Physiologisches Institut, University of Würzburg, 97070 Würzburg, Germany
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ABSTRACT |
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Epidermal growth factor (EGF) regulates cell proliferation, differentiation, and ion transport by using extracellular signal-regulated kinase (ERK)1/2 as a downstream signal. 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 suggested that steroids interact with peptide hormones, in part, by rapid, potentially nongenomic, mechanisms. Previously, we have shown that aldosterone modulates Na+/H+ exchange in Madin-Darby canine kidney (MDCK) cells by means of ERK1/2 in a way similar to growth factors. Here, we tested the hypothesis that aldosterone uses the EGF-R as a heterologous signal transducer in MDCK cells. Nanomolar concentrations of aldosterone induce a rapid increase in ERK1/2 phosphorylation, cellular Ca2+ concentration, and Na+/H+ exchange activity similar to increases induced by EGF. Furthermore, aldosterone induced a rapid increase in EGF-R-Tyr phosphorylation, and inhibition of EGF-R kinase abolished aldosterone-induced signaling. Inhibition of ERK1/2 phosphorylation reduced the Ca2+ response, whereas prevention of Ca2+ influx did not abolish ERK1/2 phosphorylation. Our data show that aldosterone uses the EGF-R-ERK1/2 signaling cascade to elicit its rapid effects in MDCK cells.
epidermal growth factor; aldosterone; extracellular signal-regulated kinase 1/2; calcium; Madin-Darby canine kidney cells
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INTRODUCTION |
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THE CLASSIC GENOMIC MECHANISM of steroid hormone action involves binding to intracellular receptors, transcription, and protein synthesis. However, aldosterone can also induce rapid responses by interfering with mechanisms of regulation of intracellular pH or Ca2+ (6, 9, 12, 30, 43, 50), intracellular generation of inositol 1,4,5-trisphosphate (IP3) (5), protein kinase C, and extracellular signal-regulated kinase (ERK)1/2 phosphorylation (9, 11, 26, 37). Former studies also revealed that aldosterone acts within several minutes on plasma membrane K+ conductance of different cells (29, 30, 41). These rapid actions of aldosterone are thought to be mediated by a plasma membrane receptor (47), although this putative receptor has not been identified. Recently, it has been shown that steroid hormones are able to interact with peptide hormone signaling (35, 46). For example, the interaction of progesterone with oxytocin signaling (17), as well as the interaction of estradiol with growth factor and angiotensin II signaling (28), has been described. In the case of aldosterone, an interaction with angiotensin II and vasopressin has been suggested (35, 48). Some of these effects have been shown to be incompatible with the classic genomic pathway. Rapid, potentially nongenomic, actions of steroids have recently been investigated more extensively, and there are several reports supporting the existence of such actions. The precise underlying mechanism and the physiological or pathophysiological significance are not yet understood. The interaction of steroids with peptide hormone signaling represents one possible mechanism for rapid steroid action while offering an explanation for the significance of these effects, i.e., modulation of peptide hormone signaling.
In the present study, we investigated the possible interaction of aldosterone with peptide hormone signaling in a cell line that has previously been shown to respond in a rapid, nongenomic way to aldosterone, Madin-Darby canine kidney (MDCK)-C11 cells (12). We determined the possible interaction with epidermal growth factor (EGF) receptor (EGF-R). The EGF-R has been shown to be involved in signaling events elicited by, for example, G protein-coupled receptors, growth hormone, and cytokines by means of a mechanism called transactivation (19, 27). Therefore, the EGF-R can serve as a central transducer of heterologous signaling systems. Moreover, a transcription-independent interaction of glucocorticoids with EGF-R has been reported (7). Furthermore, we have shown that aldosterone increases H+ affinity of Na+/H+ exchange in MDCK-C11 cells by means of ERK1/2, which is a behavior similar to that of growth factors (11, 44). Thus it is conceivable that the EGF-R represents a pathway involved in rapid aldosterone signaling similar to, for example, G protein-coupled receptors.
EGF regulates cell proliferation, differentiation, and tissue repair and, at least in part, uses mitogen-activated protein kinases as downstream signals. In addition, enhanced EGF signaling has been observed in several tumor cells (19, 27). Furthermore, it has been shown that EGF does affect epithelial salt transport in a cell-specific manner, leading to either enhanced or reduced salt reabsorption (8, 22, 23, 31, 45). Our results show that aldosterone uses the EGF-R-ERK1/2 signaling pathway to elicit its rapid effects on ERK1/2 phosphorylation, Ca2+ homeostasis, and pH homeostasis in MDCK cells. Possibly, the EGF-R represents a membrane target for rapid effects of aldosterone.
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METHODS |
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Cell culture. We used a subtype of MDCK cells, denominated C11 (MDCK-C11), which has been cloned recently in our laboratory (14). Cells were cultivated, as described previously (12, 14, 33), in MEM supplemented with 10% fetal calf serum at 37°C and 5% CO2. Serum was removed from the media 24 h before the experiment. For the experiments presented, the cells were cultivated either on permeable supports (Becton Dickinson, Heidelberg, Germany) in 96-well plates [for phosphorylated ERK (pERK)1/2-ELISA] or on poly-L-lysine-coated glass coverslips (for Ca2+ and pH measurements). Because the effects were not statistically different for the three conditions, the data could be compared directly and were pooled.
Western blot analysis. Cells were washed three times with ice-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, 200 µM sodium-orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µM pepstatin A, 40 mg/l bestatin, 2 mg/l aprotinin, and 1% Triton X-100) for 25 min at 4°C. Insoluble material was removed by centrifugation at 12,000 g for 15 min at 4°C. Cell lysates were matched for protein, separated on SDS-PAGE, and transferred to a polyvinylidene difluoride microporous membrane. Membranes were subsequently 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 and ERK2 when catalytically activated by phosphorylation at Thr202 and Tyr204. The primary antibody was detected by using horseradish peroxidase-conjugated secondary IgG visualized by the Amersham enhanced chemiluminescence system. Linearity of the signal has been verified by serial dilution, as recommended by the manufacturer. Densitometric analysis was performed by using SigmaGel 1.05 (Jandel, Corte Madera, CA).
Quantification of ERK1/2 phosphorylation by ELISA. For the quantification of ERK1/2 phosphorylation, we performed pERK1/2-ELISA according to Versteeg et al. (42). In control experiments, we compared the effect of EGF determined by Western blot and pERK1/2-ELISA and found no significant difference. Thus the results obtained by Western blot and pERK1/2-ELISA were pooled. Cells were seeded in 96-well plates (Maxisorp; Nunc) and serum starved for 24 h before the experiment. After stimulation, the cells were fixed with 4% formaldehyde in PBS for 20 min at room temperature and washed three times with PBS containing 0.1% Triton X-100. Endogenous peroxidase was quenched with 0.6% H2O2 in PBS/Triton X-100 for 20 min. Cells were washed three times in PBS/Triton X-100, blocked with 10% fetal calf serum in PBS/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 4°C. The next 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, dilution 1:1,000) in PBS/Triton X-100 with 5% BSA for 1 h at room temperature and were washed three times with PBS/Triton X-100 for 5 min and twice with PBS. Subsequently, the cells were incubated with 50 µl of a solution containing 0.4 mg/ml O-phenylenediamine, 11.8 mg/ml Na2HPO4, 7.3 mg/ml citric acid, and 0.015% H2O2 for 15 min at room temperature in the dark. The resulting signal was detected at 490 nm with a multiwell, multilabel counter (Victor2; Wallac, Turku, Finland). After the peroxidase reaction, the cells were washed twice with PBS/Triton X-100 and twice with demineralized water. After the wells were dried for 5 min, 100 µl of trypan blue solution (0.2% in PBS) were added for 5 min at room temperature. Subsequently, the cells were washed four times with demineralized water, and 100 µl of 1% SDS solution were added and incubated on a shaker for 1 h at room temperature. Finally, the absorbance was measured at 595 nm with the ELISA reader.
Immunoprecipitation. Immunoprecipitation was performed as described recently (34). Briefly, cell lysates were precleared with protein A/G-Sepharose for 20 min at 4°C. To precipitate the EGF-R, anti-EGF-R antibody [clone Ab-5 (Calbiochem-Novabiochem); Ref. 36; 10 µg/mg protein] was added for 2 h, followed by overnight incubation with protein A/G-Sepharose. Immune complexes were collected by centrifugation, washed three times with lysis buffer, and subjected to SDS/8%-PAGE, and phosphorylated EGF-R was detected by using anti-phospho-Tyr antibody (PY99). Densitometric analysis was performed by using SigmaGel 1.05.
Determination of cytosolic Ca2+. Cytosolic free Ca2+ was determined by using the Ca2+-sensitive dye fura 2 (5 µmol/l; Molecular Probes, Leiden, The Netherlands) as described previously (12) and by using an inverted Axiovert 100 TV microscope (×400 magnification, oil immersion; Zeiss, Oberkochen, Germany) and an automatic filter change device (Hamamatsu, Herrsching, Germany). In brief, after serum depletion for 24 h, cells were incubated with HEPES-Ringer containing fura 2 acetoxymethyl ester in a final concentration of 5 µmol/l for 15 min. Subsequently, the coverslips were mounted on the microscope stage. The fluorescence signal was monitored at 510 nm, with the excitation wavelength alternating between 334 and 380 nm, by using a 100-W xenon lamp. The sampling rate was one ratio every 2 s. Cytosolic Ca2+ concentration ([Ca2+]i) was calculated according to Grynkiewicz et al. (18) by using a dissociation constant (Kd) of 225 nmol/l, after subtraction of background fluorescence. The maximum and minimum ratios (Rmax and Rmin) were measured after addition of calibration solutions, which contained 1 µmol/l ionomycin and 1 mmol/l Ca2+ to determine Rmax or 1 mmol/l EGTA and no Ca2+ to determine Rmin. Possible artifacts were excluded by measurement of autofluorescence of the different substances without fura 2.
Determination of cytosolic pH. Intracellular pH of single cells was determined by using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (2 µmol/l; Molecular Probes) as described previously (12) and with the setup described in Determination of cytosolic Ca2+. In brief, cells were each incubated with MEM containing 2 µmol/l 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester for 5 min. Then, the coverslips were rinsed four times with superfusion solution to remove the dye-containing medium. The coverslips were transferred to the stage of an inverted Axiovert 100 TV microscope (Zeiss) and allowed to equilibrate for 15 min. The excitation light source was a 100-W mercury lamp. The excitation wavelengths were 450 nm/490 nm. The emitted light was filtered through a band-pass filter (515-565 nm). The data-acquisition rate was one fluorescence intensity ratio every 2 s. After background subtraction, fluorescence intensity ratios were calculated. pH calibration was performed after each experiment by the nigericin (Sigma, St. Louis, MO) technique (39, 49), by using at least three calibration solutions in the range from pH 6.8 to 7.8. The calibration solutions contained 115 mmol/l KCl and 30 mmol/l NaCl.
IP3 formation. IP3 formation was determined by anion exchange columns, as described previously (32). In brief, cells seeded in six-well plates were preincubated during 24 h in media containing 0.5 µCi/ml [3H]inositol. Before experimentation, radioactive medium is aspirated, and the cells are washed with 3 × 2 ml HEPES-Ringer and then incubated for 30 min in 2 ml HEPES-Ringer containing 15 mM LiCl (pH 7.4). This medium was replaced with 1 ml HEPES-Ringer+15 mM LiCl, and after 10 min of incubation at 37°C, 1-ml aliquots of control Ringer+15 mM LiCl with the desired agonists were added. After 15 min of incubation, cells were lysed with 1 ml of 4 mM EDTA/1% SDS (90°C), and lysates were applied to ion exchange columns prepared as follows. Dowex-AG 1X-8 (0.5 g, formate form) was laid into 5-ml pipette tips with cotton wool on the bottom. Applied samples are washed with 2-ml aliquots of H2O and 5 mM disodium tetraborate/60 mM sodium formate, and then inositol 4-monophosphate (IP1), inositol 1,4-bisphosphate (IP2), and IP3 were eluted by subsequent addition of 2 ml of 0.1 M formic acid/0.2 M ammonium formate (IP1), 0.1 M formic acid/0.4 M ammonium formate (IP2), and 0.1 M formic acid/1.0 M ammonium formate (IP3). Eluted IP1-IP3 were collected into scintillation vials, mixed with 10 ml of scintillation cocktail, and counted.
Arachidonic acid release. Arachidonic acid release was performed as described elsewhere (4; 24). In brief, cells were labeled for 24 h with 0.5 µCi of [3H]arachidonic acid (ARC, St. Louis, MO). Subsequently, the media were removed, and an aliquot was counted to determine the extent of incorporation (85-90% of arachidonic acid added to the media). The cells were rinsed three times with ice-cold control Ringer solution (see Materials) and incubated for 30 min with control Ringer solution at 37°C (equilibration period). Finally, the experimental Ringer solutions were added, and aliquots were taken after 5 and 15 min to determine the amount of radioactivity released. At the end of the experiment, the cells were lysed with 1 mol/l NaOH. The release of radioactivity into the Ringer solution was determined as the percentage of total incorporated radioactivity. To verify the contribution of phospholipase A2 (PLA2), we tested the inhibitory action of arachidonyl trifluoromethyl ketone (38).
Determination of cell number. Cell number was determined by using a Z2 series Coulter Counter. Cells were seeded on plastic dishes, grown to subconfluence (~ 40,000 cells/cm2), and made quiescent before the experiments by 24-h serum removal.
Materials.
Unless otherwise stated, all materials were from Sigma (Munich,
Germany). Ethylisopropyl amiloride was kindly provided by Dr. H. J. Lang from Aventis (Frankfurt, Germany). Control Ringer solution
was composed of (in mmol/l) 130.0 NaCl, 5.4 KCl, 1.0 CaCl2,
1.0 MgCl2, 1.0 NaH2PO4, 10 HEPES,
and 5 glucose (pH 7.4 at 37°C), plus the respective vehicles (ethanol
or DMSO 1
).
Statistics. Values are means ± SE. Significance of difference was tested by paired or unpaired Student's t-test or ANOVA, as applicable. Differences were considered significant if P < 0.05. Cells from at least two different passages were used for each experimental series; n represents the number of cells or tissue culture dish investigated. For pH determinations, at least five coverslips were investigated for each experimental condition.
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RESULTS |
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ERK1/2 phosphorylation.
As shown in Fig. 1, A and C, 10 nmol/l
aldosterone and 10 µg/l EGF stimulate ERK1/2 phosphorylation in
MDCK-C11 cells, as described by us previously for aldosterone
(11). The time of exposure was 10 min, because in a
previous study it was shown that there was no difference between 5- or
10-min exposure (11). Control solutions always contained
the appropriate amount of vehicle (ethanol or DMSO 1:1,000). To
determine whether the side of aldosterone application, apical or
basolateral, might be important, we compared the effect of aldosterone
on filter-grown cells. As shown in Fig.
1A, there was only a slight
difference for aldosterone but a marked difference for EGF. At present,
we do not know whether these data mean that aldosterone acts within the
cells or just rapidly crosses the monolayer. The effects of aldosterone
and EGF in cells grown to ~80% confluence on solid supports were
compared with the effects in cells grown on permeable supports. We did not observe significant differences. Therefore, most of the experiments were performed with cells cultivated on solid supports, and the data
were pooled with those from cells grown on permeable supports. The
reason for the effectiveness of EGF in cells grown to ~80% confluence on solid supports is most probably the lack of a complete apical-to-basolateral differentiation, as observed previously (11).
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Cytosolic Ca2+.
Mitogenic factors, such as EGF, are known to affect cytosolic
Ca2+ homeostasis because of an increased Ca2+
entry across the plasma membrane or because of the release of Ca2+ from intracellular stores (25, 40). Under
control conditions, [Ca2+]i was 91 ± 20 nmol/l (n = 200). Figure
2 shows that 10 nmol/l aldosterone or 10 µg/l EGF induced a small but comparable increase in
[Ca2+]i, corresponding to ~100 nmol/l.
These aldosterone-induced changes in [Ca2+]i
([Ca2+]i) are in agreement with previously
reported changes for MDCK-C11 or skin cells (12, 21);
however, they are smaller compared with changes observed, for example,
in M-1 cortical collecting duct cells (20). Although the
changes in MDCK-C11 cells are small, it has been shown that they
contribute to Na+/H+-exchange activation, for
example (12). Similar to the effects on ERK1/2
phosphorylation, inhibition of EGF-R kinase with compound 56 (100 nmol/l) significantly reduced the
[Ca2+]i
induced by aldosterone or EGF. Thus aldosterone and EGF increase Ca2+ by means of EGF-R signaling. To rule out the
possibility that the EGF-R inhibitors, although used at nanomolar
concentrations, reduced signaling through toxic effects, we determined
whether these substances affected the bradykinin-induced signal.
However, both substances did not affect the Ca2+ rise
induced by 100 nmol/l bradykinin, which induced a Ca2+
spike of ~1,100 nmol/l. These data indicate that the EGF-R inhibitors did not act by means of toxic impairment of signaling.
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Aldosterone action depends on EGF-R-kinase activity.
To determine whether Tyr phosphorylation of the EGF-R is affected by
aldosterone and EGF, we performed EGF-R immunoprecipitation (stimulation time = 10 min for all experiments). As shown in Fig. 3, A and B, aldosterone and EGF enhanced Tyr
phosphorylation of the EGF-R. Figure
3A again shows that there is a
certain degree of autocrine activation of the EGF-R in MDCK cells, most
probably involving transforming growth factor (TGF)-
(36). When EGF and aldosterone were added together, the
extent of EGF-R-phosphorylation was enhanced. To determine whether this
enhancement also affected downstream signaling, we determined the
effects of aldosterone+EGF on ERK1/2 phosphorylation and
Ca2+.
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Interaction of aldosterone and EGF.
In addition to its own effect on ERK1/2 phosphorylation, aldosterone
increased the effect of 10 µg/l EGF on ERK1/2 phosphorylation (stimulation time = 10 min for all experiments). Because ERK1/2 phosphorylation seems to be the signal upstream of all other events investigated in this study, except EGF-R phosphorylation, and can be
quantitated reliably by ELISA (see METHODS), we determined the ERK1/2 phosphorylation dose-response curve for EGF and
EGF+aldosterone. As shown in Fig.
4A, there was a shift to the
left of the dose-response curve (factor ~5-10) when aldosterone
and EGF were added simultaneously, compared with EGF alone. ERK1/2
phosphorylation under these conditions was again prevented by EGF-R
kinase blockade, as shown in Fig. 4B.
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Effects of aldosterone and EGF on arachidonic acid release and
cytosolic pH.
To evaluate the potential affect of the Ca2+ and ERK1/2
signals on cell functions, we investigated arachidonic acid release and
cytosolic pH. When EGF (10 µg/l) or aldosterone (10 nmol/l) were each
added to the cells, there was only a very small increase of arachidonic
acid-derived radioactivity release observable (Fig. 6A). By contrast, bradykinin
(100 nmol/l) induced a threefold increase in arachidonic acid-derived
radioactivity release, indicating that the experimental setup worked.
Simultaneous addition of EGF and aldosterone resulted in a
significantly increased release, indicating a synergistic action of the
two. Inhibition of PLA2 with 25 µmol/l arachidonyl
trifluoromethyl ketone prevented the stimulation of
radioactivity release (Fig. 6A). Thus the observed release
of radioactivity can be attributed to PLA2 activity.
Inhibition of ERK1/2 phosphorylation with 25 µmol/l PD-98059 also
prevented stimulation of arachidonic acid release (110 ± 8% of
control, n = 4), as was the case when extracellular
Ca2+ was lowered (102 ± 10% of control,
n = 4).
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Effects of aldosterone and EGF on cell proliferation.
EGF acts as mitogen in many different cell types. Thus we investigated
whether aldosterone modulates the proliferative action of EGF. Cells
were made quiescent at ~70% confluence by 24-h serum removal and
were incubated for another 48 h with EGF and/or aldosterone in
serum-free media. As shown in Fig.
7A, the number of cells remained virtually constant under control conditions during the 48-h
incubation period. Thus any increase in the number of cells under
experimental conditions must reflect cell proliferation. Figure
7A shows the effect of EGF on the number of cells. At 10 µg/l, EGF exerted a slight proliferative action in MDCK-C11 cells. When added alone at 10 nmol/l, aldosterone had no significant effect.
However, when aldosterone (10 nmol/l) was added together with EGF (10 µg/l), there was a clear potentiation of the proliferative effect.
When saturating concentrations of EGF were used (100 µg/l), aldosterone had no further effect. Thus these data nicely mirror the
responses of ERK1/2 phosphorylation and arachidonic acid release and
show that aldosterone modulates the proliferative action of EGF.
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DISCUSSION |
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During the last several years, several reports showed that steroid hormones, such as aldosterone, can elicit rapid (<10 min), potentially nongenomic, cellular responses. The underlying mechanism(s) for the rapid actions of aldosterone are still unknown. One hypothesis is based on the interaction of steroid hormones with peptide hormone signaling. For example, the interaction of progesterone with oxytocin signaling, as well as the interaction of estradiol or glucocorticoids with growth factor and angiotensin II signaling, has been described (17, 28). In the case of aldosterone, an interaction with angiotensin II and vasopressin has been suggested (35, 48). Previously, we have shown that aldosterone increases H+ affinity of Na+/H+ exchange in MDCK-C11 cells by means of ERK1/2, a behavior similar to the action of growth factors such as EGF (11, 44). Transactivation of EGF-R is involved in the transmission of signals triggered by other mediators, for example, hormones acting by means of heterotrimeric G proteins (19). The EGF-R is therefore considered a transducer of heterologous signaling. Thus it is conceivable that EGF-R also plays a role in the integration of rapid steroid signaling.
The results presented here show that aldosterone acts on ERK1/2 phosphorylation, cytosolic Ca2+, and pH homeostasis in a manner very similar to the effects observed during EGF exposure. These data allow the hypothesis that aldosterone also "uses" the EGF-R as a transducer of heterologous signaling, as already shown for other hormones (19). If this hypothesis is true, then we should expect that the effects of aldosterone are reduced when the EGF-R kinase is inhibited. As shown for ERK1/2 phosphorylation and cytosolic Ca2+, this was indeed the case. When EGF-R kinase was inhibited, the effects of aldosterone were significantly smaller (cytosolic Ca2+) or even completely abolished (ERK1/2 phosphorylation). The reason for the small remaining effect on cytosolic Ca2+ can be explained by the higher sensitivity of Ca2+ measurements compared with ERK1/2 phosphorylation. If aldosterone acts on cytosolic Ca2+ and ERK1/2 phosphorylation by means of the EGF-R kinase, we would also expect that aldosterone leads to enhanced EGF-R phosphorylation. As shown here, EGF-R phosphorylation was enhanced in the presence of aldosterone. Thus the two lines of evidence presented here support the hypothesis that rapid effects of aldosterone involve the EGF-R pathway, at least in the cell system investigated here. This is the first report indicating an interaction of aldosterone with Tyr kinase receptor signaling. The possible physiological and/or pathophysiological significance of this interaction is supported by the fact that aldosterone was effective at nanomolar concentrations, although more detailed investigations have to be performed in future studies. It is conceivable that elevated circulating aldosterone concentrations (for example, in patients with liver cirrhosis) may elicit part of its effects by means of an interaction with EGF-R.
The data presented here also indicate that there is a certain degree of
autocrine stimulation of the EGF-R in MDCK cells (see Fig. 1),
as already described (36). This autocrine activation loop
most probably results from the simultaneous expression of EGF-R and
transforming growth factor (TGF)-. Thus, even though the cells were
made quiescent by serum removal, there is always a certain
preactivation under control conditions. The autocrine preactivation is
also responsible for the fact that the observed effects of EGF are
smaller compared with other cell systems such as vascular smooth muscle
cells, for example (2).
Our data show that aldosterone enhances EGF-R phosphorylation
followed by phosphorylation of ERK1/2. Subsequently,
Ca2+ entry across the plasma membrane is increased
(Fig. 8). This conclusion is based on the
observations that prevention of Ca2+ entry did not abrogate
ERK1/2 phosphorylation, whereas inhibition of ERK1/2 phosphorylation
significantly reduced the Ca2+ signal. Moreover, inhibition
of EGF-R Tyr kinase significantly reduced ERK1/2 phosphorylation and
the Ca2+ signal. Furthermore, we did not observe a
substantial increase in IP3 formation, which argues against
the involvement of phospholipase C and subsequent Ca2+
release from IP3-sensitive stores. Finally, ERK1/2 and
Ca2+ do then transmit the aldosterone signal to further
downstream events, as in Na+/H+-exchange
activation (11, 15).
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How does the interaction of aldosterone with the EGF-R affect EGF signaling in MDCK-C11 cells? We tried to answer this question pharmacodynamically by using ERK1/2 phosphorylation as a parameter. Aldosterone and EGF could interact in a simple additive manner, leading to a shift to the left of the dose-response curve, with no change in the maximum effect when both are added simultaneously. Alternatively, aldosterone could enhance the maximum effect, either additively or in terms of a potentiation of EGF. Finally, aldosterone could shift the dose-response curve of EGF to lower concentrations, thereby sensitizing the cells for EGF. As shown in Fig. 4A, aldosterone and EGF seem not to act additively in a simple manner or with respect to maximum ERK1/2 phosphorylation. At submaximum EGF concentrations, aldosterone and EGF seem to induce an overadditive ERK1/2 phosphorylation, leading to a shift to the left of the EGF dose-response curve. Thus at submaximum concentrations of EGF, the cells seem to respond more sensitively to EGF when aldosterone is present compared with the situation where aldosterone is absent. This interpretation would also explain the enhanced EGF-induced response with respect to the Ca2+ signal, which is downstream of ERK1/2 phosphorylation. This, of course, is a pharmacodynamic description of the modulation of EGF effects, and future studies will have to focus on the underlying mechanisms. Previously, we have shown that rapid effects of aldosterone in MDCK-C11 cells are not prevented by the mineralocorticoid receptor antagonist spironolactone (13), which is similar to results obtained in other cell types (46). In some preliminary experiments, we tested the effect of spironolactone (1 µmol/l) on aldosterone-induced ERK1/2 phosphorylation and could not detect any inhibitory action of spironolactone (data not shown). Thus it is unlikely that the interaction of aldosterone with EGF-R depends on the mineralocorticoid receptor.
What is the nature of the interaction of aldosterone with EGF-R
signaling? One possibility is a direct interaction on the level of the
EGF-R. A similar mechanism for steroid-hormone and peptide-hormone
cross talk has been proposed for progesterone and oxytocin
(17). Another possible mechanism is the involvement of
additional factors, for example, Src kinase, which could link the
action of aldosterone to EGF-R phosphorylation. In this case, aldosterone would have to activate the additional factor directly or by
means of an aldosterone receptor, as proposed by Wehling et al.
(47). Several years ago, it was shown that aldosterone can
change the metabolism of membrane phospholipids, for example, leading
to an increase in the diglyceride fraction (16). Changes in the lipid environment could also affect the activation of a membrane
protein such as EGF-R (1). Finally, there exists the possibility that the interaction of aldosterone with EGF-R depends on
the endogenous EGF-R ligand TGF-, known to be expressed in MDCK
cells (36). Although the time course of the aldosterone action is not in favor of a mechanism requiring the cleavage of an
endogenous EGF-R ligand, it cannot be ruled out completely. Future
studies will focus on the mechanism underlying the interaction of
aldosterone with ERG-R signaling in more detail.
What is the cellular significance of aldosterone interaction with EGF-R signaling? To gain more information regarding the possible significance of rapid steroid effects, it is important to determine changes in cell function. Our data show that aldosterone-induced modulation of EGF-R signaling indeed affects certain cell functions. Of course there are many more aspects of cell function that could be affected and have not been determined here (for example, transepithelial ion transport) but will be subject to investigation in future studies. Aldosterone used the EGF-R signaling cascade to modulate the release of arachidonic acid and the activation of Na+/H+ exchange. Stimulation of arachidonic acid release and Na+/H+ exchange will also affect cell function and/or signaling, thereby creating a complex network.
With respect to a differentiation of the direct genomic (by means of the mineralocorticoid receptor) and primary nongenomic effects, the data reported here offer a new perspective. The interaction of aldosterone with EGF-R signaling is a primary nongenomic event that may have a secondary genomic impact, for example, by means of the nuclear factor of activated transcription and serum response element. Therefore, the primarily nongenomic responses may finally result in an alternative genomic response via pathways that do not rely on the mineralocorticoid receptor.
What is the role of interaction with EGF-R signaling in the physiological response to aldosterone? Presently, the answer to this question is not known. However, it is known that EGF-R signaling modulates transepithelial ion transport and stimulates salt reabsorption in certain cell types (8, 22). Furthermore, it is known that EGF-R signaling may exert profibrotic actions (10). Thus there are certain similarities with respect to the physiological (salt reabsorption) and the pathophysiological (fibrosis) action of aldosterone and EGF. Therefore it is conceivable that the interaction of aldosterone with EGF-R signaling may support physiological and pathophysiological responses to aldosterone. However, with respect to sodium handling in the distal nephron, the actions of EGF and aldosterone are opposed, because EGF has been reported to inhibit the epithelial sodium channel (31, 45). Thus aldosterone could limit its own stimulatory action on sodium reabsorption by means of the EGF-R signaling cascade, representing a negative feedback in this case. By contrast, with respect to intestinal sodium reabsorption, EGF and aldosterone seem to act in the same direction, i.e., stimulation of sodium absorption (23). Therefore, EGF-R signaling could support the stimulatory effect of aldosterone on intestinal sodium absorption. Of course, these hypotheses have to be verified in future studies by using EGF-R kinase inhibitors to test the importance of EGF-R signaling for aldosterone effects, for example.
In conclusion, our data show that aldosterone uses the EGF-R-ERK1/2 signaling cascade to elicit rapid effects in MDCK cells. In addition, aldosterone seems to modulate the action of EGF. Possibly, the EGF-R represents a membrane target for rapid effects of aldosterone. Of course, the data presented here do not affect the importance of genomic actions of aldosterone but do add an additional, primarily nongenomic, pathway.
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ACKNOWLEDGEMENTS |
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This study was supported by Deutsche Forschungsgemeinschaft Grants Ge 905/4-1 and SFB487/A6.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. Gekle, Physiologisches Institut, Universität Würzburg, Röntgenring 9, 97070 Würzburg, Germany (E-mail: michael.gekle{at}mail.uni-wuerzburg.de).
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.
10.1152/ajprenal.00159.2001
Received 18 May 2001; accepted in final form 23 October 2001.
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REFERENCES |
---|
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---|
1.
Barrantes, FJ,
Antollini SS,
Bouzat CB,
Garbus I,
and
Massol RH.
Nongenomic effects of steroids on the nicotinic acetycholine receptor.
Kidney Int
57:
1382-1389,
2000[ISI][Medline].
2.
Bokemeyer, D,
Schmitz U,
and
Kramer HJ.
Angiotensin II-induced growth of vascular smooth muscle cells requires an Src-dependent activation of the epidermal growth factor receptor.
Kidney Int
58:
549-558,
2000[ISI][Medline].
3.
Bridges, AJ,
Zhou H,
Cody DR,
Rewcastle GW,
McMichael A,
Showalter HD,
Fry DW,
Kraker AJ,
and
Denny WA.
Tyrosine kinase inhibitors. 8. An unusually steep structure-activity relationship for analogues of 4-(3-bromoanilino)-6,7-dimethoxyquinazoline (PD 153035), a potent inhibitor of the epidermal growth factor receptor.
J Med Chem
39:
267-276,
1996[ISI][Medline].
4.
Carroll, MA,
Schwartzman M,
Baba M,
Miller MJS,
and
McGiff JC.
Renal cytochrome P-450-related arachidonate metabolism in rabbit aortic coarctation.
Am J Physiol Renal Fluid Electrolyte Physiol
255:
F151-F157,
1988
5.
Christ, M,
Eisen C,
Aktas J,
Theisen K,
and
Wehling M.
The inositol-1,4,5-trisphosphate system is involved in rapid effects of aldosterone in human mononuclear leukocytes.
J Clin Endocrinol Metab
77:
1452-1457,
1993[Abstract].
6.
Cooper, GJ,
and
Hunter M.
Na+-H+exchange in frog early distal tubule: effect of aldosterone on the set-point.
J Physiol (Lond)
479:
423-432,
1994[Abstract].
7.
Croxtall, JD,
Choudhury Q,
and
Flower RJ.
Glucocorticoids act within minutes to inhibit recruitment of signaling factors to activated EGF receptors through a receptor-dependent, transcription-independent mechanism.
Br J Pharmacol
130:
289-298,
2000
8.
Danto, SI,
Borok Z,
Zhang XL,
Lopez MZ,
Patel P,
Crandall ED,
and
Lubman RL.
Mechanisms of EGF-induced stimulation of sodium reabsorption by alveolar epithelial cells.
Am J Physiol Cell Physiol
275:
C82-C92,
1998
9.
Doolan, CM,
and
Harvey BJ.
Modulation of cytosolic protein kinase C and calcium ion activity by steroid hormones in rat distal colon.
J Biol Chem
271:
8763-8767,
1996
10.
El Nahas, AM.
Growth factors and glomerular sclerosis.
Kidney Int
41:
S15-S20,
1992[ISI].
11.
Gekle, M,
Freudinger R,
Mildenberger S,
Schenk K,
Marschitz I,
and
Schramek H.
Rapid activation of Na+/H+-exchange in MDCK-cells by aldosterone involves MAP-kinases ERK1/2.
Pflügers Arch
441:
781-786,
2001[ISI][Medline].
12.
Gekle, M,
Golenhofen N,
Oberleithner H,
and
Silbernagl S.
Rapid activation of Na+ /H+-exchange by aldosterone in renal epithelial cells requires Ca2+ and stimulation of a plasma membrane proton conductance.
Proc Natl Acad Sci USA
93:
10500-10504,
1996
13.
Gekle, M,
Silbernagl S,
and
Oberleithner H.
The mineralocorticoid aldosterone activates a proton conductance in cultured kidney cells.
Am J Physiol Cell Physiol
273:
C1673-C1678,
1997
14.
Gekle, M,
Wünsch S,
Oberleithner H,
and
Silbernagl S.
Characterization of two MDCK-cell subtypes as a model system to study principal and intercalated cell properties.
Pflügers Arch
428:
157-162,
1994[ISI][Medline].
15.
Gomperts, BD.
GE: a GTP-binding protein mediating exocytosis.
Annu Rev Physiol
52:
591-606,
1990[ISI][Medline].
16.
Goodman, DBP,
Allen JE,
and
Rasmussen H.
Studies on the mechanism of action of aldosterone: hormone-induced changes in lipid metabolism.
Biochemistry
10:
3825-3831,
1971[ISI][Medline].
17.
Grazzini, E,
Guillon G,
Mouillac B,
and
Zingg HH.
Inhibition of oxytocin receptor function by direct binding of progesterone.
Nature
392:
509-512,
1998[ISI][Medline].
18.
Grynkiewicz, G,
Poenie M,
and
Tsien RY.
A new generation of Ca2+ indicators with greatly improved fluorescent properties.
J Biol Chem
260:
3440-3450,
1985[Abstract].
19.
Hackel, PO,
Zwick E,
Prenzel N,
and
Ullrich A.
Epidermal growth factor receptors: critical mediators of multiple receptor pathways.
Curr Opin Cell Biol
11:
184-189,
1999[ISI][Medline].
20.
Harvey, BJ,
and
Higgins M.
Nongenomic effects of aldosterone on Ca2+ in M-1 cortical collecting duct cells.
Kidney Int
57:
1395-1403,
2000[ISI][Medline].
21.
Haseroth, K,
Gerdes D,
Berger S,
Feuring M,
Günther A,
Herbst C,
Christ M,
and
Wehling M.
Rapid nongenomic effects of aldosterone in mineralocorticoid-receptor-knockout mice.
Biochem Biophys Res Commun
266:
257-261,
1999[ISI][Medline].
22.
Keely, SJ,
Uribe JM,
and
Barrett KE.
Carbachol stimulates transactivation of epidermal growth factor receptor and mitogen-activated protein kinase in T84 cells. Implications for carbachol-stimulated chloride secretion.
J Biol Chem
273:
27111-27117,
1998
23.
Khurana, S,
Nath SK,
Levine SA,
Bowser JM,
Tse CM,
Cohen ME,
and
Donowitz M.
Brush border phosphatidylinositol 3-kinase mediates epidermal growth factor stimulation of intestinal NaCl absorption and Na+/H+ exchange.
J Biol Chem
271:
9919-9927,
1996
24.
Lazarowski, ER,
Boucher RC,
and
Harden TK.
Calcium-dependent release of arachidonic acid in response to purinergic receptor activation in airway epithelium.
Am J Physiol Cell Physiol
266:
C406-C415,
1994
25.
Lovisolo, D,
Distasi C,
Antoniotti S,
and
Munaron L.
Mitogens and calcium channels.
NIPS
12:
279-285,
1997
26.
Manegold, JC,
Falkenstein E,
Wehling M,
and
Christ M.
Rapid aldosterone effects on tyrosine phosphorylation in vasular smooth muscle cells.
Cell Mol Biol (Noisy-le-grand)
45:
805-813,
1999.
27.
Moghal, N,
and
Sternberg PW.
Multiple positive and negative regulators of signaling by the EGF-receptor.
Curr Opin Cell Biol
11:
190-196,
1999[ISI][Medline].
28.
Neugarten, J,
Medve I,
Lei J,
and
Silbiger SR.
Estradiol suppresses mesangial cell type I collagen synthesis via activation of the MAP kinase cascade.
Am J Physiol Renal Physiol
277:
F875-F881,
1999
29.
Oberleithner, H,
Kersting U,
Silbernagl S,
Steigner W,
and
Vogel U.
Fusion of cultured dog kidney (MDCK) cells. II. Relationship between cell pH and K+ conductance in response to aldosterone.
J Membr Biol
111:
49-56,
1989[ISI][Medline].
30.
Oberleithner, H,
Weigt M,
Westphale HJ,
and
Wang W.
Aldosterone activates Na+/H+exchange and raises cytoplasmic pH in target cells of the amphibian kidney.
Proc Natl Acad Sci USA
84:
1464-1468,
1987[Abstract].
31.
Ookawara, S,
Tabei K,
Furuya H,
and
Asano Y.
The effect of EGF on electrolyte transport is mediated by tyrosine kinases in the rabbit cortical collecting duct.
Miner Electrolyte Metab
25:
191-198,
1999[ISI][Medline].
32.
Orlov, SN,
Dulin NO,
Gagnon F,
Gekle M,
Douglas JG,
Schwartz JH,
and
Hamet P.
Purinergic modulation of Na+,K+,Cl cotransport and MAP kinases is limited to C11-MDCK cells resembling intercalated cells from collecting ducts.
J Membr Biol
172:
225-234,
1999[ISI][Medline].
33.
Schramek, H,
Sorokin A,
Watson RD,
and
Dunn MJ.
Differential long-term regulation of MEK and of p42 MAPK in rat glomerular mesangial cells.
Am J Physiol Cell Physiol
270:
C40-C48,
1996
34.
Schramek, H,
Wilflingseder D,
Pollack V,
Freudinger R,
Mildenberger S,
and
Gekle M.
Ochratoxin A-induced stimulation of extracellular signal-regulated kinases 1/2 is associated with Madin-Darby canine kidney-C7 cell dedifferentiation.
J Pharmacol Exp Ther
283:
1460-1468,
1997
35.
Schwab, A,
and
Oberleithner H.
The early response to aldosterone in the kidney.
In: Genomic and Non-Genomic Effects of Aaldosterone, edited by Wehling M.. Boca Raton, FL: CRC, 1995, p. 51-76.
36.
Shi, W,
Fan H,
Shum L,
and
Derynck R.
The tetraspanin CD9 associates with transmembrane TGF-alpha and regualtes TGF-alpha-induced EGF receptor activation and cell proliferation.
J Cell Biol
148:
591-601,
2000
37.
Stockand, JD,
Spier BJ,
Worrell RT,
Yue G,
Al-Baldawi N,
and
Eaton DC.
Regulation of Na+ reabsorption by the aldosterone-induced small G protein K-Ras2A.
J Biol Chem
274:
35449-35454,
1999
38.
Suszták, K,
Mócsai A,
Ligeti E,
and
Kapus A.
Electrogenic H+ pathway contributes to stimulus-induced changes of internal pH and membrane potential in intact neutrophils: role of cytoplasmic phospholipase A2.
Biochem J
325:
501-510,
1997[ISI][Medline].
39.
Thomas, JA,
Buchsbaum RN,
Zimniak A,
and
Racker E.
Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ.
Biochemistry
18:
2210-2218,
1979[ISI][Medline].
40.
Tinhofer, I,
Maly K,
Dietl P,
Hochholdinger F,
Mayr S,
Obermeier A,
and
Grunicke HH.
Differential Ca2+ signaling induced by activation of the epidermal growth factor and nerve growth factor receptors.
J Biol Chem
271:
30505-30509,
1996
41.
Urbach, V,
van Kerkhove E,
Maguire D,
and
Harvey BJ.
Rapid activation of KATP channels by aldosterone in principal cells of frog skin.
J Physiol (Lond)
491:
111-120,
1996[Abstract].
42.
Versteeg, HH,
Nijhuis E,
Van den Brink GR,
Evertzen M,
Pynaert GN,
van Deventer SJ,
Coffer PJ,
and
Peppelenbosch MP.
A new phosphospecific cell-based ELISA for p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, protein kinase B and cAMP-response-element-binding protein.
Biochem J
350:
717-722,
2000[ISI][Medline].
43.
Vilella, S,
Guerra L,
Helmle-Kolb C,
and
Murer H.
Aldosterone actions on basolateral Na+/H+ exchange in Madin-Darby canine kidney cells.
Pflügers Arch
422:
9-15,
1992[ISI][Medline].
44.
Wakabayashi, S,
Shigekawa M,
and
Pouysségur J.
Molecular physiology of vertebrate Na+/H+ exchangers.
Physiol Rev
77:
51-67,
1997
45.
Warden, DH,
and
Stokes JB.
EGF and PGE2 inhibit rabbit CCD Na+ transport by different mechanisms: PGE2 inhibits Na+-K+ pump.
Am J Physiol Renal Fluid Electrolyte Physiol
264:
F670-F677,
1993
46.
Wehling, M.
Specific, nongenomic actions of steroid homones.
Annu Rev Physiol
59:
365-393,
1997[ISI][Medline].
47.
Wehling, M,
Christ M,
and
Theisen K.
Membrane receptors for aldosterone: a novel pathway for mineralocorticoid action.
Am J Physiol Endocrinol Metab
263:
E974-E979,
1992[ISI][Medline].
48.
Wehling, M,
Neylon CB,
Fullerton M,
Bobik A,
and
Funder JW.
Nongenomic effects of aldosterone on intracellular Ca2+ in vascular smooth muscle cells.
Circ Res
76:
973-979,
1995
49.
Weiner, ID,
and
Hamm LL.
Use of fluorescent dye BCECF to measure intracellular pH in cortical collecting tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
256:
F957-F964,
1989
50.
Winter, DC,
Schneider MF,
O'Sullivan GC,
Harvey BJ,
and
Geibel J.
Rapid effects of aldosterone on sodium-hydrogen exchange in isolated colonic crypts.
J Membr Biol
170:
17-26,
1999[ISI][Medline].