Human Epidermal Growth Factor Receptor-1 Expression Renders Chinese Hamster Ovary Cells Sensitive to Alternative Aldosterone Signaling*

The epidermal growth factor (EGF) regulates cell proliferation, differentiation, and ion transport using ERK1/2 as a downstream effector. Furthermore, the EGF receptor (EGFR) is involved in signaling by G-protein-coupled receptors, growth hormone, and cytokines via transactivation. It has been suggested that steroids interact with peptide hormones. Previously, we have shown that aldosterone modulates EGF responses in Madin-Darby canine kidney cells (Gekle, M., Freudinger, R., Mildenberger, S., and Silbernagl, S. (2002) Am. J. Physiol. 282, F669–F679). Here, we tested the hypothesis that human EGFR-1 can confer alternative aldosterone responsiveness with respect to ERK1/2 phosphorylation to Chinese hamster ovary cells, which do not express EGFR. Wild-type Chinese hamster ovary cells did not respond to EGF or aldosterone. After transfection of human EGFR-1, the cells responded to EGF, but not to aldosterone. However, when submaximal concentrations of EGF were used, nanomolar concentrations of aldosterone potentiated the action of EGF within minutes, resulting in a leftward shift of the EGF dose-response curve. This was not the case in mock-transfected of c-Src EGFR, an of tyrosine kinases t -butyl)pyrazolo[3,4- d prevented the action of aldosterone. Our data show that aldosterone uses the EGF-EGFR-MEK1/2-ERK1/2 signaling cascade to elicit its alternative effects. In the presence of EGF, aldosterone leads to EGFR transactivation via cytosolic tyrosine kinases of the Src family.

The classical genomic mechanism of steroid hormone action involves binding to intracellular receptors and stimulation of transcription and protein synthesis. Yet, aldosterone can also induce rapid responses by interfering with, for example, regulation of intracellular pH or calcium (1)(2)(3)(4)(5)(6), intracellular generation of inositol 1,4,5-trisphosphate (7), and protein kinase C (PKC) 1 and ERK1/2 phosphorylation (5, 8 -10). Former studies also revealed that aldosterone acts within several minutes on plasma membrane K ϩ conductance of different cells (2,11,12). These alternative actions of aldosterone have been suggested to be mediated by a plasma membrane receptor (13), although this putative receptor has not been identified. Recently, it has been shown that steroid hormones are capable of interacting with peptide hormone signaling (14,15). For example, the interaction of progesterone with oxytocin signaling has been described (16), as well as the interaction of estradiol with growth factor and angiotensin II signaling (17). In the case of aldosterone, an interaction with angiotensin II and vasopressin has been suggested (15,18).
Another attractive candidate of interaction with steroids represents the epidermal growth factor (EGF). EGF regulates cell proliferation, differentiation, and tissue repair and uses, at least in part, mitogen-activated protein kinases as downstream signals. In addition, enhanced EGF signaling has been observed in several tumor cells (19,20). Furthermore, it has been shown that EGF affects epithelial salt transport in a cellspecific manner, leading to either enhanced or reduced salt reabsorption (21)(22)(23)(24)(25). The EGF receptor (EGFR) has been shown to be involved in signaling events elicited by, for example, G-protein-coupled receptors, growth hormone, and cytokines via a mechanism called transactivation (19,20). This mechanism of transactivation involves EGFR activation by intracellular signaling components. These may include c-Src, JAK2, and phosphatases (26). Thus, EGFR can serve as a central transducer of heterologous signaling systems. A transcription-independent interaction of glucocorticoids or estrogens with EGFR has been reported (27,28). Recently, we showed that aldosterone also interacts with EGF signaling (29). In Madin-Darby canine kidney (MDCK) cells, aldosterone potentiates the effects of EGF on ERK1/2 phosphorylation and Ca 2ϩ homeostasis, two important components of cellular signaling networks. Furthermore, aldosterone potentiates the effect of EGF on Na ϩ /H ϩ exchange activity and cell proliferation.
The precise underlying mechanisms and the physiological or pathophysiological significance of this cross-talk are not yet completely understood. The interaction of steroids with peptide hormone signaling represents one possible mechanism for alternative steroid action and at the same time offers an explanation for the significance of these effects, i.e. modulation of peptide hormone signaling.
In this study, we investigated the interaction of aldosterone with human EGFR-1 (HER1) in a heterologous expression system using Chinese hamster ovary (CHO) cells. These cells do not express EGFR under control conditions (30) and are therefore an ideal model to investigate the importance of HER1 in the alternative aldosterone action. We used HER1-transfected CHO cells and tried to answer three questions. (i) Is HER1 transfection sufficient to elicit alternative aldosterone responses? (ii) Does this response depend on the presence of EGF? (iii) Which signaling components are used for the crosstalk between aldosterone and HER1?
Our results show that aldosterone requires the EGF-HER1-MEK1/2-ERK1/2 signaling cascade to elicit, at least in part, its alternative effects. This transactivating effect is mediated by cytosolic tyrosine kinases (c-Src).

EXPERIMENTAL PROCEDURES
Cell Culture-We used CHO-K1 cells from American Type Culture Collection (Manassas, VA). Cells were cultivated in Ham's F-12 medium supplemented with 10% fetal calf serum at 37°C and 5% CO 2 . 24 h prior to the experiments, serum was removed from the medium. For the experiments presented, the cells were cultivated either on Petri dishes (BD Biosciences, Heidelberg, Germany) or in 96-well plates (for pERK1/2-ELISA).
Transfection and Dilution Cloning-Transfection of the cells was performed with the QIAGEN Polyfect reagent (QIAGEN Inc., Hilden, Germany) according to the manufacturer's instructions. We used the HER1 expression vector pRK5-HER1 (31) and pBabepuro (32), which carries a puromycin resistance gene (10:1 ratio). Selection of transfected clones was performed with puromycin (5 mg/liter). Clones were isolated by limited dilution cloning and analyzed for HER1 expression by Western blotting.
Quantification of ERK1/2 Phosphorylation by ELISA-For quantification of ERK1/2 phosphorylation, we performed pERK1/2-ELISA according to Versteeg et al. (33). In control experiments, we compared the effects of EGF determined by Western blotting and pERK1/2-ELISA and found no significant difference, as already described (29). Thus, the results obtained by Western blotting and pERK1/2-ELISA were pooled. Cells were seeded in 96-well plates (Maxisorp, Nunc) and serumstarved for 24 h prior to the experiment. After stimulation as indicated below, 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 (PBS/Triton). Endogenous peroxidase was quenched with 0.6% H 2 O 2 in PBS/Triton for 20 min. Cells were washed three times with PBS/Triton, blocked with 10% fetal calf serum in PBS/Triton for 1 h, and incubated overnight with the above-described primary antibody (1:1000) in PBS/Triton containing 5% bovine serum albumin at 4°C. The next day, cells were washed three times with PBS/Triton for 5 min, incubated with peroxidase-conjugated mouse anti-rabbit secondary antibody (1:10,000) in PBS/Triton containing 5% bovine serum albumin for 1 h at room temperature, and washed three times with PBS/Triton for 5 min and twice with PBS. Subsequently, the cells were incubated with 50 l of solution containing 0.4 mg/ml o-phenylenediamine, 11.8 mg/ml Na 2 HPO 4 , 7.3 mg/ml citric acid, and 0.015% H 2 O 2 for 15 min at room temperature in the dark. The resulting signal was detected at 490 nm with a multiwell multilabel counter (Victor 2 , Wallac, Turku, Finland). After the peroxidase reaction, the cells were washed twice with PBS/Triton and twice with demineralized water. After drying the wells for 5 min, 100 l of trypan blue solution (0.2% in PBS) was added for 5 min at room temperature. Subsequently, the cells were washed four times with demineralized water, and 100 l of 1% SDS solution was added and incubated on a shaker for 1 h at room temperature. Finally, the absorbance was measured at 595 nm with the abovementioned ELISA reader.
c-Src Tyr Phosphorylation-Immunoprecipitation was performed as recently described (34). Briefly, cell lysates were precleared with protein A/G-agarose for 20 min at 4°C. To precipitate c-Src, anti-c-Src receptor antibody (sc-19, Santa Cruz Biotechnology) 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-12% PAGE, and phosphorylated c-Src receptor was detected using anti-phosphotyrosine antibody (pY99). Densitometric analysis was performed using Sigmagel Version 1.05 software.
EGFR Tyr Phosphorylation-Because wild-type CHO cells do not express EGFR and there was no EGF-affected Tyr(P) band at 170 kDa, we evaluated EGFR Tyr phosphorylation directly by Western blotting using anti-phosphotyrosine antibody (pY99) at a 1:1000 dilution as the primary antibody.
Statistics-The data are presented as means Ϯ S.E. Significance of difference was tested by paired or unpaired Student's t test or analysis of variance as applicable. Differences were considered significant if p was Ͻ0.05. Cells from at least two different passages were used for each experimental series. n represents the number of cells or tissue culture dishes investigated.

RESULTS
To test the hypothesis that alternative actions of aldosterone involve EGFR, we reconstituted CHO cells, which do not express EGFR, with the receptor to obtain a defined cell system for the investigation of the role of EGFR in alternative aldosterone signaling. Fig. 1A shows that parental cells did not respond to EGF or aldosterone with ERK1/2 phosphorylation, which was readily triggered following PKC activation with phorbol 12-myristate 13-acetate (PMA). In addition, Fig. 1B confirms that EGFR was not detectable by Western blotting in CHO cells. We then transfected HER1 into these cells and isolated six stable clones expressing HER1 (CHO-HER1 cells). Fig. 1B demonstrates the expression of HER1 in one of the clones (designated F3). After transfection with HER1, the cells responded with enhanced ERK1/2 phosphorylation after EGF exposure (Fig. 1C), indicating that the signaling cascade from HER1 to ERK1/2 was intact. Fig. 1D shows the time and concentration dependence of EGFinduced ERK1/2 phosphorylation.
We next tested the effects of short-term aldosterone treatment on ERK1/2 phosphorylation. To obtain reliable quantification, the majority of the ERK1/2 phosphorylation experiments were evaluated by an ELISA method originally described by Versteeg et al. (33) and recently adapted and evaluated for our purposes (29). This was especially important because we wanted to quantitatively investigate potential synergism between aldosterone and EGF. The immunoblot and ELISA analysis depicted in Fig. 2A show that CHO-HER1 cells did not respond to aldosterone alone. However, in the presence of submaximal EGF, aldosterone enhanced ERK1/2 phosphorylation. As shown in Fig. 2B, the responsiveness of CHO-HER1 cells to EGF could be easily confirmed by the ELISA method. Furthermore, the potentiating effect of aldosterone on EGF action was independently observed in six CHO-HER1 clones. Therefore, we believe that the action of aldosterone is due to the expression of HER1. In the presence of aldosterone and EGF, ERK1/2 phosphorylation was increased by a factor of 1.33-1.64 compared with EGF alone. The EGF-induced increase in ERK1/2 phosphorylation ((pERK1/2 aldosteroneϩEGF Ϫ pERK control )/(pERK1/2 EGF Ϫ pERK control )) was enhanced by a factor of 1.43-2.65 by aldosterone (Fig. 2). These data show that aldosterone potentiates EGF signaling via HER1. PMAinduced ERK1/2 phosphorylation was not affected by aldosterone (see Fig. 4), indicating specificity of the observed effect. Furthermore, 10 nmol/liter hydrocortisone did not enhance EGF-induced signaling (see Fig. 4). In the subsequent experiments, we focused on the CHO-HER1 clone F3. Fig. 3A shows the time course of ERK1/2 phosphorylation in the presence of 1 g/liter EGF or EGF ϩ 10 nmol/liter aldos-terone. The steroid exerted the strongest potentiating effect during the initial phase of ERK1/2 phosphorylation. We next analyzed the concentration dependence of EGF (Fig. 3B). Aldosterone led to a leftward shift in the EGF dose-response curve with no significant change in the maximal effect. Thus, aldosterone sensitizes the cells to EGF, similar to the effect observed in MDCK cells (29). This is also evident from the data in Fig. 3C, where the rate of potentiation induced by 10 nmol/ liter aldosterone is plotted against the effect of EGF at different concentrations. Finally, we tested different concentrations of aldosterone (Fig. 3D) and observed a concentration dependence with maximal effects at 10 nmol/liter. Altogether, these data show that the effects observed in MDCK cells (29) can be reproduced by transfection of HER1 in an otherwise unresponsive wild-type CHO cell. Furthermore, the data show that aldosterone modulates the sensitivity of the cell to EGF and the FIG. 1. EGFR expression and EGF responsiveness of CHO cells. A, wildtype CHO cells did not respond to EGF or aldosterone with respect to ERK1/2 phosphorylation. Application time was 5 min. B, wild-type CHO cells transfected with empty vector (CHO-mock) did not express EGFR, whereas CHO-HER1 cells stably expressed EGFR (the blot shows clone F3). C, CHO-HER1 cells responded to EGF (10 g/liter) with ERK1/2 phosphorylation. Application time was 5 min. D, the time dependence and concentration dependence of EGF-induced ERK1/2 phosphorylation in CHO-HER1 cells were characterized. The data were obtained by pERK1/2-ELISA. The cells did not respond to aldosterone alone. n ϭ 6 -12 for each plotted value.

FIG. 2. Interaction of EGF and aldosterone.
A, in CHO-HER1 cells, aldosterone (10 nmol/liter) potentiated the effect of EGF (1 g/liter) on ERK1/2 phosphorylation. Application time was 5 min. However, the cells did not respond to aldosterone alone. Representative data are from clone F3. Upper panel, Western blot analysis; lower panel, pERK1/2-ELISA analysis. n ϭ 6 -12 for each plotted value. B, the interaction of aldosterone and EGF was tested in six different CHO-HER1 clones (n ϭ 9 -15). In all clones, aldosterone had a significant effect. In mock-transfected cells, no interaction could be observed. *, p Ͼ 0.05. Application time was 5 min. The table shows the -fold increase, determined as described under "Results." time course of the EGF response. Both of these effects have the potential to alter the cell biological significance of the EGF signal, as shown by us in MDCK cells for proliferation and activation of Na ϩ /H ϩ exchange in a recent study (29).
In the next step, we applied a pharmacological approach to determine signaling components involved in aldosterone-induced potentiation of EGF signaling (Fig. 4A). Inhibition of the HER1 kinase by 100 nmol/liter tyrphostin AG1478 or inhibition of the MEK1/2 kinases by 1 mol/liter U0126 completely prevented ERK1/2 phosphorylation by either EGF or aldosterone ϩ EGF. These data show that the observed effects were indeed mediated by the HER1-MEK1/2-ERK1/2 axis. Because transactivation of EGFR has been shown to involve the cytosolic tyrosine kinase c-Src in many cases (26), we tested the c-Src inhibitor PP2 (100 nmol/liter) (Fig. 4A). In the presence of PP2, EGF still induced ERK1/2 phosphorylation. However, aldosterone was no longer able to potentiate the effect of EGF. Thus, these data indicate that aldosterone acts via c-Src. Finally, we tried to test the involvement of PKC. Inhibition of PKC by bisindolylmaleimide (100 nmol/liter) (Fig. 4A) induced per se a potentiation of EGF-induced ERK1/2 phosphorylation. These data indicate that there exits a negative feedback loop via PKC with respect to HER1 signaling under our experimental conditions, which is already known (20). This hypothesis is further confirmed by the inhibitory action of PMA on EGFinduced ERK1/2 phosphorylation (Fig. 4A, inset). Be that as it may, inhibition of PKC by bisindolylmaleimide did not prevent the potentiating action of aldosterone with respect to EGFinduced ERK1/2 phosphorylation.
In the last step, we confirmed the hypothesis of c-Src-mediated HER1 stimulation, derived from the pharmacological data, by immunoprecipitation and Western blotting. The data in Fig. 4B show that aldosterone induced HER1 Tyr phosphorylation. Furthermore, aldosterone led to Tyr phosphorylation of c-Src (Fig. 4C), and aldosterone-induced EGFR hyperphosphorylation could be prevented by PP2 (Fig. 4D). Thus, our data support the conclusion that aldosterone potentiates EGF signaling by c-Src-mediated stimulation of HER1. DISCUSSION During the last few years, several reports have shown that steroid hormones such as aldosterone can elicit rapid, so-called alternative, cellular responses. The underlying mechanism(s) for the rapid actions of aldosterone are still not well understood. One hypothesis is based on the interaction of steroid hormones with peptide hormone signaling. For example, the interaction of progesterone with oxytocin signaling has been described (16), as well as the interaction of estradiol or glucocorticoids with growth factor and angiotensin II signaling (17). In the case of aldosterone, an interaction with angiotensin II and vasopressin has been suggested (15,18). We have previously shown that aldosterone enhances EGF signaling with respect to ERK1/2 phosphorylation and Ca 2ϩ homeostasis in MDCK cells (29,35).
Transactivation of EGFR is involved in the transmission of signals triggered by other mediators such as hormones acting via heterotrimeric G-proteins (19). EGFR is therefore considered a transducer of heterologous signaling. Thus, it is conceivable that EGFR also plays a role in alternative aldosterone signaling. The questions addressed in this study were as follows. (i) Is HER1 transfection sufficient to elicit alternative aldosterone responses? (ii) Does this response depend on the FIG. 3. Concentration dependence of the interaction of EGF and aldosterone with respect to ERK1/2 phosphorylation. All data shown were obtained with pERK1/2-ELISA. A, analysis of the time course of the aldosterone-EGF interaction showed that the effect of aldosterone was strongest in the initial phase. Conditions were as follows: 1 g/liter EGF, 10 nM aldosterone, and n ϭ 6 -15. B, aldosterone (10 nmol/liter) led to a leftward shift of the EGF dose-response curve without altering the maximal effect of EGF (n ϭ 6 -15). Application time was 5 min. C, the effect of aldosterone (10 nmol/liter) correlated negatively with EGF-induced ERK1/2 phosphorylation, corresponding to the leftward shift of the dose-response curve shown in B. Application time was 5 min. D, the effect of aldosterone was concentration-dependent, with a maximum at 10 nmol/liter (n ϭ 6 -15). Application time was 5 min. EGF was used at 1 g/liter.
presence of EGF? (iii) Which signaling components are used for the cross-talk between aldosterone and HER1?
Our data show that expression of HER1 plus the addition of EGF were sufficient to reconstitute the alternative signaling network in CHO cells. The observation that the addition of EGF was necessary for the aldosterone effect is in agreement with its action in MDCK cells, where aldosterone stimulates an autocrine EGF activation loop (29,35). Thus, it seems that EGF ϩ EGFR are sufficient to reconstitute the rapid activation of ERK1/2 by aldosterone. The steroid hormone leads to a sensitization of the cells to EGF (leftward shift of the dose-response curve) without enhancing the maximal effect. Cross-talk between signaling systems and EGFR can result from receptor phosphorylation, followed by dimerization and subsequent activation of the receptor kinase (19,26,36). In another mechanistic concept, cross-communication results from the rapid activation of metalloproteinase and cleavage of proheparinbinding EGF-like growth factor (37). Finally, inhibition of phosphatase activity in the presence of high basal receptor kinase activity has been proposed as a mechanism for transactivation (26). Common to all these scenarios is the fact that the addition of exogenous EGF does not seem to be necessary for transactivation. This is in contrast to the action of aldosterone reported here. At present, the detailed underlying processes for the action of aldosterone are not known. Possibly, aldosterone stands for a new scenario in which there is no pure transactivation, but "transmodulation" of EGF actions. However, in cells with an autocrine EGF activation loop such as MDCK cells (29), the addition of aldosterone has the same effect compared with other transactivating stimuli. The concentration ranges used here for aldosterone and EGF occur in physiological and pathophysiological situations, supporting the potential relevance of our findings. The relevance at the cellular level has been demonstrated in MDCK cells, where the aldosterone-induced transactivation of the EGFR signaling network led to enhanced Na ϩ /H ϩ exchange activity and cell proliferation (29,35). Of course, the relevance in vivo has to be demonstrated in further studies using, for example, EGFR kinase inhibitors. It is known that EGFR signaling modulates transepithelial ion transport and stimulates salt reabsorption in certain cell types (21,22). Furthermore, it is known that EGFR signaling may exert profibrotic actions (38). Thus, there are certain similarities with respect to the physiological (salt reabsorption) and pathophysiological (fibrosis) actions of aldosterone and EGF. Therefore, it is conceivable that the interaction of aldosterone with EGFR signaling may support physiological and pathophysiological responses to aldosterone.
The question that now arises is related to the mechanism by which aldosterone interacts with the EGFR signaling network. It has been shown that aldosterone can activate PKC (5). Because PKC can lead to activation of ERK1/2, this pathway is a potential candidate for the interaction of aldosterone and EGF. On the other hand, PKC can also inactivate EGFR signaling. Our data indicate that the later is also true in our system. When PKC was inhibited, the effect of EGF on ERK1/2 phosphorylation was enhanced. In addition, when PKC was stimulated by PMA, the effect of EGF was reduced. Finally, inhibition of PKC did not prevent the action of aldosterone. Thus, PKC does not seem to be the signaling module that links aldosterone to EGF. Another signaling pathway known to be involved in EGFR transactivation is the cytosolic tyrosine kinase c-Src pathway (26,36,39). Activated c-Src then phosphorylates EGFR, increasing its activity. When we inhibited c-Src, the effect of aldosterone was abolished completely, indicating that the steroid indeed acts via c-Src. This hypothesis is further supported by the fact that aldosterone induced c-Src phosphorylation and therefore its activation. From these data, we can derive the following working model (Fig. 5): aldosterone stimulates c-Src, which then co-stimulates EGFR when EGF is present. Subsequently, the Raf-MEK-ERK cascade is activated. Finally, pERK1/2 can elicit various cellular responses, ranging from activation of Na ϩ /H ϩ exchange to gene expression and proliferation (29). Within this signaling network, PKC can exert a 2-fold action: inhibition of EGFR and activation of ERK1/2 phosphorylation. Although we have already shown the cell biological significance of the aldosterone-EGF interaction in MDCK cells (29), where EGFR-dependent alternative aldosterone actions affect Ca 2ϩ homeostasis, Na ϩ /H ϩ exchange, arachidonic acid release, and cell proliferation, it will be necessary in future studies to determine the cell biological consequences in CHO-HER1 cells compared with mock-transfected CHO cells.
Our data presented here help to elucidate some aspects regarding the mechanisms of alternative aldosterone actions. Nevertheless, there are issues remaining that have to be investigated in future studies. Thus, it is not clear at the moment how aldosterone leads to the activation of c-Src. Furthermore, we will have to investigate why the addition of EGF is a prerequisite for unmasking the alternative action of aldosterone. It will also be necessary to determine whether the mineralocorticoid receptor can interact with the signaling network described here. It has recently been proposed that the classical mineralocorticoid receptor may contribute to at least some alternative effects of aldosterone (40). Finally, the importance of the signaling model described here for the classical action of aldosterone (via the mineralocorticoid receptor) will be investigated. This is of special importance considering the widespread expression of EGFR, including cell types known to express the mineralocorticoid receptor such as renal epithelial and cardiac cells. For this purpose, we will used cotransfection of CHO cells with HER1 and the mineralocorticoid receptor to examine alternative and classical aldosterone signaling. In conclusion, our data show that aldosterone requires the EGF-EGFR-MEK1/2-ERK1/2 signaling cascade to elicit its alternative effects and that the EGF-EGFR system is sufficient to reconstitute some effects observed in cells with endogenous EGFR expression.