Hydrogen peroxide and epidermal growth factor activate phosphatidylinositol 3-ki - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 12:59 |只看该作者 |倒序浏览 |打印

Laboratory of Cell and Molecular Physiology and Interdisciplinary Research Institute, Université Libre de Bruxelles, Bruxelles, Belgium
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ABSTRACT

Activation of phosphatidylinositol 3-kinase (PI 3-kinase) is required for insulin stimulation of sodium transport in A6 cell monolayers. In this study, we investigate whether stimulation of the PI 3-kinase by other agents also provoked an increase in sodium transport. Both epidermal growth factor (EGF) and H2O2 provoked a rise in sodium transport that was inhibited by LY-294002, an inhibitor of PI 3-kinase activity. PI 3-kinase activity was estimated in extracts from A6 cell monolayers directly by performance of a PI 3-kinase assay. We also estimated the relative importance of the PI 3-kinase pathway by two different methods: 1) coprecipitation of the p85 regulatory subunit with anti-phosphotyrosine antibodies and 2) phosphorylation of PKB on both Ser 473 and Thr 308 residues observed by Western blotting. Since the mitogen-activated protein kinase (MAPK) pathway has also been implicated in the regulation of sodium transport, we also investigated whether this pathway is turned on by insulin, H2O2, or EGF. Phosphorylation of ERK1/2 was increased only transiently by insulin and H2O2 but quite sustainedly by EGF. Inhibitors of this pathway (U-0126 and PD-98059) failed to affect the insulin and H2O2 stimulation of sodium transport but increased substantially the stimulation induced by EGF. The latter effect was associated with an increase in PKB phosphorylation, thus suggesting that the stimulation of the MAPK pathway prevents, in part, the stimulation of the PI 3-kinase pathway in the transport of sodium stimulated by EGF.

epithelial sodium channels; mitogen-activated protein kinase! y! Q+ P$ @+ C6 b- ?0 T2 n0 z
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BY CONTROLLING SODIUM balance, the kidney regulates extracellular fluid volume and arterial blood pressure. This fine regulation mainly occurs in the distal nephron and more particularly in the collecting duct, which is sensitive to several hormones and in which the principal cells express amiloride-sensitive epithelial sodium channels (ENaC) within their luminal plasma membrane. Sodium ion enters into the cell through these channels and is then extruded at the basolateral membrane by the Na -K -ATPase (51, 55). The rate-limiting step of vectorial sodium transport is mainly attributed to the activity of ENaC, which is the target for hormonal control (7, 10, 14, 18, 20, 21, 23, 24, 27, 28, 50, 56, 70). In A6 cell (derived from Xenopus laevis kidney) monolayers, insulin has been well documented to increase sodium transport (5, 26, 32, 43) by increasing the activity of phosphatidylinositol 3-kinase (PI 3-kinase) and thereby generating phosphatidylinositol 3,4,5-trisphosphate (PIP3) within the basolateral membrane (4, 6, 8, 53). PIP3 then diffuses in the apical membrane (9), and apical addition of exogenous permeant PIP3 reproduces the stimulation of sodium transport (42, 64). Among the products of PI 3-kinase, only PIP3 and to a lesser extent PI(3,4)P2 increase sodium transport. Furthermore, overexpression of the specific PIP3 3-phosphatase referred to as phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in A6 cells reduces the stimulation of sodium transport (41, 42). Here, we have investigated the critical role of PIP3 by studying whether increased PIP3 production (resulting from increased PI 3-kinase activity), independently of insulin, also stimulates sodium transport across A6 cell monolayers.  z3 Z" V6 S( g1 m3 X
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Epidermal growth factor (EGF) and hydrogen peroxide (H2O2) are known activators of PI 3-kinase in several systems (17, 45, 52, 57). However, EGF appears to affect sodium transport quite variously: it increases sodium absorption in the airways (19) and in the intestine (34), whereas it inhibits sodium absorption in a murine collecting tubule cell line (mCT1) (59, 68), in rabbit collecting tubules (46, 66, 67), and in a C7 clone of Madin-Darby canine kidney (MDCK) cells (30). Thus the action of EGF appears quite specific to the cells involved. In renal cells, the observed decrease in sodium transport was suggested to result from activation of the mitogen-activated protein kinase (MAPK) signaling pathway (11, 59), whereas the enhanced sodium absorption observed in the intestine was attributed to the activation of a brush-border PI 3-kinase (34).% }1 P3 F9 v" Z% d' t

On the other hand, the effects of H2O2 or of other reactive oxygen species on sodium transport have not been investigated, to our knowledge, in the distal nephron except in the thick ascending limb of Henle's loop, where superoxide stimulates sodium transport (47)., J8 }8 r0 {' m& C

In this study, similarly to insulin, EGF and H2O2 were shown to increase sodium transport across A6 cell monolayers in a PI 3-kinase-dependent manner. Furthermore, we studied the possible stimulation of the MAPK pathways by insulin, EGF, and H2O2 and observed that ERK1/2 was sustainedly phosphorylated only in the case of EGF stimulation, whereby it appears to dampen the stimulation of sodium transport. The results strongly support that any increase in PI 3-kinase activity leads to the proportional stimulation of sodium transport.
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EXPERIMENTAL PROCEDURES  r+ O$ ]. Q2 z
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Materials. Porcine insulin and human EGF were purchased from Sigma (St. Louis, MO). The MEK inhibitors U-0126 and PD-98059 and the EGFR kinase inhibitor tyrphostin AG-1478 were purchased from Merck (Darmstadt, Germany). The PI 3-kinase inhibitor LY-294002 was purchased from Cell Signaling Technology (Beverly, MA). Protease inhibitor tablets were purchased from Roche Molecular Biochemicals (Mannheim, Germany). Millicell inserts were purchased from Millipore (Bedford, MA). Transwell inserts were purchased from Corning Costar (Cambridge, MA). Tissue culture flasks were purchased from Sarstedt (Nümbrecht, Germany). Media was purchased from Invitrogen Life Technologies (Carlsbad, CA). Electrophysiological measurements were realized by use of an epithelial voltohmmeter from World Precision Instruments (Sarasota, FL). The current evaluated by this technique is called an epithelial open-circuit sodium current. A monoclonal antibody to phospho-p42/p44, also called phosphoERK1/2 (used at 500-fold dilution) and a polyclonal antibody to total p42/p44, also called ERK1/2 (used at 3,000-fold dilution), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). PhosphoPKB Thr 308 and Ser 473 (at 250-fold dilution) and total PKB (at 500-fold dilution) antibodies were purchased from Cell Signaling Technology. Monoclonal antibodies to phosphotyrosine (at 1,000-fold dilution) and to the PI 3-kinase p85 (at 250-fold dilution) subunit were purchased from Upstate Biotechnology (Lake Placid, NY). Protein A-agarose was purchased from Upstate Biotechnology. Peroxidase-labeled secondary antibody was purchased from Dako (Glostrup, Denmark). Phosphatidylinositol and phosphatidylserine were purchased from Advanti Polar-Lipids (Alabaster, AL). [-32P]ATP was purchased from Amersham Biosciences (Arlington Heights, IL).: @4 W! v: \$ M, t& N
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Cell culture. A6 cells were received from Prof. W. Van Driessche (Dept. of Physiology, Katholiek Universiteit te Leuven, Leuven, Belgium) and originated from Dr. J. P. Johnson (Univ. of Pittsburgh, Pittsburgh PA). A6 cells were grown at 28°C in a humidified incubator gassed with 1% CO2 in air. Cells were cultured in a 260 mosmol/kgH2O amphibian medium without exogenous addition of aldosterone as described previously (42). For biochemical experiments, cells were subcultured onto 100-mm Costar Transwell inserts. For electrophysiological experiments, A6 cells were subcultured onto 24-mm Millicell inserts and incubated overnight in serum-free medium. Cells were used 7–14 days after seeding.

Electrophysiology. Confluent monolayers of A6 cells grown on Millicell and Transwell inserts were used only if their electrical resistance was >4,200 ﹞cm2 and mean transepithelial potential difference was >30 mV. Resistance and potential difference were measured using an epithelial voltohmmeter with chopstick electrodes made of Ag-AgCl pellets. Sodium transport was calculated from the transepithelial potential difference and resistance. Amiloride added to the apical bathing medium completely inhibited this current, validating such computation of sodium transport. Insulin (100 nM) was always added to the basolateral bathing medium. H2O2 (1 mM) was added for most experiments to the apical bathing medium, and EGF (100 ng/ml) was added to the basolateral bathing medium. MAPK inhibitors U-0126 and PD-98059 were dissolved in DMSO and used at final concentration of 10 and 50 μM, respectively, and were added bilaterally to the bathing medium. The EGF receptor (EGFR) kinase inhibitor tyrphostin AG-1478 (dissolved in DMSO) was used at a final concentration of 1 μM and was added bilaterally to the bathing medium. The PI 3-kinase inhibitor LY-294002 (dissolved in DMSO) was used at a final concentration of 50 μM and was added bilaterally to the bathing medium. The final concentration of DMSO never exceeded 0.1% (vol/vol).5 R# H; f5 C& I* B! q  V* w5 q
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Western blot analysis. Immunodetection of total PKB, phosphoPKB on Thr 308 and Ser 473 residues, total ERK1/2, and phosphoERK1/2 were performed on extracts of cells grown for a minimum of 10 days on 100-mm Transwell inserts incubated in the presence or absence of 100 nM insulin, 1 mM H2O2, and 100 ng/ml EGF added to the apical or basolateral bathing medium of A6 cell monolayers for various period of time as indicated in each experiment. Cells were scraped in ice-cold lysis buffer (150 mM KCl, 10 mM Tris﹞HCl, 2 mM EDTA, 1 mM sodium orthovanadate, 100 mM NaF, 10 nM okadaic acid, 0.5% NP-40, 0.1% -mercaptoethanol, 300 μg/ml pefabloc, and 10 μg/ml leupeptin, pH 7.4) and harvested quickly at 4°C. Cells extracts were maintained at 4°C for 1 h and centrifuged (13,000 rpm; 20 min); the pellet was then discarded. Protein concentration was determined using the Bradford method (12). SDS-PAGE was performed as described previously (42). Proteins were separated on 7 and 9% acrylamide gels. Membrane incubated overnight with anti-phosphoPKB antibodies was washed and reprobed with total PKB. Similarly, membrane incubated overnight with anti-phosphoERK1/2 antibodies was washed and reprobed with total ERK1/2. Each blot presented was performed at least three times.! a  y+ ~* S1 G( f

Immunoprecipitation. Immunoprecipitation was carried out according to the procedure previously described (42). Briefly, cell lysates were incubated overnight with anti-phosphotyrosine antibodies and incubated for a subsequent 2 h with protein A-agarose. The beads were washed three times in PBS and collected by centrifugation. After being boiled, samples were loaded onto 7% acrylamide gels, and membranes were probed with anti-p85 antibody.$ a; {  A# e6 @3 w% r7 {. p
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PI 3-kinase assay. Immunoprecipitation of phosphotyrosine-containing proteins was carried out as previously described. Beads were collected and washed three times in 50 mM Tris﹞HCl buffer (pH 7.4) and assayed for PI 3-kinase activity.3 s8 O0 J4 _, s/ a6 t2 N! E9 }( s
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Ten micrograms phosphatidinositol and 20 μg phosphatidylserine in CHCl3 were dried at room temperature and suspended in 50 μl of 50 mM Tris﹞HCl. The phospholipid mixture was vortexed and sonicated before addition of 4 μCi [-32P]ATP in 80 μl kinase buffer (100 mM Tris﹞HCl, 3 mM DTT, 200 mM NaCl, 1 mM EDTA, 10 mM MgCl2, 200 μM ATP, pH 7.4). This mixture was then added to the beads and incubated for 30 min at 37°C. The reaction was stopped with Blye & Dyer (CHCl3/CH3OH/HCl 0.9N, 5:5:1, vol/vol/vol). The mixture was vortexed and centrifuged. The organic phase was collected and evaporated under a flow of nitrogen. Samples were redissolved in 80 μl CH3OH/CHCl3 (1:1, vol/vol) and spotted under nitrogen on an oxalate-precoated TLC plate. After migration (eluant: CH3COCH3, CH3OH, CH3COOH, H2O, CHCl3, 30:26:24:14:80, vol/vol/vol/vol/vol), radioactive spots were visualized and the phosphatidylinositol 3-phosphate (PI3P) was identified.

Statistics. Electrophysiological data are presented as means ± SE. Paired t-tests were used to compare the experimental vs. control group.( U: R- V8 I9 f
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RESULTS% a0 `: Q6 L# {7 R& T9 S
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Insulin, H2O2, and EGF increase sodium transport in a LY-294002-dependent manner. Apical, basolateral, or bilateral addition of H2O2 increases sodium transport similarly (data not shown) in a dose-dependent manner, reaching a maximum after 15–30 min (Fig. 1A). The increase in sodium transport was maximum using 1 mM. Concentrations greater than 5 mM were lethal for A6 cells (as deduced from a drastic and sudden fall in epithelial resistance). The increase in sodium transport was amiloride sensitive (data not shown), and the peak effect for both H2O2 and EGF stimulation was highly significant (Fig. 1C, P $ \; n# d  l, j. ~/ ^7 L3 T5 [

When added to the basolateral bathing medium of A6 cell monolayers, EGF also increased sodium transport (Fig. 1B). Again, amiloride added at the peak of the effect completely abolished the current (data not shown). No effect was observed when EGF was added to the apical side (data not shown). A concentration of 100 ng/ml EGF was used for most experiments. Stimulation was maximum after 15 min, and then sodium transport decreased quickly (Fig. 1B).

The increase in sodium transport was higher for H2O2 and insulin than for EGF stimulation (Fig. 1)./ A+ w+ b! k4 a% X1 ~

Insulin-stimulated sodium transport in A6 cell monolayers requires PI 3-kinase activity, as established by Record et al. (53) using the specific inhibitor of PI 3-kinase LY-294002 (49, 69). Similarly, H2O2 and EGF stimulated sodium transport in a PI 3-kinase-dependent manner. Monolayers were preincubated for 30 min with 50 μM LY-294002 or solvent alone added bilaterally to the bathing medium and stimulated with insulin (Fig. 2A), H2O2 (Fig. 2B), or EGF (Fig. 2C). The stimulation of sodium transport was prevented in each case by preincubation with the PI 3-kinase inhibitor (Fig. 2, A–C).

Coprecipitation of p85 by anti-phosphotyrosine immunopreciptates. The stimulation of PI 3-kinase by insulin results from the recruitment of the p85 regulatory subunit by phosphorylated tyrosine residues on the insulin receptor substrate (8). H2O2 and EGF have also been described as activators of PI 3-kinase (17, 45, 52, 58). Like other growth factor receptors, the EGFR dimerizes on binding of its ligand and becomes phosphorylated on tyrosine residues. Phosphorylated tyrosine on the EGFR or on some associated adaptor is thought to recruit the SH2 domains of the p85 regulatory subunit of PI 3-kinase, bringing the catalytic p110 subunit close to its substrate (PIP2), allowing the production of PIP3.8 O9 m$ _& `. i

On the other hand, H2O2, which enters rapidly into cells, has also been reported to phosphorylate critical tyrosine residues (perhaps by preventing their dephosphorylation), leading also to the recruitment of the regulatory subunit of PI 3-kinase with subsequent PIP3 production (52).

We therefore immunoprecipitated proteins containing tyrosine-phosphorylated residues in extracts from A6 cells stimulated or not with insulin, H2O2, and EGF for various time and detected the amount of p85 subunit coprecipitated by Western blotting. This provides an indirect assessment of PI 3-kinase activity as previously reported (13, 16, 25, 31, 63, 65). A6 cell monolayers were stimulated with insulin (100 nM, basolateral), H2O2 (1 mM, apical), and EGF (100 ng/ml, basolateral) for 0, 2, and 5 min. The level of recruitment of the p85 subunit was higher for A6 cell extracts pretreated with insulin and H2O2 than for EGF pretreatment (Fig. 3A). In the latter case, the recruitment of p85 was weaker in correlation with a lower increase in sodium transport (Figs. 1 and 3A).! U3 C" H/ |* z0 i, x9 @* e5 F

PI 3-kinase assay. Phosphotyrosine containing proteins from lysates of A6 cell monolayers stimulated with insulin, EGF, and H2O2 for different times (2 and 5 min) were first immunoprecipitated and then added to liposomes containing phosphatidylinositol. The mixture was incubated with [-32P]ATP. After reaction and lipid extraction, the samples were spotted on silicate plates and activity of PI 3-kinase was then evaluated by detection of PI3P.; B% X; j  M0 j3 L6 q! _1 r
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Basal activity of PI 3-kinase was already observed in lysate from unstimulated A6 cell monolayers compared with samples where no lysate was added and where no PI3P was detected (Fig. 3B). This PI 3-kinase activity was further increased by insulin, H2O2, and EGF stimulation. This increase was always higher after 2-min rather than after 5-min stimulation. This increase was lower after EGF rather than after H2O2 stimulation (Fig. 3B). Treatment with 50 μM LY-294002 inhibited PI 3-kinase activity in unstimulated and stimulated A6 cell monolayers (Fig. 3C).
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PKB phosphorylation by insulin, H2O2, and EGF in A6 cells monolayers. As the PI 3-kinase pathway is usually associated with an increase in PKB activity (13), the immunodetection of phosphorylated PKB on both Thr 308 and Ser 473 was used as a readout of increased PIP3 level resulting from increased PI 3-kinase activity.

Confluent A6 cell monolayers grown onto 60-cm2 Transwell inserts were incubated overnight in serum-free medium and then stimulated with insulin (100 nM), H2O2 (1 mM), or EGF (100 ng/ml) for 0, 5, 7, 10, 15, and 30 min. For unstimulated cells, no phosphorylated PKB band was systematically detected. Each stimulatory agent induced a maximum in PKB phosphorylation 7 min after its addition. In the case of insulin or H2O2, PKB phosphorylation was more sustained than in the case of EGF stimulation (Fig. 4), although the amount of total PKB reprobed on the same membranes was identical. The lower PKB phosphorylation correlates with a lower increase in sodium transport.

MAPK downregulates EGF-stimulated sodium transport in A6 cell monolayers. Insulin, H2O2, and EGF are known to activate the MAPK signaling pathway in several systems (3, 39, 57, 71). Therefore, the possible involvement of this signaling pathway was investigated in A6 cell monolayers, with the aim of seeking some correlation between MAPK activity and modifications in sodium transport. Immunodetection by Western blotting of phosphorylated ERK1/2 was used as an assessment of MAPK activity. A6 cell monolayers were treated with the MAPK kinase (MEK) inhibitors U-0126 and PD-98059 to investigate the effect of inhibition of the MAPK pathway on sodium transport. Phosphorylated ERK1/2 was detected in extracts from unstimulated A6 cell monolayers incubated overnight in serum-free medium (Fig. 5). EGF increased the level of phosphorylated ERK1/2 after only 5 min (data not shown), and a maximum level of phosphorylation was observed after 15 min that was sustained for 45 min (Fig. 6). Insulin and H2O2 also increased the level of phosphorylated ERK1/2, but only transiently (Fig. 5). Two well-known inhibitors of the MAPK pathway, U-0126 and PD-98059, decreased ERK1/2 phosphorylation (Figs. 5 and 6) in extracts from A6 cell monolayers stimulated with EGF, H2O2, or insulin.% d' Q) |, n" K: W, r# J, G& ]6 T
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Their possible effect on insulin-, H2O2-, and EGF-stimulated sodium transport was therefore tested. To this end, A6 cell monolayers were preincubated for 30 min with U-0126 or PD-98059 and stimulated with insulin (100 nM), H2O2 (1 mM), or EGF (100 ng/ml). The increase in sodium transport induced by H2O2 and insulin was similar regardless of U-0126 or PD-98059 preteatment (Fig. 7, A, B, D, and E), whereas the preincubation with U-0126 or PD-98059 to a lesser extent drastically increased the EGF stimulation of sodium transport (Fig. 7, C and F). Therefore, it appears that the MAPK pathway downregulates sodium transport in EGF-stimulated A6 cell monolayers, as previously observed in the case of phorbol ester addition (10). Furthermore, the increase in sodium transport induced by simultaneous addition of U-0126 and EGF was correlated with an increase in PKB phosphorylation (compared with the addition of EGF alone), as detected by Western blotting (Fig. 6).( `5 g. i8 N2 u2 [
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H2O2-stimulated sodium transport does not require EGFR transactivation. Several studies have demonstrated that H2O2 induces the transactivation of EGFR (71). Therefore, we investigated whether EGFR transactivation could be involved in H2O2 stimulation of sodium transport. A6 cell monolayers were preincubated for 30 min with tyrphostin AG-1478, an EGFR kinase inhibitor, and then the monolayers were stimulated with H2O2. Inhibition of EGFR kinase induced no difference in the stimulation of sodium transport brought about by H2O2 or insulin, whereas the stimulation of sodium transport induced by EGF was completely abolished (Fig. 8).; _$ w( e+ h' k/ Z

DISCUSSION7 |  z( g5 k/ Y

Insulin stimulates sodium transport in a PI 3-kinase-dependent manner in A6 cell monolayers, and the 3-phosphorylated phosphoinositides like PIP3 or PI(3,4)P2 play a mediator role in the insulin stimulation of sodium transport. In this study, we extended our investigations on the critical role of PI 3-kinase activation in the stimulation of sodium transport by using other agents that activate PI 3-kinase. As both EGF and H2O2 activate PI 3-kinase in several cell systems (3, 17, 52, 57, 58), we investigated whether such activation also occurred in A6 cell monolayers and whether it also leads to an increase in sodium transport.
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The relevance of studying the action of EGF on renal sodium reabsorption is further illustrated by the fact that EGFRs are expressed in the basal plasma membrane of each segment of the nephron. Also, in autosomal polycystic kidney disease, mislocalization of EGFR (within the apical membrane), accompanied by hyperactivity, has been described as an early event relevant to the progression of this disease (22, 38, 54, 61, 62)./ B" j1 f7 v3 @$ b" ]

As for H2O2 and reactive oxygen species, they have been implicated in renal ischemia and reperfusion injury (1, 29, 35, 37, 48). To our knowledge, their effect on sodium transport has only been described in the thick ascending limb of Henle's loop, where superoxide stimulates salt absorption (47).  q$ |* P; @/ c5 e; ~( r
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Activation of PI 3-kinase by EGF results from the recruitment of the p85 regulatory subunit by phosphorylated tyrosine residues of the EGFR. In the case of H2O2 stimulation, the signaling pathway is not as well defined (40, 52).
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We observed here that in A6 cell monolayers, EGF, H2O2, and insulin increase the activity of PI 3-kinase estimated by the amount of PI3P generated from PI and ATP. All three agents increased its activity after 2 and 5 min, always with a maximum after 2 min. PI 3-kinase activity increased less after EGF than after H2O2, which can be correlated with the magnitude of increase in sodium transport. Furthermore, baseline PI 3-kinase activity was found to be decreased by LY-294002 as well as baseline sodium transport, further supporting the critical role of this enzyme in controlling sodium transport across A6 cell monolayers.

The p85 subunit of PI 3-kinase recruited by phosphorylated tyrosines associated with membranous receptors brings the p110 subunit close to its substrate (PIP2) and allows the catalytic reaction to occur (generation of PIP3). Therefore, the amount of p85 coprecipitated by phosphotyrosine antibodies can be interpreted as the recruitment, i.e., the movement, of both subunits of PI 3-kinase to the plasma membrane with subsequent activation. The amount of p85 coprecipitated already increased after 2-min stimulation with all three agents.

The increase in PI 3-kinase activity was also reflected by the activation of PKB, a downstream effector sensitive to the amount of PIP3 generated in most cells. It might appear surprising that we did not assess instead the phosphorylation of SGK, a related kinase that has been extensively studied in A6 cells and which has been proven to play a role in the stimulation of sodium transport induced by aldosterone (14). However, in the present experimental conditions, i.e., in the absence of aldosterone, the expression of SGK is barely detectable, unlike in the case of PKB. Therefore, PKB phosphorylation was used as a very general reporter of the amount of PIP3 generated, hence of PI 3-kinase activity. Whether PKB also plays a role in controlling ENaC activity may be raised as both kinases exhibit overlapping substrate specificity. Therefore, phosphorylation of PKB represents an attractive candidate mediating the cell action of insulin, H2O2, and EGF, although obviously this hypothesis remains to be substantiated. Thus as previously reported for insulin, both EGF and H2O2 induced the phosphorylation of PKB on both Ser 473 and Thr 308 residues. The increases in PI 3-kinase activity and PKB phosphorylation were greater for H2O2 than for EGF.8 K1 x8 b+ A% a( W4 M9 H

We have previously demonstrated that the stimulation of sodium transport provoked by insulin is reduced in A6 cell monolayers overexpressing PTEN (42). On the contrary, a reduction in PTEN activity should lead to an increased PIP3 level and increased sodium transport across A6 cell monolayers. This could potentially contribute to the effect of H2O2 observed here as in other cell systems. H2O2 inactivated 3-phosphatase PTEN with a subsequent increase in PIP3 level (15, 40, 41). However, the relative importance of PIP3 degradation by various phosphatases was not investigated in the present study. Nevertheless, regardless of an effect on PIP3 degradation, H2O2 activates PI 3-kinase in A6 cells, as previously reported for other cells (52, 58). This effect may be explained by inhibition of tyrosine phosphatases by H2O2, leading to increased phosphorylated tyrosine residues on adaptors recruited by insulin or EGF receptors.
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Both EGF and H2O2 also increased sodium transport with striking similarity, although this effect was greater for H2O2 than for EGF in correlation with a greater stimulation of PI 3-kinase. In each case, the stimulation of sodium transport was abolished by LY-294002, whereas tyrphostin AG-1478, a specific inhibitor of EGFR tyrosine kinase, abolished only the stimulation induced by EGF. Thus a correlation was observed between the level of increase in sodium transport and the level of activation of PI 3-kinase. This suggests that this kinase controls ENaC activity in A6 cell monolayers; however, the exact process linking PI 3-kinase to ENaC remains to be defined.

We also investigated the possible stimulation of the MAPK signaling pathway, as it has been shown to be stimulated by insulin, H2O2, or EGF in several other cell types (3, 39, 57, 71). In A6 cells, activation of MAPK signaling has been shown to downregulate sodium transport on PMA addition (11). Phosphorylated ERK1/2, one major kinase of this pathway, can affect the interaction between Nedd4–2 and ENaC, inducing the degradation of ENaC residing in the apical membrane (2, 60). We therefore investigated whether such downregulation also occurs in A6 cell monolayers stimulated by insulin, H2O2, or EGF. Although the MAPK pathway does not seem to be involved in the insulin and H2O2 stimulation of sodium transport, it appears to downregulate the stimulation of sodium transport induced by EGF.

To our knowledge, the effect of EGF on sodium transport has not yet been studied in A6 cell monolayers. The interest in such studies resides in the fact that in several systems, EGF has been shown to activate both pathways, MAPK and PI 3-kinase, which appear to have opposite effects on sodium transport in A6 cell monolayers. As indicated before, EGF increases sodium transport by activation of PI 3-kinase and this stimulation was drastically increased by addition of U-0126 or PD-98059, thus suggesting that the increase in sodium transport observed with EGF alone is limited by the continuous activation of the MAPK pathway. Quite interestingly, the significant increase in the EGF-stimulated sodium transport induced by inhibition of MAPK was also accompanied by an increase in PKB phosphorylation. By Western blot analysis, we observed that A6 cell extracts from U-0126-pretreated monolayers stimulated by EGF displayed not only a decrease in phosphorylated level of ERK1/2 but also an increase in phosphorylated PKB. Such an interaction between PI 3-kinase and MAPK signaling has also been suggested in epithelia chronically exposed to aldosterone. However, in this situation (aldosterone exposure for 20–24 h), the same MEK inhibitors increased short-circuit current across MDCK C7 monolayers (30, 36) but not across A6 cell monolayers (33, 63).4 n" k3 o, v  ]. Q

In MDCK C7 monolayers, EGF decreases sodium transport and simultaneously activates MAPK, suggesting a causal relationship. Furthermore, the stimulation of sodium transport induced by chronic aldosterone treatment appeared limited by activation of MAPK induced by transactivation of EGFR in these cells. Thus these authors suggested that transactivation of EGFR represents a negative feedback loop limiting aldosterone-induced sodium reabsorption. They further demonstrated that the phosphorylation of ERK1/2 was simultaneously increased and that U-0126 preteatment allowed a further rise in sodium transport. Unfortunately, the level of PI 3-kinase activity was not assessed in these studies. We would like to speculate that the preponderance of either MAPK vs. PI 3-kinase is responsible for either the decrease vs. the increase in sodium transport observed in different epithelia carrying ENaC. Such cell-specific differences in sodium transport could then be explained by differences in the efficiency of PI 3-kinase vs. MAPK activation from cell line to cell line. However, differences in expression and function of various ERK isoforms could also explain the variable efficiency of MAPK's effect on sodium transport. Particular attention should be paid to the ERK5 isoform, as it is activated by EGF (44).
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In conclusion, any increase in PI 3-kinase activity whatever the stimulatory agents leads to an increase in sodium transport across A6 cell monolayers. Furthermore, in the case of EGF, the increase in sodium transport is limited by the simultaneous stimulation of the MAPK pathway, and MEK inhibitors further raise sodium transport.# |9 J+ M- E, C( A2 e7 `
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GRANTS

This work was supported by grants from the Fonds Alphonse et Jean Forton, the Action de Recherche Concertée of the Communauté Franaise de Belgique, and the Fonds de la Recherche Scientifique Médicale. D. Blero is an aspirant of the Fond National de la Recherche Scientifique. N. Markadieu is a Boursier de l'Université Libre de Bruxelles.
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ACKNOWLEDGMENTS7 J6 Y) s3 V- z
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This work was presented in abstract form at the Transporters 2004 Meeting (Cambridge, UK, September 2004).! X8 h' p8 L* _5 Z" c
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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.9 N0 u- c) p+ _' v. H; Y' T

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