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doi:10.1152/ajprenal.00159.2001282:669-679, 2002. First published Oct 30, 2001;Am J Physiol Renal Physiol

Michael Gekle, Ruth Freudinger, Sigrid Mildenberger and Stefan Silbernagl

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Aldosterone interaction with epidermal growth factorreceptor signaling in MDCK cells

MICHAEL GEKLE, RUTH FREUDINGER, SIGRID MILDENBERGER, AND STEFAN SILBERNAGLPhysiologisches Institut, University of Wurzburg, 97070 Wurzburg, Germany

Received 18 May 2001; accepted in final form 23 October 2001

Gekle, Michael, Ruth Freudinger, Sigrid Milden-berger, and Stefan Silbernagl. Aldosterone interactionwith epidermal growth factor receptor signaling in MDCKcells. Am J Physiol Renal Physiol 282: F669F679, 2002.First published October 30, 2001; 10.1152/ajprenal.00159.2001.Epidermal growth factor (EGF) regulates cell prolif-eration, differentiation, and ion transport by using extracel-lular signal-regulated kinase (ERK)1/2 as a downstream sig-nal. Furthermore, the EGF-receptor (EGF-R) is involved in

signaling by G protein-coupled receptors, growth hormone,and cytokines by means of transactivation. It has been sug-gested that steroids interact with peptide hormones, in part,by rapid, potentially nongenomic, mechanisms. Previously,we have shown that aldosterone modulates Na/H ex-change in Madin-Darby canine kidney (MDCK) cells bymeans of ERK1/2 in a way similar to growth factors. Here, wetested the hypothesis that aldosterone uses the EGF-R as aheterologous signal transducer in MDCK cells. Nanomolarconcentrations of aldosterone induce a rapid increase inERK1/2 phosphorylation, cellular Ca2 concentration, andNa/H exchange activity similar to increases induced byEGF. Furthermore, aldosterone induced a rapid increase inEGF-R-Tyr phosphorylation, and inhibition of EGF-R kinaseabolished aldosterone-induced signaling. Inhibition of

ERK1/2 phosphorylation reduced the Ca

2

response,whereas prevention of Ca2 influx did not abolish ERK1/2phosphorylation. Our data show that aldosterone uses theEGF-R-ERK1/2 signaling cascade to elicit its rapid effects inMDCK cells.

epidermal growth factor; aldosterone; extracellular signal-regulated kinase 1/2; calcium; Madin-Darby canine kidneycells

THE CLASSIC GENOMIC MECHANISM of steroid hormone ac-tion involves binding to intracellular receptors, tran-scription, and protein synthesis. However, aldosteronecan also induce rapid responses by interfering with

mechanisms of regulation of intracellular pH or Ca2

(6, 9, 12, 30, 43, 50), intracellular generation of inositol1,4,5-trisphosphate (IP3) (5), protein kinase C, andextracellular signal-regulated kinase (ERK)1/2 phos-phorylation (9, 11, 26, 37). Former studies also re-vealed that aldosterone acts within several minutes onplasma membrane K conductance of different cells(29, 30, 41). These rapid actions of aldosterone are

thought to be mediated by a plasma membrane recep-tor (47), although this putative receptor has not beenidentified. Recently, it has been shown that steroidhormones are able to interact with peptide hormonesignaling (35, 46). For example, the interaction of pro-gesterone with oxytocin signaling (17), as well as theinteraction of estradiol with growth factor and angio-tensin II signaling (28), has been described. In the caseof aldosterone, an interaction with angiotensin II andvasopressin has been suggested (35, 48). Some of theseeffects have been shown to be incompatible with theclassic genomic pathway. Rapid, potentially non-genomic, actions of steroids have recently been inves-tigated more extensively, and there are several reportssupporting the existence of such actions. The preciseunderlying mechanism and the physiological or patho-physiological significance are not yet understood. Theinteraction of steroids with peptide hormone signalingrepresents one possible mechanism for rapid steroidaction while offering an explanation for the signifi-cance of these effects, i.e., modulation of peptide hor-mone signaling.

In the present study, we investigated the possibleinteraction of aldosterone with peptide hormone sig-naling in a cell line that has previously been shown torespond in a rapid, nongenomic way to aldosterone,Madin-Darby canine kidney (MDCK)-C11 cells (12).We determined the possible interaction with epidermalgrowth factor (EGF) receptor (EGF-R). The EGF-R hasbeen shown to be involved in signaling events elicitedby, for example, G protein-coupled receptors, growthhormone, and cytokines by means of a mechanismcalled transactivation (19, 27). Therefore, the EGF-Rcan serve as a central transducer of heterologous sig-naling systems. Moreover, a transcription-independentinteraction of glucocorticoids with EGF-R has been

reported (7). Furthermore, we have shown that aldo-sterone increases H affinity of Na/H exchange inMDCK-C11 cells by means of ERK1/2, which is a be-havior similar to that of growth factors (11, 44). Thus itis conceivable that the EGF-R represents a pathwayinvolved in rapid aldosterone signaling similar to, forexample, G protein-coupled receptors.

Address for reprint requests and other correspondence: M. Gekle,Physiologisches Institut, Universitat Wurzburg, Rontgenring 9, 97070Wurzburg, Germany (E-mail: [email protected]).

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

Am J Physiol Renal Physiol 282: F669F679, 2002.First published October 30, 2001; 10.1152/ajprenal.00159.2001.

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EGF regulates cell proliferation, differentiation, andtissue repair and, at least in part, uses mitogen-acti-vated protein kinases as downstream signals. In addi-tion, enhanced EGF signaling has been observed inseveral tumor cells (19, 27). Furthermore, it has beenshown that EGF does affect epithelial salt transport ina cell-specific manner, leading to either enhanced orreduced salt reabsorption (8, 22, 23, 31, 45). Our re-

sults show that aldosterone uses the EGF-R-ERK1/2signaling pathway to elicit its rapid effects on ERK1/2phosphorylation, Ca2 homeostasis, and pH homeosta-sis in MDCK cells. Possibly, the EGF-R represents amembrane target for rapid effects of aldosterone.

METHODS

Cell culture. We used a subtype of MDCK cells, denomi-nated C11 (MDCK-C11), which has been cloned recently inour laboratory (14). Cells were cultivated, as described pre-

viously (12, 14, 33), in MEM supplemented with 10% fetalcalf serum at 37C and 5% CO2. Serum was removed from themedia 24 h before the experiment. For the experimentspresented, the cells were cultivated either on permeable

supports (Becton Dickinson, Heidelberg, Germany) in 96-well plates [for phosphorylated ERK (pERK)1/2-ELISA] or onpoly-L-lysine-coated glass coverslips (for Ca2 and pH mea-surements). Because the effects were not statistically differ-ent for the three conditions, the data could be compareddirectly and were pooled.

Western blot analysis. Cells were washed three times withice-cold PBS and lysed in ice-cold Triton X-100 lysis buffer(50 mM Tris HCl at pH 7.5, 100 mM NaCl, 5 mM EDTA, 200M sodium-orthovanadate, 0.1 mM phenylmethylsulfonylfluoride, 1 g/ml leupeptin, 1 M pepstatin A, 40 mg/l besta-tin, 2 mg/l aprotinin, and 1% Triton X-100) for 25 min at 4C.Insoluble material was removed by centrifugation at 12,000 gfor 15 min at 4C. Cell lysates were matched for protein,separated on SDS-PAGE, and transferred to a polyvinylidene

difluoride microporous membrane. Membranes were subse-quently blotted with a rabbit anti-phospho-ERK1/2 antibody(New England Biolabs, Beverly, MA) or with anti-phospho-Tyr-antibody (PY99; Santa Cruz Biotechnology, Santa Cruz,CA). Anti-phospho-ERK1/2 antibody only detects ERK1 andERK2 when catalytically activated by phosphorylation atThr202 and Tyr204. The primary antibody was detected byusing horseradish peroxidase-conjugated secondary IgG vi-sualized by the Amersham enhanced chemiluminescence sys-tem. Linearity of the signal has been verified by serial dilu-tion, as recommended by the manufacturer. Densitometricanalysis was performed by using SigmaGel 1.05 (Jandel,Corte Madera, CA).

Quantification of ERK1/2 phosphorylation by ELISA. Forthe quantification of ERK1/2 phosphorylation, we performed

pERK1/2-ELISA according to Versteeg et al. (42). In controlexperiments, we compared the effect of EGF determined byWestern blot and pERK1/2-ELISA and found no significantdifference. Thus the results obtained by Western blot andpERK1/2-ELISA were pooled. Cells were seeded in 96-wellplates (Maxisorp; Nunc) and serum starved for 24 h beforethe experiment. After stimulation, the cells were fixed with4% formaldehyde in PBS for 20 min at room temperature andwashed three times with PBS containing 0.1% Triton X-100.Endogenous peroxidase was quenched with 0.6% H2O2 inPBS/Triton X-100 for 20 min. Cells were washed three timesin PBS/Triton X-100, blocked with 10% fetal calf serum inPBS/Triton X-100 for 1 h, and incubated overnight with the

above described primary antibody (see Western blot analysis;1:500) in PBS/Triton X-100 containing 5% BSA at 4C. Thenext day, cells were washed three times with PBS/Triton

X-100 for 5 min and incubated with secondary antibody(peroxidase-conjugated mouse anti-rabbit antibody, dilution1:1,000) in PBS/Triton X-100 with 5% BSA for 1 h at roomtemperature and were washed three times with PBS/Triton

X-100 for 5 min and twice with PBS. Subsequently, the cellswere incubated with 50 l of a solution containing 0.4 mg/mlO-phenylenediamine, 11.8 mg/ml Na2HPO4, 7.3 mg/ml citricacid, and 0.015% H2O2 for 15 min at room temperature in thedark. The resulting signal was detected at 490 nm with amultiwell, multilabel counter (Victor2; Wallac, Turku, Fin-land). After the peroxidase reaction, the cells were washedtwice with PBS/Triton X-100 and twice with demineralizedwater. After the wells were dried for 5 min, 100 l of trypanblue solution (0.2% in PBS) were added for 5 min at roomtemperature. Subsequently, the cells were washed four timeswith demineralized water, and 100 l of 1% SDS solutionwere added and incubated on a shaker for 1 h at roomtemperature. Finally, the absorbance was measured at 595nm with the ELISA reader.

Immunoprecipitation. Immunoprecipitation was per-formed as described recently (34). Briefly, cell lysates wereprecleared with protein A/G-Sepharose for 20 min at 4C. Toprecipitate the EGF-R, anti-EGF-R antibody [clone Ab-5(Calbiochem-Novabiochem); Ref. 36; 10 g/mg protein] wasadded for 2 h, followed by overnight incubation with protein

A/G-Sepharose. Immune complexes were collected by centrif-ugation, washed three times with lysis buffer, and subjectedto SDS/8%-PAGE, and phosphorylated EGF-R was detectedby using anti-phospho-Tyr antibody (PY99). Densitometricanalysis was performed by using SigmaGel 1.05.

Determination of cytosolic Ca2. Cytosolic free Ca2 wasdetermined by using the Ca2-sensitive dye fura 2 (5 mol/l;Molecular Probes, Leiden, The Netherlands) as describedpreviously (12) and by using an inverted Axiovert 100 TVmicroscope (400 magnification, oil immersion; Zeiss,Oberkochen, Germany) and an automatic filter change device

(Hamamatsu, Herrsching, Germany). In brief, after serumdepletion for 24 h, cells were incubated with HEPES-Ringercontaining fura 2 acetoxymethyl ester in a final concentra-tion of 5 mol/l for 15 min. Subsequently, the coverslips weremounted on the microscope stage. The fluorescence signalwas monitored at 510 nm, with the excitation wavelengthalternating between 334 and 380 nm, by using a 100-Wxenon lamp. The sampling rate was one ratio every 2 s.Cytosolic Ca2 concentration ([Ca2]i) was calculated accord-ing to Grynkiewicz et al. (18) by using a dissociation constant(Kd) of 225 nmol/l, after subtraction of background fluores-cence. The maximum and minimum ratios (Rmax and Rmin)were measured after addition of calibration solutions, whichcontained 1 mol/l ionomycin and 1 mmol/l Ca2 to deter-mine Rmax or 1 mmol/l EGTA and no Ca

2 to determine Rmin.

Possible artifacts were excluded by measurement of autofluo-rescence of the different substances without fura 2.

Determination of cytosolic pH. Intracellular pH of singlecells was determined by using the pH-sensitive dye 2,7-bis(2-carboxyethyl)-5(6)-carboxy fluorescein (2 mol/l; Mo-lecular Probes) as described previously (12) and with thesetup described in Determination of cytosolic Ca2. In brief,cells were each incubated with MEM containing 2 mol/l2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethylester for 5 min. Then, the coverslips were rinsed four timeswith superfusion solution to remove the dye-containing me-dium. The coverslips were transferred to the stage of aninverted Axiovert 100 TV microscope (Zeiss) and allowed to

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equilibrate for 15 min. The excitation light source was a100-W mercury lamp. The excitation wavelengths were 450nm/490 nm. The emitted light was filtered through a band-pass filter (515565 nm). The data-acquisition rate was onefluorescence intensity ratio every 2 s. After background sub-traction, fluorescence intensity ratios were calculated. pHcalibration was performed after each experiment by the ni-gericin (Sigma, St. Louis, MO) technique (39, 49), by using atleast three calibration solutions in the range from pH 6.8 to7.8. The calibration solutions contained 115 mmol/l KCl and30 mmol/l NaCl.

IP3 formation. IP3 formation was determined by anionexchange columns, as described previously (32). In brief, cellsseeded in six-well plates were preincubated during 24 h inmedia containing 0.5 Ci/ml [3H]inositol. Before experimen-tation, radioactive medium is aspirated, and the cells arewashed with 3 2 ml HEPES-Ringer and then incubated for30 min in 2 ml HEPES-Ringer containing 15 mM LiCl (pH7.4). This medium was replaced with 1 ml HEPES-Ring-er15 mM LiCl, and after 10 min of incubation at 37C, 1-mlaliquots of control Ringer15 mM LiCl with the desiredagonists were added. After 15 min of incubation, cells werelysed with 1 ml of 4 mM EDTA/1% SDS (90C), and lysateswere applied to ion exchange columns prepared as follows.

Dowex-AG 1X-8 (0.5 g, formate form) was laid into 5-mlpipette tips with cotton wool on the bottom. Applied samplesare washed with 2-ml aliquots of H2O and 5 mM disodiumtetraborate/60 mM sodium formate, and then inositol4-monophosphate (IP1), inositol 1,4-bisphosphate (IP2), andIP3 were eluted by subsequent addition of 2 ml of 0.1 Mformic acid/0.2 M ammonium formate (IP1), 0.1 M formicacid/0.4 M ammonium formate (IP2), and 0.1 M formic acid/1.0 M ammonium formate (IP3). Eluted IP1IP3 were col-lected into scintillation vials, mixed with 10 ml of scintilla-tion cocktail, and counted.

Arachidonic acid release. Arachidonic acid release wasperformed as described elsewhere (4; 24). In brief, cells werelabeled for 24 h with 0.5 Ci of [3H]arachidonic acid (ARC,St. Louis, MO). Subsequently, the media were removed, and

an aliquot was counted to determine the extent of incorpora-tion (8590% of arachidonic acid added to the media). Thecells were rinsed three times with ice-cold control Ringersolution (see Materials) and incubated for 30 min with con-trol Ringer solution at 37C (equilibration period). Finally,the experimental Ringer solutions were added, and aliquotswere taken after 5 and 15 min to determine the amount ofradioactivity released. At the end of the experiment, the cellswere lysed with 1 mol/l NaOH. The release of radioactivityinto the Ringer solution was determined as the percentage oftotal incorporated radioactivity. To verify the contribution ofphospholipase A2 (PLA2), we tested the inhibitory action ofarachidonyl trifluoromethyl ketone (38).

Determination of cell number. Cell number was deter-mined by using a Z2 series Coulter Counter. Cells were

seeded on plastic dishes, grown to subconfluence ( 40,000cells/cm2), and made quiescent before the experiments by24-h serum removal.

Materials. Unless otherwise stated, all materials werefrom Sigma (Munich, Germany). Ethylisopropyl amiloridewas kindly provided by Dr. H. J. Lang from Aventis (Frank-furt, Germany). Control Ringer solution was composed of (inmmol/l) 130.0 NaCl, 5.4 KCl, 1.0 CaCl2, 1.0 MgCl2, 1.0NaH2PO4, 10 HEPES, and 5 glucose (pH 7.4 at 37C), plusthe respective vehicles (ethanol or DMSO 1).

Statistics. Values are means SE. Significance of differ-ence was tested by paired or unpaired Students t-test or

ANOVA, as applicable. Differences were considered signifi-

cant if P 0.05. Cells from at least two different passageswere used for each experimental series; n represents thenumber of cells or tissue culture dish investigated. For pHdeterminations, at least five coverslips were investigated foreach experimental condition.

RESULTS

ERK1/2 phosphorylation. As shown in Fig. 1, A and

C, 10 nmol/l aldosterone and 10 g/l EGF stimulateERK1/2 phosphorylation in MDCK-C11 cells, as de-scribed by us previously for aldosterone (11). The timeof exposure was 10 min, because in a previous study itwas shown that there was no difference between 5- or10-min exposure (11). Control solutions always con-tained the appropriate amount of vehicle (ethanol orDMSO 1:1,000). To determine whether the side ofaldosterone application, apical or basolateral, might beimportant, we compared the effect of aldosterone onfilter-grown cells. As shown in Fig. 1A, there was onlya slight difference for aldosterone but a marked differ-ence for EGF. At present, we do not know whetherthese data mean that aldosterone acts within the cellsor just rapidly crosses the monolayer. The effects ofaldosterone and EGF in cells grown to 80% conflu-ence on solid supports were compared with the effectsin cells grown on permeable supports. We did notobserve significant differences. Therefore, most of theexperiments were performed with cells cultivated onsolid supports, and the data were pooled with thosefrom cells grown on permeable supports. The reason forthe effectiveness of EGF in cells grown to 80% con-fluence on solid supports is most probably the lack of acomplete apical-to-basolateral differentiation, as ob-served previously (11).

We tested whether inhibition of the EGF-R-kinase

by compound 56 [c56; 100 nmol/l 4-(3-bromoanilino)-6,7-dimethoxyquinazoline] (3) or tyrphostin AG1478(100 nmol/l) reduced the aldosterone- and EGF-in-duced phosphorylation of ERK1/2. As shown in Fig. 1,B and C, this was indeed the case, indicating that bothsubstances use the EGF-R for signaling. Furthermore,the data in Fig. 1, B and C, show that there is anautocrine activation loop of the EGF-R signaling cas-cade in MDCK-C11 cells, as already shown, becauseEGF-R-kinase inhibition was effective in controls (36).Thus the cells are already in a certain state of preac-tivation; therefore, the observed effects are smallercompared with cells that are completely silent. Bycontrast to aldosterone, hydrocortisone (10 nmol/l) did

not affect ERK1/2-phosphorylation (Fig. 1D). Thesedata again show that the vehicle (ethanol) in which thesteroids were dissolved is not responsible for the ob-served effects.

Cytosolic Ca2. Mitogenic factors, such as EGF, areknown to affect cytosolic Ca2 homeostasis because ofan increased Ca2 entry across the plasma membraneor because of the release of Ca2 from intracellularstores (25, 40). Under control conditions, [Ca2]i was91 20 nmol/l (n 200). Figure 2 shows that 10 nmol/laldosterone or 10 g/l EGF induced a small but com-parable increase in [Ca2]i, corresponding to 100

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nmol/l. These aldosterone-induced changes in [Ca2]i([Ca2]i) are in agreement with previously reportedchanges for MDCK-C11 or skin cells (12, 21); however,they are smaller compared with changes observed, forexample, in M-1 cortical collecting duct cells (20). Al-

though the changes in MDCK-C11 cells are small, ithas been shown that they contribute to Na/H-ex-change activation, for example (12). Similar to theeffects on ERK1/2 phosphorylation, inhibition ofEGF-R kinase with compound 56 (100 nmol/l) signifi-

Fig. 1. Aldosterone- and epidermal growth factor (EGF)-induced extracellular signal-regulated kinase (ERK)1/2phosphorylation. A: aldosterone (10 nmol/l) or EGF (10 g/l) induce phosphorylation of ERK1/2. Apical vs.basolateral comparison of the effect of the addition of 10 nmol/l aldosterone on ERK1/2 phosphorylation. Exposuretime was 10 min. Previous experiments showed that the effect during 5- or 10-min exposure was identical (11). B:inhibition of the EGF-receptor (EGF-R) kinase with compound 56 (c56; 100 nmol/l) prevents ERK1/2 phosphory-lation. Representative blot of 2 experiments is shown. Exposure time was 10 min. aldo, Aldosterone. C: summaryof aldosterone- (10 nmol/l) and EGF-induced (10 g/l) ERK1/2 phophorylation. Data from Western blots andphosphorylated ERK (pERK)1/2-ELISA were pooled, because they were not different. Furthermore, cells grown in96-well plates behaved identically to cells grown on permeable supports. The data show that besides c56 another

inhibitor of the EGF-R kinase, tyrphostin AG-1478 (100 nmol/l), also abolishes the effects of aldosterone and EGF;n 6 for each bar. Data indicate that there is an autocrine activation loop of EGF-R, as already described (36),leading to certain background phosphorylation of ERK1/2. *P 0.05 vs. control. D: hydrocortisone (hydro; 10nmol/l) did not affect ERK1/2 phosphorylation. Representative blot from 2 individual experiments is shown.Exposure time 10 min. con, Control.

Fig. 2. Aldosterone- and EGF-inducedCa2 signaling. A: EGF (10 g/l) oraldosterone alone (10 nmol/l) exerts aslight increase in baseline cytosolicCa2 concentration [Ca2]i. B: summaryof the Ca2 changes elicited by aldoste-rone or EGF. The inhibitor of EGF-Rkinase, c56 (100 nmol/l), reduces theCa2 changes significantly; n 60120 for each bar. *P 0.05 vs. aldoste-rone or EGF, respectively.



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cantly reduced the [Ca2]i induced by aldosterone orEGF. Thus aldosterone and EGF increase Ca2 bymeans of EGF-R signaling. To rule out the possibilitythat the EGF-R inhibitors, although used at nanomolarconcentrations, reduced signaling through toxic effects,we determined whether these substances affected thebradykinin-induced signal. However, both substancesdid not affect the Ca2 rise induced by 100 nmol/l

bradykinin, which induced a Ca2 spike of 1,100nmol/l. These data indicate that the EGF-R inhibitorsdid not act by means of toxic impairment of signaling.

Aldosterone action depends on EGF-R-kinase activ-ity. To determine whether Tyr phosphorylation of theEGF-R is affected by aldosterone and EGF, we per-formed EGF-R immunoprecipitation (stimulationtime 10 min for all experiments). As shown in Fig. 3,A andB, aldosterone and EGF enhanced Tyr phosphor-ylation of the EGF-R. Figure 3A again shows that thereis a certain degree of autocrine activation of the EGF-Rin MDCK cells, most probably involving transforminggrowth factor (TGF)- (36). When EGF and aldosterone

were added together, the extent of EGF-R-phosphory-lation was enhanced. To determine whether this en-hancement also affected downstream signaling, we de-termined the effects of aldosteroneEGF on ERK1/2phosphorylation and Ca2.

Interaction of aldosterone and EGF. In addition to itsown effect on ERK1/2 phosphorylation, aldosteroneincreased the effect of 10 g/l EGF on ERK1/2 phos-

phorylation (stimulation time 10 min for all experi-ments). Because ERK1/2 phosphorylation seems to bethe signal upstream of all other events investigated inthis study, except EGF-R phosphorylation, and can bequantitated reliably by ELISA (see METHODS), we deter-mined the ERK1/2 phosphorylation dose-responsecurve for EGF and EGFaldosterone. As shown in Fig.4A, there was a shift to the left of the dose-responsecurve (factor 510) when aldosterone and EGF wereadded simultaneously, compared with EGF alone.ERK1/2 phosphorylation under these conditions wasagain prevented by EGF-R kinase blockade, as shownin Fig. 4B.

When EGF (10 g/l) was added in the presence ofaldosterone, [Ca2]i (400500 nmol/l) values weresignificantly greater when compared with the sum ofthe individual effects (Fig. 4, C and D). Furthermore,we observed a Ca2 peak in the majority of the cellsbefore a new plateau was reached. The order of appli-cation (i.e., aldosterone then EGF or EGF then aldo-sterone) had no significant effect on the observed ef-fects. To determine whether the appearance of theCa2 peak was the result of enhanced IP3 formation,we determined the effect of aldosterone and EGF onIP3 formation. As shown in Fig. 5A, aldosterone or EGFdid not induce a substantial increase in IP3 formation. ATP (100 mol/l) was used as a positive control. An

enhancement of the Ca2 changes was also observedwith 1 nmol/l aldosterone (Ca2 for 1 nmol/l aldoste-rone10 g/l EGF 350 56 nmol/l; n 75). When100 g/l EGF was used, the enhancement of the Ca2

response by aldosterone was substantially smallercompared with the response when using 10 g/l EGF(Fig. 4D), indicating that aldosterone did not induce achange in the maximum Ca2 response, which is inagreement with the effects observed for ERK1/2 phos-phorylation. In the presence of 10 mol/l La3 or whenextracellular [Ca2] was lowered to 5 mol/l, thechanges in [Ca2]i were almost completely abrogated(Fig. 4D), indicating the dependence on extracellular

Ca2

. As already mentioned, all control solutions con-tained the respective vehicles (either ethanol orDMSO 1). Thus the effects observed cannot beexplained by the vehicles. Inhibition of ERK1/2 phos-phorylation with PD-98059 (25 mol/l, a concentrationthat prevented ERK1/2 phosphorylation; data notshown) reduced the Ca2 signal significantly (Fig. 5B).These data indicate that ERK1/2 phosphorylation isupstream of the Ca2 influx. By contrast, loweringextracellular Ca2 to prevent aldosterone- and EGF-induced Ca2 signaling did not affect ERK1/2 phos-phorylation (Fig. 5C).

Fig. 3. Aldosterone and EGF-induced EGF-R phosphorylation. A:aldosterone (10 nmol/l) and EGF (10 g/l) enhance EGF-R phosphor-ylation (10-min exposure). Moreover, aldosterone enhances the EGF-induced Tyr phosphorylation. Blot also shows that there is a certaindegree of autocrine phosphorylation of the EGF-R, as already de-scribed for Madin-Darby canine kidney cells (36) and is representa-tive of 4 experiments. Exposure time 10 min. B: summary ofEGF-R phosphorylation from 4 individual experiments. pEGFR,phosphorylated EGF-R. *P 0.05 vs. control.



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Effects of aldosterone and EGF on arachidonic acidrelease and cytosolic pH. To evaluate the potentialaffect of the Ca2 and ERK1/2 signals on cell functions,we investigated arachidonic acid release and cytosolicpH. When EGF (10 g/l) or aldosterone (10 nmol/l)were each added to the cells, there was only a verysmall increase of arachidonic acid-derived radioactiv-ity release observable (Fig. 6A). By contrast, bradyki-

nin (100 nmol/l) induced a threefold increase in arachi-donic acid-derived radioactivity release, indicatingthat the experimental setup worked. Simultaneous ad-dition of EGF and aldosterone resulted in a signifi-cantly increased release, indicating a synergistic actionof the two. Inhibition of PLA2 with 25 mol/l arachido-nyl trifluoromethyl ketone prevented the stimulationof radioactivity release (Fig. 6A). Thus the observed

Fig. 4. Interaction of EGF and aldosterone of ERK1/2 phosphorylation and Ca2 signaling. A: dose-response curveof ERK1/2 phosphorylation. Addition of EGF in the presence of 10 nmol/l aldosterone led to a shift to the left of theEGF dose-response curve. Exposure time 10 min; n 510. B: ERK1/2 phosphorylation induced byaldosteroneEGF is prevented by inhibitors of EGF-R kinase, tyrphostin AG-1478 (tyropho; 100 nmol/l), or c56(100 nmol/l). C: addition of EGF (10 g/l) in the presence of aldosterone (10 nM) led to an enhanced change incytosolic Ca2 concentration ([Ca2]i compared with the addition of EGF alone (compare with Fig. 2). Furthermore,we observed a Ca2 peak in the majority of the cells before the plateau was reached. Order of application (i.e.,aldosterone then EGF or EGF then aldosterone) had no significant effect on the observed effects. D: summary of

the maximum changes in cytosolic Ca2

. Bars, means of 55120 cells from at least 4 coverslips from at least 2different passages. c56 100 nmol/l; La3 10 mol/l LaCl3; low Ca2 Ca2 5 mol/l. *P 0.05 vs. respectivecontrol.



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release of radioactivity can be attributed to PLA2 ac-tivity. Inhibition of ERK1/2 phosphorylation with 25mol/l PD-98059 also prevented stimulation of arachi-donic acid release (110 8% of control, n 4), as wasthe case when extracellular Ca2 was lowered (102 10% of control, n 4).

Fig. 6. Arachidonic acid (AA) release and cytosolic pH. A: arachi-donic acid release is significantly greater when aldosterone (10nmol/l) and EGF (10 g/l) are simultaneously present compared withthe situation when each substance is added alone. The release wasabolished by the inhibitor of phospholipase A2, arachidonyl triflu-oromethyl ketone (AACOCF; 25 mol/l). Bradykinin (BK; 100 nmol/l)served as a positive control; n 6 for each bar. *P 0.05 vs. control.B: aldosterone (10 nmol/l) or EGF (10 g/l) induced a comparablecytosolic alkalinization (pH). In the presence of aldosteroneEGF,alkalinization was significantly enhanced. Order of application (i.e.,aldosterone then EGF or EGF then aldosterone) had no significanteffect on the observed effects. Because the alkalinization was almostcompletely prevented by 10 mol/l ethylisopropyl amiloride, it re-sulted from Na/H-exchange activation; n 60 for each bar.

Fig. 5. Mechanism of Ca2 increase. A: aldosterone (10 nmol/l) orEGF (10 g/l) do not stimulate inositol-1,4,5-trisphosphate (IP3)formation. ATP (100 mol/l) was used as a positive control; n 6 foreach bar. B: inhibition of ERK1/2 phosphorylation by 25 mol/lPD-98059 (PD) reduced the Ca2 signal (Ca2) elicited by aldoste-

rone (10 nmol/l) or aldosteroneEGF significantly; n 3550. C:prevention of changes in cytosolic Ca2 by incubation in low-Ca2

solution (5 mol/l Ca2) did not reduce ERK1/2 phosphorylation.Representative blot from 2 individual experiments. Exposure time 10 min.



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Previously, we have shown that aldosterone stimu-lates Na/H exchange in MDCK-C11 cells by meansof ERK1/2 and leads to cytosolic alkalinization (11). Asshown in Fig. 6B, 10 g/l EGF led to an alkalinizationsimilar to that when aldosterone (10 nmol/l) was used(pH under control conditions was 7.22 0.06, n 75). Again, aldosterone enhanced the effect of EGF. TheNa/H-exchange inhibitor ethylisopropyl amiloride

(10 mol/l) prevented alkalinization almost completely(Fig. 6B). Moreover, no significant alkalinization couldbe observed in the presence of 25 mol/l PD-98059(pH 0.021 0.013, n 30) or when Ca2 influx wasprevented (pH0.040 0.011, n 30). These dataindicate that aldosterone and EGF act on Na/H

exchange by means of ERK1/2 and Ca2.Effects of aldosterone and EGF on cell proliferation.

EGF acts as mitogen in many different cell types. Thuswe investigated whether aldosterone modulates theproliferative action of EGF. Cells were made quiescentat 70% confluence by 24-h serum removal and wereincubated for another 48 h with EGF and/or aldoste-rone in serum-free media. As shown in Fig. 7A, thenumber of cells remained virtually constant under con-trol conditions during the 48-h incubation period. Thusany increase in the number of cells under experimentalconditions must reflect cell proliferation. Figure 7Ashows the effect of EGF on the number of cells. At 10g/l, EGF exerted a slight proliferative action inMDCK-C11 cells. When added alone at 10 nmol/l, al-dosterone had no significant effect. However, whenaldosterone (10 nmol/l) was added together with EGF(10 g/l), there was a clear potentiation of the prolifer-ative effect. When saturating concentrations of EGFwere used (100 g/l), aldosterone had no further effect.Thus these data nicely mirror the responses of ERK1/2

phosphorylation and arachidonic acid release and showthat aldosterone modulates the proliferative actionof EGF.

DISCUSSION

During the last several years, several reports showedthat steroid hormones, such as aldosterone, can elicit

rapid (10 min), potentially nongenomic, cellular re-sponses. The underlying mechanism(s) for the rapidactions of aldosterone are still unknown. One hypoth-esis is based on the interaction of steroid hormoneswith peptide hormone signaling. For example, the in-teraction of progesterone with oxytocin signaling, aswell as the interaction of estradiol or glucocorticoidswith growth factor and angiotensin II signaling, has

been described (17, 28). In the case of aldosterone, aninteraction with angiotensin II and vasopressin hasbeen suggested (35, 48). Previously, we have shownthat aldosterone increases H affinity of Na/H ex-change in MDCK-C11 cells by means of ERK1/2, abehavior similar to the action of growth factors such asEGF (11, 44). Transactivation of EGF-R is involved inthe transmission of signals triggered by other media-tors, for example, hormones acting by means of hetero-trimeric G proteins (19). The EGF-R is therefore con-sidered a transducer of heterologous signaling. Thus itis conceivable that EGF-R also plays a role in theintegration of rapid steroid signaling.

The results presented here show that aldosteroneacts on ERK1/2 phosphorylation, cytosolic Ca2, andpH homeostasis in a manner very similar to the effectsobserved during EGF exposure. These data allow thehypothesis that aldosterone also uses the EGF-R as atransducer of heterologous signaling, as already shownfor other hormones (19). If this hypothesis is true, thenwe should expect that the effects of aldosterone arereduced when the EGF-R kinase is inhibited. As shownfor ERK1/2 phosphorylation and cytosolic Ca2, thiswas indeed the case. When EGF-R kinase was inhib-ited, the effects of aldosterone were significantlysmaller (cytosolic Ca2) or even completely abolished(ERK1/2 phosphorylation). The reason for the small

remaining effect on cytosolic Ca2

can be explained bythe higher sensitivity of Ca2measurements comparedwith ERK1/2 phosphorylation. If aldosterone acts oncytosolic Ca2 and ERK1/2 phosphorylation by meansof the EGF-R kinase, we would also expect that aldo-sterone leads to enhanced EGF-R phosphorylation. Asshown here, EGF-R phosphorylation was enhanced in

Fig. 7. Cell proliferation. Cells were made

quiescent by 24-h incubation in serum-freemedia. A: subsequent 48-h incubation in se-rum-free media did not significantly alter thenumber of cells. B: 48-h incubation with 10g/l EGF induced a slight increase in thenumber that was potentiated significantly by10 nmol/l aldosterone; n 9 for each bar.(10) 10 g/l; (100) 100 g/l. *P 0.05 vs.control.



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the presence of aldosterone. Thus the two lines ofevidence presented here support the hypothesis thatrapid effects of aldosterone involve the EGF-R path-way, at least in the cell system investigated here. Thisis the first report indicating an interaction of aldoste-rone with Tyr kinase receptor signaling. The possiblephysiological and/or pathophysiological significance ofthis interaction is supported by the fact that aldoste-

rone was effective at nanomolar concentrations, al-though more detailed investigations have to be per-formed in future studies. It is conceivable that elevatedcirculating aldosterone concentrations (for example, inpatients with liver cirrhosis) may elicit part of itseffects by means of an interaction with EGF-R.

The data presented here also indicate that there is acertain degree of autocrine stimulation of the EGF-R inMDCK cells (see Fig. 1), as already described (36). Thisautocrine activation loop most probably results fromthe simultaneous expression of EGF-R and transform-ing growth factor (TGF)-. Thus, even though the cellswere made quiescent by serum removal, there is al-ways a certain preactivation under control conditions.The autocrine preactivation is also responsible for thefact that the observed effects of EGF are smaller com-pared with other cell systems such as vascular smoothmuscle cells, for example (2).

Our data show that aldosterone enhances EGF-Rphosphorylation followed by phosphorylation of ERK1/2.Subsequently, Ca2 entry across the plasma mem-brane is increased (Fig. 8). This conclusion is based onthe observations that prevention of Ca2 entry did notabrogate ERK1/2 phosphorylation, whereas inhibitionof ERK1/2 phosphorylation significantly reduced theCa2 signal. Moreover, inhibition of EGF-R Tyr kinasesignificantly reduced ERK1/2 phosphorylation and the

Ca2

signal. Furthermore, we did not observe a sub-stantial increase in IP3 formation, which arguesagainst the involvement of phospholipase C and sub-

sequent Ca2 release from IP3-sensitive stores. Fi-nally, ERK1/2 and Ca2 do then transmit the aldoste-rone signal to further downstream events, as in Na/H-exchange activation (11, 15).

How does the interaction of aldosterone with theEGF-R affect EGF signaling in MDCK-C11 cells? Wetried to answer this question pharmacodynamically byusing ERK1/2 phosphorylation as a parameter. Aldo-

sterone and EGF could interact in a simple additivemanner, leading to a shift to the left of the dose-response curve, with no change in the maximum effectwhen both are added simultaneously. Alternatively,aldosterone could enhance the maximum effect, eitheradditively or in terms of a potentiation of EGF. Finally,aldosterone could shift the dose-response curve of EGFto lower concentrations, thereby sensitizing the cellsfor EGF. As shown in Fig. 4A, aldosterone and EGFseem not to act additively in a simple manner or withrespect to maximum ERK1/2 phosphorylation. At sub-maximum EGF concentrations, aldosterone and EGFseem to induce an overadditive ERK1/2 phosphoryla-tion, leading to a shift to the left of the EGF dose-response curve. Thus at submaximum concentrationsof EGF, the cells seem to respond more sensitively toEGF when aldosterone is present compared with thesituation where aldosterone is absent. This interpreta-tion would also explain the enhanced EGF-inducedresponse with respect to the Ca2 signal, which isdownstream of ERK1/2 phosphorylation. This, ofcourse, is a pharmacodynamic description of the mod-ulation of EGF effects, and future studies will have tofocus on the underlying mechanisms. Previously, wehave shown that rapid effects of aldosterone in MDCK-C11 cells are not prevented by the mineralocorticoidreceptor antagonist spironolactone (13), which is simi-

lar to results obtained in other cell types (46). In somepreliminary experiments, we tested the effect of spi-ronolactone (1 mol/l) on aldosterone-induced ERK1/2phosphorylation and could not detect any inhibitoryaction of spironolactone (data not shown). Thus it isunlikely that the interaction of aldosterone withEGF-R depends on the mineralocorticoid receptor.

What is the nature of the interaction of aldosteronewith EGF-R signaling? One possibility is a direct in-teraction on the level of the EGF-R. A similar mecha-nism for steroid-hormone and peptide-hormone crosstalk has been proposed for progesterone and oxytocin(17). Another possible mechanism is the involvement ofadditional factors, for example, Src kinase, which could

link the action of aldosterone to EGF-R phosphoryla-tion. In this case, aldosterone would have to activatethe additional factor directly or by means of an aldo-sterone receptor, as proposed by Wehling et al. (47).Several years ago, it was shown that aldosterone canchange the metabolism of membrane phospholipids, forexample, leading to an increase in the diglyceride frac-tion (16). Changes in the lipid environment could alsoaffect the activation of a membrane protein such asEGF-R (1). Finally, there exists the possibility that theinteraction of aldosterone with EGF-R depends on theendogenous EGF-R ligand TGF-, known to be ex-

Fig. 8. Present hypothesis for the interaction of aldosterone andEGF. Aldosterone activates EGF-R activation by an unknown mech-anism. Subsequently, ERK1/2 phosphorylation increases and stim-ulates Ca2 entry. These two signals lead to, e.g., Na/H-exchangeactivation and arachidonic acid release. PLA2, phospholipase A2;TRK, tyrosine kinase.



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pressed in MDCK cells (36). Although the time courseof the aldosterone action is not in favor of a mechanismrequiring the cleavage of an endogenous EGF-R ligand,it cannot be ruled out completely. Future studies willfocus on the mechanism underlying the interaction ofaldosterone with ERG-R signaling in more detail.

What is the cellular significance of aldosterone inter-action with EGF-R signaling? To gain more informa-

tion regarding the possible significance of rapid steroideffects, it is important to determine changes in cellfunction. Our data show that aldosterone-inducedmodulation of EGF-R signaling indeed affects certaincell functions. Of course there are many more aspectsof cell function that could be affected and have not beendetermined here (for example, transepithelial iontransport) but will be subject to investigation in futurestudies. Aldosterone used the EGF-R signaling cascadeto modulate the release of arachidonic acid and theactivation of Na/H exchange. Stimulation of arachi-donic acid release and Na/H exchange will also af-fect cell function and/or signaling, thereby creating a

complex network.With respect to a differentiation of the directgenomic (by means of the mineralocorticoid receptor)and primary nongenomic effects, the data reportedhere offer a new perspective. The interaction of aldo-sterone with EGF-R signaling is a primary nongenomicevent that may have a secondary genomic impact, forexample, by means of the nuclear factor of activatedtranscription and serum response element. Therefore,the primarily nongenomic responses may finally resultin an alternative genomic response via pathways thatdo not rely on the mineralocorticoid receptor.

What is the role of interaction with EGF-R signalingin the physiological response to aldosterone? Presently,

the answer to this question is not known. However, it isknown that EGF-R signaling modulates transepithe-lial ion transport and stimulates salt reabsorption incertain cell types (8, 22). Furthermore, it is known thatEGF-R signaling may exert profibrotic actions (10).Thus there are certain similarities with respect to thephysiological (salt reabsorption) and the pathophysio-logical (fibrosis) action of aldosterone and EGF. There-fore it is conceivable that the interaction of aldosteronewith EGF-R signaling may support physiological andpathophysiological responses to aldosterone. However,with respect to sodium handling in the distal nephron,the actions of EGF and aldosterone are opposed, be-

cause EGF has been reported to inhibit the epithelialsodium channel (31, 45). Thus aldosterone could limitits own stimulatory action on sodium reabsorption bymeans of the EGF-R signaling cascade, representing anegative feedback in this case. By contrast, with re-spect to intestinal sodium reabsorption, EGF and aldo-sterone seem to act in the same direction, i.e., stimu-lation of sodium absorption (23). Therefore, EGF-Rsignaling could support the stimulatory effect of aldo-sterone on intestinal sodium absorption. Of course,these hypotheses have to be verified in future studiesby using EGF-R kinase inhibitors to test the impor-

tance of EGF-R signaling for aldosterone effects, forexample.

In conclusion, our data show that aldosterone usesthe EGF-R-ERK1/2 signaling cascade to elicit rapideffects in MDCK cells. In addition, aldosterone seemsto modulate the action of EGF. Possibly, the EGF-Rrepresents a membrane target for rapid effects of aldo-sterone. Of course, the data presented here do not

affect the importance of genomic actions of aldosteronebut do add an additional, primarily nongenomic, path-way.

This study was supported by Deutsche ForschungsgemeinschaftGrants Ge 905/41 and SFB487/A6.

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