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Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

Published on: November 15, 2013

The mineralocorticoid receptor is a constitutive nuclear factor in cardiomyocytes due to hyperactive nuclear

Iván Hernández-Díaz¹, Teresa Giraldez, María Rosa Arnau

¹Departamento de Fisiología, Facultad de Medicina, Universidad de La Laguna, La Laguna 38071, Spain.

|May 21, 2010

Abstract:

The mineralocorticoid receptor (MR), a member of the nuclear receptor family, mediates the action of aldosterone in target epithelia, enhancing sodium reabsorption. In addition, MR may have other physiological functions in nonepithelial tissues. Altered expression or inappropriate activation of cardiac MR is directly linked to the development of cardiac fibrosis, and MR blockade is beneficial for the treatment of heart failure. However, the physiological role, activation status, and target genes of MR in the heart are poorly known. Because ligand-free steroid receptors are typically cytoplasmic and translocate to the nucleus upon ligand binding, we examined the subcellular localization of MR under different corticosteroid levels using subcellular fractionation and immunostaining. Our results demonstrate that MR is a chromatin-bound factor in mouse left ventricle and in a cultured model of cardiomyocytes, HL-1 cells, regardless of circulating corticosteroid levels. Immunohistochemical localization of MR in human heart confirms the subcellular localization pattern. Mutation of nuclear localization signals (NLSs) demonstrates that MR constitutive nuclear localization mainly depends on the synergistic contribution of NLS0 and NLS1. Constitutive nuclear localization in HL-1 cells can be reverted by cotransfection of heat shock protein 90. Heat shock protein 90 expression levels in the mouse heart and HL-1 cells are lower than those found in other tissues, suggesting that low levels of cochaperones render MR NLSs hyperactive in cardiomyocytes. Even though MR is constitutively nuclear, corticosteroids still control the transactivation properties of the receptor in a model promoter, although other MR ligand-independent activities cannot be excluded.

Insights

The mineralocorticoid receptor (MR) is constitutively nuclear in heart cells, independent of aldosterone. This finding suggests novel roles for MR in cardiac function and disease, potentially independent of its canonical signaling pathway.

Area of Science:

Cardiovascular Biology
Molecular Endocrinology
Nuclear Receptor Signaling

Background:

The mineralocorticoid receptor (MR) mediates aldosterone's effects on sodium reabsorption in epithelia.
Cardiac MR is implicated in cardiac fibrosis and heart failure, but its physiological role and activation status in the heart are poorly understood.
Steroid receptors typically reside in the cytoplasm and translocate to the nucleus upon ligand binding.

Purpose of the Study:

To investigate the subcellular localization of the mineralocorticoid receptor (MR) in cardiac tissue under varying corticosteroid levels.
To elucidate the mechanisms governing MR's localization and activation in cardiomyocytes.
To explore potential ligand-independent functions of MR in the heart.

Main Methods:

Subcellular fractionation and immunostaining in mouse left ventricle and HL-1 cardiomyocytes.
Immunohistochemistry in human heart tissue.
Mutation analysis of nuclear localization signals (NLSs) and co-transfection with heat shock protein 90 (HSP90).

Main Results:

MR is constitutively chromatin-bound in mouse and human heart cells, irrespective of corticosteroid levels.
Constitutive nuclear localization is primarily dependent on NLS0 and NLS1, and can be reversed by HSP90.
Lower HSP90 expression in cardiomyocytes may contribute to hyperactive MR NLSs.
Corticosteroids modulate MR transactivation, but ligand-independent activities are possible.

Conclusions:

Cardiac MR exhibits constitutive nuclear localization, challenging the traditional model of ligand-dependent nuclear translocation.
This nuclear localization is regulated by specific NLSs and influenced by HSP90 levels.
The findings suggest novel mechanisms of MR regulation and function in the heart, potentially involving ligand-independent pathways relevant to cardiovascular disease.

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