Targeted degradation of ENaC in response to PKC activation ofthe ERK1/2 cascade - 《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 13:36 |只看该作者 |倒序浏览 |打印

作者：Rachell E.Booth and James D.Stockand作者单位：Department of Physiology, University Health Science Center, SanAntonio, Texas 78229-3900 4 N: j7 c! e# D6 o

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      【摘要】
   Renal A6 epithelial cells were used todetermine the mechanism by which protein kinase C (PKC) decreasesepithelial Na channel (ENaC) activity. Activation of PKCreduced relative Na reabsorption to over the next 24-48 h. In response toPKC signaling, -, -, and -ENaC levels were 0.97, 0.36, and0.39, respectively, after 24 h, with the levels of the latter twosubunits being significantly decreased. The PKC-mediated decreases in - and -ENaC were significantly reversed by simultaneous additionof the mitogen-activated protein kinase (MAPK)/extracellularsignal-regulated kinase-1/2 inhibitors U-0126 and PD-98059. Theseinhibitors, in addition, protected Na reabsorption fromPKC, demonstrating that the MAPK1/2 cascade, in some instances, plays acentral role in downregulation of ENaC activity. The effects of PKC on - and -ENaC levels were additive with those of inhibitors oftranscription (actinomycin D) and translation (emetine andcycloheximide), suggesting that PKC promotes subunit degradation anddoes not affect subunit synthesis. The bulk of whole cell -ENaC wasdegraded within 1 h after treatment with inhibitors of synthesis;however, a significant pool was "protected" from inhibitors for upto 12 h. PKC affected this protected pool of -ENaC. Moreover,proteosome inhibitors (MG-132 and lactacystin) reversed PKC effects onthis protected pool of -ENaC. Thus PKC signaling via MAPK1/2 cascadeactivation in A6 cells promotes degradation of -ENaC.
      【关键词】 proteosome hypertension sodium transport MG MG lactacystin9 R6 w8 ]) E/ {/ X0 u
   INTRODUCTION
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ACTIVITY OF THE AMILORIDE-SENSITIVE epithelial Na channel (ENaC) israte limiting for transepithelial Na (re)absorption(reviewed in Refs. 7, 9, 12, 21, 23, 27 ). Active ENaC islocated in the luminal plasma membrane of many epithelia, includingsalivary glands, lung, distal colon, and nephron. Phenotypic analysisof ENaC knockout mice and rare forms of genetic hyper- and hypotensionin humans associated with improper salt conservation and wasting,respectively, demonstrates that this channel and its proper regulationplay a pivotal role in blood pressure control (reviewed in Refs. 3, 15, 34 ).

ENaC is a heteromeric channel consisting of three homologous butdistinct subunits:,, and. Each subunit has twomembrane-spanning regions: one large extracellular loop and twocytosolic domains. The -subunit is believed to form the channelpore, with - and -ENaC serving as accessory regulatory subunits( 20 ). The cytosolic COOH-terminal tails of - and -ENaC are effector sites for channel regulation ( 12, 23 ).+ b6 b+ _; ?+ ~  X6 T8 P
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Several endocrine factors, such as the mineralocorticoid aldosterone,and disparate cellular signaling cascades impinge on ENaC activity tofine tune Na balance ( 7, 9, 21 ). Similar toother ion channels, ENaC activity is controlled at the level of channelgating and number of active channels in the luminal plasma membrane.Although detailed examination of posttranslational modification,membrane insertion and retrieval, and protein degradation has providedclues about ENaC regulation, a complete understanding of ENaCmodulation remains elusive.2 {. V" M$ @* v  y& _

Yanase and Handler ( 36 ) were the first to demonstrate thatprotein kinase C (PKC) inhibits Na transport by affectingamiloride-sensitive channels in renal A6 epithelial cells. Severalinvestigators subsequently confirmed that PKC inhibitsamiloride-sensitive ENaC ( 2, 8, 17 ). The initial decreasein ENaC activity most likely results from a decrease in openprobability and/or withdrawal of ENaC protein from the luminalmembrane. We recently demonstrated in renal epithelia that a laterPKC-dependent, long-term downregulation of ENaC results from decreasesin total cellular ENaC pools, with kinase decreasing - and -, butnot -, ENaC levels ( 29 ). Lin et al. ( 16 )and Zentner et al. ( 37 ) found in salivary epithelia asimilar action of PKC, but in this instance, kinase decreases -ENaClevels through transcriptional interference mediated by PKC-activatedmitogen-activated protein (MAP) kinase (MAPK)-1/2 signaling. Themechanism by which long-term activation of PKC decreases - and -ENaC levels has not been investigated.
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All three ENaC subunits contain well-conserved PY (PPPXY) motifs intheir cytosolic COOH termini ( 25, 26 ). This motif bindsNedd4 ubiquitin ligases, including Nedd4-2, which ultimately facilitatechannel retrieval and degradation and, thus, decrease ENaC activity( 12, 23 ). Indeed, gain of function mutations in - and -ENaC resulting from disruption/deletion of the PY motif leads tothe inheritable, monogenic hypertensive disease Liddle's syndrome(reviewed in Refs. 3, 15, 34 ).MAPK1/2-mediated phosphorylation of threonine-623 and -613 in - and -ENaC, respectively, which are located just proximal to the PYmotif, facilitates Nedd4 binding ( 22 ). This implies thatthese residues are important in the regulation of ENaC activity,possibly by impinging on channel retrieval. Supporting this contentionare recent findings showing that alanine substitution for theseconserved threonine residues increases ENaC activity in some instancesin a reconstituted system ( 22 ). PKC signaling impingeson the MAPK1/2 cascade by activating Raf ( 32 ). BecauseMAPK1/2 may play a role in the regulation of ENaC via posttranslationalmodification and PKC is a known activator of MAPK1/2 signaling, wehypothesized that activation of PKC promotes degradation of - and -ENaC through activation of the MAPK1/2 cascade. The presentfindings support such a mechanism., J. O* Y& c1 h/ G
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METHODS

Materials and reagents. All chemicals and enzymes were of reagent grade and were purchased fromSigma (St. Louis, MO) and BioMol (Plymouth Meeting, PA) unless notedotherwise. The immortalized amphibian distal tubule A6 epithelial cellline was obtained from American Type Culture Collection. The PKCactivator phorbol 12-myristate 13-acetate (PMA) and its negativecontrol 4 -PMA were prepared fresh in DMSO as 1 mg/ml stock solutionsand used at a final concentration of 100 ng/ml (162 nM). TheMAPK/extracellular signal-regulated kinase (MEK) inhibitors PD-98059and U-0126, as well as its negative control, U-0124, were stored frozen(in DMSO) as 10, 5, and 5 mM stock solutions and used at finalconcentrations of 10, 0.5, and 0.5 µM, respectively. The translationinhibitors cycloheximide (Chx, in methanol) and emetine (Emt, inH 2 O) were stored at 4°C as 1.0 mg/ml stock solutions andused at final concentrations of 3.5 and 1.8 µM, respectively. Thetranscription inhibitor actinomycin D (ActD, in methanol) was stored at4°C as a 1 mg/ml stock solution and used at a final concentration of790 nM. The proteosome inhibitors MG-132, MG-262, and lactacystin werestored frozen (in DMSO) as 6.0, 10, and 10 mM stock solutions and usedat final concentrations of 6.0, 1.0, and 10 µM, respectively.) g0 i+ @9 }6 N4 H* j! u! I
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All reagents used for Western blot analysis, unless noted otherwise,were obtained from Bio-Rad (Hercules, CA) and Pierce (Rockford, IL).For each lysate, protein concentration was determined with thebicinchoninic acid protein assay. Affinity-purified rabbit polyclonal anti- Xenopus ENaC (xENaC) antibodies (Ab 586 for -ENaC, Ab 592 for -ENaC, and Ab 2102 for -ENaC) have beendescribed previously ( 18, 29 ). The affinity-purifiedchicken polyclonal anti- -xENaC antibody LLC2 has been describedpreviously ( 29, 35 ). These antibodies are subunitspecific, in that they show no improper cross-reactivity, and recognizethe appropriate native and recombinant ENaC subunits. Therabbit polyclonal anti-MAPK1/2 and rabbit polyclonal anti-MEK1/2 andmonoclonal phospho-MAPK1/2 antibodies were obtained from UpstateBiotechnology (Waltham, MA) and Cell Signaling Technologies (Beverly,MA), respectively. Anti-rabbit and anti-mouse horseradishperoxidase-conjugated secondary antibodies were obtained fromKirkegaard and Perry Laboratories (Gaithersburg, MD). Kodak BioMaxLight-1 film and Chemiluminescence Reagents Plus (NEN Life ScienceProducts, Boston, MA) were used to develop Western blots.( `" ?  a* h* v6 m

Cell culture. All experiments were performed on renal A6 epithelial cells( passages 75-81 ). Cells were cultured on polycarbonatesupports (Costar Transwell-Clear inserts; 0.4-µm pore size,4.7-cm 2 growth area) using standard methods describedpreviously ( 29, 31 ). Briefly, cells were maintained at26°C in 1% CO 2 with complete amphibian medium [26.2%L-15 Leibovitz, 26.2% Ham's F-12, 7.6% fetal bovine serum, 1.5% L -glutamine (200 mM solution), 0.3%penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/mlstreptomycin), and 0.3% of a 7% sodium bicarbonate solution].Double-distilled H 2 O was added (~38%) for a finalsolution osmolarity of ~200 mosM. The medium was also supplementedwith 1.5 µM aldosterone. High-resistance polarized A6 cell monolayerswere used for all experiments. With these culture conditions, theamiloride-sensitive ENaC mediates Na reabsorption.

Western blot analysis. All immunochemistry was performed on whole A6 cell lysate with gelsroutinely loaded with lysate at 60 µg/well. Whole A6 cell lysate wasextracted after three washes with Tris-buffered saline using standardprocedures ( 31 ). Cells were scraped and then 2 h at 4°C in RIPA lysis buffer (10 mM NaPO 4, 150 mM NaCl, 1% deoxycholate, 1% Triton X-100, and 0.1% SDS, pH 7.2)supplemented with the protease inhibitor phenylmethylsulfonyl fluoride(1 µM). After cellular debris was cleared, standardization of totalprotein concentration, and addition of Laemmli sample buffer (0.005%bromphenol blue, 10% glycerol, 3% SDS, 1 mM EDTA, 77 mMTris · HCl, and 20 mM dithiothreitol), lysates were heated to85°C for 10 min. Proteins were then separated by standard SDS-PAGE (7.5% gels) and subsequently electrophoretically transferred to nitrocellulose (0.45 µM). Western blot analysis was performed usingstandard techniques and appropriate antibodies ( 29, 31 ), with primary and secondary antibodies used at 1:1,000 and 1:20,000, respectively. Tween 20 (0.1%) and 5% dried milk (Nestle,Wilkes-Barre, PA) were used as blocking reagents. Band intensity wasquantified with densitometric scanning using Sigmagel (JandelScientific, San Rafael, CA). When possible, the flood configurationwith the highest practical threshold was used to measure band density.1 H0 m9 j) ^" X
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Western blots were often stripped of primary and secondary antibody tofacilitate subsequent reprobing with distinct antibodies. All Westernblots were stripped in 100 mM 2-mercaptoethanol, 62.5 mMTris · HCl (pH 6.7), and 2% SDS for 30 min at 55°C withconstant agitation. After removal of antibodies, nonspecificinteractions were reblocked by incubation in Tris-buffered saline-Tween20 and 5% milk for 2 h before the blots were reprobed withprimary antibody.! L7 B$ t7 y8 C$ w1 v& [

Electrophysiology. Transepithelial Na current was calculated, as describedpreviously ( 28-31 ), from Ohm's law as the ratio oftransepithelial voltage to transepithelial resistance underopen-circuit conditions using a Millicel Electrical Resistance Systemwith dual Ag-AgCl pellet electrodes (Millipore, Billerica, MA) tomeasure voltage and resistance.

Experimental design. All experiments were performed on A6 cells grown on permeable supportsmaintained in the presence of aldosterone and serum. Cells were usedonly after formation of electrically tight monolayers. With theseconditions, each monolayer served as its own control; effect ofexperimental maneuvers on relative current was one end point, andassessment of ENaC subunit levels after treatment was the other endpoint. Changes in ENaC subunit levels were usually normalized to theeffects of vehicle at the same time point. All reagents, including PMAand inhibitors, were added simultaneously unless noted otherwise.Typically, starting voltages and resistances were measured, andmonolayers were subsequently treated with vehicle, PMA alone, PMA inthe presence of inhibitor, and inhibitor alone from 0 to 24 h. Atthe culmination of each experiment, voltages and current werereevaluated and cells were extracted. The levels of ENaC subunits intreated lysates were then established with immunochemistry. Thisexperimental design facilitated quantitation of the effects of PMA inthe presence and absence of inhibitors on changes in ENaC subunitlevels and transepithelial current.

Statistics. Values are means ± SE. Statistical significance ( P 0.05) was determined using the t -test for differences inmean values and a one-way analysis of variance in conjunction with theStudent-Newman-Keuls test for multiple comparisons.

RESULTS
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PKC decreases Na transport and -and -ENaC levels. Figure 1 shows the effects of adding thePKC activator PMA on Na transport across A6 epithelialcell monolayers as well as on the levels of -, -, and -ENaC inthese cells. Addition of PMA, in contrast to 4 -PMA, whichhad no effect, significantly decreased current to 0.12 ± 0.02 and0.16 ± 0.04 by 2 and 24 h, respectively.' f) K+ W$ A+ }( m3 W" t' T; S1 e1 N
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Fig. 1. Protein kinase C (PKC) decreases Na transport and -and -subunit epithelial Na channel (ENaC) levels. A : relative (to pretreatment) current levels across A6 cellmonolayers after treatment with phorbol 12-myristate 13-acetate (PMA)and its inactive analog (4 -PMA). *Significant decrease vs. startinglevels and 4 -PMA. B : typical Western blots probed withanti- (~90 kDa)-, anti- (~95 kDa)-, and anti- (~95kDa)- Xenopus ENaC (xENaC) antibodies. Each lane contains thesame amount of lysate from cells treated with vehicle, PMA, and itsinactive analog for 2 or 24 h. C : effects of 2 and24 h of PMA treatment on xENaC subunit levels. *Significantdecrease vs. starting values. D : typical Western blotcontaining lysate from A6 cells treated with vehicle and PMA in theabsence and presence of PKC inhibitors2,2',3,3',4,4'-hexahydroxy-1,1'-biphenyl-6,6'-dimethonol dimethyl ether(HBBDE) and Gö-6976. Blot was probed with anti- -xENaCantibody. E : typical Western blot containing lysate from A6cells treated with vehicle and PMA in the absence and presence of PKCinhibitors HBBDE and calphostin C. Blot was probed with anti- -xENaCantibody.3 G) x8 z) F' T+ o% x( {( D0 \
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Figure 1 B shows that PMA, but not its inactive analog,decreases - and -ENaC levels after 24 h of treatment. Thetypical Western blots of Fig. 1 B contained whole A6 celllysate and were probed with rabbit polyclonal anti- -xENaC andanti- -xENaC antibodies and the chicken polyclonal anti- -xENaCantibody. The rabbit polyclonal anti- -xENaC antibody Ab 2102 andLLC2 produced identical results (Figs. 1 E and 2 A ). Figure 1 C summarizes the effects of 2 and 24 h of PMA treatment on ENaCsubunit levels. At 2 h, the relative levels of - and -ENaCof 0.94 ± 0.24 and 1.1 ± 0.06, respectively, wereunaffected by PMA, whereas those of -ENaC were already markedly decreased to 0.71 ± 0.12 ( n = 6). At 24 h,the relative level of -ENaC (0.97 ± 0.26) was unaffected,whereas levels of - and -ENaC were significantly decreased to0.36 ± 0.04 and 0.39 ± 0.04 ( n = 6),respectively.

Fig. 2. Inhibitors of the mitogen-activated protein kinase (MAPK)-1/2cascade protect Na transport and - and -ENaC fromeffects of PKC. A : typical Western blot containing lysatefrom cells treated with vehicle, PMA, and PMA in the presence of themitogen-activated protein kinase (MAPK)/extracellular signal-regulatedkinase (MEK)-1/2 inhibitors PD-98059 and U-0126. Inhibitors wereadded simultaneously with PMA. Blots were probed with anti- -xENaC(Ab 592), anti- -xENaC (Ab 2102), phospho-MEK1/2, phospho-MAPK1/2,and MAPK1/2 antibodies, respectively. B : relative currentshowing effects of 6 and 24 h of treatment with vehicle (DMSO),PMA, PMA PD-98059, and PMA U-0126. *Significantdecrease from starting values and vehicle at the same time point.**Significantly greater than PMA at the same time point. C :relative density of -ENaC in cells treated with vehicle, PMA,PMA PD-98059, and PMA U-0126. *Significant decreasecompared with vehicle at the same time point. **Significant increaseover PMA at the same time point. D : relative density of -ENaC in cells treated with vehicle, PMA, PMA PD-98059, andPMA U-0126. *Significant decrease compared with vehicle at thesame time point.

The typical Western blots in Fig. 1, D and E,demonstrate that PKC inhibitors lessen the PMA-dependent decrease in - (Fig. 1 D ) and -ENaC (Fig. 1 E ) levels. Forthese experiments, monolayers were treated with vehicle, PMA, andPMA PKC inhibitor (45 µM 2,2',3,3',4,4'-hexahydroxy-1,1'-biphenyl-6,6'-dimethonol dimethyl ether, 5 nM Gö-6976, and 50 nM calphostin C) for 16 h.. v6 f) ~1 e! m. a/ V8 P

PKC decreases Na transport and ENaClevels through activation of the MAPK1/2 cascade. The experiments reported in Fig. 2 tested whether PKC decreases -and -ENaC levels and Na reabsorption via activation ofthe MAPK1/2 cascade. The typical Western blots in Fig. 2 A contain lysate extracted from cells treated for 6 and 24 h withvehicle and PMA in the absence and presence of the structurallyunrelated MEK1/2 inhibitors PD-98059 (10 µM) and U-0126 (0.5 µM).For these experiments, inhibitors were added simultaneously with PMA.We reported previously that MAPK1/2 levels in A6 cells are relativelyconstant and unaffected by many treatments ( 11 ). ThusMAPK1/2 level (Fig. 2 A ) was assessed to ensure equal loading.
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The effects of PMA in the absence and presence of MEK1/2 inhibitors onNa transport at 6 and 24 h are summarized in Fig. 2 B ( n = 18). PMA significantly decreasedrelative (to pretreatment) current to 0.29 ± 0.07 and 0.10 ± 0.03 at 6 and 24 h, respectively. Vehicle was without effect ateither time point. At 6 h, PMA in the presence of U-0126 hadsignificantly less effect on current, with relative current being0.65 ± 0.10. After 24 h, MEK1/2 inhibitors significantly protected current from PMA, with relative current levels of 1.2 ± 0.17 and 0.77 ± 0.06 for PMA PD-98059 and PMA U-0126, respectively.

Figure 2 C summarizes the effects of PMA in the absence andpresence of MEK1/2 inhibitors on -ENaC levels. After 24 h oftreatment, -ENaC levels in the presence of vehicle and PMA were0.96 ± 0.05 and 0.26 ± 0.05, with significantly lowerlevels in the PMA group ( n = 9). PD-98059 and U-0126significantly countered the effect of PMA to decrease -ENaC levelsat 24 h, with relative levels of 0.76 ± 0.12 and 0.69 ± 0.08, respectively ( n = 6). Although the effect isnot as robust, MEK1/2 inhibitors also protect -ENaC levels at 6 h. At this time, PMA significantly decreased relative -ENaC levelsfrom 1.01 ± 0.02 to 0.15 ± 0.05, with significantly greaterlevels in the presence of PMA PD-98059 and PMA U-0126 (0.57 ± 0.04 and 0.41 ± 0.08, respectively) than in thepresence of PMA alone ( n = 3). The actions of PMA inthe absence and presence of MEK1/2 inhibitors on -ENAC levels after6 and 24 h are summarized in Fig. 2 D ( n = 3). Although relative changes in -ENaC levels were more difficultto quantify, it was clear that, similar to their effects on -ENaC,MEK1/2 inhibitors tended to counter PMA-dependent decreases in -ENaC. Neither MEK1/2 inhibitor when added alone affected current orENaC subunit levels (Fig. 3 ), and thenegative control U-0124 was without effect (not shown). Moreover, asshown in Fig. 3, the p38 MAP kinase inhibitor SB-203580 had no effect on PMA-dependent decreases in -ENaC ( n = 2).
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Fig. 3. p44/42 MAPK (MAPK1/2), but not p38 MAPK, signalingdecreases -ENaC. Typical Western blot contained lysate from cellsgrown on filtered supports and treated with vehicle, PMA alone, and PMAin the presence of PD-98059, U-0126, and SB-203580. Blot was probedwith anti- -xENaC antibody ( top ) and then stripped andreprobed with anti-MAPK1/2 antibody ( bottom ). Black arrow,~95-kDa protein; gray arrow, faster-migrating more-diffuse band./ q0 I- [6 X8 u" B) f5 c2 N
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In addition to showing that PKC affects ENaC levels and Na transport in A6 cells via the MAPK1/2 cascade, results in Fig. 2 showthat the MAPK1/2 cascade is transiently activated in these cells byPKC, with MEK1/2 and MAPK1/2 being activated (phosphorylated) at 6 h and deactivated, most likely by negative-feedback pathways, by24 h. As expected, MEK1/2 inhibitors blocked activation(phosphorylation) of MAPK1/2. This can relieve MEK1/2 of feedbackinhibition, resulting in apparent hyperactivation of this upstream kinase.

Figure 3 shows that the anti- -ENaC antibody used in the presentstudy recognizes a ~95-kDa protein and a faster-migrating more-diffuse band in A6 cell lysate. Others postulated that the extracellular loop of the -subunit in active ENaC is cleaved byextracellular proteases, leading to a protein that runs on SDS-PAGE asa broad-band ~70-kDa protein ( 19, 33 ). PMA and otherexperimental maneuvers reproducibly affected the ~95-kDa protein, butnot the more-diffuse, faster-migrating protein (Figs. 4, 5, and 6 ). This, in combination withthe finding that the levels of the faster-migrating protein did notcorrelate well with current, led us to focus exclusively on the~95-kDa protein., s% J' t& i8 {% `
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Fig. 4. Effects of PKC on Na transport and - and -ENaCare additive with inhibitors of synthesis. A : typicalWestern blot containing lysate from cells treated with vehicle (DMSO),PMA, and cycloheximide (Chx) and emetine (Emt) in the absence andpresence of PMA. Blots were probed with anti- - and anti- -xENaCand MAPK1/2 antibodies, respectively. B : typical Westernblot containing lysate from cells treated with vehicle, PMA, andactinomycin D (ActD) in the absence and presence of PMA. Blots wereprobed with anti- (Ab 592)- and anti- -xENaC (Ab 2102) and MAPK1/2antibodies, respectively. C : relative density of -ENaC incells treated with vehicle (Con), PMA, Chx, Emt, and ActD in theabsence and presence of PMA. *Significantly lower than vehicle.**Significantly lower than PMA alone and respective inhibitor alone. D : relative density of -ENaC in cells treated withvehicle, PMA, Chx, Emt, and ActD in the absence and presence of PMA.4 u  S' T- [) W
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Fig. 5. A6cells contain a pool of -ENaC that is "protected" frominhibitors of synthesis. A : typical Western blots from cellstreated with vehicle, PMA, and ActD in the absence and presence of PMA.Blots were probed with anti- -xENaC antibody. B : typicalWestern blots containing lysate from cells treated with inhibitors. V,vehicle. Blots were probed with anti- -xENaC antibody (Ab 2102). C : -ENaC decay over time in response to inhibitors oftranscription and translation. Decay line for inhibitors oftranscription was calculated starting at 2 h./ {- q2 K5 a, `! C4 k& T9 z

Fig. 6. PKC decreases a protected pool of -ENaC in a manner that issensitive to inhibitors of the proteosome. A : typicalWestern blot of lysate from cells treated for 2 h with vehicle andChx and for an additional 4 h with Chx in the presence and absenceof PMA with and without MG-132 (132) and lactacystin (LC).Blot was probed with anti- -xENaC antibody. B : -ENaClevels in A6 cells treated with vehicle (Con), Chx for 2 hfollowed by an additional 4 h of treatment with Chx alone,Chx PMA, and CHX proteosome inhibitors (PI) in theabsence and presence of PMA. * P P C :relative current in A6 cells treated with vehicle (Con), Chx for 2 h followed by an additional 4 h of treatment with Chx alone,Chx PMA, and Chx PI in the absence and presence of PMA.* P
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Inhibition of transcription and translation is additive with PKC todecrease ENaC subunit levels. The experiments reported in Fig. 4 were performed to elucidate themechanism of PKC action on ENaC testing whether this kinase impinges onchannel synthesis or degradation. Figure 4 A shows representative Western blots containing lysate from cells treated withvehicle (DMSO), PMA, inhibitors of translation (3.5 µM Chx and 1.8 µM Emt), and PMA inhibitors of translation for ~10 h. Theseblots were probed with Ab 592 (for -ENaC), Ab 2102 (for -ENaC),and anti-MAPK1/2 antibodies. Activation of PKC clearly was additivewith inhibitors of translation with respect to decreasing -ENaClevels (measured as the ~95-kDa band). PMA also decreased -ENaClevels in an additive manner with inhibitors of translation. 6 h) andinhibitors of translation ( -ENaC, this was oftenmore difficult to consistently demonstrate. For the (first) blot inFig. 4 A, the effects of Emt and PMA are clearly additive on -ENaC, but in this experiment the effects of Chx have already saturated.

The inhibitor of transcription, ActD, was also additive with PMA.Typical Western blots containing lysate extracted from cells treatedwith vehicle, PMA, ActD (25 nM), and PMA ActD for 10 h areshown in Fig. 4 B.

The effects of PMA, transcription and translation inhibitors, and PMAin the presence of these inhibitors on - and -ENaC levels areshown in Fig. 4, C and D. For -ENaC, PMAtreatment significantly decreased levels to 0.34 ± 0.07. Chx,Emt, and ActD alone significantly decreased levels to 0.20 ± 0.03, 0.44 ± 0.11, and 0.42 ± 0.12, respectively.Simultaneous addition of PMA with Chx, Emt, and ActD significantlydecreased -ENaC levels to 0.08 ± 0.03, 0.12 ± 0.04, and0.09 ± 0.04, respectively, all of which are significantly lowerthan values with PMA and inhibitor alone ( n 7). Similarto -ENaC, PMA decreased -ENaC levels in an additive manner withinhibitors of transcription and translation ( n = 3).The effects of transcription and translation inhibitors on relativecurrents are reported in Table 1.

Table 1. Relative currents across A6 cells treated with PMA and transcriptionand translation inhibitors; t' y1 d; Y3 _  \9 y  S

PKC decreases -ENaC levels by promoting subunit degradation. The results in Figs. 1-3 demonstrate that PKC via MAPK1/2signaling decreases -ENaC levels. The results in Fig. 4 showing PKC to be additive with inhibitors of synthesis suggest that the mechanism of action is an increase in subunit degradation. Because MAPK1/2 phosphorylates the -ENaC subunit in a manner consistent with targeted degradation ( 22 ), we tested whether PKC doesindeed promote -ENaC degradation.

The representative Western blots in Fig. 5 A, which wereprobed with Ab 2102, contained lysate from cells treated with vehicle, PMA, ActD, and PMA ActD. PMA and ActD decreased -ENaC levels, with these actions clearly being additive by 3 h. PMA decreased -ENaC levels before ActD affected subunit levels.

Figure 5 B is a representative experiment (1 of 5) showing anextended time course for the actions of Chx, Emt, and ActD on -ENaC(probed with Ab 2102). The decay in -ENaC levels over time inresponse to inhibitors of transcription and translation is summarizedin Fig. 5 C. The blots in Fig. 5 B contained lysate from cells treated with inhibitors. Inhibitors of translation, as wellas the inhibitor of transcription, showed two phases of action: anearly effect with a time constant for ActD startingwith 2 h) and a later action with a time constant 6 h. Ourinterpretation of these results is that there are two pools of -ENaCin A6 cells: one that turns over rapidly with a short half-life andanother that is somewhat protected with a longer half-life.From the experiments in Figs. 4 and 5 A (and also Fig. 6 ),PMA clearly influenced the protected pool of channels. Although PMAdecreases current before 30 min, with current remaining 24 h (Fig. 1 A ), inhibitors of translation and transcription began to affect current only after ~4 h (Table 1 ). Indeed, at 2 h, -ENaC remained in cells treated with inhibitors of translation, although no decrease in Na transport was observed in these cells. Thus inhibitors of synthesis have a major influence on total cellular -ENaC pools before they affect current. This suggests that the protected pool of -ENaC correlates better with active channels than does the pool that turnsover more quickly.- C3 h/ n8 G. E+ B  w/ ~- h

To begin to determine whether this PMA-sensitive protected pool of -ENaC was possibly in the plasma membrane and sensitive toPKC/MAPK1/2-directed degradation, we performed the experiments described in Fig. 6. For these experiments, A6 cell monolayers weretreated with Chx for 2 h and then treated for an additional 4 h with fresh Chx alone and in combination with PMA in the absence andpresence of the proteosome inhibitors MG-132 (6.0 µM) and lactacystin(10 µM). MG-262 (1.0 µM) was also used and produced resultsidentical to MG-132 and lactacystin (not shown). The representative blot ( n = 6) in Fig. 6 A contained lysatefrom the respective groups and was probed with Ab 2102. In Fig. 6 B, the effects of proteosome inhibitors were pooled toallow for comparison with Chx alone and Chx PMA. Proteosomeinhibitors significantly reversed the effects of Chx on -ENaC, withlevels being 0.12 ± 0.05 and 0.34 ± 0.05 with Chx in theabsence and presence of proteosome inhibitors, respectively. Similarly,in the presence of PMA Chx, proteosome inhibitors significantlyprotected -ENaC, with levels of 0.07 ± 0.04 and 0.33 ± 0.06, respectively. Interestingly, although proteosome inhibitorsprotected -ENaC levels in the presence of Chx alone or in additionto PMA, proteosome inhibitors protected transport only in the absenceof PMA (Fig. 6 C ).
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Figure 6 C shows relative current across A6 cells treatedwith Chx for 2 h followed by further treatment for 4 h withChx in the presence and absence of PMA with and without proteosomeinhibitors ( n = 4). Proteosome inhibitors did notaffect decreases in current in response to Chx PMA, withrelative currents of 1.04 ± 0.04, 0.27 ± 0.04, 0.06 ± 0.05, 0.05 ± 0.04, 0.07 ± 0.07, and 0.04 ± 0.03 forvehicle, Chx, Chx PMA, Chx PMA MG-132, Chx PMA MG-262, and Chx PMA lactacystin, respectively.Addition of MG-132, MG-262, and lactacystin alone had no effect oncurrent (not shown) but significantly lessened the effects of Chx, with relative current of 0.45 ± 0.06, 0.46 ± 0.05, and 0.41 ± 0.04 for Chx MG-132, MG-262, and lactacystin, respectively.Moreover, we were unable to detect a protective effect on -ENaClevels or current by any proteosome inhibitor when they were added to cells simultaneously with PMA in the absence of Chx pretreatment (notshown, n = 3). Thus proteosome inhibitors protectedcurrent and -ENaC levels in the presence of decreased synthesis;however, in the combined presence of decreased synthesis and activated PKC, proteosome inhibitors protected only -ENaC levels and not current. We interpret this as PKC promoting retrieval of ENaC from themembrane and ultimate targeting of this channel for degradation at theproteosome, with PKC acting at a site upstream of the proteosome, possibly on the channel itself or on proteins involved in channel retrieval.1 t/ D, P) s$ _

DISCUSSION" E1 C# E6 r: N3 D) m; H: k# J
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We reported previously that activation of PKC leads to decreasesin - and -ENaC levels in renal A6 epithelia ( 29 ).These decreases result in long-term suppression of Na transport. The present results are consistent with these earlier findings and expand on them by defining the cellular signaling cascadeand mechanisms underpinning decreased ENaC activity. Figure 7 shows the simplest model consistentwith our present and past findings. Also shown in Fig. 7 is thecellular signaling cascade activated by PKC that we believe impinges onENaC. The present study demonstrates for the first time that thelong-term effects of PKC on - and -ENaC levels, as well astransport, are mediated by activation of the MAPK1/2 cascade, withdecreases in - and -ENaC levels in response to PKC-activatedMAPK1/2 signaling resulting from targeted degradation at theproteosome. Moreover, the present results in the context of theprevious findings of others are consistent with the possibility thatPKC-MAPK1/2 signaling acts directly on a pool of channels resident inthe plasma membrane.% W, a4 a+ S& b
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Fig. 7. Simplest model for PKC-MAPK1/2 regulation of ENaC,consistent with present results. MAPK1/2 signaling promotes ENaCretrieval and subsequent degradation via an endosomal compartment, withinhibitors of the proteosome affecting degradation at a step downstreamof retrieval. ENaC mem and ENaC endo, plasmamembrane and endosomal fractions of ENaC, respectively; Aldo,aldosterone; Sgk, serum- and glucocorticoid-induced protein kinase.Gray and black arrows, vesicular movement and signaling steps,respectively. Inhibitors are positioned to identify steps theyimpede., Y) E/ K9 N9 i  _8 W4 D; N1 p
( G5 c5 w) g4 L$ Y1 {+ f- |) E
Several other signaling cascades/proteins that are known to regulateENaC activity and levels, such as Nedd4-2, serum and glucocorticoid-induced protein kinase (Sgk), and N4WBP5A ( 6, 14, 24 ), also target ENaC retrieval and degradation. Thus retrievalmay be a particularly important point for physiological regulation ofENaC activity. Indeed, the human hypertensive diseases associated withabnormal ENaC retrieval support this contention ( 15 ).
6 A/ \5 `" J) f. j- L
Role of the MAPK1/2 cascade in PKC-mediated decreases in - and -ENaC and Na transport. The results in Figs. 2 and 3 showing that PKC actions on ENaC subunitlevels and Na transport are countered by two distinctinhibitors of MEK1/2 strongly imply that the MAPK1/2 cascade plays acentral role in negative regulation of channel activity. Consistentwith this implication are findings from Shi and colleagues( 22 ) showing that MAPK1/2 phosphorylates - and -ENaCon residues that directly influence interactions with Nedd4. IncreasedNedd4 binding to - and -ENaC promotes retrieval of the channelfrom the membrane and subsequent degradation ( 25, 26 ). Shiand colleagues also demonstrated that alanine substitution of thesecritical residues increases channel activity ~3.5-fold. The salientfeature of this MAPK1/2 regulation of ENaC activity is that it is aposttranslational event that modifies existing channels in a mannerthat facilitates their targeted retrieval and, ultimately, degradation.This mechanism is distinct from that proposed by Lin and co-workers( 16 ) and Zentner et al. ( 37 ) for MAPK1/2 andPKC regulation of -ENaC in salivary epithelia. This latter mechanisminvolves transcriptional interference. Thus PKC-MAPK1/2 signalinginfluences ENaC activity through at least two distinct mechanisms in asubunit-specific manner: transcriptional interference for -ENaC andposttranslational targeting for degradation of - and -ENaC.Exactly which mechanism is used to regulate ENaC in response to PKCmust also then be tissue and, possibly, species specific, inasmuch asour results, as well as those of Shi and colleagues, excludetranscriptional interference in renal A6 cells and in certainreconstituted systems.
5 [8 L" E# O3 `/ t" t
Support for posttranslational control of ENaC in response to PKC. The results in Fig. 4 showing the effects of PKC to be additive withinhibitors of transcription and translation are most consistent withthis kinase ultimately affecting - and -ENaC levels, as well asNa transport, at a site other than channel synthesis, suchas targeting channels for retrieval and, ultimately, degradation.Alternatively, both subunits could have alternative routes fortranscription and translation that are resistant to ActD, and Chx andEmt, respectively, but sensitive to PKC. Although we cannotdefinitively exclude this latter possibility, we believe that it isextremely unlikely. One other possibility that we cannot definitivelyexclude with the present results but suspect to be unlikely is that theeffects of PKC-MAPK1/2 signaling are indirect and mediated by aprotein, such as Sgk or N4WP5A ( 1, 14 ), that protects thechannel from degradation., @( F5 P" }! t& q, x
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The observation that activation of PKC influences -ENaC levels at atime between inhibitors of translation and transcription (Figs. 3 and 4 ) ( 29 ) provides additional support, albeit superficial, for the idea that PKC decreases subunit levels at a site distinct fromeither step of synthesis. Inhibitors of transcription and translation,in addition, affected -ENaC levels within 2-4 h, which is muchfaster than the actions of PKC on this subunit ( 29 ). Again, this suggests that PKC must act on ENaC subunits at a site distinct from synthesis.

The results in Fig. 6 demonstrate that when -ENaC levels are loweredby blocking synthesis with Chx for 2 h, the subsequent PMA-dependent decrease in -ENaC levels is sensitive to proteosome inhibitors. Because, as shown in Fig. 5, Chx has the greatest effect on -ENaC levels before 1 h and has little additional effect onsubunit levels between 2 and 4 h, we argue then that, in the experiments of Fig. 6, proteosome inhibition did not merely lessen normal channel turnover but actually countered targeted degradation initiated by PKC-MAPK1/2 signaling. Others have shown that inhibition of the proteosome protects the bulk of ENaC from rapid turnover ( 5, 18, 26 ). The present experiments differed from these earlier experiments, because we allowed degradation of the bulk of -ENaC before determining whether proteosomal blockade impinged onthe actions of PKC to decrease the protected pool of -ENaC. Such anapproach enabled us to focus specifically on this subunit pool in theabsence of the high background noise contributed by the turnover ofthat pool, which has a much shorter half-life.' V1 R( c7 V! b) `' v

There are two pools of -ENaC: one turns over quickly, and one isprotected but sensitive to PKC. Close inspection of the results in Fig. 5 shows two pools of -ENaC.One pool is quickly ( inhibitors. Similarly, blockade of transcription also quickly ( -ENaC, although it ismarkedly less abundant, is more resistant to blockade of transcriptionand translation, with significant levels being measurable for up to8-12 h after addition of inhibitor. We argue that the first poolcontains -ENaC, which is quickly turned over, and the second pool isprotected or somehow removed from the normal route of degradation,leading to the rapid turnover of the first pool. An alternative that wecannot exclude, but believe is unlikely, is that ENaC degradation issuppressed by some protein that itself has a very short half-life(e.g., Sgk) ( 4 ), and it is this latter protein that isaffected by transcription and translation inhibitors, as well asPKC-MAPK1/2 signaling. Because translation inhibitors decrease -ENaClevels before affecting current, we argue further that this protectedpool is more closely associated with active channels in the plasmamembrane. These observations are intriguing and merit furtherinvestigation but, in the context of the findings of others ( 5, 10, 13, 18, 35 ), enable us to speculate that this protected poolmight reflect a membrane-resident or supapical pool of -ENaC. Weiszand colleagues ( 35 ) reported that the half-life of thetotal cellular pools of - and -ENaC in A6 cells is ~2 h, butthe half-life of the pool that reaches 24 h.Kleyman and colleagues ( 13 ) report a similar half-life formembrane-resident -ENaC subunits in A6 cells. In contrast with thesestudies are the findings of De La Rosa and colleagues ( 5 )showing that in A6 cells whole cell and membrane-resident channels havea half-life of 60 min. Similarly, heterologously expressed ENaC has ashort half-life ( 26 ). Clearly, the present results showinga decreased but abundant level of -ENaC after 8-12 h oftreatment with inhibitors of synthesis contrast with these latterstudies and are more consistent with the findings showing that someportion of ENaC has a half-life 6 h.
  H6 `" C5 K2 }3 r
An intriguing aspect of the present research not fully understood isthe apparent discordant regulation of - and -ENaC levels by PKC,with PKC affecting the former subunit much more quickly than thelatter. Although this possibly could reflect differences in therelative abundance of each subunit, others reported previously that thethree ENaC subunits are noncoordinately regulated in A6 cells( 35 ).

In summary, the present results are consistent with the mechanism wherethe bulk of freshly synthesized ENaC is quickly degraded, with therates of synthesis and degradation being much more rapid than those forchannel insertion and retrieval into/from the plasma membrane. PKC viaMAPK1/2 then would simply increase the rate of channel retrieval,ultimately promoting degradation of this newly retrieved channel poolbecause of the very rapid degradation rate for ENaC. With such amechanism, blocking channel synthesis, as in the present study, withall other factors remaining unaffected, would lead to a decrease inENaC levels before it would affect transport because of the slow rateof channel retrieval. Moreover, blocking channel synthesis anddegradation in the presence of increased channel retrieval, as wespeculate is the case when A6 cells are treated with Chx, PMA, and aproteosome inhibitor, would then affect only ENaC levels and nottransport. In contrast to this, blocking channel degradationsimultaneously with retrieval would influence, as we found, ENaC levelsand transport.* f8 `, C$ y. I
- X0 A  Q1 O( s* b  C' P9 `" N
Relationship between aldosterone and PKC signaling. We recently reported that aldosterone activates MAPK1/2 signaling inrenal A6 epithelial cells ( 11 ). This genomic activation ofthe MAPK1/2 cascade was via transcriptional control of Ki-RasA, resulting in prolonged MAPK1/2 signaling. In consideration of thepresent results, aldosterone activation of the MAPK1/2 cascade wouldappear to be a negative-feedback response that might temper prolongedavid Na reabsorption.
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ACKNOWLEDGEMENTS

We thank Drs. D. C. Eaton, B. Malik, and J. P. Johnsonfor sharing anti-xENaC antibodies.
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