Targeted degradation of ENaC in response to PKC activation of the ERK1/2 cascade

Rachell E. Booth and James D. Stockand

Department of Physiology, University Health Science Center, San Antonio, Texas 78229-3900


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Renal A6 epithelial cells were used to determine the mechanism by which protein kinase C (PKC) decreases epithelial Na+ channel (ENaC) activity. Activation of PKC reduced relative Na+ reabsorption to <20% within 60 min. This decrease was sustained over the next 24-48 h. In response to PKC signaling, alpha -, beta -, and gamma -ENaC levels were 0.97, 0.36, and 0.39, respectively, after 24 h, with the levels of the latter two subunits being significantly decreased. The PKC-mediated decreases in beta - and gamma -ENaC were significantly reversed by simultaneous addition of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase-1/2 inhibitors U-0126 and PD-98059. These inhibitors, in addition, protected Na+ reabsorption from PKC, demonstrating that the MAPK1/2 cascade, in some instances, plays a central role in downregulation of ENaC activity. The effects of PKC on beta - and gamma -ENaC levels were additive with those of inhibitors of transcription (actinomycin D) and translation (emetine and cycloheximide), suggesting that PKC promotes subunit degradation and does not affect subunit synthesis. The bulk of whole cell gamma -ENaC was degraded within 1 h after treatment with inhibitors of synthesis; however, a significant pool was "protected" from inhibitors for up to 12 h. PKC affected this protected pool of gamma -ENaC. Moreover, proteosome inhibitors (MG-132 and lactacystin) reversed PKC effects on this protected pool of gamma -ENaC. Thus PKC signaling via MAPK1/2 cascade activation in A6 cells promotes degradation of gamma -ENaC.

proteosome; hypertension; sodium transport; MG-132; MG-262; lactacystin


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ACTIVITY OF THE AMILORIDE-SENSITIVE epithelial Na+ channel (ENaC) is rate limiting for transepithelial Na+ (re)absorption (reviewed in Refs. 7, 9, 12, 21, 23, 27). Active ENaC is located in the luminal plasma membrane of many epithelia, including salivary glands, lung, distal colon, and nephron. Phenotypic analysis of ENaC knockout mice and rare forms of genetic hyper- and hypotension in humans associated with improper salt conservation and wasting, respectively, demonstrates that this channel and its proper regulation play a pivotal role in blood pressure control (reviewed in Refs. 3, 15, 34).

ENaC is a heteromeric channel consisting of three homologous but distinct subunits: alpha , beta , and gamma . Each subunit has two membrane-spanning regions: one large extracellular loop and two cytosolic domains. The alpha -subunit is believed to form the channel pore, with beta - and gamma -ENaC serving as accessory regulatory subunits (20). The cytosolic COOH-terminal tails of beta - and gamma -ENaC are effector sites for channel regulation (12, 23).

Several endocrine factors, such as the mineralocorticoid aldosterone, and disparate cellular signaling cascades impinge on ENaC activity to fine tune Na+ balance (7, 9, 21). Similar to other ion channels, ENaC activity is controlled at the level of channel gating and number of active channels in the luminal plasma membrane. Although detailed examination of posttranslational modification, membrane insertion and retrieval, and protein degradation has provided clues about ENaC regulation, a complete understanding of ENaC modulation remains elusive.

Yanase and Handler (36) were the first to demonstrate that protein kinase C (PKC) inhibits Na+ transport by affecting amiloride-sensitive channels in renal A6 epithelial cells. Several investigators subsequently confirmed that PKC inhibits amiloride-sensitive ENaC (2, 8, 17). The initial decrease in ENaC activity most likely results from a decrease in open probability and/or withdrawal of ENaC protein from the luminal membrane. We recently demonstrated in renal epithelia that a later PKC-dependent, long-term downregulation of ENaC results from decreases in total cellular ENaC pools, with kinase decreasing gamma - and beta -, but not alpha -, ENaC levels (29). Lin et al. (16) and Zentner et al. (37) found in salivary epithelia a similar action of PKC, but in this instance, kinase decreases alpha -ENaC levels through transcriptional interference mediated by PKC-activated mitogen-activated protein (MAP) kinase (MAPK)-1/2 signaling. The mechanism by which long-term activation of PKC decreases beta - and gamma -ENaC levels has not been investigated.

All three ENaC subunits contain well-conserved PY (PPPXY) motifs in their cytosolic COOH termini (25, 26). This motif binds Nedd4 ubiquitin ligases, including Nedd4-2, which ultimately facilitate channel retrieval and degradation and, thus, decrease ENaC activity (12, 23). Indeed, gain of function mutations in beta - and gamma -ENaC resulting from disruption/deletion of the PY motif leads to the inheritable, monogenic hypertensive disease Liddle's syndrome (reviewed in Refs. 3, 15, 34). MAPK1/2-mediated phosphorylation of threonine-623 and -613 in gamma - and beta -ENaC, respectively, which are located just proximal to the PY motif, facilitates Nedd4 binding (22). This implies that these residues are important in the regulation of ENaC activity, possibly by impinging on channel retrieval. Supporting this contention are recent findings showing that alanine substitution for these conserved threonine residues increases ENaC activity in some instances in a reconstituted system (22). PKC signaling impinges on the MAPK1/2 cascade by activating Raf (32). Because MAPK1/2 may play a role in the regulation of ENaC via posttranslational modification and PKC is a known activator of MAPK1/2 signaling, we hypothesized that activation of PKC promotes degradation of beta - and gamma -ENaC through activation of the MAPK1/2 cascade. The present findings support such a mechanism.


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Materials and reagents. All chemicals and enzymes were of reagent grade and were purchased from Sigma (St. Louis, MO) and BioMol (Plymouth Meeting, PA) unless noted otherwise. The immortalized amphibian distal tubule A6 epithelial cell line was obtained from American Type Culture Collection. The PKC activator phorbol 12-myristate 13-acetate (PMA) and its negative control 4alpha -PMA were prepared fresh in DMSO as 1 mg/ml stock solutions and used at a final concentration of 100 ng/ml (162 nM). The MAPK/extracellular signal-regulated kinase (MEK) inhibitors PD-98059 and 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 final concentrations of 10, 0.5, and 0.5 µM, respectively. The translation inhibitors cycloheximide (Chx, in methanol) and emetine (Emt, in H2O) were stored at 4°C as 1.0 mg/ml stock solutions and used at final concentrations of 3.5 and 1.8 µM, respectively. The transcription inhibitor actinomycin D (ActD, in methanol) was stored at 4°C as a 1 mg/ml stock solution and used at a final concentration of 790 nM. The proteosome inhibitors MG-132, MG-262, and lactacystin were stored frozen (in DMSO) as 6.0, 10, and 10 mM stock solutions and used at final concentrations of 6.0, 1.0, and 10 µM, respectively.

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 the bicinchoninic acid protein assay. Affinity-purified rabbit polyclonal anti-Xenopus ENaC (xENaC) antibodies (Ab 586 for alpha -ENaC, Ab 592 for beta -ENaC, and Ab 2102 for gamma -ENaC) have been described previously (18, 29). The affinity-purified chicken polyclonal anti-gamma -xENaC antibody LLC2 has been described previously (29, 35). These antibodies are subunit specific, in that they show no improper cross-reactivity, and recognize the appropriate native and recombinant ENaC subunits. The rabbit polyclonal anti-MAPK1/2 and rabbit polyclonal anti-MEK1/2 and monoclonal phospho-MAPK1/2 antibodies were obtained from Upstate Biotechnology (Waltham, MA) and Cell Signaling Technologies (Beverly, MA), respectively. Anti-rabbit and anti-mouse horseradish peroxidase-conjugated secondary antibodies were obtained from Kirkegaard and Perry Laboratories (Gaithersburg, MD). Kodak BioMax Light-1 film and Chemiluminescence Reagents Plus (NEN Life Science Products, Boston, MA) were used to develop Western blots.

Cell culture. All experiments were performed on renal A6 epithelial cells (passages 75-81). Cells were cultured on polycarbonate supports (Costar Transwell-Clear inserts; 0.4-µm pore size, 4.7-cm2 growth area) using standard methods described previously (29, 31). Briefly, cells were maintained at 26°C in 1% CO2 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/ml streptomycin), and 0.3% of a 7% sodium bicarbonate solution]. Double-distilled H2O was added (~38%) for a final solution osmolarity of ~200 mosM. The medium was also supplemented with 1.5 µM aldosterone. High-resistance polarized A6 cell monolayers were used for all experiments. With these culture conditions, the amiloride-sensitive ENaC mediates Na+ reabsorption.

Western blot analysis. All immunochemistry was performed on whole A6 cell lysate with gels routinely loaded with lysate at 60 µg/well. Whole A6 cell lysate was extracted after three washes with Tris-buffered saline using standard procedures (31). Cells were scraped and then maintained for >2 h at 4°C in RIPA lysis buffer (10 mM NaPO4, 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 total protein concentration, and addition of Laemmli sample buffer (0.005% bromphenol blue, 10% glycerol, 3% SDS, 1 mM EDTA, 77 mM Tris · HCl, and 20 mM dithiothreitol), lysates were heated to 85°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 using standard 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 was quantified with densitometric scanning using Sigmagel (Jandel Scientific, San Rafael, CA). When possible, the flood configuration with the highest practical threshold was used to measure band density.

Western blots were often stripped of primary and secondary antibody to facilitate subsequent reprobing with distinct antibodies. All Western blots were stripped in 100 mM 2-mercaptoethanol, 62.5 mM Tris · HCl (pH 6.7), and 2% SDS for 30 min at 55°C with constant agitation. After removal of antibodies, nonspecific interactions were reblocked by incubation in Tris-buffered saline-Tween 20 and 5% milk for 2 h before the blots were reprobed with primary antibody.

Electrophysiology. Transepithelial Na+ current was calculated, as described previously (28-31), from Ohm's law as the ratio of transepithelial voltage to transepithelial resistance under open-circuit conditions using a Millicel Electrical Resistance System with dual Ag-AgCl pellet electrodes (Millipore, Billerica, MA) to measure voltage and resistance.

Experimental design. All experiments were performed on A6 cells grown on permeable supports maintained in the presence of aldosterone and serum. Cells were used only after formation of electrically tight monolayers. With these conditions, each monolayer served as its own control; effect of experimental maneuvers on relative current was one end point, and assessment of ENaC subunit levels after treatment was the other end point. Changes in ENaC subunit levels were usually normalized to the effects of vehicle at the same time point. All reagents, including PMA and inhibitors, were added simultaneously unless noted otherwise. Typically, starting voltages and resistances were measured, and monolayers were subsequently treated with vehicle, PMA alone, PMA in the presence of inhibitor, and inhibitor alone from 0 to 24 h. At the culmination of each experiment, voltages and current were reevaluated and cells were extracted. The levels of ENaC subunits in treated lysates were then established with immunochemistry. This experimental design facilitated quantitation of the effects of PMA in the presence and absence of inhibitors on changes in ENaC subunit levels and transepithelial current.

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


    RESULTS
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ABSTRACT
INTRODUCTION
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PKC decreases Na+ transport and beta - and gamma -ENaC levels. Figure 1 shows the effects of adding the PKC activator PMA on Na+ transport across A6 epithelial cell monolayers as well as on the levels of alpha -, beta -, and gamma -ENaC in these cells. Addition of PMA, in contrast to 4alpha -PMA, which had no effect, significantly decreased current to 0.12 ± 0.02 and 0.16 ± 0.04 by 2 and 24 h, respectively.


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Fig. 1.   Protein kinase C (PKC) decreases Na+ transport and beta - and gamma -subunit epithelial Na+ channel (ENaC) levels. A: relative (to pretreatment) current levels across A6 cell monolayers after treatment with phorbol 12-myristate 13-acetate (PMA) and its inactive analog (4alpha -PMA). *Significant decrease vs. starting levels and 4alpha -PMA. B: typical Western blots probed with anti-alpha (~90 kDa)-, anti-beta (~95 kDa)-, and anti-gamma (~95 kDa)-Xenopus ENaC (xENaC) antibodies. Each lane contains the same amount of lysate from cells treated with vehicle, PMA, and its inactive analog for 2 or 24 h. C: effects of 2 and 24 h of PMA treatment on xENaC subunit levels. *Significant decrease vs. starting values. D: typical Western blot containing lysate from A6 cells treated with vehicle and PMA in the absence and presence of PKC inhibitors 2,2',3,3',4,4'-hexahydroxy-1,1'-biphenyl-6,6'-dimethonol dimethyl ether (HBBDE) and Gö-6976. Blot was probed with anti-beta -xENaC antibody. E: typical Western blot containing lysate from A6 cells treated with vehicle and PMA in the absence and presence of PKC inhibitors HBBDE and calphostin C. Blot was probed with anti-gamma -xENaC antibody.

Figure 1B shows that PMA, but not its inactive analog, decreases beta - and gamma -ENaC levels after 24 h of treatment. The typical Western blots of Fig. 1B contained whole A6 cell lysate and were probed with rabbit polyclonal anti-alpha -xENaC and anti-beta -xENaC antibodies and the chicken polyclonal anti-gamma -xENaC antibody. The rabbit polyclonal anti-gamma -xENaC antibody Ab 2102 and LLC2 produced identical results (Figs. 1E and 2A). Figure 1C summarizes the effects of 2 and 24 h of PMA treatment on ENaC subunit levels. At 2 h, the relative levels of alpha - and beta -ENaC of 0.94 ± 0.24 and 1.1 ± 0.06, respectively, were unaffected by PMA, whereas those of gamma -ENaC were already markedly decreased to 0.71 ± 0.12 (n = 6). At 24 h, the relative level of alpha -ENaC (0.97 ± 0.26) was unaffected, whereas levels of beta - and gamma -ENaC were significantly decreased to 0.36 ± 0.04 and 0.39 ± 0.04 (n = 6), respectively.


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

The typical Western blots in Fig. 1, D and E, demonstrate that PKC inhibitors lessen the PMA-dependent decrease in beta - (Fig. 1D) and gamma -ENaC (Fig. 1E) levels. For these experiments, monolayers were treated with vehicle, PMA, and PMA + 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.

PKC decreases Na+ transport and ENaC levels through activation of the MAPK1/2 cascade. The experiments reported in Fig. 2 tested whether PKC decreases beta - and gamma -ENaC levels and Na+ reabsorption via activation of the MAPK1/2 cascade. The typical Western blots in Fig. 2A contain lysate extracted from cells treated for 6 and 24 h with vehicle and PMA in the absence and presence of the structurally unrelated 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 relatively constant and unaffected by many treatments (11). Thus MAPK1/2 level (Fig. 2A) was assessed to ensure equal loading.

The effects of PMA in the absence and presence of MEK1/2 inhibitors on Na+ transport at 6 and 24 h are summarized in Fig. 2B (n = 18). PMA significantly decreased relative (to pretreatment) current to 0.29 ± 0.07 and 0.10 ± 0.03 at 6 and 24 h, respectively. Vehicle was without effect at either time point. At 6 h, PMA in the presence of U-0126 had significantly less effect on current, with relative current being 0.65 ± 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 2C summarizes the effects of PMA in the absence and presence of MEK1/2 inhibitors on gamma -ENaC levels. After 24 h of treatment, gamma -ENaC levels in the presence of vehicle and PMA were 0.96 ± 0.05 and 0.26 ± 0.05, with significantly lower levels in the PMA group (n = 9). PD-98059 and U-0126 significantly countered the effect of PMA to decrease gamma -ENaC levels at 24 h, with relative levels of 0.76 ± 0.12 and 0.69 ± 0.08, respectively (n = 6). Although the effect is not as robust, MEK1/2 inhibitors also protect gamma -ENaC levels at 6 h. At this time, PMA significantly decreased relative gamma -ENaC levels from 1.01 ± 0.02 to 0.15 ± 0.05, with significantly greater levels in the presence of PMA + PD-98059 and PMA + U-0126 (0.57 ± 0.04 and 0.41 ± 0.08, respectively) than in the presence of PMA alone (n = 3). The actions of PMA in the absence and presence of MEK1/2 inhibitors on beta -ENAC levels after 6 and 24 h are summarized in Fig. 2D (n = 3). Although relative changes in beta -ENaC levels were more difficult to quantify, it was clear that, similar to their effects on gamma -ENaC, MEK1/2 inhibitors tended to counter PMA-dependent decreases in beta -ENaC. Neither MEK1/2 inhibitor when added alone affected current or ENaC subunit levels (Fig. 3), and the negative control U-0124 was without effect (not shown). Moreover, as shown in Fig. 3, the p38 MAP kinase inhibitor SB-203580 had no effect on PMA-dependent decreases in gamma -ENaC (n = 2).


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Fig. 3.   p44/42 MAPK (MAPK1/2), but not p38 MAPK, signaling decreases gamma -ENaC. Typical Western blot contained lysate from cells grown on filtered supports and treated with vehicle, PMA alone, and PMA in the presence of PD-98059, U-0126, and SB-203580. Blot was probed with anti-gamma -xENaC antibody (top) and then stripped and reprobed with anti-MAPK1/2 antibody (bottom). Black arrow, ~95-kDa protein; gray arrow, faster-migrating more-diffuse band.

In addition to showing that PKC affects ENaC levels and Na+ transport in A6 cells via the MAPK1/2 cascade, results in Fig. 2 show that the MAPK1/2 cascade is transiently activated in these cells by PKC, with MEK1/2 and MAPK1/2 being activated (phosphorylated) at 6 h and deactivated, most likely by negative-feedback pathways, by 24 h. As expected, MEK1/2 inhibitors blocked activation (phosphorylation) of MAPK1/2. This can relieve MEK1/2 of feedback inhibition, resulting in apparent hyperactivation of this upstream kinase.

Figure 3 shows that the anti-gamma -ENaC antibody used in the present study recognizes a ~95-kDa protein and a faster-migrating more-diffuse band in A6 cell lysate. Others postulated that the extracellular loop of the gamma -subunit in active ENaC is cleaved by extracellular proteases, leading to a protein that runs on SDS-PAGE as a broad-band ~70-kDa protein (19, 33). PMA and other experimental maneuvers reproducibly affected the ~95-kDa protein, but not the more-diffuse, faster-migrating protein (Figs. 4, 5, and 6). This, in combination with the finding that the levels of the faster-migrating protein did not correlate well with current, led us to focus exclusively on the ~95-kDa protein.


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Fig. 4.   Effects of PKC on Na+ transport and beta - and gamma -ENaC are additive with inhibitors of synthesis. A: typical Western blot containing lysate from cells treated with vehicle (DMSO), PMA, and cycloheximide (Chx) and emetine (Emt) in the absence and presence of PMA. Blots were probed with anti-beta - and anti-gamma -xENaC and MAPK1/2 antibodies, respectively. B: typical Western blot containing lysate from cells treated with vehicle, PMA, and actinomycin D (ActD) in the absence and presence of PMA. Blots were probed with anti-beta (Ab 592)- and anti-gamma -xENaC (Ab 2102) and MAPK1/2 antibodies, respectively. C: relative density of gamma -ENaC in cells treated with vehicle (Con), PMA, Chx, Emt, and ActD in the absence and presence of PMA. *Significantly lower than vehicle. **Significantly lower than PMA alone and respective inhibitor alone. D: relative density of beta -ENaC in cells treated with vehicle, PMA, Chx, Emt, and ActD in the absence and presence of PMA.



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Fig. 5.   A6 cells contain a pool of gamma -ENaC that is "protected" from inhibitors of synthesis. A: typical Western blots from cells treated with vehicle, PMA, and ActD in the absence and presence of PMA. Blots were probed with anti-gamma -xENaC antibody. B: typical Western blots containing lysate from cells treated with inhibitors. V, vehicle. Blots were probed with anti-gamma -xENaC antibody (Ab 2102). C: gamma -ENaC decay over time in response to inhibitors of transcription and translation. Decay line for inhibitors of transcription was calculated starting at 2 h.



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Fig. 6.   PKC decreases a protected pool of gamma -ENaC in a manner that is sensitive to inhibitors of the proteosome. A: typical Western blot of lysate from cells treated for 2 h with vehicle and Chx and for an additional 4 h with Chx in the presence and absence of PMA with and without MG-132 (132) and lactacystin (LC). Blot was probed with anti-gamma -xENaC antibody. B: gamma -ENaC levels 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 + proteosome inhibitors (PI) in the absence and presence of PMA. *P < 0.05 vs. Con; **P < 0.05 vs. Chx or Chx + PMA. 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 < 0.05 vs. Con. **Significantly less than Chx.

Inhibition of transcription and translation is additive with PKC to decrease ENaC subunit levels. The experiments reported in Fig. 4 were performed to elucidate the mechanism of PKC action on ENaC testing whether this kinase impinges on channel synthesis or degradation. Figure 4A shows representative Western blots containing lysate from cells treated with vehicle (DMSO), PMA, inhibitors of translation (3.5 µM Chx and 1.8 µM Emt), and PMA + inhibitors of translation for ~10 h. These blots were probed with Ab 592 (for beta -ENaC), Ab 2102 (for gamma -ENaC), and anti-MAPK1/2 antibodies. Activation of PKC clearly was additive with inhibitors of translation with respect to decreasing gamma -ENaC levels (measured as the ~95-kDa band). PMA also decreased beta -ENaC levels in an additive manner with inhibitors of translation. However, because of the disparity in time of action for PMA (>6 h) and inhibitors of translation (<2 h) to decrease beta -ENaC, this was often more difficult to consistently demonstrate. For the (first) blot in Fig. 4A, the effects of Emt and PMA are clearly additive on beta -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 treated with vehicle, PMA, ActD (25 nM), and PMA + ActD for 10 h are shown in Fig. 4B.

The effects of PMA, transcription and translation inhibitors, and PMA in the presence of these inhibitors on gamma - and beta -ENaC levels are shown in Fig. 4, C and D. For gamma -ENaC, PMA treatment 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 significantly decreased gamma -ENaC levels to 0.08 ± 0.03, 0.12 ± 0.04, and 0.09 ± 0.04, respectively, all of which are significantly lower than values with PMA and inhibitor alone (n >=  7). Similar to gamma -ENaC, PMA decreased beta -ENaC levels in an additive manner with inhibitors of transcription and translation (n = 3). The effects of transcription and translation inhibitors on relative currents are reported in Table 1.

                              
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Table 1.   Relative currents across A6 cells treated with PMA and transcription and translation inhibitors

PKC decreases gamma -ENaC levels by promoting subunit degradation. The results in Figs. 1-3 demonstrate that PKC via MAPK1/2 signaling decreases gamma -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 gamma -ENaC subunit in a manner consistent with targeted degradation (22), we tested whether PKC does indeed promote gamma -ENaC degradation.

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

Figure 5B is a representative experiment (1 of 5) showing an extended time course for the actions of Chx, Emt, and ActD on gamma -ENaC (probed with Ab 2102). The decay in gamma -ENaC levels over time in response to inhibitors of transcription and translation is summarized in Fig. 5C. The blots in Fig. 5B contained lysate from cells treated with inhibitors. Inhibitors of translation, as well as the inhibitor of transcription, showed two phases of action: an early effect with a time constant <0.5 h (calculated for ActD starting with 2 h) and a later action with a time constant >6 h. Our interpretation of these results is that there are two pools of gamma -ENaC in A6 cells: one that turns over rapidly with a short half-life and another that is somewhat protected with a longer half-life. From the experiments in Figs. 4 and 5A (and also Fig. 6), PMA clearly influenced the protected pool of channels. Although PMA decreases current before 30 min, with current remaining suppressed for >24 h (Fig. 1A), inhibitors of translation and transcription began to affect current only after ~4 h (Table 1). Indeed, at 2 h, <30% of total gamma -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 gamma -ENaC pools before they affect current. This suggests that the protected pool of gamma -ENaC correlates better with active channels than does the pool that turns over more quickly.

To begin to determine whether this PMA-sensitive protected pool of gamma -ENaC was possibly in the plasma membrane and sensitive to PKC/MAPK1/2-directed degradation, we performed the experiments described in Fig. 6. For these experiments, A6 cell monolayers were treated 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 and presence of the proteosome inhibitors MG-132 (6.0 µM) and lactacystin (10 µM). MG-262 (1.0 µM) was also used and produced results identical to MG-132 and lactacystin (not shown). The representative blot (n = 6) in Fig. 6A contained lysate from the respective groups and was probed with Ab 2102. In Fig. 6B, the effects of proteosome inhibitors were pooled to allow for comparison with Chx alone and Chx + PMA. Proteosome inhibitors significantly reversed the effects of Chx on gamma -ENaC, with levels being 0.12 ± 0.05 and 0.34 ± 0.05 with Chx in the absence and presence of proteosome inhibitors, respectively. Similarly, in the presence of PMA + Chx, proteosome inhibitors significantly protected gamma -ENaC, with levels of 0.07 ± 0.04 and 0.33 ± 0.06, respectively. Interestingly, although proteosome inhibitors protected gamma -ENaC levels in the presence of Chx alone or in addition to PMA, proteosome inhibitors protected transport only in the absence of PMA (Fig. 6C).

Figure 6C shows relative current across A6 cells treated with Chx for 2 h followed by further treatment for 4 h with Chx in the presence and absence of PMA with and without proteosome inhibitors (n = 4). Proteosome inhibitors did not affect decreases in current in response to Chx + PMA, with relative 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 for vehicle, 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 on current (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 gamma -ENaC levels or current by any proteosome inhibitor when they were added to cells simultaneously with PMA in the absence of Chx pretreatment (not shown, n = 3). Thus proteosome inhibitors protected current and gamma -ENaC levels in the presence of decreased synthesis; however, in the combined presence of decreased synthesis and activated PKC, proteosome inhibitors protected only gamma -ENaC levels and not current. We interpret this as PKC promoting retrieval of ENaC from the membrane and ultimate targeting of this channel for degradation at the proteosome, with PKC acting at a site upstream of the proteosome, possibly on the channel itself or on proteins involved in channel retrieval.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We reported previously that activation of PKC leads to decreases in beta - and gamma -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 cascade and mechanisms underpinning decreased ENaC activity. Figure 7 shows the simplest model consistent with our present and past findings. Also shown in Fig. 7 is the cellular signaling cascade activated by PKC that we believe impinges on ENaC. The present study demonstrates for the first time that the long-term effects of PKC on beta - and gamma -ENaC levels, as well as transport, are mediated by activation of the MAPK1/2 cascade, with decreases in beta - and gamma -ENaC levels in response to PKC-activated MAPK1/2 signaling resulting from targeted degradation at the proteosome. Moreover, the present results in the context of the previous findings of others are consistent with the possibility that PKC-MAPK1/2 signaling acts directly on a pool of channels resident in the plasma membrane.


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Fig. 7.   Simplest model for PKC-MAPK1/2 regulation of ENaC, consistent with present results. MAPK1/2 signaling promotes ENaC retrieval and subsequent degradation via an endosomal compartment, with inhibitors of the proteosome affecting degradation at a step downstream of retrieval. ENaCmem and ENaCendo, plasma membrane 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 they impede.

Several other signaling cascades/proteins that are known to regulate ENaC 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 retrieval may be a particularly important point for physiological regulation of ENaC activity. Indeed, the human hypertensive diseases associated with abnormal ENaC retrieval support this contention (15).

Role of the MAPK1/2 cascade in PKC-mediated decreases in beta - and gamma -ENaC and Na+ transport. The results in Figs. 2 and 3 showing that PKC actions on ENaC subunit levels and Na+ transport are countered by two distinct inhibitors of MEK1/2 strongly imply that the MAPK1/2 cascade plays a central role in negative regulation of channel activity. Consistent with this implication are findings from Shi and colleagues (22) showing that MAPK1/2 phosphorylates beta - and gamma -ENaC on residues that directly influence interactions with Nedd4. Increased Nedd4 binding to beta - and gamma -ENaC promotes retrieval of the channel from the membrane and subsequent degradation (25, 26). Shi and colleagues also demonstrated that alanine substitution of these critical residues increases channel activity ~3.5-fold. The salient feature of this MAPK1/2 regulation of ENaC activity is that it is a posttranslational event that modifies existing channels in a manner that 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 and PKC regulation of alpha -ENaC in salivary epithelia. This latter mechanism involves transcriptional interference. Thus PKC-MAPK1/2 signaling influences ENaC activity through at least two distinct mechanisms in a subunit-specific manner: transcriptional interference for alpha -ENaC and posttranslational targeting for degradation of beta - and gamma -ENaC. Exactly which mechanism is used to regulate ENaC in response to PKC must also then be tissue and, possibly, species specific, inasmuch as our results, as well as those of Shi and colleagues, exclude transcriptional interference in renal A6 cells and in certain reconstituted systems.

Support for posttranslational control of ENaC in response to PKC. The results in Fig. 4 showing the effects of PKC to be additive with inhibitors of transcription and translation are most consistent with this kinase ultimately affecting beta - and gamma -ENaC levels, as well as Na+ transport, at a site other than channel synthesis, such as targeting channels for retrieval and, ultimately, degradation. Alternatively, both subunits could have alternative routes for transcription and translation that are resistant to ActD, and Chx and Emt, respectively, but sensitive to PKC. Although we cannot definitively exclude this latter possibility, we believe that it is extremely unlikely. One other possibility that we cannot definitively exclude with the present results but suspect to be unlikely is that the effects of PKC-MAPK1/2 signaling are indirect and mediated by a protein, such as Sgk or N4WP5A (1, 14), that protects the channel from degradation.

The observation that activation of PKC influences gamma -ENaC levels at a time 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 from either step of synthesis. Inhibitors of transcription and translation, in addition, affected beta -ENaC levels within 2-4 h, which is much faster 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 gamma -ENaC levels are lowered by blocking synthesis with Chx for 2 h, the subsequent PMA-dependent decrease in gamma -ENaC levels is sensitive to proteosome inhibitors. Because, as shown in Fig. 5, Chx has the greatest effect on gamma -ENaC levels before 1 h and has little additional effect on subunit 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 gamma -ENaC before determining whether proteosomal blockade impinged on the actions of PKC to decrease the protected pool of gamma -ENaC. Such an approach enabled us to focus specifically on this subunit pool in the absence of the high background noise contributed by the turnover of that pool, which has a much shorter half-life.

There are two pools of gamma -ENaC: one turns over quickly, and one is protected but sensitive to PKC. Close inspection of the results in Fig. 5 shows two pools of gamma -ENaC. One pool is quickly (<1 h) decreased by addition of translation inhibitors. Similarly, blockade of transcription also quickly (<4 h) decreases this pool. The other pool of gamma -ENaC, although it is markedly less abundant, is more resistant to blockade of transcription and translation, with significant levels being measurable for up to 8-12 h after addition of inhibitor. We argue that the first pool contains gamma -ENaC, which is quickly turned over, and the second pool is protected or somehow removed from the normal route of degradation, leading to the rapid turnover of the first pool. An alternative that we cannot exclude, but believe is unlikely, is that ENaC degradation is suppressed by some protein that itself has a very short half-life (e.g., Sgk) (4), and it is this latter protein that is affected by transcription and translation inhibitors, as well as PKC-MAPK1/2 signaling. Because translation inhibitors decrease gamma -ENaC levels before affecting current, we argue further that this protected pool is more closely associated with active channels in the plasma membrane. These observations are intriguing and merit further investigation but, in the context of the findings of others (5, 10, 13, 18, 35), enable us to speculate that this protected pool might reflect a membrane-resident or supapical pool of gamma -ENaC. Weisz and colleagues (35) reported that the half-life of the total cellular pools of alpha - and gamma -ENaC in A6 cells is ~2 h, but the half-life of the pool that reaches the apical membrane is >24 h. Kleyman and colleagues (13) report a similar half-life for membrane-resident alpha -ENaC subunits in A6 cells. In contrast with these studies are the findings of De La Rosa and colleagues (5) showing that in A6 cells whole cell and membrane-resident channels have a half-life of <= 60 min. Similarly, heterologously expressed ENaC has a short half-life (26). Clearly, the present results showing a decreased but abundant level of gamma -ENaC after 8-12 h of treatment with inhibitors of synthesis contrast with these latter studies and are more consistent with the findings showing that some portion of ENaC has a half-life of >6 h.

An intriguing aspect of the present research not fully understood is the apparent discordant regulation of gamma - and beta -ENaC levels by PKC, with PKC affecting the former subunit much more quickly than the latter. Although this possibly could reflect differences in the relative abundance of each subunit, others reported previously that the three ENaC subunits are noncoordinately regulated in A6 cells (35).

In summary, the present results are consistent with the mechanism where the bulk of freshly synthesized ENaC is quickly degraded, with the rates of synthesis and degradation being much more rapid than those for channel insertion and retrieval into/from the plasma membrane. PKC via MAPK1/2 then would simply increase the rate of channel retrieval, ultimately promoting degradation of this newly retrieved channel pool because of the very rapid degradation rate for ENaC. With such a mechanism, blocking channel synthesis, as in the present study, with all other factors remaining unaffected, would lead to a decrease in ENaC levels before it would affect transport because of the slow rate of channel retrieval. Moreover, blocking channel synthesis and degradation in the presence of increased channel retrieval, as we speculate is the case when A6 cells are treated with Chx, PMA, and a proteosome inhibitor, would then affect only ENaC levels and not transport. In contrast to this, blocking channel degradation simultaneously with retrieval would influence, as we found, ENaC levels and transport.

Relationship between aldosterone and PKC signaling. We recently reported that aldosterone activates MAPK1/2 signaling in renal A6 epithelial cells (11). This genomic activation of the MAPK1/2 cascade was via transcriptional control of Ki-RasA, resulting in prolonged MAPK1/2 signaling. In consideration of the present results, aldosterone activation of the MAPK1/2 cascade would appear to be a negative-feedback response that might temper prolonged avid Na+ reabsorption.


    ACKNOWLEDGEMENTS

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


    FOOTNOTES

This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-59594 (to J. D. Stockand), American Heart Association Grant SDG-0130008N, the American Society of Nephrology Carl W. Gottschalk Research Scholar Grant, the American Physiological Society Lazaro J. Mandel Young Investigator Award, intramural support from the University of Texas Health Science Center at San Antonio, and American Heart Association-Texas Affiliate Grant 0225048Y (to R. E. Booth).

Address for reprint requests and other correspondence: J. D. Stockand, UTHSCSA, Dept. of Physiology-7756, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900 (E-mail: stockand{at}uthscsa.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 21, 2003;10.1152/ajprenal.00373.2002

Received 16 October 2002; accepted in final form 20 January 2003.


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