Human mineralocorticoid receptor (MR) gene haplotypes modulate MR expression and transactivation: Implication for the stress response

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Summary

Stress causes activation of the hypothalamic–pituitary–adrenal (HPA) axis, resulting in secretion of corticosteroids which facilitate behavioural adaptation. These effects exerted by corticosteroids are mediated by two brain corticosteroid receptor types, the mineralocorticoid receptor (MR), with a high affinity already occupied under basal conditions and the glucocorticoid receptor (GR), with a low affinity only activated during stress.

Here, we studied MR gene haplotypes constituted by the two single nucleotide polymorphisms MR-2G/C (rs2070951) and MRI180V (rs5522). The haplotypes showed differences in cortisol-induced gene transcription and protein expression while the structural variant MRI180V did not affect ligand binding.

Moreover, in a well characterized cohort of 166 school teachers these haplotypes have been associated with perceived chronic stress (Trier Inventory for the Assessment of Chronic Stress, TICS) and, in a subgroup of 47 subjects, with ACTH, cortisol and heart rate responses to acute psychosocial stress (Trier Social Stress Test, TSST). MR haplotypes were significantly associated with the TICS scales “excessive demands at work” and “social overload”. Subjects homozygous for haplotype MR-2C/MRI180, which in vitro showed highest expression and transactivational activity, displayed the highest salivary cortisol (p < 0.001), plasma cortisol (p = 0.010), plasma ACTH (p = 0.003) and heart rate (p = 0.018) responses.

It is concluded that the investigated MR haplotypes modulate cortisol-induced gene transcription in vitro. Moreover, these haplotypes may contribute to individual differences in perceived chronic stress as well as neuroendocrine and cardiovascular stress responses.

Introduction

Cortisol has profound effects in the brain, underlying behavioural adaptation to stress and feedback regulation of the hypothalamic–pituitary–adrenal (HPA) axis. These actions exerted by cortisol are mediated by a high affinity brain corticosteroid receptor, the mineralocorticoid receptor (MR; NR3C2) and a lower affinity glucocorticoid receptor (GR). The GR is widely expressed while the MR predominantly occurs in limbic brain areas including the hippocampus. Animal studies have shown that MR occupation is maintained at basal pulsatile cortisol levels while the GR becomes only activated with rising cortisol levels in response to stress and at the peaks of the corticosterone pulses (Conway-Campbell et al., 2007, Lightman et al., 2008, Sarabdjitsingh et al., 2009). The MR and GR operate as transcription factors in the regulation of gene transcription, but recently these receptors were also found to mediate fast membrane-mediated actions (Di et al., 2003, Karst et al., 2005). Through the MR cortisol regulates basal HPA axis pulsatility (Atkinson et al., 2008) and the threshold or onset of the HPA axis response to stress (Ratka et al., 1989, Dodt et al., 1993, Arvat et al., 2001, Wellhoener et al., 2004) while the GR facilitates the suppression of stress-induced HPA axis activation and promotes adaptation.

Two functional single nucleotide polymorphisms (SNPs) in the MR have been previously identified, namely MR-2G/C (rs2070951) located 2 nucleotides before the translation start site and MRI180V (rs5522), a SNP resulting in an amino acid change in the N-terminal domain of the protein. Both SNPs affect transactivation in vitro (DeRijk et al., 2006, van Leeuwen et al., 2010). MR-2G/C is located outside the coding region of the MR but inside the Kozac translation regulatory sequence, and is expected to influence brain function via changes in MR protein expression. The structural variant MRI180V was previously found to be associated with HPA axis and autonomic nervous system reactivity (DeRijk et al., 2006). This effect exerted by MRI180V may occur through differences in ligand binding, translocation to the nucleus, dimerization or recruitment of coactivators. Furthermore, these two SNPs in the MR are in linkage disequilibrium resulting in three common haplotypes and one very rare (frequency less than 0.1%) haplotype (DeRijk et al., 2008). The in vitro and in vivo effects of these haplotypes are currently not known.

The main objective of the current study was to measure transactivation, ligand binding and protein expression of MRI180V, MR-2G/C and the resulting haplotypes. In addition, we sought to evaluate the association between these haplotypes and valid (endo)phenotypes for psychobiological stress regulation in a cohort that is indepentdent of the samples that have previously been studied by our group (DeRijk et al., 2006, van Leeuwen et al., 2010). Therefore, we performed a genetic association analysis in a cohort of school teachers that has been characterized with the Trier Inventory for the Assessment of Chronic Stress (TICS) and the Trier Social Stress Test (TSST).

Section snippets

Functional characterization in vitro

The applied methods have been used previously to illustrate functional consequences of genetic variation in other nuclear receptors. Russcher et al. (2005) demonstrated with similar assays that genetic variation in the GR influences the transactivational capacity and protein expression. The GR SNPs that were functional in the in vitro functionality assays appeared to be associated with several parameters in vivo thereby validating the in vitro assays (Van Rossum et al., 2004).

Construction of the hMR plasmids

The expression

Functional characterization in vitro

All four MR haplotypes were tested in vitro. According to the observed frequency in the population (DeRijk et al., 2008) the haplotypes are referred to as Hap 1 (GA), constituted by MR-2 G and MRI180V A, Hap 2 (CA), constituted by MR-2C and MRI180V A, Hap 3 (CG), constituted by MR-2C and MRI180V G and the in vivo rarely observed Hap 4 (GG), constituted by MR-2 G and MRI180V G.

Transactivation assay

The four different MR haplotypes showed differential cortisol-induced luciferase transcription from a triple tyrosine

Discussion

Here we described neuroendocrine and behavioural consequences of two common functional polymorphisms in the human MR, MRI180V and MR-2G/C, both in vitro and in vivo. The haplotypes of the two SNPs showed differences in cortisol-induced transcription of the reporter gene. From protein analysis of the haplotypes it can be concluded that MR-2G/C changes protein expression while MRI180V did not have this effect. Furthermore, MRI180V did not affect ligand binding. Our data suggest that the

Role of funding source

This study was supported by Psychiatric Hospital Rivierduinen, the Netherlands Brain Foundation, the Royal Netherlands Academy for Arts and Sciences and the Emmy Noether research grant KU 1401/4-1 and KU 1401/4-2 of the German Research Foundation (DFG) awarded to Brigitte M. Kudielka. Authors NvL, SB, ERdK, RHD, BMK, & SW are members of the International Research Training Group (IRTG) funded by the DFG (GRH 1389/1) and the NWO (grant DN95-420). The sponsors had no further role in study design;

Conflict of interest

None declared.

Acknowledgements

The support by GGZ Rivierduinen and the Royal Netherlands Academy for Arts and Sciences is gratefully acknowledged. We would like to thank J. Kellner, C. Jones, M. Hilkhuijsen and D. Schoonderwoerd for their technical support.

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      Two SNPs, rs2070951 and rs5522, are located in the 5′ region of the NR3C2 gene; rs2070951 is located in the non-coding region and has two alleles of G/C, while rs5522 is located in the coding region, has two alleles of A/G, and leads to missense mutations encoding isoleucine and valine (A/G, I180V). Leeuwen and Klok et al. found that CA haplotypes increased MR activity (Leeuwen et al., 2011; Klok et al., 2011a), and the increase in MR activity was associated with heightened dispositional optimism, fewer desperate thoughts, and a lower risk of depression, although all effects were limited to women (Klok et al., 2011a). Consistent with these results, carriers of the G allele of rs5522 showed decreased MR activity and increased vulnerability to depressive symptoms among the elderly population (Kuningas et al., 2007).

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    Both these authors contributed equally to this work.

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