Presence of urotensin-II receptors at the cell nucleus: Specific tissue distribution and hypoxia-induced modulation

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Abstract

Urotensin II (UII) and its receptor UT, are widely expressed in the cardiovascular and central nervous system, where they exert regulatory actions under both physiological and pathological conditions. Our study, aimed at investigating the presence of functional nuclear UT in various rat and monkey tissues as well as in human cell lines, demonstrated for the first time by Western blot analysis and confocal immunofluorescence a tissue-specific nuclear expression of this receptor (heart and central nervous system). This nuclear UT was further characterized pharmacologically through radioligand binding studies using specific ligands of the urotensinergic system, as well as somatostatin. In 2D-gel experiments, we observed the presence of different post-translational modifications between membrane and nuclear UT receptors in brain extracts. Transcription initiation assays showed de novo RNA synthesis caused by UII and Urotensin-related peptide (URP) which were inhibited by an UT antagonist urantide. In hypoxic/ischemic conditions, UT receptors were differentially modulated in regard to subcellular localization. Thus, the unique regiospecificity of the nuclear UT receptor along with its particular modulation under hypoxic conditions could indicate a specific and complementary physiological role that could be correlated with pro-angiogenic and/or neuromodulatory actions of UII, both in the cardiovascular and central nervous system.

Introduction

In the classical view, peptide hormones bind to cell surface receptors, mostly G protein-coupled receptors (GPCR), and subsequently activate intracellular mediators of signal transduction. Over the last three decades, evidence has accumulated to indicate that at least some extracellular signalling molecules also bind and act within cells either after internalization into target cells or retention inside the producing cell (Re and Cook, 2006, Duverger et al., 1995, Gobeil et al., 2006). Defined as intracrines, such factors are quite diverse in structure and function (Re and Cook, 2008). Demonstration of an intracellular localization and trafficking of GPCRs both in vivo and in vitro was achieved (Koenig and Edwardson, 1997, Roth et al., 1981). However, only a few reports demonstrated the presence of functional GPCRs at the nucleus. Indeed, angiotensin II receptor (AT1), endothelin receptors (ETA) and (ETB) within the large family 1 of rhodopsin-like GPCRs, have been characterized in cell nuclei. In addition, the parathyroid hormone receptor and the metabotropic glutamate mGluR5 receptor, belonging respectively to family 2 and 3 of GPCRs, were also observed in nuclei (O’Malley et al., 2003). Interestingly, it was demonstrated that AT1 and endothelin-1 receptors present in the nuclei were able to induce intranuclear calcium mobilization and mRNA synthesis (Boivin et al., 2003, Eggena et al., 1993). These results suggested that nuclear receptors might represent a complementary system that could participate to the regulation of specific physiological action.

Urotensin II (UII), a disulfide bridged peptide originally isolated from the caudal neurosecretory system of a teleost fish (Pearson et al., 1980), is currently considered as the most potent mammalian vasoconstrictor identified so far (Douglas and Ohlstein, 2000). The urotensinergic system, composed of the urotensin II receptor (UT), UII and a paralogue peptide named urotensin II-related peptide (URP) (Vaudry et al., 2010), is not restricted to cardiac tissues. In fact, UT is widely expressed in the central nervous system as well as in various peripheral organs including kidney, liver, lung, skeletal muscle, pancreas, and adrenal gland (Jegou et al., 2006, Onan et al., 2004). UT activation is associated with multiple physiological actions such as vasoconstriction, vasodilation, osmoregulation, inhibition of glucose-induced insulin release, and cell proliferation (Vaudry et al., 2010). The multiple effects of UII and the broad expression pattern of its receptor indicate that the urotensinergic system could be involved in physiopathological processes. Indeed, correlations between different pathological states, i.e. chronic renal failure (Totsune et al., 2001), congestive heart failure (Gruson et al., 2006), hypertension (Cheung et al., 2004), diabetes mellitus (Totsune et al., 2003), and elevated concentration of plasmatic UII have been demonstrated.

The similarities existing between UII, endothelin-1 and angiotensin II, such as a strong contractile activity and receptors belonging to subclass 1A of GPCRs, prompted us to investigate the presence of UT at the surface of heart isolated nuclei. As we had hypothesized, our investigation confirmed the ectopic localization of functional nuclear UT receptors in heart (Doan et al., 2011). Consequently, based on our previous findings and the broad expression pattern of the urotensinergic system, we hypothesized that the presence of nuclear UT would not be limited to cardiac tissues.

In this paper, we investigated the presence of specific urotensin II binding sites both at the cellular and nuclear level in various rat and monkey tissues. Surprisingly, from a variety of tissues, nuclear UT receptors, as observed by Western blot and characterized by competitive binding assays, were only found in the central nervous system (CNS). Moreover, these nuclear receptors were functional as evaluated by a transcription initiation assay. Finally, we observed that cellular UT distribution was modulated under hypoxic conditions. Altogether, these results suggest a precise and complementary role for nuclear UT receptors.

Section snippets

Materials and methods

Materials, Total protein isolation, Isolation of membrane and nuclei from heart, Electrophoresis and Immunoblotting, Peptide Iodination, Nuclear Receptor Binding Assay, Confocal Microscopy, Transcription Initiation Assay, Effect of Cobalt chloride on UT receptor expression, Data Analysis are described in Supplemental Information.

Biochemical characterization of nuclear UII binding sites in rat tissues

A few years ago, the presence at the nuclear level of functional neuropeptide Y, endothelin-1 (ET-1), angiotensin II (AT), apelin and beta-adrenergic receptors suggested that nuclei were in fact a cell inside a cell (Boivin et al., 2008). The similarity between UII, AT and ET-1 in terms of physiological function led our group to investigate the presence of UII receptors at the nuclear surface. As such, we recently reported the presence of functional UT receptors in isolated rat heart nuclei (

Discussion

Hundreds of neurotransmitters regulate cellular development and function through G protein-coupled receptor signalling. GPCRs exert their effects by regulating ion channels, second messenger production, and protein kinase cascades, which in turn control cellular activity, gene expression, plasticity, differentiation, morphogenesis, and migration. In the recent years, the presence of endosomal receptors that could regulate complementary physiological action has almost become “a classic GPCR

Acknowledgements

This research was supported by the Canadian Institutes of Health Research. N.T.T.M is the recipient of a studentship from the Fondation Armand-Frappier.

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