Direct GPCR-EGFR interaction enables synergistic membrane-to-nucleus information transfer.

We addressed the heteromerization of the epidermal growth factor receptor (EGFR) with G-protein coupled receptors (GPCR) on the basis of angiotensin-II-receptor-subtype-1(AT1R)-EGFR interaction as proof-of-concept and show its functional relevance during synergistic nuclear information transfer, beyond ligand-dependent EGFR transactivation. Following in silico modelling, we generated EGFR-interaction deficient AT1R-mutants and compared them to AT1R-wildtype. Receptor interaction was assessed by co-immunoprecipitation (CoIP), Förster resonance energy transfer (FRET) and fluorescence-lifetime imaging microscopy (FLIM). Changes in cell morphology, ERK1/2-phosphorylation (ppERK1/2), serum response factor (SRF)-activation and cFOS protein expression were determined by digital high content microscopy at the single cell level. FRET, FLIM and CoIP confirmed the physical interaction of AT1R-wildtype with EGFR that was strongly reduced for the AT1R-mutants. Responsiveness of cells transfected with AT1R-WT or -mutants to angiotensin II or EGF was similar regarding changes in cell circularity, ppERK1/2 (direct and by ligand-dependent EGFR-transactivation), cFOS-expression and SRF-activity. By contrast, the EGFR-AT1R-synergism regarding these parameters was completely absent for in the interaction-deficient AT1R mutants. The results show that AT1R-EGFR heteromerisation enables AT1R-EGFR-synergism on downstream gene expression regulation, modulating the intensity and the temporal pattern of nuclear AT1R/EGFR-information transfer. Furthermore, remote EGFR transactivation, via ligand release or cytosolic tyrosine kinases, is not sufficient for the complete synergistic control of gene expression.

EGFR activation differentially affects the inflammatory profiles of female human aortic and coronary artery endothelial cells.

Endothelial cells (EC) are key players in vascular function, homeostasis and inflammation. EC show substantial heterogeneity due to inter-individual variability (e.g. sex-differences) and intra-individual differences as they originate from different organs or vessels. This variability may lead to different responsiveness to external stimuli. Here we compared the responsiveness of female human primary EC from the aorta (HAoEC) and coronary arteries (HCAEC) to Epidermal Growth Factor Receptor (EGFR) activation. EGFR is an important signal integration hub for vascular active substances with physiological and pathophysiological relevance. Our transcriptomic analysis suggested that EGFR activation differentially affects the inflammatory profiles of HAoEC and HCAEC, particularly by inducing a HCAEC-driven leukocyte attraction but a downregulation of adhesion molecule and chemoattractant expression in HAoEC. Experimental assessments of selected inflammation markers were performed to validate these predictions and the results confirmed a dual role of EGFR in these cells: its activation initiated an anti-inflammatory response in HAoEC but a pro-inflammatory one in HCAEC. Our study highlights that, although they are both arterial EC, female HAoEC and HCAEC are distinguishable with regard to the role of EGFR and its involvement in inflammation regulation, what may be relevant for vascular maintenance but also the pathogenesis of endothelial dysfunction.

Synergy of epidermal growth factor (EGFR) and angiotensin II (AT1R) receptor determines composition and temporal pattern of transcriptome variation.

The tyrosine kinase receptor EGFR and the G-protein-coupled receptor AT1R induce essential cellular responses, in part via receptor crosstalk with an unknown role in nuclear information transfer and transcription regulation. We investigated whether this crosstalk results in linear, EGFR-mediated nuclear signalling or in parallel, synergistic information transfer leading to qualitative and temporal variations, relevant for gene expression and environment interaction. AT1R and EGFR synergistically activate SRF via the ERK1/2-TCF and actin-MRTF pathways. Synergism, comprised of switch-like and graded single cell response, converges on the transcription factors AP1 and EGR, resulting in synergistic transcriptome alterations, in qualitative (over-additive number of genes), quantitative (over-additive expression changes of individual genes) and temporal (more late onset and prolonged expressed genes) terms. Gene ontology and IPA® pathway analysis indicate prolonged cell stress (e.g. hypoxia-like) and dysregulated vascular biology. Synergism occurs during separate but simultaneous activation of both receptors and during AT1R-induced EGFR transactivation. EGFR and AT1R synergistically regulate gene expression in qualitative, quantitative and temporal terms with (patho)physiological relevance, extending the importance of EGFR-AT1R crosstalk beyond cytoplasmic signalling.

Endothelial epidermal growth factor receptor is of minor importance for vascular and renal function and obesity-induced dysfunction in mice.

Vascular EGF receptors (EGFR) influence function and structure of arterial vessels. In genetic mouse models we described the role of vascular smooth muscle (VSMC) EGFR for proper physiological function and structure as well as for pathophysiological alterations by obesity or angiotensin II. As the importance of endothelial (EC) EGFR in vivo is unknown, we analyzed the impact of EC-EGFR knockout in a conditional mouse model on vascular and renal function under control condition as well as in obesity and in comparison to VSMC-KO. Heart and lung weight, blood pressure and aortic transcriptome (determined by RNA-seq) were not affected by EC-EGFR-KO. Aortic reactivity to α1-adrenergic stimulation was not affected by EC-EGFR-KO contrary to VSMC-EGFR-KO. Endothelial-induced relaxation was reduced in abdominal aorta of EC-EGFR-KO animals, whereas it was enhanced in VSMC-EGFR-KO animals. Mesenteric arteries of EC-EGFR-KO animals showed enhanced sensitivity to α1-adrenergic stimulation, whereas endothelial-induced relaxation and vessel morphology were not affected. Renal weight, histomorphology, function (albumin excretion, serum creatinine, fractional water excretion) or transcriptome were not affected by EC-EGFR-KO, likewise in VSMC-EGFR-KO. High fat diet (HFD) over 18 weeks induced arterial wall thickening, renal weight increase, creatininemia, renal and aortic transcriptome alterations with a similar pattern in EC-EGFR-WT and EC-EGFR-KO animals by contrast to the previously reported impact of VSMC-EGFR-KO. HFD induced endothelial dysfunction in abdominal aortae of EC-EGFR-WT, which was not additive to the EC-EGFR-KO-induced endothelial dysfunction. As shown before, VSMC-EGFR-KO prevented HFD-induced endothelial dysfunction. HFD-induced albuminuria was less pronounced in EC-EGFR-KO animals and abrogated in VSMC-EGFR-KO animals. Our results indicate that EC-EGFR, in comparison to VSMC-EGFR, is of minor and opposite importance for basal renovascular function as well as for high fat diet-induced vascular remodeling and renal end organ damage.

Knockout of vascular smooth muscle EGF receptor in a mouse model prevents obesity-induced vascular dysfunction and renal damage in vivo.

AIMS/HYPOTHESIS: Obesity causes type 2 diabetes leading to vascular dysfunction and finally renal end-organ damage. Vascular smooth muscle (VSM) EGF receptor (EGFR) modulates vascular wall homeostasis in part via serum response factor (SRF), a major regulator of VSM differentiation and a sensor for glucose. We investigated the role of VSM-EGFR during obesity-induced renovascular dysfunction, as well as EGFR-hyperglycaemia crosstalk. METHODS: The role of VSM-EGFR during high-fat diet (HFD)-induced type 2 diabetes was investigated in a mouse model with inducible, VSM-specific EGFR-knockout (KO). Various structural and functional variables as well as transcriptome changes, in vivo and ex vivo, were assessed. The impact of hyperglycaemia on EGFR-induced signalling and SRF transcriptional activity and the underlying mechanisms were investigated at the cellular level. RESULTS: We show that VSM-EGFR mediates obesity/type 2 diabetes-induced vascular dysfunction, remodelling and transcriptome dysregulation preceding renal damage and identify an EGFR-glucose synergism in terms of SRF activation, matrix dysregulation and mitochondrial function. EGFR deletion protects the animals from HFD-induced endothelial dysfunction, creatininaemia and albuminuria. Furthermore, we show that HFD leads to marked changes of the aortic transcriptome in wild-type but not in KO animals, indicative of EGFR-dependent SRF activation, matrix dysregulation and mitochondrial dysfunction, the latter confirmed at the cellular level. Studies at the cellular level revealed that high glucose potentiated EGFR/EGF receptor 2 (ErbB2)-induced stimulation of SRF activity, enhancing the graded signalling responses to EGF, via the EGFR/ErbB2-ROCK-actin-MRTF pathway and promoted mitochondrial dysfunction. CONCLUSIONS/INTERPRETATION: VSM-EGFR contributes to HFD-induced vascular and subsequent renal alterations. We propose that a potentiated EGFR/ErbB2-ROCK-MRTF-SRF signalling axis and mitochondrial dysfunction underlie the role of EGFR. This advanced working hypothesis will be investigated in mechanistic depth in future studies. VSM-EGFR may be a therapeutic target in cases of type 2 diabetes-induced renovascular disease. DATA AVAILABILITY: The datasets generated during and/or analysed during the current study are available in: (1) share_it, the data repository of the academic libraries of Saxony-Anhalt ( https://doi.org/10.25673/32049.2 ); and (2) in the gene expression omnibus database with the study identity GSE144838 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE144838 ). Graphical abstract.

The selective mineralocorticoid receptor antagonist eplerenone prevents decompensation of the liver in cirrhosis.

BACKGROUND AND PURPOSE: The mineralocorticoid receptor (MR) contributes to fibrosis in various tissues, and MR antagonists, like eplerenone, are used to prevent fibrosis. The role of MR antagonists in hepatic fibrosis and cirrhosis is unknown. Here, we investigated the role of MRs and eplerenone in cirrhosis development. EXPERIMENTAL APPROACH: Liver fibrosis (5 weeks) and cirrhosis, without (8 weeks) and with ascites (12 weeks), were induced by CCl4 in rats and comprehensively analysed. The effect of eplerenone on the development of cirrhosis with ascites was assessed. MR expression, cellular and subcellular distribution and impact of hypoxia were investigated in vivo and ex vivo. Primary rat hepatocytes and cell lines were used to investigate MR trafficking and transcriptional activity mechanistically. KEY RESULTS: In cirrhosis with ascites, MR mRNA and protein expressions were reduced in hepatocytes of hypoxic areas. While in normoxic areas MRs were mainly cytosolic, the remaining MRs in hypoxic areas were mainly localized in the nuclei, indicating activation followed by translocation and degradation. Accordingly, eplerenone treatment prevented nuclear MR translocation and the worsening of cirrhosis. Exposing hepatocytes ex vivo to hypoxia induced nuclear MR translocation and enhanced transcriptional MR activity at response elements of the NF-κB pathway. CONCLUSIONS AND IMPLICATIONS: We showed for the first time that hypoxia leads to a pathogenetic ligand-independent activation of hepatic MRs during cirrhosis resulting in their nuclear translocation and transcriptional activation of the NF-κB pathway. Treatment with eplerenone prevented the worsening of cirrhosis by blocking this ligand-independent activation of the MR.

Substance-specific importance of EGFR for vascular smooth muscle cells motility in primary culture.

Besides their importance for the vascular tone, vascular smooth muscle cells (VSMC) also contribute to pathophysiological vessel alterations. Various G-protein coupled receptor ligands involved in vascular dysfunction and remodeling can transactivate the epidermal growth factor receptor (EGFR) of VSMC, yet the importance of EGFR transactivation for the VSMC phenotype is incompletely understood. The aims of this study were (i) to characterize further the importance of the VSMC-EGFR for proliferation, migration and marker gene expression for inflammation, fibrosis and reactive oxygen species (ROS) homeostasis and (ii) to test the hypothesis that vasoactive substances (endothelin-1, phenylephrine, thrombin, vasopressin and ATP) rely differentially on the EGFR with respect to the abovementioned phenotypic alterations. In primary, aortic VSMC from mice without conditional deletion of the EGFR, proliferation, migration, marker gene expression (inflammation, fibrosis and ROS homeostasis) and cell signaling (ERK 1/2, intracellular calcium) were analyzed. VSMC-EGFR loss reduced collective cell migration and single cell migration probability, while no difference between the genotypes in single cell velocity, chemotaxis or marker gene expression could be observed under control conditions. EGF promoted proliferation, collective cell migration, chemokinesis and chemotaxis and leads to a proinflammatory gene expression profile in wildtype but not in knockout VSMC. Comparing the impact of five vasoactive substances (all reported to transactivate EGFR and all leading to an EGFR dependent increase in ERK1/2 phosphorylation), we demonstrate that the importance of EGFR for their action is substance-dependent and most apparent for crowd migration but plays a minor role for gene expression regulation.

Consequences of postnatal vascular smooth muscle EGFR deletion on acute angiotensin II action.

Epi dermal growth factor (EGF) receptor (EGFR) is activated by its canonical ligands and transactivated by various vasoactive substances, e.g. angiotensin II (Ang II). Vascular EGFR has been proposed to be involved in vascular tissue homoeostasis and remodelling. Thus, most studies have focused on its role during long-term vascular changes whereas the relevance for acute regulation of vascular function in vivo and ex vivo is insufficiently understood. To investigate the postnatal role of VSMCs (vascular smooth muscle cells) EGFR in vivo and ex vivo, we generated a mouse model with cell-specific and inducible deletion of VSMC EGFR and studied the effect on basal blood pressure, acute pressure response to, among others, Ang II in vivo as well as ex vivo, cardiovascular tissue homoeostasis and vessel morphometry in male mice. In knockout (KO) animals, systolic, diastolic and mean blood pressures were reduced compared with wild-type (WT). Furthermore, Ang II-induced pressure load was lower in KO animals, as was Ang II-induced force development and extracellular-signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in aortic rings from KO animals. By contrast, we observed no difference in force development during application of serotonin, KCl, endothelin-1 or endothelin-1-induced pressure load in KO animals. In addition, nitric oxide (NO)-mediated vasodilation was not affected. Heart weight (HW) increase and up-regulation of aortic and cardiac expression of Ccl2 (chemoattractant protein-2) and serpinE1 (plasminogen activator inhibitor 1) during the transition from 4- to 10-months of age were prevented by VSMC EGFR KO. We conclude that VSMC EGFR is involved in basal blood pressure homoeostasis and acute pressure response to Ang II, and thereby contributes to maturation-related remodelling.

Moderate inappropriately high aldosterone/NaCl constellation in mice: cardiovascular effects and the role of cardiovascular epidermal growth factor receptor.

Non-physiological activation of the mineralocorticoid receptor (MR), e.g. by aldosterone under conditions of high salt intake, contributes to the pathogenesis of cardiovascular diseases, although beneficial effects of aldosterone also have been described. The epidermal growth factor receptor (EGFR) contributes to cardiovascular alterations and mediates part of the MR effects. Recently, we showed that EGFR is required for physiological homeostasis and function of heart and arteries in adult animals. We hypothesize that moderate high aldosterone/NaCl, at normal blood pressure, affects the cardiovascular system depending on cardiovascular EGFR. Therefore we performed an experimental series in male and female animals each, using a recently established mouse model with EGFR knockout in vascular smooth muscle cells and cardiomyocytes and determined the effects of a mild-high aldosterone-to-NaCl constellation on a.o. marker gene expression, heart size, systolic blood pressure, impulse conduction and heart rate. Our data show that (i) cardiac tissue of male but not of female mice is sensitive to mild aldosterone/NaCl treatment, (ii) EGFR knockout induces stronger cardiac disturbances in male as compared to female animals and (iii) mild aldosterone/NaCl treatment requires the EGFR in order to disturb cardiac tissue homeostasis whereas beneficial effects of aldosterone seem to be independent of EGFR.

The nephrotoxic Ifosfamide-metabolite chloroacetaldehyde interferes with renal extracellular matrix homeostasis.

BACKGROUND/AIMS: Chronic renal proximal tubule dysfunction after therapy with the antineoplastic agent ifosfamide (IFO) is often attributed to the metabolite chloroacetaldehyde (CAA). Chronic IFO-nephropathy is reported to result in tubulointerstitial fibrosis and inflammation. METHODS: To elucidate possible effects of CAA on extracellular matrix homeostasis, we investigated the action of CAA on markers of extracellular matrix (ECM) homeostasis in human proximal tubule cells (RPTEC) by use of direct ELISA for extracellular collagens and gelatin zymography. RESULTS: An increase in type III collagen and a decrease in type IV collagen abundance in the media of RPTEC could be observed after exposure to CAA in clinically relevant concentrations. CAA increased intracellular type III and decreased intracellular type IV collagen. MMP-2 activity was decreased but MMP-9 activity unchanged. The enhanced CAA-induced collagen III formation could be attenuated by the intracellular Ca(2+)-chelator BAPTA-AM, the PKA-antagonist H-89 and by extracellular acidification. CAA-induced collagen III abundance was enhanced by db-cAMP and IBMX and by protein overload. CONCLUSIONS: CAA exerts profibrotic effects on RPTEC dependent on Ca(2+) and cAMP/PKA-signaling. These effects are enhanced by additional protein burden and attenuated by acidification. © 2014 S. Karger AG, Basel.

Influence of aldosterone and salt or ouabain in a10 rat aorta smooth muscle cells.

BACKGROUND/AIMS: It is currently under debate whether aldosterone is able to induce fibrosis or whether it acts only as a cofactor under pathological conditions, e.g. as an elevated salt (NaCl) load. METHODS: We tested the interaction of 10 nM aldosterone, 15 mM NaCl and 1 µM ouabain using rat aorta smooth muscle cells (A10) with respect to the following parameters: necrosis, apoptosis, glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase activity, glutathione (GSH) content, collagen and fibronectin homeostasis and intracellular calcium distribution. RESULTS: Necrosis rates were increased after 48 h of incubation with aldosterone, salt or ouabain and in the combination of aldosterone and salt or ouabain. Apoptosis rates were decreased. A reduced defense capacity against oxidative stress was mirrored in the decreased G6PD activity and GSH content. Collagen III or fibronectin synthesis rates were unchanged, but gelatinase activity was increased resulting in a decreased media collagen III and fibronectin content. Calcium stores were increased by aldosterone in combination with ouabain. CONCLUSION: Aldosterone and salt per se can lead to cell injury that is aggravated in combination or with cardiotonic steroids. In cooperation with other vascular cells, this can generate a permissive milieu enabling aldosterone or salt to promote more extensive vascular injury.

Colocalization of mineralocorticoid and EGF receptor at the plasma membrane.

The mineralocorticoid receptor (MR), a ligand-activated transcription factor expressed in various cell types (e.g. epithelial cells, neurons, smooth muscle cells, immune cells), plays important roles in neurohumoral, neuronal, cardiovascular, renal and intestinal function. Pathophysiological relevant signaling mechanisms include nongenomic pathways involving the EGF receptor (EGFR). We investigated whether a MR-EGFR colocalization may underlie the functional MR-EGFR interaction by coimmunoprecipitation, fluorescence resonance energy transfer (FRET) and confocal microscopy in a heterologous expression system. EGFR and a small fraction of MR colocalize at the cell membrane, independently of short time exposure (

Mineralocorticoid receptor inhibits CREB signaling by calcineurin activation.

We investigated the interaction of MR with cAMP-response element binding protein (CREB) and provide a mechanistic explanation and insights into the cellular relevance. MR --> CREB crosstalk was assessed in vascular smooth muscle cells and heterologous expression systems. Experiments were designed in a way that only one variable changed at a time and the respective vehicles served as controls. MR, but not GR, activation (aldosterone or hydrocortisone, IC(50), approximately 0.3 nM) inhibits CREB transcriptional activity induced by stimulation of beta1/2-adrenoceptors and adenylyl cyclase or addition of membrane-permeable cAMP up to 70% within 2 h after addition. The MR DNA-binding domain is not required for this inhibition. cAMP formation is virtually unchanged, whereas MR exerts a robust inhibition of CREB(S133) phosphorylation via calcineurin/PP2B activation without changes in PP2B-Aalpha or beta expression. In parallel, the PP2B-sensitive NFaT-pathway is activated. The inhibitory crosstalk attenuates CREB-induced glucose-6-phosphate dehydrogenase expression. Overall, transcriptional relevant MR --> CREB crosstalk occurs at the level of CREB phosphorylation by enhanced calcineurin activity, enables GRE-independent genomic signaling of MR, and is of potential pathophysiological relevance.

Aldosterone-induced EGFR expression: interaction between the human mineralocorticoid receptor and the human EGFR promoter.

Aldosterone plays a key role in cardiovascular and renal injury. The underlying mechanisms are not completely understood. Because the epidermal growth factor receptor (EGFR) is involved in the development of fibrosis and vascular dysfunction, upregulation of EGFR expression by aldosterone-bound mineralocorticoid receptor (MR) is an attractive hypothesis. We investigated the effect of aldosterone on EGFR expression in the aorta of adrenalectomized rats and in human aorta smooth muscle cells (HAoSMC) in primary culture. Aldosterone, but not dexamethasone, stimulated EGFR expression in vivo in the aorta as well as in HAoSMC. EGFR degradation was not affected. Aldosterone-induced EGFR expression in HAoSMC was dose dependent and prevented by spironolactone. Furthermore, incubation of HAoSMC with aldosterone led to enhanced EGF-induced ERK1/2 phosphorylation and an EGFR-dependent increase in media fibronectin. EGFR promoter reporter gene assay as well as chromatin immunoprecipitation data indicate that MR interacts with the EGFR promoter. With deletion constructs we gained evidence that this interaction takes place between the hMR and the EGFR promoter regions 316-163 (stronger activation site, EC50 approximately 1.0 nM) and 163-1 (weaker activation site, EC50 approximately 0.7 nM), which do not comprise canonical glucocorticoid response elements and are not activated by the human glucocorticoid receptor. The interactions require in part the NH2-terminal domains of MR. ELISA-based transcription factor DNA binding assay with in vitro synthesized hMR suggest direct binding to region 163-1. Our results indicate that aldosterone leads to enhanced EGFR expression via an interaction with the EGFR promoter, which is MR specific and could contribute to the aldosterone-induced increase in fibronectin abundance.

Altered collagen homeostasis in human aortic smooth muscle cells (HAoSMCs) induced by aldosterone.

The importance of aldosterone for cardiovascular diseases is well established. Most of the adverse effects seem to originate from its ability to produce vascular injury, including fibrosis. It is currently under debate whether aldosterone per se is able to induce fibrosis or whether it acts as a cofactor under pathological conditions. We tested whether aldosterone per se and in the presence of reactive oxygen stress (H(2)O(2)) enhances collagen abundance in human aortic smooth muscle cell (HAoSMC) media in primary culture and, if so, by which means. Collagen abundance, as well as epidermal growth factor receptor (EGFR) expression and ERK1/2 phosphorylation, was investigated by ELISA and Western blot. Collagenase activity and H(2)O(2) formation were determined by fluorometry and luminometry. Aldosterone alone did not affect collagen abundance but potentiated the stimulatory effect of low concentrations of H(2)O(2) (1-10 micromol/l). This effect disappeared when shedding of membrane-bound EGFR ligands was prevented by GM6001. EGFR expression and cellular EGF responsiveness were enhanced by aldosterone. Inhibition of the EGFR kinase (tyrphostin AG1478) prevented the increase of collagen. The increase in collagen abundance was prevented by blockade of the mineralocorticoid receptor (MR) and could be reproduced by MR transfection into Chinese hamster ovary cells. We conclude that aldosterone sensitizes HAoSMC for H(2)O(2)-induced increase of collagen abundance at least in part by enhanced EGFR expression.

Chloroacetaldehyde as a sulfhydryl reagent: the role of critical thiol groups in ifosfamide nephropathy.

Chloroacetaldehyde (CAA) is a metabolite of the alkylating agent ifosfamide (IFO) and putatively responsible for renal damage following anti-tumor therapy with IFO. Depletion of sulfhydryl (SH) groups has been reported from cell culture, animal and clinical studies. In this work the effect of CAA on human proximal tubule cells in primary culture (hRPTEC) was investigated. Toxicity of CAA was determined by protein content, cell number, LDH release, trypan blue exclusion assay and caspase-3 activity. Free thiols were measured by the method of Ellman. CAA reduced hRPTEC cell number and protein, induced a loss in free intracellular thiols and an increase in necrosis markers. CAA but not acrolein inhibited the cysteine proteases caspase-3, caspase-8 and cathepsin B. Caspase activation by cisplatin was inhibited by CAA. In cells stained with fluorescent dyes targeting lysosomes, CAA induced an increase in lysosomal size and lysosomal leakage. The effects of CAA on cysteine protease activities and thiols could be reproduced in cell lysate. Acidification, which slowed the reaction of CAA with thiol donors, could also attenuate effects of CAA on necrosis markers, thiol depletion and cysteine protease inhibition in living cells. Thus, CAA directly reacts with cellular protein and non-protein thiols, mediating its toxicity on hRPTEC. This effect can be reduced by acidification. Therefore, urinary acidification could be an option to prevent IFO nephropathy in patients.

Disturbed Ca2+-signaling by chloroacetaldehyde: a possible cause for chronic ifosfamide nephrotoxicity.

BACKGROUND: Renal damage following chemotherapy with ifosfamide is attributed to the metabolic activation of the drug and the generation of chloroacetaldehyde (CAA). Little is known about the mechanism by which CAA impairs renal function. In this study the effect of CAA on intracellular Ca(2+) homeostasis in human renal proximal tubule cells (RPTEC) in primary culture was investigated. METHODS: Intracellular Ca(2+) was measured using the Ca(2+)-sensitive dye fura-2. Cell viability was determined by protein content and cell number. Oncotic and apoptotic cell death was assayed using trypan blue exclusion, caspase-3 activity, and 4',6-diamino-2-phenylindole (DAPI) staining. RESULTS: CAA (1.5 to 150 micromol/L) induced sustained elevations of intracellular free calcium ([Ca(2+)](i)) from 75 +/- 3 nmol/L to maximal 151 +/- 6 nmol/L. This effect was dependent on extracellular Ca(2+), but not Ca(2+) entry. The rise in [Ca(2+)](i) mediated by CAA could be attributed to inhibition of Na(+)-dependent extrusion of intracellular Ca(2+), indicating an inhibitory action of CAA on Na(+)/Ca(2+) exchange. Modulation of protein kinase A (PKA), but not protein kinase C (PKC) blunted the effect of CAA. Thus, CAA seems to inhibit Na(+)/Ca(2+) exchange by interaction with cyclic adenosine monophosphate (cAMP)-PKA-signaling. A 48-hour exposure to 15 micromol/L CAA significantly reduced cell number and protein content of RPTEC by induction of necrosis. This effect of 15 micromol/L CAA could be overcome by coadministration of the intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM). CONCLUSION: First, CAA inhibits the Na+/Ca2+-exchanger. Second, this effect is dependent on PKA. Third, CAA induces necrotic rather than apoptotic cell death. Finally, disturbed Ca(2+) homeostasis via Na(+)/Ca(2+) exchange contributes to the nephrotoxic action of CAA in RPTEC.

Evidence for epidermal growth factor receptor as negative-feedback control in aldosterone-induced Na+ reabsorption.

Aldosterone enhances Na(+) reabsorption via epithelial Na(+) channels (ENaC). Aldosterone also stimulates the protein kinase ERK1/2- and the epidermal growth factor (EGF) receptor (EGFR)-signaling pathway. Yet EGF and ERK1/2 are known inhibitors of ENaC-mediated Na(+) reabsorption. In the present study, using the well-established Madin-Darby canine kidney C7 cell line, we tested the hypothesis that EGFR represents a negative-feedback control for chronic aldosterone-induced Na(+) reabsorption [amiloride-inhibitable short-circuit current (I(sc))]. Mineralocorticoid receptor expression was confirmed by RT-PCR and Western blot analysis. Aldosterone enhanced ERK1/2 phosphorylation in an EGFR-dependent way. Furthermore, aldosterone stimulated EGFR expression. Aldosterone (10 nmol/l) induced a small transient increase in I(sc) under control conditions. Inhibition of ERK1/2 phosphorylation with U-0126 (10 micromol/l) stimulated I(sc), indicating constitutive ENaC inhibition. Aldosterone exerted a significantly larger effect in the presence of U-0126 than without U-0126. EGF (10 microg/l) inhibited I(sc), whereas inhibition of EGFR kinase by tyrphostin AG-1478 (100 nmol/l) enhanced I(sc). Aldosterone was more effective in the presence of AG-1478 than without AG-1478. In summary, we propose that the EGFR-signaling cascade can serve as a negative-feedback control to limit the effect of aldosterone-induced Na(+) reabsorption.

Aldosterone stimulates epidermal growth factor receptor expression.

The steroid hormone aldosterone plays an important role during pathological tissue modifications, similar to cardiovascular or renal fibrosis. The underlying mechanisms for the pathological actions are not understood. Interaction of aldosterone with the epidermal growth factor (EGF) receptor is an attractive hypothesis to explain pathological tissue remodeling elicited by aldosterone, because (i) mineralocorticoids can sensitize cells for EGF, (ii) mineralocorticoid receptor (MR)-antagonists reduce EGFR-mRNA expression, (iii) EGFR itself supports the development of cardiovascular or renal fibrosis, and (iv) signaling elements involved in the pathological action of aldosterone (similar to ERK1/2 or NFkB) are typical downstream modules during EGF signaling. In addition, an interaction of aldosterone and EGF with respect to ERK1/2 activation has been described. Here we show that aldosterone stimulates EGFR expression in renal tissue of adrenalectomized rats and in human renal primary cell cultures. Furthermore, Chinese hamster ovary (CHO) cells normally devoid of EGFR or MR express EGFR after transfection with human MR (CHO-MR cells) but not after transfection with human glucocorticoid receptor (CHO-GR cells). In CHO-MR cells, EGFR-expression is up-regulated by aldosterone and inhibited by spironolactone. CHO-MR cells but not CHO-GR cells respond with ERK1/2 phosphorylation to EGF exposure. The responsiveness to other peptide hormones was virtually not affected. These data suggest that EGFR is an aldosterone-induced protein and is involved in the manifold (patho)biological actions of aldosterone.

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 cells. The EGFR kinase inhibitor tyrphostin AG1478 or the MEK1/2 inhibitor U0126 completely prevented the effect. Furthermore, aldosterone enhanced Tyr phosphorylation of c-Src and EGFR, and an inhibitor of cytosolic tyrosine kinases (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyriociaine) 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.