Elsevier

Free Radical Biology and Medicine

Volume 53, Issue 5, 1 September 2012, Pages 1088-1100
Free Radical Biology and Medicine

Original Contribution
Modulation of transcriptional mineralocorticoid receptor activity by nitrosative stress

https://doi.org/10.1016/j.freeradbiomed.2012.06.028Get rights and content

Abstract

The mineralocorticoid receptor (MR) plays an important role in salt and water homeostasis and pathological tissue modifications, such as cardiovascular and renal fibrosis. Importantly, MR activation by aldosterone per se is not sufficient for the deleterious effects but requires the additional presence of a certain pathological milieu. Phenomenologically, this milieu could be generated by enhanced nitrosative stress. However, little is known regarding the modulation of MR transcriptional activity in a pathological milieu. The glucocorticoid receptor (GR), the closest relative of the MR, binds to the same hormone-response element but elicits protective effects on the cardiovascular system. To investigate the possible modulation of MR and GR by nitrosative stress under controlled conditions we used human embryonic kidney (HEK) cells and measured MR and GR transactivation after stimulation with the nitric oxide (NO)-donor SNAP and the peroxynitrite-donor Sin-1. In the presence of corticosteroids NO led to a general reduced corticosteroid receptor activity by repression of corticosteroid receptor–DNA interaction. The NO-induced diminished transcriptional MR activity was most pronounced during stimulation with physiological aldosterone concentrations, suggesting that NO treatment prevented its pathophysiological overactivation. In contrast, single peroxynitrite administration specifically induced the MR transactivation activity whereas genomic GR activity remained unchanged. Mechanistically, peroxynitrite permitted nuclear MR translocation whereas the cytosolic GR distribution was unaffected. Consequently, peroxynitrite represents a MR-specific aldosterone mimetic. In summary, our data indicate that the genomic function of corticosteroid receptors can be modulated by nitrosative stress which may induce the shift from physiological toward pathophysiological MR effects.

Highlights

▸ Nitrosative stress influences genomic corticosteroid receptor activity. ▸ NO reduces genomic mineralocorticoid (MR) and glucocorticoid receptor (GR) activity. ▸ Mechanistically, NO reduces corticosteroid receptor–DNA interaction. ▸ Peroxynitrite acts as a ligand-independent activator of MR but not of GR. ▸ Mechanistically, peroxynitrite enhances nuclear import of the MR.

Introduction

Cardiovascular diseases are the leading cause of mortality and morbidity in the industrial countries [1].

It is well established that increased amounts of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are implicated as mediators in these pathophysiological processes. Also, oxidative stress is responsible for the proinflammatory and fibrogenic phenotype and abnormal reactivity of the vasculature [2].

On the other hand, in recent years the importance of the mineralocorticoid receptor (MR) for reno-cardiovascular pathophysiology has become increasingly recognized. The MR and its closest relative the glucocorticoid receptor (GR) belong to the corticosteroid receptors (CR). Classically, in epithelial tissue the MR is involved in Na+ retention and thereby long-term regulation of blood pressure [3]. Independently of its effects on blood pressure and electrolyte control, the activated MR promotes in nonepithelial cells numerous pathophysiological effects such as inflammation, fibrosis, and hypertrophy, while the GR induces predominantly protective/anti-inflammatory actions [4], [5], [6].

The mechanisms/conditions responsible for the switch from physiological (beneficial) to pathological MR actions are not completely understood but seem to involve alterations in local tissue homeostasis (parainflammation). Numerous in vivo studies confirmed that the damaging effects of aldosterone in the cardiovascular tissue depend on a high salt diet which leads to a milieu characterized by enhanced oxidative stress conditions. A very important aspect of the deleterious MR actions is the fact that the activated receptor per se is not capable of eliciting damage but requires a certain pathological milieu (e.g., high salt diet) and only this coincidence leads to pathophysiological MR activation (coincidence model) [7], [8]. Furthermore, pathophysiological MR effects are also observed in the absence of elevated levels of its agonist aldosterone, indicating ligand-independent receptor activation or enhanced MR reactivity due to additional factors [7]. Recently, Nagata et al. showed that MR blockade is beneficial even in low-aldosterone salt-sensitive hypertension and prevents cardiac hypertrophy as well as failure [9]. There is evidence that shifts in the redox state and/or altered radical formation (RNS, ROS) can induce pathophysiological MR activation [8], [10].

According to the concept of classical steroid action the cytosolic MR [11] acts as a ligand-dependent transcription factor which is activated by corticosteroids such as aldosterone and hydrocortisone and influences the expression of various genes [11], [12], [13]. In epithelial tissue such as kidney, colon, sweat glands, and vascular tissue MR is particularly protected against the about ∼100-fold higher plasma free glucocorticoid, compared to aldosterone levels due to the expression of the 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) which oxidizes the glucocorticoid cortisol into the inactive metabolite cortisone [14]. According to a hypothesis of Funder in cardiovascular tissue the MR is mainly occupied by glucocorticoids due to the low or missing expression of 11β-HSD2 acting as MR antagonists as long as the level of oxidative/nitrosative conditions is adjusted to the cellular level of antioxidative compounds. Under pathophysiological conditions (elevated levels of oxidative/nitrosative compounds) it is conceivable that MR is activated by glucocorticoids leading to the noted pathophysiolocial MR effects [15].

Furthermore, there are indications of a functional cross talk between these classical MR actions and primary nongenotropic signaling modules such as cAMP, PKC, ERK1/2, CDK5, and cytosolic Ca2+ [16], [17], [18], [19]. Thus, corticosteroid-mediated gene induction does not seem to be controlled by simple on/off switches but by the interaction of various players acting in a spatially and temporally coordinated manner [20]. Understanding the transcriptional action of CR therefore requires information regarding the modulation of this action by other signaling pathways as well as by altered tissue homeostasis such as elevated radical formation occurring during inflammation. Unfortunately, only little information is available with respect to the MR.

There are some indications for MR modulation by oxidative stress [21], [22] or the cellular redox state [23]. Treatment of mice with glutathione-depleting agents affected the urinary Na+/K+ ratio after aldosterone administration, indicating that oxidative stress may impair MR-mediated actions [22]. Additionally, cis-diamminedichloroplatinum (II) impairs MR function in COS-7 cells at least in part by a redox-dependent mechanism [21]. However, when investigating the effect of hydrogen peroxide per se on aldosterone-induced transactivation activity of human MR expressed in HEK cells, using a GRE (glucocorticoid-response-element) reporter, we were unable to detect a modulatory effect [18]. In light of the clinical implications of pathological MR actions it is important to identify and understand these mechanisms and conditions directing deleterious MR effects in more detail.

Therefore, in the present study we investigated the effects of an established NO donor S-nitroso-N-acteyl-DL-penicillamine (SNAP) and the effect of an established peroxynitrite (ONOO-) donor 3-morpholinosydnonimine hydrochloride (Sin-1) on genomic MR and GR activity in comparison to aldosterone/hydrocortisone as well as combined treatment (corticosteroid+SNAP/Sin-1). Furthermore, we performed mechanistic analyses to determine whether the NO-/ONOO--induced modulation of CR activity depend on (i) the AB domain of MR which represents the coactivator and corepressor-binding domain, (ii) activation of signaling, (iii) altered cytosolic-nuclear shuttling, (iv) changed MR-DNA-binding behavior, or (vi) posttranslational modification by cysteine nitrosylation and/or tyrosine nitration [24].

Section snippets

Materials

Unless stated otherwise all materials were obtained from Sigma, Munich, Germany.

Cell culture

HEK-293 cells which do not express detectable MR [25] nor 11β-HSD2 [26] were acquired from American Type Culture Collection (Rockville, MD) and cultivated in DMEM/Ham's F-12 medium supplemented with 10% fetal calf serum at 37 °C with 5% CO2 as described previously [27], [28]. The bovine aortic endothelial cell line GM7373 [29] was provided by Prof. Dr. H. Oberleithner (University of Münster, Germany) and cultivated

Formation of NO, ONOO- and tyrosine nitration in the experimental system

For characterization of our experimental system we measured NO and peroxynitrite (ONOO-) formation in the presence of the donors Sin-1 and SNAP under cell culture conditions, using the Griess reagent assay [35]. In the presence of Sin-1 NO formation increased rapidly and reached a plateau phase after 6 h with 200 μmol/L and after 24 h with 1000 μmol/L in cell culture media (Fig. 1A). SNAP-induced NO formation was slightly slower and reached the plateau phase at ≥24 h. At a concentration of 200 μmol/L

Discussion

In the present work we describe the influence of RNS on the transcriptional activity of CRs. While SNAP is a pure NO donor, Sin-1 releases NO and O2.-, leading to the formation of ONOO- [37], [40], [45], [46] which allows us to differentiate between different aspects of nitrosative stress.

Conclusion

According to our results NO and ONOO- are two MR modulators of pathological relevance. Whereas NO acts as a nonspecific attenuator of MR and GR, most probably at the step of receptor–DNA interaction, ONOO- acts as a ligand-independent activator of MR but not GR. ONOO--induces MR trafficking and activity possibly by posttranscriptional protein modification, such as carbonylation. Because the formation of ONOO- is enhanced in diseased cardiovascular tissue and the nonphysiological activation of

Acknowledgments

This study was supported by the Deutsche Forschungsgemeinschaft, DFG (Grants Ge 905/13-1, GR 3415/1–2), and the Wilhelm-Roux program of the Medical Faculty University of Halle-Wittenberg. The rMR1-18 1D5 monoclonal antibody developed by C. Gomez-Sanchez was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biology, Iowa City, Iowa

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