RESUMEN
Cortisol resistance has also been reported in the degu, Octodon degus, a New World hystricomorph endemic to central Chile. The degu is used as a model for studies of stress and diurnal rhythms, parental behaviour and female masculinization. Another New World hystricomorph, the guinea pig, also exhibits glucocorticoid resistance, a result of amino acid sequences that differ from other mammalian glucocorticoid receptors (GR). Mutations in the ligand-binding domain of the human GR have been identified in familial or sporadic generalised cortisol resistance as have variants in the guinea pig. To address the possibility that the high levels of cortisol observed in the degu are a result of the same or similar sequence variations observed in the guinea pig GR, we have cloned, expressed and characterised the ligand-binding domain (LBD) of the degu GR. Somewhat unexpectedly, neither the amino acids nor the region involved in the resistance observed in the guinea pig GR are relevant in the degu GR. The relative resistance to cortisol observed in the degu GR is conferred by the substitution of two isoleucine residues, which are highly conserved in the GR across species, with a valine doublet. These amino acids lie in the region between helices 5 and 6 of the GR LBD, a region known to be important in determining the affinity of ligand-binding in steroid receptors.
Asunto(s)
Octodon , Aminoácidos , Animales , Femenino , Cobayas , Hidrocortisona/metabolismo , Ligandos , Octodon/metabolismo , Receptores de Glucocorticoides/genéticaRESUMEN
The mineralocorticoid receptor (MR) plays a central role in sodium homoeostasis by transducing the response to aldosterone in the distal nephron and other sodium transporting epithelia. The MR is a member of the nuclear receptor family of ligand-dependent transcription factors; it is unusual in being the receptor for two steroid hormones aldosterone and cortisol (which also binds to the closely related glucocorticoid receptor). Less well recognised is that progesterone also binds to the MR with high affinity. The conformation of the ligand-bound receptor is determined by the ligand including whether the conformation is agonist or antagonist. An agonist MR conformation then enables interactions with DNA, other MR (homodimerization) and coregulatory molecules to regulate gene expression. Insights into the structural determinants of an agonist response to ligand come from studies of the evolution of the MR. Progesterone is an agonist in the fish MR, but antagonist in the MR of terrestrial vertebrates; this switch results from the loss of a critical leucine that mediates a leucine:leucine interaction between helix 1 and helix 8 which enables the agonist response to progesterone. The insights into the intramolecular dynamics of activation suggest novel ways in which MR antagonism may be achieved beyond the current, progesterone-based antagonists in clinical use.
Asunto(s)
Aldosterona , Receptores de Mineralocorticoides , Animales , Humanos , Hidrocortisona , Ligandos , Antagonistas de Receptores de MineralocorticoidesRESUMEN
The mineralocorticoid receptor (MR) is highly conserved across vertebrate evolution. In terrestrial vertebrates, the MR mediates sodium homeostasis by aldosterone and also acts as a receptor for cortisol. Although the MR is present in fish, they lack aldosterone. The MR binds progesterone and spironolactone as antagonists in human MR but as agonists in zebrafish MR. We have defined the molecular basis of these divergent responses using MR chimeras between the zebrafish and human MR coupled with reciprocal site-directed mutagenesis and molecular dynamic (MD) simulation based on the crystal structures of the MR ligand-binding domain. Substitution of a leucine by threonine in helix 8 of the ligand-binding domain of the zebrafish MR confers the antagonist response. This leucine is conserved across fish species, whereas threonine (serine in rodents) is conserved in terrestrial vertebrate MR. MD identified an interaction of the leucine in helix 8 with a highly conserved leucine in helix 1 that stabilizes the agonist conformation including the interaction between helices 3 and 5, an interaction which has previously been characterized. This switch in the MR coincides with the evolution of terrestrial vertebrates and of aldosterone synthesis. It was perhaps mandatory if the appearance of aldosterone as a specific mediator of the homeostatic salt retention was to be tolerated. The conformational changes also provide insights into the structural basis of agonism versus antagonism in steroid receptors with potential implications for drug design in this important therapeutic target.
Asunto(s)
Evolución Molecular , Progesterona/metabolismo , Dominios y Motivos de Interacción de Proteínas/genética , Receptores de Mineralocorticoides/genética , Espironolactona/metabolismo , Aldosterona/biosíntesis , Sustitución de Aminoácidos , Animales , Homeostasis , Humanos , Leucina/genética , Ligandos , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica en Hélice alfa/genética , Receptores de Mineralocorticoides/metabolismo , Roedores/genética , Roedores/metabolismo , Serina/genética , Relación Estructura-Actividad , Treonina/genética , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
BACKGROUND: Most colorectal cancer (CRC) patients are diagnosed at an advanced or metastatic stage with poor prognosis. Ubiquitin-specific protease 6 N-terminal-like protein (USP6NL) with high expression in CRC tissues regulates CRC cell proliferation via Wnt/ß-catenin pathway. We hypothesized that USP6NL impacts CRC growth and inhibition of USP6NL may be a novel treatment strategy to improve CRC therapy. METHODS: USP6NL level in human CRC tissues and its association with tumor growth and metastasis were examined. Its roles and potential mechanisms in regulating tumor growth were studied by genetic and pharmacological manipulation of CRC cells in vitro and in vivo. RESULTS: Herein, we found that USP6NL was up-regulated in tumorous tissues of CRC patients. Our data suggested that knockdown of USP6NL in human CRC cell lines (HCT116 and LOVO cells) inhibited cell proliferation, induced G0/G1 cell cycle arrest, and prevented the tumorigenicity of HCT116 cells in nude mice, and which was associated with the prevention of Wnt/ß-catenin pathway. On the contrary, USP6NL overexpression in human CRC cells (SW480) showed the opposite result. Our data suggested that the promoted cell proliferation, G1/S cell cycle progression, and the enhanced expression of ß-catenin Cyclin D1 and C-myc while reduced P27 induced by the overexpression of USP6NL were significantly reversed by additional treatment of XAV939, indicating that activating Wnt/ß-catenin pathway was the mechanism, by which USP6NL exerted carcinogenesis in CRC in vitro. Besides, our data suggested that knockdown of USP6NL increased the ubiquitination of ß-catenin, indicating that USP6NL may serve as a deubiquitinase that regulated ß-catenin accumulation in this process. Furthermore, 10058-F4 down-regulated USP6NL, inhibited CRC cell proliferation and induced cell cycle arrest. The result demonstrated a possible feedback loop between USP6NL, ß-catenin and C-myc in regulating CRC cell growth. CONCLUSION: USP6NL was an oncogene in CRC, and it may be a potential target for the treatment of CRC.
RESUMEN
The classic role of mineralocorticoid receptor (MR) is to promote sodium transport in epithelial tissues. However, the MR is also expressed in a range of tissues in which its role appears unrelated to sodium transport, and under normal physiological conditions, it may be responding to cortisol (corticosterone in rodents) rather than aldosterone. The relative importance of transcriptional mechanisms such as classical genomic signaling via a hormone response element, transrepression of other transcription factors, and nongenomic signaling is not clear, particularly in nonepithelial tissues. The goal of the present study was to define the role of the different signaling pathways for the MR by separating the functional role of classic genomic signaling, mediated by DNA binding, from these two other mechanisms. We used gene targeting to generate mice in which serine is substituted for cysteine at codon 603 in the MR; this mutation precludes DNA binding. These MR C603S mutant mice either die at birth or fail to thrive, lose weight, and die between days 10 and 13 in a manner similar to that observed previously for mice null for the MR gene. Renal expression and cellular localization of MR C603S by immunohistochemistry was equivalent to control mice. MR C603S mice were rescued by twice-daily saline injections. Despite increased aldosterone levels, renal expression of aldosterone-induced genes was not increased. This unique mouse model demonstrates that DNA binding is essential for the epithelial MR response and will provide the basis for analysis of nonclassical signaling of the MR in nonepithelial tissues.
Asunto(s)
Aldosterona/farmacología , ADN/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Receptores de Mineralocorticoides/metabolismo , Sodio/metabolismo , Secuencia de Aminoácidos , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Sustitución del Gen , Transporte Iónico/efectos de los fármacos , Transporte Iónico/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Missense , Unión Proteica/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas/genética , Receptores de Mineralocorticoides/genética , Dedos de Zinc/genéticaRESUMEN
Aldosterone administration in rats results in several cardiac alterations. Previous studies have demonstrated that proanthocyanidins, phenolic bioactive compounds, have cardioprotective effects. We studied the potential beneficial effects of the proanthocyanidin-rich almond skin extract (PASE) on the cardiac alterations induced by aldosterone-salt treatment, their effects in mineralocorticoid receptor activity and we sought to confirm proanthocyanidins as the specific component of the extract involved in the beneficial cardiac effects. Male Wistar rats received aldosterone (1 mg/Kg/day) +1% NaCl for 3 weeks. Half of the animals in each group were simultaneously treated with either PASE (100 mg/Kg/day) or spironolactone (200 mg/Kg/day). The ability of PASE to act as an antagonist of the mineralocorticoid receptor was examined using a transactivation assay. High performance liquid chromatography was used to identify and to isolate proanthocyanidins. Hypertension and diastolic dysfunction induced by aldosterone were abolished by treatment with PASE. Expression of the aldosterone mediator SGK-1, together with fibrotic, inflammatory and oxidative mediators were increased by aldosterone-salt treatment; these were reduced by PASE. Aldosterone-salt induced transcriptional activity of the mineralocorticoid receptor was reduced by PASE. HPLC confirmed proanthocyanidins as the compound responsible for the beneficial effects of PASE. The effects of PASE were comparable to those seen with the mineralocorticoid antagonist, spironolactone. The observed responses in the aldosterone-salt treated rats together with the antagonism of transactivation at the mineralocorticoid receptor by PASE provides evidence that the beneficial effect of this proanthocyanidin-rich almond skin extract is via as a mineralocorticoid receptor antagonist with proanthocyanidins identified as the compounds responsible for the beneficial effects of PASE.
Asunto(s)
Corazón/efectos de los fármacos , Hipertensión/prevención & control , Antagonistas de Receptores de Mineralocorticoides/farmacología , Extractos Vegetales/farmacología , Proantocianidinas/farmacología , Receptores de Mineralocorticoides/metabolismo , Aldosterona/toxicidad , Animales , Línea Celular , Chlorocebus aethiops , Hipertensión/etiología , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Masculino , Antagonistas de Receptores de Mineralocorticoides/uso terapéutico , Extractos Vegetales/uso terapéutico , Proantocianidinas/uso terapéutico , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Prunus/química , Ratas , Ratas Wistar , Receptores de Mineralocorticoides/genética , Espironolactona/farmacología , Espironolactona/uso terapéuticoRESUMEN
The mineralocorticoid receptor (MR) is unique in responding to 2 physiological ligands: aldosterone and cortisol. In epithelial tissues, aldosterone selectivity is determined by the activity of 11ß-hydroxysteroid dehydrogenase type 2. In other tissues, cortisol is the primary ligand. To understand the structural determinants of ligand-specific MR activation, we sought to identify coregulatory molecules that interact with the ligand-binding domain (LBD) of the MR. A yeast-2-hybrid (Y2H) kidney cDNA library was screened with the human MR-LBD in the presence of aldosterone and cortisol. One clone, identified as aldosterone-specific in the Y2H assay, exhibited a 7-fold greater response, aldosterone vs. cortisol, in a mammalian-2-hybrid (M2H) assay. This clone encodes the region of the tesmin gene that has 2 leucine-x-x-leucine-leucine (LxxLL) motifs. Full-length tesmin coactivates (>2-fold) MR-mediated transactivation in the presence of aldosterone, but not of cortisol; this specificity is observed with a range of promoters. GST pulldown and coimmunoprecipitation of the MR by tesmin supports a direct interaction, mediated by the 2 LxxLL motifs. Tesmin thus represents a novel MR coregulator that exhibits a differential interaction, providing further evidence of the adoption of ligand-dependent conformations by the MR-LBD.
Asunto(s)
Metalotioneína/metabolismo , Coactivadores de Receptor Nuclear/metabolismo , Receptores de Mineralocorticoides/metabolismo , Aldosterona/metabolismo , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Humanos , Hidrocortisona/metabolismo , Metalotioneína/química , Metalotioneína/genética , Coactivadores de Receptor Nuclear/química , Coactivadores de Receptor Nuclear/genética , Regiones Promotoras Genéticas , Unión Proteica , Receptores de Mineralocorticoides/genética , Especificidad por Sustrato , Técnicas del Sistema de Dos HíbridosRESUMEN
Comparison between evolutionarily distant receptors can provide critical insights into both structure and function. Sequence comparison between the mineralocorticoid receptors (MR) of the zebrafish (zMR) and human (hMR) reveals a high degree of sequence conservation in the major functional domains. We isolated a zMR cDNA to contrast the transcriptional response to a range of ligands and to establish whether a teleost MR exhibits the amino/carboxyl-terminal interaction (N/C-interaction) previously reported for the hMR. Aldosterone, deoxycorticosterone (DOC) and cortisol induced zMR transcriptional activity with similar efficacy to that observed with the hMR. The hMR antagonist, spironolactone, acted as an agonist with the zMR. The zMR exhibited an N/C-interaction in response to aldosterone but, in contrast to the hMR, cortisol and DOC predominantly stimulated the interaction in the zMR. Conservation of the N/C-interaction between evolutionarily distant MR provides evidence of functional significance.
Asunto(s)
Receptores de Mineralocorticoides/metabolismo , Pez Cebra/metabolismo , Aldosterona/metabolismo , Aldosterona/farmacología , Secuencia de Aminoácidos , Animales , Antihipertensivos/metabolismo , Antihipertensivos/farmacología , Línea Celular , Desoxicorticosterona/metabolismo , Desoxicorticosterona/farmacología , Humanos , Hidrocortisona/metabolismo , Hidrocortisona/farmacología , Antagonistas de Receptores de Mineralocorticoides/metabolismo , Antagonistas de Receptores de Mineralocorticoides/farmacología , Datos de Secuencia Molecular , Nimodipina/metabolismo , Nimodipina/farmacología , Receptores de Mineralocorticoides/agonistas , Receptores de Mineralocorticoides/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Espironolactona/metabolismo , Espironolactona/farmacología , Activación Transcripcional/efectos de los fármacos , Pez Cebra/anatomía & histología , Pez Cebra/genéticaAsunto(s)
Mutación , Seudohipoaldosteronismo/genética , Receptores de Mineralocorticoides/genética , Aldosterona/química , Aldosterona/metabolismo , Análisis Mutacional de ADN , Humanos , Masculino , Persona de Mediana Edad , Modelos Moleculares , Unión Proteica , Receptores de Mineralocorticoides/química , Receptores de Mineralocorticoides/metabolismoRESUMEN
The amino acids that confer aldosterone binding specificity to the mineralocorticoid receptor (MR) remain to be determined. We had previously analyzed a panel of chimeras created between the MR and the glucocorticoid receptor and determined that amino acids 804-874 of the MR ligand binding domain are critical for aldosterone binding. In the present study a further series of chimeras was created within this region. The chimeras were analyzed by a transactivation assay and [(3)H]aldosterone binding, and the critical region was narrowed down to amino acids 820-844. Site-directed mutagenesis was used to create single and multiple amino acid substitutions in this region. These studies identified 12 of the 16 amino acids that differ in the MR and the glucocorticoid receptor in this region as being critical to conferring aldosterone responsivity. The amino acids that differ in the region 820-844 lie on the surface of the molecule and, therefore, it appears that MR ligand binding selectivity is conferred by residues that do not form part of the ligand binding pocket. Other studies have found that the corresponding regions of the androgen and glucocorticoid receptors are critical for the binding of natural and synthetic ligands, suggesting a common mechanism governing ligand binding specificity. The new chimeras also displayed, as previously reported, a dissociation between cortisol binding and transactivation and, intriguingly, only those that bound aldosterone with high affinity were activated by cortisol, suggesting a common mechanism that underlies specificity of aldosterone binding and the ability of cortisol to activate the MR.