a% n0 U% A$ ^
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ACKNOWLEDGEMENTS
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We thank Dr. Suzanne D. Conzen for providing the HA-taggedfull-length, N-60, and K127M SGK1 constructs.
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