Departments of Pediatrics and Physiology and Biophysics, Rainbow Center for Childhood PKD, Case Western Reserve University, Cleveland, Ohio
Submitted 29 April 2004 ; accepted in final form 29 December 2004
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ABSTRACT |
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mitogen-activated protein kinase; epithelial ion transport; epithelial sodium channel
Corticosteroids, insulin, and vasopressin cause acute and/or chronic stimulation of sodium transport in epithelial cells (45) by several mechanisms, including alterations in ENaC open probability (23), increased synthesis of channel subunits (2), enhanced trafficking of the channel to the plasma membrane (1), and decreased retrieval of channels from the apical membrane (11, 35). Several receptor agonists cause acute and/or chronic downregulation of Na+ absorption, including epidermal growth factor (EGF) (10, 32, 43, 46), acetylcholine and PGE2 (6, 15, 17, 46), and extracellular ATP (26). Several intracellular signaling pathways (i.e., PKC, calcium, Src kinase, ERK1/2) have been implicated (17, 18, 21, 24, 43, 47); however, the precise molecular mechanisms responsible for inhibition of ENaC function have not been fully elucidated.
ENaC belongs to the degenerin/ENaC gene superfamily, and functional channels are thought to be composed of some combination of three homologous subunits: -,
-, and
-ENaC (7). Each subunit has the predicted membrane topology of two hydrophobic membrane-spanning regions, a highly conserved large extracellular cysteine-rich loop, and intracellular NH2 and COOH termini. The COOH termini of all three subunits of ENaC contain well-conserved PY (PPPxY) motifs that represent potential binding sites for Nedd4 ubiquitin ligases, and disruption/deletion of the PY motif in
- or
-ENaC leads to enhanced ENaC function (19, 34). Garty and coworkers (8, 33) recently showed that ERK2-mediated phosphorylation of Thr613 (
-ENaC) and Thr623 (
-ENaC), residues located near the PY motifs, facilitates Nedd4 binding and mutation of these residues and also increases amiloride-sensitive current in Xenopus oocytes expressing these channels.
EGF and extracellular ATP inhibit amiloride-sensitive sodium absorption in several epithelia, including renal collecting duct (26, 43), airway (13), colon, and endometrium (10). There are conflicting results with regard to whether elevation of intracellular calcium (12, 13, 21, 43), activation of PKC (24), or neither (26, 40) is required for the inhibitory response. Whereas activation of ERK1/2 by EGF is common to many cell types, there is no direct demonstration that ERK1/2 plays a role in acute receptor-mediated downregulation of sodium transport in epithelia. Because both EGF and extracellular ATP are known to induce phosphorylation and activation of ERK1/2 (32, 42), the present study was undertaken to determine the role of the MAP kinase cascade as a negative regulator of sodium transport. The results of these studies demonstrate that phosphorylation and activation of ERK1/2 is an important step in ATP- and EGF-induced inhibition of amiloride-sensitive sodium transport.
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METHODS |
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CT media were composed of the following: a 1:1 mix of Dulbecco's modified Eagle's medium and Ham's F-12 medium (Life Technologies) supplemented with 1.3 µg/l sodium selenite, 1.3 µg/l 3,3',5-triiodo-L-thyronine, 5 mg/l insulin, 5 µg/l transferrin, 25 µg/l prostaglandin E1, 2.5 mM glutamine, 50 nM dexamethasone, 50 mg/l streptomycin, and 30 mg/l penicillin G. Interferon- (10,000 U/l) was added to the CT media for mCT12 cells.
Transepithelial electrical measurements. Cells were seeded (25 x 105 cells/filter) onto collagen-coated, permeable supports (modified 12 mm Millicell-CM filters; Millipore) as described previously (39). Confluent cell monolayers were mounted vertically in a thermostatically controlled Ussing chamber equipped with gas inlets and separate reservoirs for the perfusion of apical and basolateral compartments. Both sides of the cell monolayer were bathed with equal volumes of Krebs-Ringer bicarbonate solution containing (in mM): 115 NaCl, 25 NaHCO3, 5 KCl, 2.5 Na2HPO, 1.8 CaCl2, 2 MgSO4, and 10 glucose. Some experiments were done with cell monolayers bathed by CT media. The solutions were circulated through the water-jacketed glass reservoir by gas lifts (95% O2-5% CO2) to maintain solution temperature at 37°C and pH at 7.4. Transepithelial voltage difference (VT) was measured between two Ringer-agar bridges, and calomel half-cells connected the bridges to a high impedance voltmeter. Current from an external direct current source was passed by silver-silver chloride electrodes and Ringer-agar bridges to clamp the spontaneous VT to 0 mV. The current required [short-circuit current (Isc)] was corrected for solution and filter series resistance and recorded. Monolayers were maintained under short-circuit conditions except for brief 3- to 5-s intervals when the current necessary to clamp the voltage to a nonzero value (210 mV) was measured to calculate transepithelial resistance.
Western blots. Cell lysates were prepared from confluent culture filters (24 mm Transwell, Clear tissue culture treated; Corning Costar) in lysis buffer containing 50 mM Tris·HCl (pH 7.5), 1% Triton X-100, 3 mM EDTA, and 1 mM EGTA. Protein concentrations were measured by bicinchoninic acid protein assay (Pierce). Western blot analysis for total and phosphorylated ERK1/2 (p42/p44) was performed as described previously (32).
Statistical analysis. All results are expressed as means ± SE. Statistical significance was evaluated by either paired or unpaired Student's t-test. P < 0.05 was considered significant.
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RESULTS |
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DISCUSSION |
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EGF-mediated inhibition of Isc. Studies of in vitro perfused cortical collecting duct (43, 46) and primary cultures of endometrial epithelial cells (10) found that EGF caused rapid inhibition of sodium transport. On the basis of changes in membrane potential and direct measurements of apical membrane sodium conductance in permeabilized monolayers, the primary site of action of EGF-induced inhibition of sodium absorption was localized to the apical sodium channel (10, 46). We were able to reproduce the effects of EGF on sodium absorption in mouse collecting duct cells and in immortalized mouse collecting duct principal cells. The studies cited above concluded that elevation of intracellular calcium directly inhibited sodium channels and/or activated PKC to cause EGF-induced inhibition of sodium absorption. Whereas permissive changes in calcium or activation of specific PKC isoforms cannot be ruled out, our results are not consistent with such a mechanism of action, because 1) the inhibitory effect of EGF on Isc was completely blocked by pretreatment with inhibitors of MEK (PD-98059 and U0126), 2) inhibition of Isc in response to calcium mobilization (DBHQ) was insensitive to MEK inhibition and could be explained by a reduction in basolateral Na+-K+-ATPase function, and 3) EGF-induced inhibition of Isc was insensitive to a general PKC inhibitor (BIM).
ATP-mediated inhibition of Isc.
Extracellular nucleotide triphosphates are known to inhibit amiloride-sensitive sodium absorption in airway epithelium (13, 21), endometrial cells (30), and distal renal tubules (24, 26, 40). A variety of studies have been conducted in freshly isolated tissues, primary cultures, and cell lines from multiple species, but there is not a consensus as to the mechanism(s) responsible for inhibition of sodium transport. Most of the results support a role for calcium mobilization (13, 21), but not PKC activation, despite evidence that direct activation of PKC with phorbol esters can lead to inhibition of amiloride-sensitive sodium absorption in airways (18, 25). O'Grady and coworkers (30) recently reported that extracellular UTP elicited a transient stimulation of Cl secretion, followed by a sustained inhibition of amiloride-sensitive sodium absorption in cultured porcine endometrial epithelial cells. They found that the inhibition of sodium transport was due to a PKC-dependent decrease in apical sodium conductance, mediated by both calcium-dependent and calcium-independent PKC isoforms. Studies of ATP- and UTP-mediated inhibition of sodium transport in renal connecting tubules and collecting duct cells have also yielded mixed results. Koster et al. (24) concluded that extracellular ATP-mediated inhibition of sodium absorption in primary cultures of rabbit cortical collecting duct (CCD) and connecting tubule was due to PKC activation; however, Leipziger and coworkers (26, 40) found no evidence of a role for calcium or PKC activation in ATP-induced inhibition of sodium transport in immortalized murine collecting duct principal cells (M1 cells) or in perfused mouse CCD. In contrast, the present studies of immortalized collecting duct principal cells (mCT12) clearly showed that elevation of intracellular calcium or activation of PKC (Figs. 5 and 6) can elicit inhibition of amiloride-sensitive sodium absorption. Furthermore, PKC-mediated inhibition is almost completely prevented by pretreatment with a PKC inhibitor (Fig. 6). The importance of these two pathways in ATP-induced inhibition of apical sodium entry is less certain because the PKC inhibitor did not alter the ATP-induced decrease in Isc (Fig. 6) and the effect of calcium mobilization on sodium absorption can be explained by inhibition of basolateral Na+-K+-ATPase (Fig. 7). Recent studies by Eaton and coworkers (48) demonstrated that phosphatidylinositol 4,5-bisphosphate stimulates ENaC, and they speculated that local depletion of phosphatidylinositol 4,5-bisphosphate in response to purinergic stimulation of PLC could underlie the ATP-induced inhibition of ENaC activity. Our results do not address the mechanism of ATP-induced inhibition of sodium absorption, but they do support a role for the MAP kinase signaling cascade because 1) exposure to extracellular ATP elicited rapid phosphorylation of ERK1/2 (p42/44), 2) ATP-induced phosphorylation of ERK1/2 was prevented by an inhibitor of ERK kinase, and 3) inhibition of ERK1/2 phosphorylation partially blocked (50%) the inhibitory effects of ATP on sodium absorption.
Mechanism of ERK1/2-mediated inhibition of amiloride-sensitive sodium absorption.
Several recent studies bear directly on the mechanisms of MAP kinase-mediated regulation of ENaC. Garty and coworkers (33) showed that ERK2 could phosphorylate COOH terminal peptides from the - and
-subunits of ENaC. They suggested that ERK2-mediated phosphorylation of
-Thr613 and
-Thr623, amino acids adjacent to PY motifs implicated in channel endocytosis, could promote ENaC-Nedd4 interaction and facilitate channel retrieval. Lysine residues in the
- and
-subunits of heterologously expressed ENaC can be ubiquitinated and mutations in the critical lysines increase ENaC activity, presumably by decreasing channel endocytosis (33, 37). A role for MAP kinase in PKC-mediated degradation of ENaC in A6 cells was recently proposed (3). The authors used immunodetection of ENaC subunits to argue that PKC caused enhanced degradation of
- and
-, but not
-ENaC. The evidence for involvement of ERK1/2 activation in the process was based on partial inhibition of PKC induced loss of
- and
-ENaC by MEK inhibitors. Unfortunately, the earliest time point at which inhibitors of MAP kinase signaling were tested was 6 h, so it is difficult to unequivocally assign a role for ERK1/2 in the early decrease in sodium transport after PKC activation (i.e., PKC causes
90% inhibition of Isc within 60 min in A6 cells). They also showed that in the presence of cycloheximide, to block channel synthesis, proteosome inhibitors prevented the PMA-induced loss of
-ENaC, but failed to prevent the PMA-induced decrease in Isc. Previous studies of PKC-induced inhibition of ENaC function have suggested that PKC rapidly decreases open probability with a slower decrease in the number of active channels; however, the role of ERK1/2 activation was not evaluated (14). Nicod et al. (27) reported that the vasopressin-induced transcript VIT32, when coexpressed with ENaC in oocytes, caused MAP kinase activation and ENaC downregulation. Despite a 50% decrease in ENaC current, there was no detectable change in surface expression of ENaC, suggesting a decrease in channel open probability rather than endocytosis. The studies described in this article do not directly address the mechanisms by which ERK1/2 activation leads to a decrease in amiloride-sensitive sodium absorption, but the rapid reversibility of the EGF-induced decrease in Isc is relevant. Reversal of EGF-induced inhibition of sodium absorption by addition of a MEK inhibitor implies that 1) ERK1/2 does not exert tonic inhibition on sodium absorption under the conditions of our experiments, 2) sustained activation of ERK1/2 is required for acute inhibition of sodium absorption by EGF, 3) ERK1/2 is rapidly dephosphorylated, and 4) ERK1/2-dependent changes in ENaC activity are rapidly reversible. These observations suggest that the early effects of ERK1/2 activation may not involve channel degradation but may be due to reversible endocytosis (recycling endosomes) or channel gating.
Physiological significance of ERK1/2-mediated regulation of sodium absorption.
Garty and coworkers (33) speculated that hypotonic shock, TGF-, and calcium mobilization might be upstream signaling pathways that could utilize ERK2 phosphorylation of ENaC to regulate sodium absorption. The results of our studies with EGF and extracellular ATP demonstrate that at least two physiologically relevant receptor-signaling pathways in epithelia may regulate sodium transport via activation of ERK1/2. A wide range of physiological and pathophysiological conditions influences MAP kinase signaling in epithelial cells (41). This is particularly relevant in polycystic kidney disease, in which abnormalities in the EGF/EGFR axis have been implicated in disease progression (29). Because of the extent to which MAP kinase activation contributes to inhibition of sodium transport by other receptor agonists and signaling pathways is undetermined and because the acute inhibition of sodium transport by EGF is readily reversible, phosphatase activity may represent another potential step for regulation.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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