Claudia Grossmann

New aspects of rapid aldosterone signaling.

Aldosterone, the endogenous ligand of the mineralocorticoid receptor (MR) in humans, is a steroid hormone that regulates salt and water homeostasis. Recently, additional pathophysiological effects in the renocardiovascular system have been identified. Besides genomic effects mediated by activated MR, rapid aldosterone actions that are independent of translation and transcription have been documented. While these nongenomic actions influence electrolyte homeostasis, pH and cell volume in classical MR target organs, they also participate in pathophysiological effects in the renocardiovascular system causing endothelial dysfunction, inflammation and remodeling. The mechanisms conveying these rapid effects consist of a multitude of signaling molecules and include a cross-talk with genomic aldosterone effects as well as with angiotensin II and epidermal growth factor receptor signaling. Rapid corticosteroid signaling via the MR has also been demonstrated in the brain. Altogether, the function of nongenomic aldosterone effects seems to be to modulate other signaling cascades, depending on the surrounding milieu.

Actions of aldosterone in the cardiovascular system: the good, the bad, and the ugly?

Aldosterone and its receptor, the mineralocorticoid receptor (MR), play a key role in the regulation of reno-cardiovascular function as well as in the regulation of normal and abnormal reno-cardiovascular function, which are responsible for the variety of its functional responses. The underlying mechanisms are of genomic and nongenotropic nature. Prevention of critical arterial hypotension by NaCl retention and regulation of potassium homeostasis, which is of eminent importance for cardiovascular electrophysiology and rhythmogenesis, represent the good face of aldosterone in the cardiovascular system. Triggering of persistent arterial hypertension with all the detrimental secondary effects on heart, kidney, vessels, and brain represents the bad face of aldosterone/MR in the cardiovascular system. Blood pressure-independent reno-cardiovascular end organ damage represents the ugly face of MR activation and does not depend on elevated aldosterone concentrations. In this way, aldosterone/MR induces or facilitates inflammatory and fibrotic processes in a permissive milieu, created for example by angiotensin II or NaCl and characterized by enhanced oxidative stress, in vascular walls.

EF Domains Are Sufficient for Nongenomic Mineralocorticoid Receptor Actions

The mineralocorticoid receptor (MR) is important for salt homeostasis and reno-cardiovascular pathophysiology. Signaling mechanisms include, besides classical genomic pathways, nongenomic pathways with putative pathophysiological relevance involving the mitogen-activated protein kinases ERK1/2. We determined the MR domains required for nongenomic signaling and their potential to elicit pathophysiological effects in cultured cells under defined conditions. The expression of full-length human MR or truncated MR consisting of the domains CDEF (MRCDEF), DEF (MRDEF), or EF (MREF) renders cells responsive for the MR ligand aldosterone with respect to nongenomic ERK1/2 phosphorylation, whereas only full-length MR and MRCDEF conferred genomic responsiveness. ERK1/2 phosphorylation depends on the EGF receptor and cSRC kinase. MREF expression is sufficient to evoke the aldosterone-induced increase of collagen III levels, similar to full-length MR expression. Our data suggest that nongenomic MR signaling is mediated by the EF domains and present the first proof of principle showing that nongenomic signaling can be sufficient for some pathophysiological effects. The minimum amino acid motif required for nongenomic MR signaling and its importance in various effects have yet to be determined.

Long-term application of the aldosterone antagonist spironolactone prevents stiff endothelial cell syndrome

Aldosterone triggers the stiff endothelial cell syndrome (SECS), characterized by an up‐regulation of epithelial sodium channels (ENaCs) and mechanical stiffening of the endothelial cell cortex accompanied by endothelial dysfunction. In vivo, aldosterone antagonism exerts sustained protection on the cardiovascular system. To illuminate the molecular mechanisms of this time‐dependent effect, a study on endothelial cells in vitro and ex vivo was designed to investigate SECS over time. Endothelia (from human umbilical veins, bovine aortae, and explants of human arteries) were cultured in aldosterone‐supplemented medium with or without the mineralocorticoid receptor (MR) antagonist spironolactone. MR expression, ENaC expression, cortical stiffness, and shear‐mediated nitric oxide (NO) release were determined after 3 d (short term) and up to 24 d (long term). Over time, MR expression increased by 129%. ENaC expression and surface abundance increased by 32% and 42% (13.8 to 19.6 molecules per cell surface), paralleled by a 49% rise in stiffness. Spironolactone prevented this development and, after 3 wk of treatment, increased NO release by 50%. Thus, spironolactone improves endothelial function long‐lastingly by preventing a time‐dependent manifestation of SECS. This emphasizes the key role of vascular endothelium as a therapeutical target in cardiovascular disorders and might explain blood pressure independent actions of MR antagonism.—Drüppel, V., Kusche‐Vihrog, K., Grossmann, C., Gekle, M., Kasprzak, B., Brand, E., Pavenstädt, H., Oberleithner, H., Kliche, K., Long‐term application of the aldosterone antagonist spironolactone prevents stiff endothelial cell syndrome. FASEB J. 27, 3652–3659 (2013). www.fasebj.org

Nuclear Shuttling Precedes Dimerization in Mineralocorticoid Receptor Signaling

The mineralocorticoid receptor (MR), a member of the steroid receptor superfamily, regulates water-electrolyte balance and mediates pathophysiological effects in the renocardiovascular system. Previously, it was assumed that after binding aldosterone, the MR dissociates from HSP90, forms homodimers, and then translocates into the nucleus where it acts as a transcription factor (Guiochon-Mantel et al., 1989; Robertson et al., 1993; Savory et al., 2001). We found that, during aldosterone-induced nuclear translocation, MR is bound to HSP90 both in the cytosol and the nucleus. Homodimerization measured by eBRET and FRET takes place when the MR is already predominantly nuclear. In vitro binding of MR to DNA was independent of ligand but could be partially inhibited by geldanamycin. Overall, here we provide insights into classical MR signaling necessary for elucidating the mechanisms of pathophysiological MR effects and MR specificity.

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 (</=60min) to receptor ligands. Twenty-four-hour-exposure to saturating concentrations of aldosterone (10nmol/l) resulted in an almost complete nuclear translocation of MR and disappearance of MR-EGFR colocalization. EGFR transactivation is enhanced only after MR stimulation. Inhibition of HSP90 by geldanamycin did not reduce the fraction of MR interacting with EGFR. Disruption of cholesterol-rich membrane domains by cyclodextrin reduced MR-EGFR interaction. In conclusion, a subfraction of MR interacts with EGFR at the plasma membrane in our heterologous expression system, possibly at cholesterol-rich domains, to form a steroid receptor/growth factor receptor signaling module.

Loss of Epidermal Growth Factor Receptor in Vascular Smooth Muscle Cells and Cardiomyocytes Causes Arterial Hypotension and Cardiac Hypertrophy

The epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, contributes to parainflammatory dysregulation, possibly causing cardiovascular dysfunction and remodeling. The physiological role of cardiovascular EGFR is not completely understood. To investigate the physiological importance of EGFR in vascular smooth muscle cells and cardiomyocytes, we generated a mouse model with targeted deletion of the EGFR using the SM22 (smooth muscle-specific protein 22) promoter. While the reproduction of knockout animals was not impaired, life span was significantly reduced. Systolic blood pressure was not different between the 2 genotypes—neither in tail cuff nor in intravascular measurements—whereas total peripheral vascular resistance, diastolic blood pressure, and mean blood pressure were reduced. Loss of vascular smooth muscle cell-EGFR results in a dilated vascular phenotype with minor signs of fibrosis and inflammation. Echocardiography, necropsy, and histology revealed a dramatic eccentric cardiac hypertrophy in knockout mice (2.5-fold increase in heart weight), with increased stroke volume and cardiac output as well as left ventricular wall thickness and lumen. Cardiac hypertrophy is accompanied by an increase in cardiomyocyte volume, a strong tendency to cardiac fibrosis and inflammation, as well as enhanced NADPH-oxidase 4 and hypertrophy marker expression. Thus, in cardiomyocytes, EGFR prevents excessive hypertrophic growth through its impact on reactive oxygen species balance, whereas in vascular smooth muscle cells EGFR contributes to the appropriate vascular wall architecture and vessel reactivity, thereby supporting a physiological vascular tone.

Nongenotropic aldosterone effects and the EGFR: interaction and biological relevance.

Aldosterone, the endogenous mineralocorticoid in humans, classically binds to the cytoplasmic mineralocorticoid receptor (MR), which then acts as a transcription factor to regulate salt and water homeostasis. Besides this traditional signal transduction, a number of rapid effects have been described for aldosterone/MR, which are not sensitive to translation or transcription inhibitors and are, therefore, nongenotropic and not the result of a direct genomic action. However, due to their variability these effects have been highly controversial. When recently alternative pathophysiological effects of aldosterone-stimulated MR were identified that included cardiovascular remodeling and endothelial dysfunction, a revived interest in the mineralocorticoids and their genomic and also nongenomic signaling occurred. Because the only known DNA-binding site of the MR is a common hormone-response-element shared by MR and the glucocorticoid receptor (GR), the nongenotropic effects are candidates for mediating the MR specific pathophysiological effects. Inspired by the findings for progesterone and estrogen receptor, an interaction between the epidermal growth factor receptor (EGFR) signaling pathway and aldosterone/MR was identified as a likely molecular mechanism for the alternative aldosterone effects with potential therapeutical implications.

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, IC50, ~0.3 nM) inhibits CREB transcriptional activity induced by stimulation of ß1/2‐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 phosphorylation via calcineurin/ PP2B activation without changes in PP2B‐Aα or ß expression. In parallel, the PP2B‐sensitive NFaT‐path‐way 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.—Grossmann, C, Wuttke, M., Ruhs, S., Seiferth, A., Mildenberger, S., Rabe, S., Schwerdt, G., Gekle, M. Mineralocorticoid receptor inhibits CREB signaling by calcineurin activation. FASEB J. 24, 2010–2019 (2010). www.fasebj.org

Ca2+ but not H2O2 modulates GRE-element activation by the human mineralocorticoid receptor in HEK cells

The mineralocorticcoid receptor (MR) plays an important role in salt and water homeostasis as well as during cardiovascular and renal fibrosis but little is known regarding its modulation by other signaling pathways. To investigate a possible modulation under controlled conditions we used human embryonic kidney (HEK) cells (devoid of endogenous MR) transfected with the human MR and measured transactivation with a GRE-SEAP-reporter construct. MR was compared to the glucocorticoid receptor (GR) as well as to MR lacking the N-terminal domains AB (MR(CDEF)). Chelation of cytosolic Ca2+ enhanced MR activity and SGK1-expression, whereas elevation of cytosolic Ca2+ with ionomycin or thapsigargin reduced MR activity. GR activity was not affected by ionomycin or thapsigargin. MR(CDEF) activity was not affected by chelation or elevation of cytosolic Ca2+. Inhibition of ERK1/2 activation by U0126 or activation of PKA by cAMP, previously shown to modulate MR and GR activity, did not affect MR(CDEF) activity either. H2O2<500micromol/l did not affect basal nor hormone-induced reporter activity. Higher concentrations exerted the same relative inhibitory effect on GRE-SEAP-activity under basal conditions as in the presence of aldosterone-stimulated MR and elicited cytotoxic effects. Our data indicate that the genomic function of MR can be modulated by cytosolic Ca2+, PKA and ERK1/2 via an interaction with the AB-domain. H2O2 seems not to affect relative MR activity directly under our experimental conditions.