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Am J Physiol Renal Physiol 282:669-679, 2002. First published Oct 30, 2001; doi:10.1152/ajprenal.00159.2001 You might find this additional information useful... This article has been cited by 6 other HighWire hosted articles, the first 5 are: Aldosterone-induced EGFR expression: interaction between the human mineralocorticoid receptor and the human EGFR promoter C. Grossmann, A. W. Krug, R. Freudinger, S. Mildenberger, K. Voelker and M. Gekle Am J Physiol Endocrinol Metab, June 1, 2007; 292 (6): E1790-E1800. [Abstract] [Full Text] [PDF] Transactivation of the IGF-1R by aldosterone J. L. Holzman, L. Liu, B. J. Duke, A. E. Kemendy and D. C. Eaton Am J Physiol Renal Physiol, April 1, 2007; 292 (4): F1219-F1228. [Abstract] [Full Text] [PDF] Nongenomic Actions of Aldosterone on the Renal Tubule D. W. Good Hypertension, April 1, 2007; 49 (4): 728-739. [Full Text] [PDF] Involvement of Aldosterone and Mineralocorticoid Receptors in Rat Mesangial Cell Proliferation and Deformability A. Nishiyama, L. Yao, Y. Fan, M. Kyaw, N. Kataoka, K. Hashimoto, Y. Nagai, E. Nakamura, M. Yoshizumi, T. Shokoji, S. Kimura, H. Kiyomoto, K. Tsujioka, M. Kohno, T. Tamaki, F. Kajiya and Y. Abe Hypertension, April 1, 2005; 45 (4): 710-716. [Abstract] [Full Text] [PDF] Evidence for epidermal growth factor receptor as negative-feedback control in aldosterone-induced Na+ reabsorption C. Grossmann, R. Freudinger, S. Mildenberger, A. W. Krug and M. Gekle Am J Physiol Renal Physiol, June 1, 2004; 286 (6): F1226-F1231. [Abstract] [Full Text] [PDF] Updated information and services including high-resolution figures, can be found at: http://ajprenal.physiology.org/cgi/content/full/282/4/F669 Additional material and information about AJP - Renal Physiology can be found at: http://www.the-aps.org/publications/ajprenal
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AJP - Renal Physiology publishes original manuscripts on a broad range of subjects relating to the kidney, urinary tract, and their respective cells and vasculature, as well as to the control of body fluid volume and composition. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2005 by the American Physiological Society. ISSN: 0363-6127, ESSN: 1522-1466. Visit our website at http://www.the-aps.org/.
1152/ajprenal. we tested the hypothesis that aldosterone uses the EGF-R as a heterologous signal transducer in MDCK cells.C. It has been suggested that steroids interact with peptide hormones. epidermal growth factor. 48). intracellular generation of inositol 1. it has been shown that steroid hormones are able to interact with peptide hormone signaling (35. Some of these effects have been shown to be incompatible with the classic genomic pathway. 10. differentiation. cellular Ca2 concentration. Furthermore. SIGRID MILDENBERGER. which is a behavior similar to that of growth factors (11. 2008 Address for reprint requests and other correspondence: M. Rapid.e. by rapid. accepted in ﬁnal form 23 October 2001 Gekle.gekle@mail. 2002. as well as the interaction of estradiol with growth factor and angiotensin II signaling (28). The costs of publication of this article were defrayed in part by the payment of page charges. potentially nongenomic. Former studies also revealed that aldosterone acts within several minutes on plasma membrane K conductance of different cells (29. First published October 30. i. transcription. G protein-coupled receptors.2001. 46). we have shown that aldosterone increases H afﬁnity of Na /H exchange in MDCK-C11 cells by means of ERK1/2. and cytokines by means of a mechanism called transactivation (19. and protein synthesis.00159. F669 Downloaded from ajprenal.org 0363-6127/02 $5. 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. 50). 11. and Na /H exchange activity similar to increases induced by EGF. Moreover. Madin-Darby canine kidney (MDCK)-C11 cells (12). Furthermore.de). aldosterone. In the case of aldosterone. Section 1734 solely to indicate this fact.physiology. the EGF-receptor (EGF-R) is involved in signaling by G protein-coupled receptors. Thus it is conceivable that the EGF-R represents a pathway involved in rapid aldosterone signaling similar to.org on February 28. 2001.5-trisphosphate (IP3) (5). Ruth Freudinger. Our data show that aldosterone uses the EGF-R-ERK1/2 signaling cascade to elicit its rapid effects in MDCK cells. Rontgenring 9. G protein-coupled receptors. 97070 ¨t ¨ ¨ Wurzburg. Nanomolar concentrations of aldosterone induce a rapid increase in ERK1/2 phosphorylation. and there are several reports supporting the existence of such actions. 12. Furthermore. 2002. calcium. These rapid actions of aldosterone are thought to be mediated by a plasma membrane receptor (47). 44).00 Copyright © 2002 the American Physiological Society . Aldosterone interaction with epidermal growth factor receptor signaling in MDCK cells MICHAEL GEKLE. In the present study. 9. The precise underlying mechanism and the physiological or pathophysiological signiﬁcance are not yet understood. and extracellular signal-regulated kinase (ERK)1/2 phosphorylation (9. 43. Universita Wurzburg.00159. Aldosterone interaction with epidermal growth factor receptor signaling in MDCK cells. Michael. 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. Gekle. and cytokines by means of transactivation. Germany ¨ ¨ Received 18 May 2001. 30. the EGF-R can serve as a central transducer of heterologous signaling systems.S. We determined the possible interaction with epidermal growth factor (EGF) receptor (EGF-R).1152/ajprenal. Here. Am J Physiol Renal Physiol 282: F669–F679. in part. extracellular signalregulated kinase 1/2. for example. and Stefan Silbernagl. Recently.ajprenal. 30.Am J Physiol Renal Physiol 282: F669–F679. whereas prevention of Ca2 inﬂux did not abolish ERK1/2 phosphorylation. an interaction with angiotensin II and vasopressin has been suggested (35. 2001. mechanisms. 37). 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. and inhibition of EGF-R kinase abolished aldosterone-induced signaling. a transcription-independent interaction of glucocorticoids with EGF-R has been reported (7). aldosterone induced a rapid increase in EGF-R-Tyr phosphorylation. although this putative receptor has not been identiﬁed. 27).—Epidermal growth factor (EGF) regulates cell proliferation. Previously. 97070 Wurzburg. 41). the interaction of progesterone with oxytocin signaling (17). The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U. has been described. growth hormone. protein kinase C. University of Wurzburg. growth hormone. The EGF-R has been shown to be involved in signaling events elicited by.4. actions of steroids have recently been investigated more extensively. and ion transport by using extracellular signal-regulated kinase (ERK)1/2 as a downstream signal. aldosterone can also induce rapid responses by interfering with mechanisms of regulation of intracellular pH or Ca2 (6. Madin-Darby canine kidney cells THE CLASSIC GENOMIC MECHANISM of steroid hormone action involves binding to intracellular receptors. for example. Physiologisches Institut. nongenomic way to aldosterone. potentially nongenomic. Therefore. Inhibition of ERK1/2 phosphorylation reduced the Ca2 response. 26. ¨ http://www. RUTH FREUDINGER. modulation of peptide hormone signaling. 10. However. Sigrid Mildenberger. First published October 30. For example. AND STEFAN SILBERNAGL Physiologisches Institut.uni-wuerzburg. Germany (E-mail: michael.. 2001.
45). The coverslips were transferred to the stage of an inverted Axiovert 100 TV microscope (Zeiss) and allowed to www. it has been shown that EGF does affect epithelial salt transport in a cell-speciﬁc manner. Wallac. Beverly. Possibly. the cells were ﬁxed with 4% formaldehyde in PBS for 20 min at room temperature and washed three times with PBS containing 0. Germany).1 mM phenylmethylsulfonyl ﬂuoride. the coverslips were mounted on the microscope stage. After the wells were dried for 5 min. and incubated overnight with the AJP-Renal Physiol • VOL above described primary antibody (see Western blot analysis. and phosphorylated EGF-R was detected by using anti-phospho-Tyr antibody (PY99). 27). 22. In addition.F670 EGF-R AND ALDOSTERONE EGF regulates cell proliferation. and 0. (18) by using a dissociation constant (Kd) of 225 nmol/l. and transferred to a polyvinylidene diﬂuoride microporous membrane. Oberkochen. uses mitogen-activated protein kinases as downstream signals. The next day. separated on SDS-PAGE. which has been cloned recently in our laboratory (14).6% H2O2 in PBS/Triton X-100 for 20 min.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. Immune complexes were collected by centrifugation. Ca2 homeostasis. Our results show that aldosterone uses the EGF-R-ERK1/2 signaling pathway to elicit its rapid effects on ERK1/2 phosphorylation.org Downloaded from ajprenal. (42). after subtraction of background ﬂuorescence. 36. we compared the effect of EGF determined by Western blot and pERK1/2-ELISA and found no signiﬁcant difference. Cytosolic free Ca2 was determined by using the Ca2 -sensitive dye fura 2 (5 mol/l. as described previously (12. Finland). and pH homeostasis in MDCK cells. dilution 1:1. Furthermore. anti-EGF-R antibody [clone Ab-5 (Calbiochem-Novabiochem). 5 mM EDTA. and 100 l of 1% SDS solution were added and incubated on a shaker for 1 h at room temperature. and tissue repair and. The primary antibody was detected by using horseradish peroxidase-conjugated secondary IgG visualized by the Amersham enhanced chemiluminescence system. the data could be compared directly and were pooled. 31. CA). For the quantiﬁcation of ERK1/2 phosphorylation. the cells were incubated with 50 l of a solution containing 0. Anti-phospho-ERK1/2 antibody only detects ERK1 and ERK2 when catalytically activated by phosphorylation at Thr202 and Tyr204. in MEM supplemented with 10% fetal calf serum at 37°C and 5% CO2.000 g for 15 min at 4°C. the EGF-R represents a membrane target for rapid effects of aldosterone. Cell lysates were matched for protein. The ﬂuorescence signal was monitored at 510 nm. 40 mg/l bestatin. METHODS Cell culture. We used a subtype of MDCK cells. enhanced EGF signaling has been observed in several tumor cells (19. Molecular Probes. by using a 100-W xenon lamp.015% H2O2 for 15 min at room temperature in the dark.ajprenal. multilabel counter (Victor2. The Netherlands) as described previously (12) and by using an inverted Axiovert 100 TV microscope ( 400 magniﬁcation. the cells were cultivated either on permeable supports (Becton Dickinson. Linearity of the signal has been veriﬁed by serial dilution. Cells were washed three times in PBS/Triton X-100. after serum depletion for 24 h. and subjected to SDS/8%-PAGE. 33). 7.05 (Jandel. Brieﬂy. 2 mg/l aprotinin. blocked with 10% fetal calf serum in PBS/Triton X-100 for 1 h.8 mg/ml Na2HPO4. Possible artifacts were excluded by measurement of autoﬂuorescence of the different substances without fura 2. oil immersion. Then. Densitometric analysis was performed by using SigmaGel 1. In brief. Densitometric analysis was performed by using SigmaGel 1. 23. 100 mM NaCl. Finally. as recommended by the manufacturer. Nunc) and serum starved for 24 h before the experiment. Membranes were subsequently blotted with a rabbit anti-phospho-ERK1/2 antibody (New England Biolabs. 2008 282 • APRIL 2002 • . Determination of cytosolic pH. denominated C11 (MDCK-C11). Endogenous peroxidase was quenched with 0. 14. 1 M pepstatin A. Serum was removed from the media 24 h before the experiment. 10 g/mg protein] was added for 2 h. Immunoprecipitation. washed three times with lysis buffer.3 mg/ml citric acid. Germany) and an automatic ﬁlter change device (Hamamatsu. Immunoprecipitation was performed as described recently (34). The sampling rate was one ratio every 2 s. Corte Madera. The maximum and minimum ratios (Rmax and Rmin) were measured after addition of calibration solutions. Heidelberg. the cells were washed four times with demineralized water. cell lysates were precleared with protein A/G-Sepharose for 20 min at 4°C. In control experiments. 11. Thus the results obtained by Western blot and pERK1/2-ELISA were pooled. and 1% Triton X-100) for 25 min at 4°C.4 mg/ml O-phenylenediamine. differentiation. 1 g/ml leupeptin. Because the effects were not statistically different for the three conditions. Herrsching. at least in part. the absorbance was measured at 595 nm with the ELISA reader. Subsequently. CA). we performed pERK1/2-ELISA according to Versteeg et al. 1:500) in PBS/Triton X-100 containing 5% BSA at 4°C. 200 M sodium-orthovanadate. Leiden. cells were each incubated with MEM containing 2 mol/l 2 . Subsequently. To precipitate the EGF-R. Ref. Molecular Probes) as described previously (12) and with the setup described in Determination of cytosolic Ca2 . Insoluble material was removed by centrifugation at 12. 0. Turku. After the peroxidase reaction.5. cells were incubated with HEPES-Ringer containing fura 2 acetoxymethyl ester in a ﬁnal concentration of 5 mol/l for 15 min. the cells were washed twice with PBS/Triton X-100 and twice with demineralized water. After stimulation. In brief. Quantiﬁcation of ERK1/2 phosphorylation by ELISA.05. Santa Cruz Biotechnology.1% Triton X-100. Subsequently.2% in PBS) were added for 5 min at room temperature. with the excitation wavelength alternating between 334 and 380 nm. Cells were cultivated. the coverslips were rinsed four times with superfusion solution to remove the dye-containing medium. Western blot analysis. leading to either enhanced or reduced salt reabsorption (8. Zeiss. Determination of cytosolic Ca2 . 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. MA) or with anti-phosphoTyr-antibody (PY99.org on February 28.7 -bis(2-carboxyethyl)-5(6)-carboxyﬂuorescein acetoxymethyl ester for 5 min.physiology. Cells were seeded in 96-well plates (Maxisorp. cells were washed three times with PBS/Triton X-100 for 5 min and incubated with secondary antibody (peroxidase-conjugated mouse anti-rabbit antibody. Intracellular pH of single cells was determined by using the pH-sensitive dye 2 . Germany) in 96well plates [for phosphorylated ERK (pERK)1/2-ELISA] or on poly-L-lysine-coated glass coverslips (for Ca2 and pH measurements). followed by overnight incubation with protein A/G-Sepharose. The resulting signal was detected at 490 nm with a multiwell. Santa Cruz. which contained 1 mol/l ionomycin and 1 mmol/l Ca2 to determine Rmax or 1 mmol/l EGTA and no Ca2 to determine Rmin. 100 l of trypan blue solution (0. Cytosolic Ca2 concentration ([Ca2 ]i) was calculated according to Grynkiewicz et al.7 bis(2-carboxyethyl)-5(6)-carboxyﬂuorescein (2 mol/l. For the experiments presented.
1A. Control Ringer solution was composed of (in mmol/l) 130. Differences were considered signiﬁAJP-Renal Physiol • VOL cant if P 0. H. The calibration solutions contained 115 mmol/l KCl and 30 mmol/l NaCl. 1.EGF-R AND ALDOSTERONE F671 equilibrate for 15 min. Cells were seeded on plastic dishes.8 to 7. Materials. and lysates were applied to ion exchange columns prepared as follows. Thus the cells are already in a certain state of preactivation. therefore. apical or basolateral.0 NaCl. Dowex-AG 1X-8 (0.and EGF-induced phosphorylation of ERK1/2. Germany). we do not know whether these data mean that aldosterone acts within the cells or just rapidly crosses the monolayer. Eluted IP1 –IP3 were collected into scintillation vials. To verify the contribution of phospholipase A2 (PLA2). 24). Cytosolic Ca2 . Cell number was determined by using a Z2 series Coulter Counter. indicating that both substances use the EGF-R for signaling. Mitogenic factors. Louis. there was only a slight difference for aldosterone but a marked difference for EGF. A and C. hydrocortisone (10 nmol/l) did not affect ERK1/2-phosphorylation (Fig. inositol 1.0 M ammonium formate (IP3). the media were removed. After background subtraction. cells were labeled for 24 h with 0. Signiﬁcance of difference was tested by paired or unpaired Student’s t-test or ANOVA. cells seeded in six-well plates were preincubated during 24 h in media containing 0. 2008 282 • APRIL 2002 • . as described by us previously for aldosterone (11). and aliquots were taken after 5 and 15 min to determine the amount of radioactivity released. and made quiescent before the experiments by 24-h serum removal. After 15 min of incubation. 10 nmol/l aldosterone and 10 g/l EGF stimulate ERK1/2 phosphorylation in MDCK-C11 cells. 100 nmol/l 4-(3-bromoanilino)6. Figure 2 shows that 10 nmol/l aldosterone or 10 g/l EGF induced a small but comparable increase in [Ca2 ]i. 0. 10 HEPES.1 M formic acid/0. corresponding to 100 www. St. This medium was replaced with 1 ml HEPES-Ringer 15 mM LiCl. As shown in Fig. MO) technique (39. this was indeed the case. The data-acquisition rate was one ﬂuorescence intensity ratio every 2 s. Cells from at least two different passages were used for each experimental series.0 CaCl2. 40).4). 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.000 cells/cm2).000).physiology. We did not observe signiﬁcant differences. Germany). St. grown to subconﬂuence ( 40. Arachidonic acid release was performed as described elsewhere (4.5 g. 1.8. because in a previous study it was shown that there was no difference between 5.4 M ammonium formate (IP2). radioactive medium is aspirated.or 10-min exposure (11).1 M formic acid/0. pH calibration was performed after each experiment by the nigericin (Sigma. 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). 1D). At the end of the experiment. 1.1 M formic acid/ 1. 5. MO). such as EGF. IP3 formation. 1. The emitted light was ﬁltered through a bandpass ﬁlter (515–565 nm). n represents the number of cells or tissue culture dish investigated.7-dimethoxyquinazoline] (3) or tyrphostin AG1478 (100 nmol/l) reduced the aldosterone.4 at 37°C). 1-ml aliquots of control Ringer 15 mM LiCl with the desired agonists were added. Unless otherwise stated. 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. all materials were from Sigma (Munich. RESULTS ERK1/2 phosphorylation. show that there is an autocrine activation loop of the EGF-R signaling cascade in MDCK-C11 cells. plus the respective vehicles (ethanol or DMSO 1‰). and the data were pooled with those from cells grown on permeable supports.org on February 28. B and C. ﬂuorescence intensity ratios were calculated. The time of exposure was 10 min. the data in Fig. B and C. and IP3 were eluted by subsequent addition of 2 ml of 0. 1.0 MgCl2. Arachidonic acid release. By contrast to aldosterone. and 0. and counted. we compared the effect of aldosterone on ﬁlter-grown cells. 1. 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. the experimental Ringer solutions were added. Furthermore. and an aliquot was counted to determine the extent of incorporation (85–90% of arachidonic acid added to the media). As shown in Fig. Values are means SE.5 Ci/ml [3H]inositol. As shown in Fig. as already shown. In brief. Control solutions always contained the appropriate amount of vehicle (ethanol or DMSO 1:1. 49). Subsequently. mixed with 10 ml of scintillation cocktail.org Downloaded from ajprenal. These data again show that the vehicle (ethanol) in which the steroids were dissolved is not responsible for the observed effects. the observed effects are smaller compared with cells that are completely silent. as observed previously (11). at least ﬁve coverslips were investigated for each experimental condition. most of the experiments were performed with cells cultivated on solid supports. and 5 glucose (pH 7. Determination of cell number.2 M ammonium formate (IP1). the cells were lysed with 1 mol/l NaOH. by using at least three calibration solutions in the range from pH 6.5 Ci of [3H]arachidonic acid (ARC. To determine whether the side of aldosterone application. The excitation light source was a 100-W mercury lamp. we tested the inhibitory action of arachidonyl triﬂuoromethyl ketone (38). Therefore. 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. The release of radioactivity into the Ringer solution was determined as the percentage of total incorporated radioactivity. Louis. Ethylisopropyl amiloride was kindly provided by Dr. Statistics.4 KCl. cells were lysed with 1 ml of 4 mM EDTA/1% SDS (90°C). J. The excitation wavelengths were 450 nm/490 nm. and then inositol 4-monophosphate (IP1). might be important. and after 10 min of incubation at 37°C.ajprenal. Lang from Aventis (Frankfurt.05.0 NaH2PO4.4-bisphosphate (IP2). as applicable. Before experimentation. In brief. Applied samples are washed with 2-ml aliquots of H2O and 5 mM disodium tetraborate/60 mM sodium formate. formate form) was laid into 5-ml pipette tips with cotton wool on the bottom. Under control conditions. because EGF-R-kinase inhibition was effective in controls (36). For pH determinations. Finally. We tested whether inhibition of the EGF-R-kinase by compound 56 [c56. [Ca2 ]i was 91 20 nmol/l (n 200). At present. IP3 formation was determined by anion exchange columns. as described previously (32).
inhibition of EGF-R kinase with compound 56 (100 nmol/l) signiﬁ- Fig. as already described (36). C: summary of aldosterone.ajprenal. These aldosterone-induced changes in [Ca2 ]i ( [Ca2 ]i) are in agreement with previously reported changes for MDCK-C11 or skin cells (12. A: EGF (10 g/l) or aldosterone alone (10 nmol/l) exerts a slight increase in baseline cytosolic Ca2 concentration [Ca2 ]i. however. n 60– 120 for each bar. Exposure time was 10 min. control. Exposure time 10 min. leading to certain background phosphorylation of ERK1/2. respectively. * P 0. Data indicate that there is an autocrine activation loop of EGF-R.05 vs. aldo. Aldosterone.(10 nmol/l) and EGF-induced (10 g/l) ERK1/2 phophorylation.or 10-min exposure was identical (11). B: inhibition of the EGF-receptor (EGF-R) kinase with compound 56 (c56.and EGF-induced Ca2 signaling. 10 nmol/l) did not affect ERK1/2 phosphorylation. 2008 Fig. A: aldosterone (10 nmol/l) or EGF (10 g/l) induce phosphorylation of ERK1/2. Representative blot of 2 experiments is shown. 21).F672 EGF-R AND ALDOSTERONE Downloaded from ajprenal. nmol/l.org on February 28. Previous experiments showed that the effect during 5. cells grown in 96-well plates behaved identically to cells grown on permeable supports. for example (12). con. Representative blot from 2 individual experiments is shown. The data show that besides c56 another inhibitor of the EGF-R kinase. Aldosterone. 100 nmol/l) prevents ERK1/2 phosphorylation. Similar to the effects on ERK1/2 phosphorylation. Al- though the changes in MDCK-C11 cells are small.physiology.05 vs. basolateral comparison of the effect of the addition of 10 nmol/l aldosterone on ERK1/2 phosphorylation. in M-1 cortical collecting duct cells (20). Apical vs. c56 (100 nmol/l). 1. Exposure time was 10 min. for example. D: hydrocortisone (hydro. it has been shown that they contribute to Na /H -exchange activation. n 6 for each bar. Data from Western blots and phosphorylated ERK (pERK)1/2-ELISA were pooled. reduces the Ca2 changes signiﬁcantly. 2. The inhibitor of EGF-R kinase. tyrphostin AG-1478 (100 nmol/l). * P 0. Control.and epidermal growth factor (EGF)-induced extracellular signal-regulated kinase (ERK)1/2 phosphorylation.org . B: summary of the Ca2 changes elicited by aldosterone or EGF. Furthermore. aldosterone or EGF. Aldosterone. AJP-Renal Physiol • VOL 282 • APRIL 2002 • www. because they were not different. also abolishes the effects of aldosterone and EGF. they are smaller compared with changes observed.
These data indicate that ERK1/2 phosphorylation is upstream of the Ca2 inﬂux. a concentration that prevented ERK1/2 phosphorylation. 4D). Figure 3A again shows that there is a certain degree of autocrine activation of the EGF-R in MDCK cells.and EGFinduced Ca2 signaling did not affect ERK1/2 phosphorylation (Fig. When 100 g/l EGF was used. control. 4. aldosterone increased the effect of 10 g/l EGF on ERK1/2 phosphorylation (stimulation time 10 min for all experiments). 3. the extent of EGF-R-phosphorylation was enhanced. When EGF and aldosterone Fig. 4B. pEGFR. In addition to its own effect on ERK1/2 phosphorylation. we determined the effects of aldosterone EGF on ERK1/2 phosphorylation and Ca2 . and can be quantitated reliably by ELISA (see METHODS). As shown in Fig. n 75). except EGF-R phosphorylation. phosphorylated EGF-R. we observed a Ca2 peak in the majority of the cells before a new plateau was reached. A: aldosterone (10 nmol/l) and EGF (10 g/l) enhance EGF-R phosphorylation (10-min exposure). To rule out the possibility that the EGF-R inhibitors. Moreover. we determined the ERK1/2 phosphorylation dose-response curve for EGF and EGF aldosterone. indicating the dependence on extracellular Ca2 . C and D). An enhancement of the Ca2 changes was also observed with 1 nmol/l aldosterone ( Ca2 for 1 nmol/l aldosterone 10 g/l EGF 350 56 nmol/l. aldosterone and EGF enhanced Tyr phosphorylation of the EGF-R. 5B). B: summary of EGF-R phosphorylation from 4 individual experiments. Thus the effects observed cannot be explained by the vehicles. A and B. 4D). aldosterone enhances the EGFinduced Tyr phosphorylation. aldosterone then EGF or EGF then aldosterone) had no signiﬁcant effect on the observed effects. aldosterone or EGF did not induce a substantial increase in IP3 formation.. we determined whether these substances affected the bradykinin-induced signal. Furthermore. Inhibition of ERK1/2 phosphorylation with PD-98059 (25 mol/l.physiology. 5C). Exposure time 10 min. Because ERK1/2 phosphorylation seems to be the signal upstream of all other events investigated in this study.100 nmol/l. as already described for Madin-Darby canine kidney cells (36) and is representative of 4 experiments. the changes in [Ca2 ]i were almost completely abrogated (Fig. In the presence of 10 mol/l La3 or when extracellular [Ca2 ] was lowered to 5 mol/l. As shown in Fig. By contrast.EGF-R AND ALDOSTERONE F673 cantly reduced the [Ca2 ]i induced by aldosterone or EGF. As already mentioned. reduced signaling through toxic effects. 4A. 5A. which is in agreement with the effects observed for ERK1/2 phosphorylation. there was a shift to the left of the dose-response curve (factor 5–10) when aldosterone and EGF were added simultaneously.org on February 28. The order of application (i. These data indicate that the EGF-R inhibitors did not act by means of toxic impairment of signaling. 3. To determine whether this enhancement also affected downstream signaling. both substances did not affect the Ca2 rise induced by 100 nmol/l bradykinin. all control solutions contained the respective vehicles (either ethanol or DMSO 1‰). compared with EGF alone.ajprenal.e. Interaction of aldosterone and EGF.org Downloaded from ajprenal.(36). the enhancement of the Ca2 response by aldosterone was substantially smaller compared with the response when using 10 g/l EGF (Fig. as shown in Fig. we performed EGF-R immunoprecipitation (stimulation time 10 min for all experiments). Aldosterone action depends on EGF-R-kinase activity. [Ca2 ]i ( 400–500 nmol/l) values were signiﬁcantly greater when compared with the sum of the individual effects (Fig. most probably involving transforming growth factor (TGF). Aldosterone and EGF-induced EGF-R phosphorylation.05 vs. Blot also shows that there is a certain degree of autocrine phosphorylation of the EGF-R. which induced a Ca2 spike of 1. data not shown) reduced the Ca2 signal signiﬁcantly (Fig. ATP (100 mol/l) was used as a positive control. When EGF (10 g/l) was added in the presence of aldosterone. As shown in Fig. * P 0. we determined the effect of aldosterone and EGF on IP3 formation. To determine whether the appearance of the Ca2 peak was the result of enhanced IP3 formation. lowering extracellular Ca2 to prevent aldosterone. www. AJP-Renal Physiol • VOL were added together. To determine whether Tyr phosphorylation of the EGF-R is affected by aldosterone and EGF. indicating that aldosterone did not induce a change in the maximum Ca2 response. However. although used at nanomolar concentrations. Thus aldosterone and EGF increase Ca2 by means of EGF-R signaling. ERK1/2 phosphorylation under these conditions was again prevented by EGF-R kinase blockade. 2008 282 • APRIL 2002 • .
* P 0. A: dose-response curve of ERK1/2 phosphorylation.org 282 • APRIL 2002 • . 2). 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. n 5–10. tyrphostin AG-1478 (tyropho.physiology. bradykiAJP-Renal Physiol • VOL nin (100 nmol/l) induced a threefold increase in arachidonic acid-derived radioactivity release. C: addition of EGF (10 g/l) in the presence of aldosterone (10 nM) led to an enhanced change in cytosolic Ca2 concentration ([Ca2 ]i compared with the addition of EGF alone (compare with Fig. Furthermore. La3 10 mol/l LaCl3. Interaction of EGF and aldosterone of ERK1/2 phosphorylation and Ca2 signaling. Inhibition of PLA2 with 25 mol/l arachidonyl triﬂuoromethyl ketone prevented the stimulation of radioactivity release (Fig. 100 nmol/l).05 vs.F674 EGF-R AND ALDOSTERONE Downloaded from ajprenal. Order of application (i. we observed a Ca2 peak in the majority of the cells before the plateau was reached.e.ajprenal. or c56 (100 nmol/l). respective control. indicating that the experimental setup worked. we investigated arachidonic acid release and cytosolic pH. 6A). Bars. Thus the observed www.org on February 28. Exposure time 10 min. B: ERK1/2 phosphorylation induced by aldosterone EGF is prevented by inhibitors of EGF-R kinase. low Ca2 Ca2 5 mol/l. means of 55–120 cells from at least 4 coverslips from at least 2 different passages. indicating a synergistic action of the two.. Simultaneous addition of EGF and aldosterone resulted in a signiﬁcantly increased release. D: summary of the maximum changes in cytosolic Ca2 . Addition of EGF in the presence of 10 nmol/l aldosterone led to a shift to the left of the EGF dose-response curve. 6A). 2008 Fig. aldosterone then EGF or EGF then aldosterone) had no signiﬁcant effect on the observed effects. By contrast. c56 100 nmol/l. there was only a very small increase of arachidonic acid-derived radioactivity release observable (Fig. 4. When EGF (10 g/l) or aldosterone (10 nmol/l) were each added to the cells.
A: aldosterone (10 nmol/l) or EGF (10 g/l) do not stimulate inositol-1. C: prevention of changes in cytosolic Ca2 by incubation in low-Ca2 solution ( 5 mol/l Ca2 ) did not reduce ERK1/2 phosphorylation. Order of application (i. In the presence of aldosterone EGF.. it resulted from Na /H -exchange activation. aldosterone then EGF or EGF then aldosterone) had no signiﬁcant effect on the observed effects. 282 • APRIL 2002 • www.05 vs. n 6 for each bar.EGF-R AND ALDOSTERONE F675 Downloaded from ajprenal. 5.org on February 28. B: aldosterone (10 nmol/l) or EGF (10 g/l) induced a comparable cytosolic alkalinization ( pH). Exposure time 10 min. control. alkalinization was signiﬁcantly enhanced. Because the alkalinization was almost completely prevented by 10 mol/l ethylisopropyl amiloride. ATP (100 mol/l) was used as a positive control. * P 0. arachidonyl triﬂuoromethyl ketone (AACOCF. The release was abolished by the inhibitor of phospholipase A2. Mechanism of Ca2 increase. 2008 Fig. n 4). 100 nmol/l) served as a positive control. Representative blot from 2 individual experiments. A: arachidonic acid release is signiﬁcantly greater when aldosterone (10 nmol/l) and EGF (10 g/l) are simultaneously present compared with the situation when each substance is added alone. as was the case when extracellular Ca2 was lowered (102 10% of control.e. release of radioactivity can be attributed to PLA2 activity. 6.ajprenal. n 4).org .5-trisphosphate (IP3) formation. n 35–50.4. Inhibition of ERK1/2 phosphorylation with 25 mol/l PD-98059 also prevented stimulation of arachidonic acid release (110 8% of control. Arachidonic acid (AA) release and cytosolic pH. AJP-Renal Physiol • VOL Fig. 25 mol/l). Bradykinin (BK. B: inhibition of ERK1/2 phosphorylation by 25 mol/l PD-98059 (PD) reduced the Ca2 signal ( Ca2 ) elicited by aldosterone (10 nmol/l) or aldosterone EGF signiﬁcantly.physiology. n 6 for each bar. n 60 for each bar.
Thus any increase in the number of cells under experimental conditions must reﬂect cell proliferation. as well as the interaction of estradiol or glucocorticoids with growth factor and angiotensin II signaling.011. such as aldosterone. n 75). n 30). When EGF-R kinase was inhibited. DISCUSSION During the last several years. When saturating concentrations of EGF were used (100 g/l). As shown in Fig. A: subsequent 48-h incubation in serum-free media did not signiﬁcantly alter the number of cells.06. a behavior similar to the action of growth factors such as EGF (11.021 0. These data allow the hypothesis that aldosterone also “uses” the EGF-R as a transducer of heterologous signaling. hormones acting by means of heterotrimeric G proteins (19). 2008 Fig. we have shown that aldosterone increases H afﬁnity of Na /H exchange in MDCK-C11 cells by means of ERK1/2. (10) 10 g/l. 48). when aldosterone (10 nmol/l) was added together with EGF (10 g/l). Effects of aldosterone and EGF on cell proliferation. * P 0. the effects of aldosterone were signiﬁcantly smaller (cytosolic Ca2 ) or even completely abolished (ERK1/2 phosphorylation). Transactivation of EGF-R is involved in the transmission of signals triggered by other mediators. EGF exerted a slight proliferative action in MDCK-C11 cells. no signiﬁcant alkalinization could be observed in the presence of 25 mol/l PD-98059 ( pH 0. If aldosterone acts on cytosolic Ca2 and ERK1/2 phosphorylation by means of the EGF-R kinase. Thus we investigated whether aldosterone modulates the proliferative action of EGF. can elicit rapid ( 10 min). the number of cells remained virtually constant under control conditions during the 48-h incubation period. cytosolic Ca2 .physiology. Cell proliferation. Previously. Thus it is conceivable that EGF-R also plays a role in the integration of rapid steroid signaling.org on February 28. As shown for ERK1/2 phosphorylation and cytosolic Ca2 .013. n 9 for each bar. 44). 6B).040 0. an interaction with angiotensin II and vasopressin has been suggested (35. as already shown for other hormones (19). cellular responses. AJP-Renal Physiol • VOL 282 • APRIL 2002 • www. for example. 10 g/l EGF led to an alkalinization similar to that when aldosterone (10 nmol/l) was used (pH under control conditions was 7.ajprenal. EGF-R phosphorylation was enhanced in Downloaded from ajprenal.F676 EGF-R AND ALDOSTERONE Previously. (100) 100 g/l.22 0. Again. In the case of aldosterone. 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. 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. 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. When added alone at 10 nmol/l. EGF acts as mitogen in many different cell types. However. has been described (17. The Na /H -exchange inhibitor ethylisopropyl amiloride (10 mol/l) prevented alkalinization almost completely (Fig. several reports showed that steroid hormones. 28). As shown here. control. Figure 7A shows the effect of EGF on the number of cells. and pH homeostasis in a manner very similar to the effects observed during EGF exposure. Cells were made quiescent by 24-h incubation in serum-free media. The underlying mechanism(s) for the rapid actions of aldosterone are still unknown. aldosterone had no signiﬁcant effect. At 10 g/l. 7A. we have shown that aldosterone stimulates Na /H exchange in MDCK-C11 cells by means of ERK1/2 and leads to cytosolic alkalinization (11).org . the interaction of progesterone with oxytocin signaling. this was indeed the case. 7. These data indicate that aldosterone and EGF act on Na /H exchange by means of ERK1/2 and Ca2 . The results presented here show that aldosterone acts on ERK1/2 phosphorylation. For example. there was a clear potentiation of the proliferative effect. One hypothesis is based on the interaction of steroid hormones with peptide hormone signaling. we would also expect that aldosterone leads to enhanced EGF-R phosphorylation. The EGF-R is therefore considered a transducer of heterologous signaling. As shown in Fig. aldosterone had no further effect. Moreover. then we should expect that the effects of aldosterone are reduced when the EGF-R kinase is inhibited. 6B. aldosterone enhanced the effect of EGF. potentially nongenomic. n 30) or when Ca2 inﬂux was prevented ( pH 0. If this hypothesis is true.05 vs. B: 48-h incubation with 10 g/l EGF induced a slight increase in the number that was potentiated signiﬁcantly by 10 nmol/l aldosterone.
Alternatively. known to be exwww. 2008 282 • APRIL 2002 • . Aldosterone activates EGF-R activation by an unknown mechanism. A similar mechanism for steroid-hormone and peptide-hormone cross talk has been proposed for progesterone and oxytocin (17). either additively or in terms of a potentiation of EGF. Another possible mechanism is the involvement of additional factors.. there is always a certain preactivation under control conditions. ERK1/2 and Ca2 do then transmit the aldosterone signal to further downstream events. tyrosine kinase. Several years ago. This interpretation would also explain the enhanced EGF-induced response with respect to the Ca2 signal. 15). Ca2 entry across the plasma membrane is increased (Fig. As shown in Fig. which is similar to results obtained in other cell types (46). Subsequently. phospholipase A2. This conclusion is based on the observations that prevention of Ca2 entry did not abrogate ERK1/2 phosphorylation. although more detailed investigations have to be performed in future studies. 8. Src kinase.physiology. thereby sensitizing the cells for EGF. Changes in the lipid environment could also affect the activation of a membrane protein such as EGF-R (1). as already described (36). aldosterone and EGF seem not to act additively in a simple manner or with respect to maximum ERK1/2 phosphorylation. aldosterone would have to activate the additional factor directly or by means of an aldosterone receptor. Na /H -exchange activation and arachidonic acid release. TRK. Aldosterone and EGF could interact in a simple additive manner. with no change in the maximum effect when both are added simultaneously. which argues against the involvement of phospholipase C and sub- Fig. the cells seem to respond more sensitively to EGF when aldosterone is present compared with the situation where aldosterone is absent. In this case. 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. in patients with liver cirrhosis) may elicit part of its effects by means of an interaction with EGF-R. and future studies will have to focus on the underlying mechanisms. Thus it is unlikely that the interaction of aldosterone with EGF-R depends on the mineralocorticoid receptor. as in Na / H -exchange activation (11. Finally. at least in the cell system investigated here.org Downloaded from ajprenal.. Thus at submaximum concentrations of EGF. inhibition of EGF-R Tyr kinase signiﬁcantly reduced ERK1/2 phosphorylation and the Ca2 signal.EGF-R AND ALDOSTERONE F677 the presence of aldosterone. 1). At submaximum EGF concentrations. is a pharmacodynamic description of the modulation of EGF effects. These two signals lead to. which could link the action of aldosterone to EGF-R phosphorylation. Present hypothesis for the interaction of aldosterone and EGF. for example (2). 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. Thus the two lines of evidence presented here support the hypothesis that rapid effects of aldosterone involve the EGF-R pathway. Finally. The data presented here also indicate that there is a certain degree of autocrine stimulation of the EGF-R in MDCK cells (see Fig. Thus. 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. Subsequently. we tested the effect of spironolactone (1 mol/l) on aldosterone-induced ERK1/2 phosphorylation and could not detect any inhibitory action of spironolactone (data not shown). Finally. AJP-Renal Physiol • VOL sequent Ca2 release from IP3-sensitive stores. we did not observe a substantial increase in IP3 formation. (47). we have shown that rapid effects of aldosterone in MDCKC11 cells are not prevented by the mineralocorticoid receptor antagonist spironolactone (13). of course. Our data show that aldosterone enhances EGF-R phosphorylation followed by phosphorylation of ERK1/2. leading to a shift to the left of the doseresponse curve.org on February 28. aldosterone could shift the dose-response curve of EGF to lower concentrations. e. PLA2.g. Moreover. leading to an increase in the diglyceride fraction (16). Previously. This. It is conceivable that elevated circulating aldosterone concentrations (for example. as proposed by Wehling et al. there exists the possibility that the interaction of aldosterone with EGF-R depends on the endogenous EGF-R ligand TGF. In some preliminary experiments. aldosterone and EGF seem to induce an overadditive ERK1/2 phosphorylation. for example. Furthermore.ajprenal.. ERK1/2 phosphorylation increases and stimulates Ca2 entry. The possible physiological and/or pathophysiological signiﬁcance of this interaction is supported by the fact that aldosterone was effective at nanomolar concentrations. leading to a shift to the left of the EGF doseresponse curve. which is downstream of ERK1/2 phosphorylation. for example. This is the ﬁrst report indicating an interaction of aldosterone with Tyr kinase receptor signaling. 4A. This autocrine activation loop most probably results from the simultaneous expression of EGF-R and transforming growth factor (TGF). whereas inhibition of ERK1/2 phosphorylation signiﬁcantly reduced the Ca2 signal. 8). even though the cells were made quiescent by serum removal. aldosterone could enhance the maximum effect. it was shown that aldosterone can change the metabolism of membrane phospholipids.
it is known that EGF-R signaling modulates transepithelial ion transport and stimulates salt reabsorption in certain cell types (8.org on February 28. and Denny WA. Kraker AJ. Br J Pharmacol 130: 289– 298. 13. Although the time course of the aldosterone action is not in favor of a mechanism requiring the cleavage of an endogenous EGF-R ligand. Rewcastle GW. In conclusion. Mechanisms of EGF-induced stimulation of sodium reabsorption by alveolar epithelial cells. and Silbernagl S. What is the cellular signiﬁcance of aldosterone interaction with EGF-R signaling? To gain more information regarding the possible signiﬁcance of rapid steroid effects.e. the data presented here do not affect the importance of genomic actions of aldosterone but do add an additional. 1994. Barrantes FJ. Gekle M.5-trisphosphate system is involved in rapid effects of aldosterone in human mononuclear leukocytes. 8. Of course there are many more aspects of cell function that could be affected and have not been determined here (for example. Miller MJS.ajprenal. Furthermore. 3. Therefore. Golenhofen N. Glucocorticoids act within minutes to inhibit recruitment of signaling factors to activated EGF receptors through a receptor-dependent. Angiotensin II-induced growth of vascular smooth muscle cells requires an Src-dependent activation of the epidermal growth factor receptor. Therefore. Kidney Int 58: 549–558.4. J Biol Chem 271: 8763–8767. 1993. 1992. Gekle M. with respect to sodium handling in the distal nephron. EGF-R signaling could support the stimulatory effect of aldosterone on intestinal sodium absorption. and Oberleithner H. EGF and aldosterone seem to act in the same direction. 2000. 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