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Department of Physiology, University Health Science Center, San Antonio, Texas 78229-3900
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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, 
-, 
-, and 
-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
- and -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
- and -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 -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 -ENaC. Moreover,
proteosome inhibitors (MG-132 and lactacystin) reversed PKC effects on
this protected pool of -ENaC. Thus PKC signaling via MAPK1/2 cascade
activation in A6 cells promotes degradation of -ENaC.
proteosome; hypertension; sodium transport; MG-132; MG-262; lactacystin
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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: , , and . Each subunit has two
membrane-spanning regions: one large extracellular loop and two
cytosolic domains. The -subunit is believed to form the channel
pore, 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).
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 - and -, but
not -, 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 -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 - and
-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 - and
-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 - and
-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 - and
-ENaC through activation of the MAPK1/2 cascade. The present
findings support such a mechanism.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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 4-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.
-ENaC, Ab 592 for -ENaC, and Ab 2102 for -ENaC) have been
described previously (18, 29). The affinity-purified
chicken polyclonal anti--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.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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PKC decreases Na+ transport and -
and -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 -, -, and -ENaC in
these cells. Addition of PMA, in contrast to 4-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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- and -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--xENaC and
anti--xENaC antibodies and the chicken polyclonal anti--xENaC
antibody. The rabbit polyclonal anti--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 - and -ENaC
of 0.94 ± 0.24 and 1.1 ± 0.06, respectively, were
unaffected 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 to
0.36 ± 0.04 and 0.39 ± 0.04 (n = 6),
respectively.
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- (Fig. 1D) and -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 -
and -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.
-ENaC levels. After 24 h of
treatment, -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 -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 -ENaC levels at 6 h. At this time, PMA significantly decreased relative -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 -ENAC levels after
6 and 24 h are summarized in Fig. 2D (n = 3). Although relative changes in -ENaC levels were more difficult
to 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 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 -ENaC (n = 2).
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-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 -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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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 -ENaC), Ab 2102 (for -ENaC),
and anti-MAPK1/2 antibodies. Activation of PKC clearly was additive
with inhibitors of translation with respect to decreasing -ENaC
levels (measured as the ~95-kDa band). PMA also decreased -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 -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
-ENaC, but in this experiment the effects of Chx have already saturated.
- and -ENaC levels are
shown in Fig. 4, C and D. For -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 -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 -ENaC, PMA decreased -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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PKC decreases -ENaC levels by promoting subunit degradation.
The results in Figs. 1-3 demonstrate that PKC via MAPK1/2
signaling 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 does
indeed promote -ENaC degradation.
-ENaC levels, with these actions clearly being additive by 3 h. PMA decreased -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 -ENaC
(probed with Ab 2102). The decay in -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 -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 -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 turns
over more quickly.
To begin to determine whether this PMA-sensitive protected pool of
-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 -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 -ENaC, with levels of 0.07 ± 0.04 and 0.33 ± 0.06, respectively. Interestingly, although proteosome inhibitors
protected -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 -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 -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 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.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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We reported previously that activation of PKC leads to decreases
in - 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 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 - and -ENaC levels, as well as
transport, are mediated by activation of the MAPK1/2 cascade, with
decreases in - and -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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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 - and
-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 - and -ENaC
on residues that directly influence interactions with Nedd4. Increased
Nedd4 binding to - and -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 -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 -ENaC and
posttranslational targeting for degradation of - and -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 - and -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.
-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 -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 -ENaC levels are lowered
by 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 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
-ENaC before determining whether proteosomal blockade impinged on
the actions of PKC to decrease the protected pool of -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 -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 -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 -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 -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 -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 -ENaC. Weisz
and colleagues (35) reported that the half-life of the
total cellular pools of - and -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 -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 -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.
- and -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.
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ACKNOWLEDGEMENTS |
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We thank Drs. D. C. Eaton, B. Malik, and J. P. Johnson for sharing anti-xENaC antibodies.
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FOOTNOTES |
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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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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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