Regulation of Na+ transport by aldosterone: signaling convergence and cross talk between the PI3-K and MAPK1/2 cascades

Qiusheng Tong,1 Rachell E. Booth,2 Roger T. Worrell,3 and James D. Stockand1

1Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio 78229-3900; 2Department of Chemistry and Biochemistry, Texas State University, San Marcos, Texas 78666; and 3Epithelial Pathobiology Group, Department of Surgery, University of Cincinnati, Cincinnati, Ohio 45219

Submitted 26 September 2003 ; accepted in final form 18 February 2004


    ABSTRACT
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 ABSTRACT
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 DISCUSSION
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Cross talk between the phosphatidylinositol 3-kinase (PI3-K) and mitogen-activating protein kinase (MAPK)1/2 signaling cascades in response to aldosterone-induced K-RasA was investigated in renal A6 epithelial cells. In addition, the contribution of these signaling pathways to aldosterone-stimulated Na+ transport was investigated. Aldosterone increased active K-RasA levels in A6 cells resulting in activation of downstream effectors in both the MAPK1/2 and PI3-K cascades with K-RasA directly interacting with the catalytic p110 subunit of PI3-K in a steroid-dependent manner. Aldosterone-stimulated PI3-K signaling impinged on the MAPK1/2 cascade at the level of Akt-mediated phosphorylation of c-Raf at an established negative regulatory site. Aldosterone also increased Sgk levels as well as stimulated phosphorylation of this kinase in a PI3-K- and K-RasA-dependent manner. Blockade of MAPK1/2 signaling had little effect on Na+ transport. Conversely, inhibition of PI3-K markedly suppressed transport. Likewise, suppression of K-RasA induction decreased transport. However, Na+ transport was subsequently stimulated under these conditions with the PLA2 inhibitor aristolochic acid, an established positive modulator of Na+ transport, suggesting that K-RasA signaling through PI3-K does not directly affect epithelial sodium channel (ENaC) levels but the activity of this channel. Consistent with this possibility, activity of ENaC reconstituted in Chinese hamster ovary cells was increased by coexpression of constitutively active PI3-K. The current study demonstrates that aldosterone increases Na+ transport, in part, by stimulating PI3-K signaling and that during aldosterone actions, there is both signaling convergence between the two aldosterone-induced proteins, K-RasA and Sgk, as well as cross talk between the PI3-K and MAPK1/2 cascades with the prior but not latter cascade enhancing ENaC activity.

epithelial sodium channel; downstream effectors; Ras; serum and glucocorticoid-induced kinase; hypertension


THE EPITHELIAL SODIUM CHANNEL (ENaC) is a centrally positioned effector modulating systemic blood pressure (8). This amiloride-sensitive, noninactivating, voltage-independent, Na+-selective ion channel resides in the apical plasma membrane of distal colonic and renal nephron epithelial cells responsible for discretionary Na+ reabsorption. ENaC activity is rate limiting for vectorial Na+ transport with the mineralocorticoid aldosterone stimulating transport by increasing the activity of this channel (23, 26). Both gain- and loss-of-function mutations in ENaC and its upstream regulatory pathways are well established, manifest in improper electrolyte and water handling by the kidney leading to several forms of inappropriate salt conservation and loss, respectively (11). In humans, as well as in several mammalian models, this aberrant salt water handling by the kidney results in blood pressure abnormalities.

Aldosterone, as mentioned above, is a critical endocrine modulator of ENaC activity. Studies from our laboratory (9, 25), as well as that of Verrey et al. (13, 21, 22), demonstrated that aldosterone increases K-RasA levels in A6 epithelial cells through a mechanism dependent on transcriptional control of the K-ras gene. We also made similar observations in cardiac fibroblasts from adult rats (24). Aldosterone not only increased K-RasA levels in A6 cells but also increased the amount of active GTP-complexed K-RasA (9). Induction of K-RasA is necessary and possibly sufficient for aldosterone action in some instances, especially in A6 cells (25). In addition, overexpression of constitutively active K-RasA with ENaC in Xenopus laevis oocytes increases channel open probability (13).

Aldosterone activates two established downstream effector-signaling pathways of K-RasA, the mitogen-activating protein kinase (MAPK)1/2 and phosphatidylinositol 3-kinase (PI3-K) cascades, in renal A6 epithelial cells (2, 9, 17, 27). Similar to K-RasA, PI3-K signaling is necessary for Na+ transport in this model (2, 17, 27). In contrast, activation of the MAPK1/2 cascade independent of aldosterone suppresses Na+ transport by targeting ENaC subunits for degradation (3). The downstream PI3-K target Sgk is also an aldosterone-induced protein with aldosterone signaling, in addition, activating this kinase in a PI3-K-dependent manner (6, 15, 27).

Although the MAPK1/2 and PI3-K signaling cascades, as well as a common initiator of both cascades, K-RasA, are known modulators of Na+ transport, the relationship between these cascades and their possible common activator in aldosterone-activated epithelial cells has not been investigated. In addition, a direct link between PI3-K and ENaC activity has not been established. Possible signaling convergence involving the aldosterone-induced proteins K-RasA and Sgk, moreover, has not been investigated. The current studies confirm that aldosterone activates both PI3-K and MAPK1/2 signaling in A6 cells but importantly demonstrate that it can do so through induction of K-RasA. In addition, we demonstrate cross-talk between these two cascades in response to aldosterone, as well as signaling convergence between Sgk and K-RasA. Finally, and we believe most importantly, the current study demonstrates directly that PI3-K activates ENaC with the PI3-K-dependent signaling cascade, but not the MAPK1/2 cascade, being a powerful mediator of aldosterone action on Na+ transport in native epithelia.


    METHODS
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Materials and reagents. All reagents, unless noted otherwise, were from Sigma (St. Louis, MO), Calbiochem (San Diego, CA), BioMol (Plymouth Meeting, PA), and Fisher Scientific (Pittsburgh, PA). Renal A6 epithelial cells were from American Type Culture Collection (Manassas, VA). Full-length human {alpha}-, {beta}-, and {gamma}-hENaC cDNA in the pMT3 plasmid have been described previously (14, 19) and were a gift from Dr. P. Snyder (Univ. of Iowa College of Medicine, Iowa City, Iowa). The adenovirus major late promoter drives expression of each cDNA. These constructs were used without modification. The construct encoding constitutive active PI3-K (p110-{alpha}) was from BD Transduction Laboratory. Dominant-negative Ras (DNRasN17) was a kind gift from Dr. G. Firestone (Univ. of California at Berkeley, Berkeley, CA) and has been used previously by our laboratory (9). The glutathione S-transferase (GST)-Sgk fusion protein used in the current study was created by subcloning X. laevis Sgk in a frame behind GST in the pGEX-KG plasmid. The donor xSgk plasmid and the pGEX-KG plasmid were kind gifts from Drs. D. Pearce (Univ. of California at San Francisco, San Francisco, CA) and A. Firulli (Univ. of Texas Health Science Center at San Antonio, San Antonio, TX), respectively. Phosphorothioate antisense and sense K-ras oligonucleotides have been described previously (9, 25) and were from the Emory Univ. (Atlanta, GA) microchemical facility. The rabbit polyclonal anti-MAPK1/2 and anti-PI3-K p110 antibodies and mouse monoclonal anti-PI3-K p85 antibody were from Upstate Biotechnology. The sheep polyclonal anti-SGK antibody was also from Upstate Biotechnology. This antibody has been discontinued. The mouse monoclonal anti-c-Raf-1 antibody was from Transduction Laboratories. The mouse monoclonal anti-v-Ha-Ras antibody was from Oncogene. The rabbit polyclonal anti-K-Ras2A and goat polyclonal anti-PKB kinase (PDK1) antibodyies were from Santa Cruz Biotechnology. All phospho-specific rabbit polyclonal antibodies were from Cell Signaling Technologies, except anti-phospho-MBP antibody, which was from Upstate Biotechnology. All secondary horseradish peroxidase-conjugated antibodies were from Kirkegaard & Perry Laboratories.

Cell culture and cell transfection. Renal A6 cells were maintained in culture as described previously (9, 25). For Western blot analysis and assessment of open-circuit current, cells were grown to confluence on polycarbonate supports (Transwell-Clear Inserts, pore size 0.4 µM, 4.7 cm2; Costar, Cambridge, MA) in the presence of serum and aldosterone (1.5 µM). Two days before experiments, cells were maintained in minimal media depleted of serum and steroids. Chinese hamster ovary (CHO) cells were maintained in tissue culture as described previously (4). For patch-clamp analysis, cells were plated on coverglass chips treated with 0.01% poly-lysine and transfected using the PolyFect reagent (Qiagen, Valencia, CA) per the manufacturer's recommendations. Initially, cells ~20–60% confluent were treated with 2.0 µg total plasmid cDNA [0.5, 0.5, 0.5, 0.5 µg of {alpha}-, {beta}-, and {gamma}-hENaC, and green fluorescent protein (GFP) cDNA]. To investigate PI3-K regulation, cells were transfected with 0.1, 0.1, 0.1, 1.0, and 0.5 µg of {alpha}-, {beta}-, {gamma}-hENaC, PI3-K, and GFP cDNA. Cells were used for up to 72 h after transfection and were maintained in culture in the presence of 10 µM amiloride replenished daily. Plasmid maxiprep cDNA was prepared using anion-exchange resin and isoproponal precipitation (Qiagen) with cDNA solubilized in water.

Western blot analysis. Western blot analysis closely followed methods described previously (3, 9, 25). In brief, cells were extracted with gentle lysis buffer [76 mM NaCl, 50 mM Tris·HCl, 2 mM EGTA, plus 1% Nonidet P-40, 10% glycerol (pH 7.4), and 1 mM phenylmethylsulfonyl fluoride] with equal amounts of total cellular protein (~80 µg) loaded per lane. In some instances, whole cell lysates were prepared in the presence of standard protein phosphatase inhibitors (in mM): 0.1 NaPPi, 0.5 NaF, 0.1 Na2MoO4, 0.1 ZnCl2, and 0.04 Na3VO4. For immunoprecipitation experiments, starting samples had equal amounts of total cellular protein (~400 µg). The methodology used in the current study for pulldown experiments using the Ras-binding-domain of Raf (RBD) and the in vitro MAPK1/2 activity assay was identical to that described previously (9). All Western blot analysis was performed using material and reagents from Pierce (Rockford, IL) and Bio-Rad (Hercules, CA).

Electrophysiology. Open-circuit current across A6 cell monolayers was quantified as described previously (3, 25). Activity of ENaC reconstituted in CHO cells was quantified using voltage-clamp analysis in the whole cell configuration as described previously (4). For these experiments, the major cations in the bath and pipette solutions were Na+ and Cs+, respectively, with symmetrical Cl. For bi-ionic selectivity studies, bath and pipette solutions were 50 mM NaCl + 100 mM NMDGCl, and 150 mM NMDGCl, respectively, with bath Na+ completely replaced with Cs+, K+, and Li+.

Statistics. Data are reported as means ± SE. Statistical significance (P <= 0.05) was determined using the t-test for differences in mean values.


    RESULTS
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 ABSTRACT
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Aldosterone-dependent MAPK1/2 and PI3-K signaling. Results presented in Figs. 13 are from experiments investigating activation of the MAPK1/2 and PI3-K signaling cascade by aldosterone and possible cross talk between these cascades in renal A6 epithelial cells. We tested possible activation and cross talk with Western blot analysis of the absolute and active levels of several constituent proteins within these cascades in response to aldosterone in the absence and presence of PI3-K and MAPK1/2 inhibitors. All Western blotting was performed at least three times. Figure 1A shows a flow chart of possible aldosterone signaling in A6 cells with this steroid hormone activating K-RasA with subsequent activation of MAPK1/2 and PI3-K signaling. Inhibitors of each branch are also indicated in this flow chart. Raf, Mek 1/2, MAPK1/2, PDK1, Sgk, Akt, and PI3-K are kinases, with the prior being protein kinases and the last a lipid kinase. Thus MAPK1/2 and PI3-K signaling can be measured by assessing phosphorylation of MAPK1/2 and Sgk protein. In addition, Akt is known to phosphorylate c-Raf at Ser259 (18, 33). This is a negative regulatory phosphorylation of previously activated c-Raf. Figure 1B shows a representative Western blot containing the Ras-binding-domain of Raf (RBD) precipitant from whole cell lysates of A6 cell monolayers treated with vehicle (DMSO) and aldosterone (1.5 µM) for 4 h. (Two lysates for each group are shown.) This blot (top) was probed with anti-K-RasA antibody. A loading control for precipitation probed with anti-MAPK antibody is shown in the bottom blot in Fig. 1B. We demonstrated previously that aldosterone has no effect on the absolute level of MAPK and thus have used this protein as a loading control (3, 9). Consistent with previous studies (9), aldosterone increased the levels of activated, GTP-complexed K-RasA.



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Fig. 1. Aldosterone (ALDO) activation of phosphatidylinositol 3-kinase (PI3-K) and mitogen-activating protein kinase (MAPK)1/2 signaling cascades. A: flow chart depicting the possible constituents of the aldosterone-induced signaling cascade in renal A6 epithelial cells. Black arrows denote positive regulation; gray arrows note negative regulation. Inhibitors of Raf, Mek 1/2, and LY-294002 are noted in gray. B: typical Western blot containing the RBD precipitant from whole cell lysates from cells treated with vehicle and aldosterone probed with anti-K-RasA antibody. Two lysates for each group are shown. Bottom blot: same lysates probed with anti-MAPK1/2 antibody as a loading control for precipitations. C: typical Western blots containing the whole cell lysates from A6 cells treated with vehicle and steroid and transfected with green fluorescent protein (GFP) alone and plus DNRasN17, supplemented with unphosphorylated MBP, ATP, and several kinase inhibitors to isolate MAPK1/2 activity. Top and bottom blots were probed with anti-phospho-MBP and anti-GFP antibodies, respectively. D: typical Western blots containing the anti-p110 PI3-K subunit (top), Ras (middle), and p85 PI3-K subunit (bottom) immunoprecipitants from the same whole cell lysates, from cells not treated with aldosterone, and from those treated with steroid for 30 and 60 min. These blots were probed with either anti-K-RasA (top) or anti-p85 PI3-K antibodies (middle and bottom). The PI3-K p85 subunit is the slower migrating band in these blots. ENaC, epithelial sodium channel; CON, control.

 


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Fig. 3. Cross talk and signaling convergence between aldosterone-induced MAPK1/2 and PI3-K cascades. A: typical Western blots containing the same whole cell lysates from A6 cells pretreated for 24 h with vehicle and sense and antisense K-ras oligonucleotides followed by aldosterone. Blot 1: blot probed with anti-K-RasA antibody. Blots 2 and 3: blots probed with anti-phospho-Raf and anti-phospho-MAPK1/2 antibody, respectively. Blot 4: blot probed with anti-MAPK1/2 antibody. B: typical Western blots containing the whole cell lysates from untreated (CON) A6 cells and cells treated with aldosterone in the absence and presence of LY-294002 for 4 h, as well as lysate from cells pretreated for 24 h with sense and antisense K-ras oligonucleotides followed by aldosterone for 4 h. Both blots contain the same lysates. Top and bottom: blots were probed with anti-Sgk and anti-MAPK1/2 antibody, respectively. C: typical Western blots containing whole cell lysate from A6 cells treated with vehicle and aldosterone in the absence and presence of Mek 1/2 and PI3-K inhibitors. Blots 1 and 2: blots are the same blot cut in half with the top and bottom halves probed with anti-phospho-Raf and anti-phospho-MAPK1/2 antibodies, respectively. Blots 3 and 4: blots are of the same blot cut in half with the top and bottom halves probed with anti-phospho-Raf and anti-MAPK antibodies, respectively. Probing with anti-MAPK antibody served as a loading control.

 
We then asked whether aldosterone also activated MAPK1/2 in a K-RasA-dependent manner. The Western blot in Fig. 1C was probed with anti-phospho-MBP (top), stripped, and then probed with anti-GFP (bottom) antibodies. This blot contained the total reaction of an in vitro MAPK assay. We have described this assay previously (9). In brief, whole A6 cell lysate was prepared from cells transfected with a plasmid encoding GFP in the absence and presence of an additional plasmid encoding dominant-negative RasN17. Monolayers were treated with vehicle and aldosterone (2 h). Lysate was supplemented with exogenous MPB, ATP, and kinase inhibitors to specifically isolate MAPK1/2 activity. These results demonstrate that aldosterone increases MAPK1/2 activity in A6 cells and that this increase can be attenuated by sequestering c-Raf in an inactive state with DNRasN17.

We next asked whether aldosterone-activated K-RasA influenced the PI3-K cascade in A6 cells. Shown in Fig. 1D are typical Western blots containing the anti-p110 PI3-K (top), anti-Ras (middle), and anti-p85 PI3-K (bottom) precipitants from the same whole cell lysates of A6 cell monolayers in the absence and presence of aldosterone for 30 and 60 min. The top, middle, and bottom blots were probed with anti-K-RasA, anti-p85, and anti-p85 antibody, respectively. The results demonstrate that K-RasA associated with the catalytic p110 subunit of PI3-K in an aldosterone-dependent manner. Interestingly, we also observed an aldosterone-dependent decrease in the amount of regulatory p85 subunit associated with K-RasA and presumably p110.

To investigate aldosterone action on the downstream PI3-K effector Sgk in amphibian A6 cells, we first characterized a commercially available anti-Sgk antibody using a GST-xSgk fusion protein (Fig. 2A). The blot on the left contains the glutathione-agarose precipitant from bacterial lysate from cells transformed with empty pGEX-EZ plasmid (lane 1) and this plasmid containing the cDNA encoding xSgk positioned in frame behind GST (pSgk: lane 2). This Western blot was probed with anti-GST antibody. Next, we took lysate from bacteria expressing pSgk precipitated with glutathione-agarose and then exposed an aliquot of this precipitant to thrombin to cleave xSgk from GST. Shown in the Western blot on the right are the uncleaved (lane 1) and cleaved (lane 2) products from this experiment. This blot was probed with anti-Sgk antibody. These results demonstrate that the antibody used in this study recognizes amphibian Sgk.



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Fig. 2. Aldosterone increases active Sgk levels via PI3-K and PDK1 signaling. A: typical Western blots containing the glutathione-agarose precipitant from control bacterial lysates and lysates from E. coli overexpressing GST-xSgk. Left: blot was probed with anti-GST antibody. Right: blot contained GST-xSgk in the absence and presence of thrombin cleavage of GST. This blot was probed with anti-Sgk antibody. B: typical Western blot containing the anti-PDK1 immunoprecipitant and supernatant from whole cell lysate from cells treated with vehicle and aldosterone. Blot probed with anti-Sgk antibody. C: typical Western blot containing whole cell lysate from cells not exposed to aldosterone and from those exposed to steroid for 0.5, 1, 2, 6, and 8 h. This blot was probed with anti-Sgk antibody. D: typical Western blot containing whole cell lysate from cells not exposed to aldosterone and from those exposed to steroid for 0.5, 1, 2, 4, and 6 h. This blot was probed initially with anti-Sgk antibody (top) and then stripped and reprobed with anti-MAPK antibody as a loading control.

 
Figure 2B shows that the upstream kinase activator of Sgk, PDK1, associates with Sgk in an aldosterone-sensitive manner. This typical Western blot contains the PDK1 immunoprecipitant and corresponding supernatant from whole cell lysate from A6 cell monolayers treated with vehicle (DMSO) and aldosterone (4 h). The blot was probed with the anti-Sgk antibody characterized in Fig. 2A. These results are consistent with aldosterone activating PI3-K, which in turn activates PDK1 with this latter kinase then associating and activating (phosphorylating; note the upward shift) Sgk (see Ref. 16).

Figure 2, C and D, tested the effects of aldosterone on Sgk. These representative Western blots contain the whole cell lysate from A6 cell monolayers treated with aldosterone from 0 to 8 h. Lysates were prepared in the presence of phosphatase inhibitors. The blots were probed with anti-Sgk antibody. Clearly shown is that aldosterone increases Sgk levels by 1 h with levels tending downward after 2 h. Similar to other groups (16, 27), we were able to distinguish phosphorylated (slower migrating band) from unphosphorylated (faster migrating band) Sgk in our experiments. This is particularly apparent in Fig. 2D. The bottom blot in Fig. 2D shows a loading control and is the top blot (probed with anti-Sgk antibody) stripped and subsequently reprobed with anti-MAPK antibody. Aldosterone preferentially increased phosphorylated Sgk consistent with activation of the upstream PI3-K-PDK1 cascade (see Ref. 16). These results demonstrate that aldosterone has two effects on Sgk, increasing absolute levels as well as active levels, a finding similar to aldosterone action on K-RasA (25, 27).

Western blots in Fig. 3A further tested whether aldosterone activates the MAPK1/2 cascade in a K-RasA-sensitive manner. These typical blots contained whole A6 cell lysate from cells treated with aldosterone (4 h) following a 24-h pretreatment with sense and antisense K-ras oligonucleotides (10 µM) identical to the area around the translation start site of X. laevis K-ras. Use of these oligonucleotides has been described previously (9, 25). In figure 3A, blot 1, blot 2, blot 3, and blot 4 were probed with anti-K-RasA, phospho-c-Raf, phospho-MAPK1/2, and MAPK1/2 antibodies, respectively. Blot 4 is simply blot 3 stripped of anti-phospho-MAPK1/2 and reprobed with anti-MAPK1/2. Consistent with our previous findings (9), suppression of K-RasA levels with antisense oligonucleotide decreased aldosterone-sensitive increases in K-RasA levels, as well as active (phospho)-MAPK1/2 levels, but not total MAPK1/2 levels. Interestingly, we also saw K-RasA-sensitive phosphorylation of c-Raf at Ser259. Akt is known to phosphorylate this site on active c-Raf, resulting in negative regulation of c-Raf kinase (18, 33). Thus measuring phosphorylation of c-Raf on Ser259 is an indirect measurement of Akt activity. We interpret these results as demonstrating that aldosterone simultaneously activates both the MAPK1/2 and PI3-K signaling cascades with cross talk between these cascades happening at the level of c-Raf and Akt. The results in the blot 1 of Fig. 3C are consistent with this interpretation.

Results shown in Fig. 3B were from experiments testing whether aldosterone increased active levels of Sgk in a K-RasA- and PI3-K-dependent manner. The typical blot in Fig. 3B probed with anti-Sgk (top) antibody contains whole A6 cell lysate from untreated cells (CON) and monolayers treated with aldosterone in the absence and presence of the PI3-K inhibitor LY-294002 (50 µM) for 4 h, as well as lysates from cells pretreated with sense and antisense K-ras oligonucleotides (24 h) followed by a 4-h aldosterone treatment. Figure 3B, bottom, is a loading control containing the same lysates and probed with anti-MAPK1/2 antibody. These results showing that, in the presence of antisense K-ras and LY-294002, aldosterone has less of an effect to increase phospho-Sgk levels are consistent with both K-RasA and PI3-K signaling being necessary for steroid-dependent activation of Sgk.

Figure 3C shows results from experiments testing aldosterone-dependent cross talk between the PI3-K and MAPK1/2 signaling cascades. The top two blots in Fig. 3C contain the same lysate and are actually the same blot cut in half and probed with anti-phospho(Ser259)-Raf (blot 1) and anti-phospho-MAPK1/2 (blot 2) antibodies. The bottom two blots in Fig. 3C are the same blot probed first with anti-phospho(Ser259)-Raf antibody (blot 3) and then stripped and reprobed with anti-MAPK (blot 4) antibody as a loading control. Whole A6 cell lysates were from monolayers treated with vehicle and aldosterone in the absence and presence of Mek 1/2 inhibitors (10 µM PD-98059; 0.5 µM U-0126), an inactive analog of the Mek 1/2 inhibitor U-0126 (0.5 µM U-0124), LY-294002 (50 µM), and this PI3-K inhibitor plus the U-0126 Mek 1/2 inhibitor for 4 h. Aldosterone activated (phosphorylated) MAPK1/2 only in the presence of uninhibited Mek 1/2 signaling. Similarly, aldosterone resulted in Akt-dependent phosphorylation of c-Raf only in the presence of functional PI3-K. These results support the idea that aldosterone activates both MAPK1/2 and PI3-K signaling in A6 cells with cross talk between these cascades at the level of Akt and c-Raf. As a whole, results in Figs. 13 are consistent with the signal transduction scheme depicted in Fig. 1A.

We next determined the contributions of K-RasA, MAPK1/2, and PI3-K signaling to aldosterone-induced Na+ transport across A6 cell monolayers. Figure 4A shows the relative (time 4 h/time 0) open-circuit current across A6 cell monolayers in response to treatment with aldosterone in the absence and presence of Mek 1/2, c-Raf (1 µM ZM-336372), and PI3-K inhibitors. Although aldosterone significantly increased current at this time point, as expected, only inhibition of PI3-K signaling affected this increase, suggesting that this pathway and not the MAPK1/2-dependent pathway plays a role in the positive actions of aldosterone on transport. The results in Fig. 4B determined whether K-RasA was necessary for aldosterone action on transport. This figure reports the aldosterone-sensitive (4 h) open-circuit current from A6 cell monolayers pretreated with sense and antisense K-ras oligonucleotide. As observed previously (25), suppression of K-RasA expression reduces the ability of steroid to induce the current. Interestingly, subsequent treatment of the antisense group with the PLA2 inhibitor aristolochic acid (200 µM; 5 min) significantly increased the current to levels similar to that observed in the aldosterone-treated sense group. Because this was a rapid response and inhibition of PLA2 has been reported previously to stimulate transport (5, 29), we interpret these results as showing that suppression of K-RasA signaling does not overtly affect expression levels of ENaC but merely reduces the activity of this channel. In summary, the results in Fig. 4 demonstrate that K-RasA and PI3-K but not MAPK1/2 signaling are necessary for aldosterone stimulation of transport in A6 cells.



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Fig. 4. PI3-K but not MAPK1/2 signaling is necessary for aldosterone-stimulated Na+ reabsorption. A: summary graph of relative (to time 0 h) open-circuit current for A6 cell monolayers treated for 4 h with aldosterone in the absence and presence of Mek 1/2 (PD-98059 and U-0126), Raf (ZM-336372), and PI3-K (LY-294002) inhibitors. *P <= 0.05 vs. aldosterone. B: summary graph of the change in open-circuit current in response to 4-h aldosterone following a 24-h pretreatment with sense and antisense K-ras oligonucleotide (10 µM). *P <= 0.05 vs. sense. Also included in this figure is the change following a 5-min application of aristolochic acid (ARIST) to the antisense (ANTI) group. **P <= 0.05 vs. antisense.

 
Similar to our results in Fig. 4, others have indirectly implicated PI3-K signaling as a positive regulator of ENaC activity (2, 17, 27). However, the effects of PI3-K on ENaC have not been directly studied. To do so, we reconstituted ENaC in CHO cells. Results presented in Fig. 5 demonstrate that overexpression of all three ENaC subunits in CHO cells results in reconstitution of functional ENaC channels. For these experiments, ENaC activity was assessed under voltage-clamp (I-V) conditions using the patch-clamp method in the whole cell configuration (4). Shown in Fig. 5A are I-V relationships for reconstituted ENaC (0.5 µg ea. subunit) under bi-ionic conditions with the bath being 50 mM Na+, Cs+, K+, and Li+. Consistent with ENaC in native epithelia and overexpressed in X. laevis oocytes, the channel was highly selective for Na+ and Li+ over other cations showing the hallmark of greater Li+ compared with Na+ conductance. Reconstituted ENaC was also sensitive to amiloride. CHO cells had very little leak current, of which none was amiloride sensitive. Figure 5B summarizes results (n = 6) from a control experiment testing the effects of coexpression of Nedd4–2 (1.0 µg) on ENaC (0.5 µg ea. subunit) activity. Similar to that reported previously (19) and as expected, Nedd4–2 suppressed ENaC activity. These results demonstrate that we can directly assess ENaC function when this channel is reconstituted in CHO cells.



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Fig. 5. Regulation of ENaC reconstituted in Chinese hamster ovary (CHO) cells by Nedd4–2 and PI3-K. A: current-voltage (I-V) relationship of macroscopic currents in CHO cells overexpressing ENaC in the presence of extracellular Li+, Na+, K+, Cs+, and Na+ + amiloride. B: summary graph of the amiloride (amil.)-sensitive inward current at –80 mV in CHO cells overexpressing ENaC (0.5 µg ea. subunit) in the absence and presence of Nedd4–2. *P <= 0.05 vs. ENaC alone. C: summary graph of the amiloride-sensitive inward current at –80 mV in CHO cells overexpressing ENaC (0.1 µg ea. subunit) in the absence and presence of constitutive active p110 PI3-K. *P <= 0.05 vs. ENaC alone.

 
Summarized results in Fig. 5C are from whole cell voltage-clamp experiments (n = 10) testing the effects of coexpression of constitutively active PI3-K (0.5 µg) on ENaC (0.1 µg ea. subunit) activity. Reported here are the amiloride-sensitive, inward Na+ currents at –80 mV for cells transfected with ENaC alone and with PI3-K. In this population study, coexpression of PI3-K significantly increased current greater than threefold. These results directly demonstrate that PI3-K increases ENaC activity.


    DISCUSSION
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 ABSTRACT
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 RESULTS
 DISCUSSION
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 REFERENCES
 
Insulin and aldosterone stimulate Na+ reabsorption. Blazer-Yost and colleagues (2, 17) and Pearce and colleagues (27) showed that PI3-K activity is necessary for insulin and aldosterone action on transport with the latter group showing that PI3-K must be active for proper posttranslational modification of the aldosterone-induced protein Sgk. In addition, aldosterone increases the levels of phospholipid products of PI3-K (2). It is believed that activated Sgk sustains ENaC in the luminal plasma membrane (1, 7, 20). Several critical questions, however, remain unanswered concerning PI3-K signaling to ENaC. First, it is unclear how aldosterone activates PI3-K signaling. Second, experiments hitherto have demonstrated that PI3-K signaling is necessary for aldosterone-stimulated as well as basal Na+ reabsorption but have not directly demonstrated that ENaC is the ultimate target of PI3-K signaling. The current results addressed both outstanding issues. We demonstrate that K-RasA associates with the catalytic p110 subunit of PI3-K in an aldosterone-dependent manner. This is consistent with Ras activation of PI3-K in other systems (10, 28, 30). Concomitantly, Sgk associates with PDK1 in an aldosterone-sensitive manner and is phosphorylated in a steroid-dependent manner only in the presence of K-RasA, suggesting that K-RasA is the impetus for aldosterone action on PI3-K signaling in A6 cells. Indeed, suppression of K-RasA expression during aldosterone signaling had the same effect as blockade of PI3-K with respect to both Sgk processing and transport. We believe these results provide strong evidence that in some instances and in certain cell types, PI3-K is positioned between K-RasA and Sgk, enabling signaling convergence between these two aldosterone-induced proteins.

The current study also addressed the second question. We demonstrate directly for the first time that PI3-K affects ENaC activity. Patch-clamp experiments demonstrate that ENaC reconstituted in CHO cells is about three times more active in the presence of constitutively active PI3-K. The mechanisms by which PI3-K stimulates ENaC in native epithelia and the reconstituted system used in the current study remain to be definitively determined. At this moment, two mechanisms appear most attractive. PI3-K signaling could sustain ENaC in the membrane as others suggested through the actions of Sgk (7, 20). However, as shown here and previously by others (6, 15, 27), Sgk is induced and activated by aldosterone in a transient manner peaking between 1 and 2 h leading to the supposition that PI3-K signaling must affect ENaC activity through multiple pathways, one of which can function in a sustained manner coupling PI3-K activity to ENaC activity over a prolonged period. The recent findings that ENaC is activated by anionic phospholipids (12, 32) are exciting and lead to the possibility that ENaC activity in some manner directly reflects the cellular levels of the phospholipids produced by PI3-K.

In addition to aldosterone stimulating PI3-K signaling and inducing and activating Sgk, aldosterone induces and activates K-RasA as well as its downstream effector MAPK1/2 cascade (9, 13, 21, 22, 25). Interestingly, PI3-K is one of only a few well-established first effectors of Ras signaling proteins with these small G proteins directly interacting with the p110 subunit of this kinase to stimulate activity (10, 28, 30, 31). Thus aldosterone-activated K-RasA could sit at the top of a bifurcating signaling pathway, leading to stimulation of both MAPK1/2 and PI3-K cascades. Our results are consistent with aldosterone activating such a cascade in A6 cells. Two questions arose from this aldosterone response during the course of the current study. Was there cross talk between these cascades, and which cascade played a major role in mediating aldosterone action on Na+ transport and ENaC activity? The current results demonstrate that there is cross talk between aldosterone-sensitive PI3-K and MAPK1/2 signaling at the level of Akt and Raf with the prior phosphorylating the latter at a site established to be involved in negative regulation of Raf (18, 33). Activation of MAPK1/2 signaling independent of aldosterone is known to decrease ENaC activity by promoting degradation of ENaC subunit protein (3). This is a slowly developing response to MAPK1/2 activation leading to decreased activity after 6 h. The current results demonstrate that aldosterone only stimulates transport in the presence of uninhibited PI3-K signaling and that MAPK1/2 signaling plays little overt role in the positive actions this steroid has on transport. With these results in mind and considering our earlier findings regarding suppression of ENaC activity by MAPK1/2, we propose that during aldosterone signaling to ENaC, the PI3-K cascade plays a major stimulatory role at the same time as suppressing negative feedback via aldosterone-activated MAPK1/2 signaling.


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This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-59594, American Heart Association-Texas Affiliate Grant 0355012Y, and the American Society of Nephrology Carl W. Gottschalk Research Scholar Grant (to J. D. Stockand).


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. E. Booth, Dept. of Chemistry and Biochemistry, Texas State Univ., 601 University Dr., CHEM 216, San Marcos, TX (E-mail: rbooth{at}txstate.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.


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