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1.
Mol Endocrinol ; 24(1): 104-13, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19887648

RESUMEN

Glucocorticoids are synthesized locally in adipose tissue and contribute to metabolic disease through the facilitation of adipose tissue expansion. Here we report that exposure of human primary preadipocytes to glucocorticoids increases their sensitivity to insulin and enhances their subsequent response to stimuli that promote differentiation. This effect was observed in primary human preadipocytes but not in immortalized 3T3-L1 murine preadipocytes or in fully differentiated primary human adipocytes. Stimulation of insulin signaling was mediated through induction of insulin receptor (IR), IR substrate protein 1 (IRS1), IRS2, and the p85 regulatory subunit of phosphoinositide-3-3-kinase, which led to enhanced insulin-mediated activation of Akt. Although induction of IRS2 was direct, induction of IR and IRS1 by glucocorticoids occurred subsequent to primary induction of the forkhead family transcription factors FoxO1A and FoxO3A. These results reveal a new role for glucocorticoids in preparing preadipocytes for differentiation.


Asunto(s)
Adipocitos/efectos de los fármacos , Adipogénesis/efectos de los fármacos , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Insulina/farmacología , Células 3T3-L1 , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Factores de Transcripción Forkhead/genética , Perfilación de la Expresión Génica , Glucocorticoides/fisiología , Humanos , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Factores de Tiempo
2.
J Biol Chem ; 282(15): 10963-71, 2007 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-17314103

RESUMEN

The glucocorticoid receptor (GR) cycles between a naive chaperone-complexed form in the cytoplasm and a transcriptionally active steroid-bound nuclear form. Nuclear import of GR occurs rapidly and is mediated through the importin alpha/beta karyopherin import pathway. By contrast, nuclear export of GR occurs only slowly under most conditions, despite a dependence on active signaling. In this study we have defined a nuclear retention signal (NRS) in the hinge region of GR that actively opposes the nuclear export of GR as well as the nuclear export mediated through an ectopic CRM1-dependent nuclear export signal (NES). The GR NRS overlaps closely with the basic NL1 nuclear localization signal (NLS) but can be distinguished from NL1 by targeted mutagenesis. Substitution of the classical NLS from SV40 T antigen for the GR NL1 results in a receptor in which nuclear export is accelerated. Remarkably, although the SV40-modified GR remains predominantly nuclear in the presence of steroid and is recruited to transcriptional regulatory regions indistinguishably from wild-type GR, the substitution dramatically weakens the ability of GR to activate transcription of a mouse mammary tumor virus reporter gene. These results suggest that active nuclear retention of GR plays an integral role in glucocorticoid signaling.


Asunto(s)
Receptores de Glucocorticoides/metabolismo , Transducción de Señal , Activación Transcripcional/genética , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Aminoácidos/genética , Aminoácidos/metabolismo , Animales , Células COS , Chlorocebus aethiops , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Ratas , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/genética
3.
J Cell Biol ; 170(5): 733-44, 2005 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-16129783

RESUMEN

Cellular pathways relay information through dynamic protein interactions. We have assessed the kinetic properties of the murine double minute protein (MDM2) and von Hippel-Lindau (VHL) ubiquitin ligases in living cells under physiological conditions that alter the stability of their respective p53 and hypoxia-inducible factor substrates. Photobleaching experiments reveal that MDM2 and VHL are highly mobile proteins in settings where their substrates are efficiently degraded. The nucleolar architecture converts MDM2 and VHL to a static state in response to regulatory cues that are associated with substrate stability. After signal termination, the nucleolus is able to rapidly release these proteins from static detention, thereby restoring their high mobility profiles. A protein surface region of VHL's beta-sheet domain was identified as a discrete [H+]-responsive nucleolar detention signal that targets the VHL/Cullin-2 ubiquitin ligase complex to nucleoli in response to physiological fluctuations in environmental pH. Data shown here provide the first evidence that cells have evolved a mechanism to regulate molecular networks by reversibly switching proteins between a mobile and static state.


Asunto(s)
Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Nucléolo Celular/ultraestructura , Recuperación de Fluorescencia tras Fotoblanqueo , Humanos , Concentración de Iones de Hidrógeno , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Señales de Clasificación de Proteína , Transporte de Proteínas/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2 , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau
4.
J Biol Chem ; 280(17): 17549-61, 2005 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-15737989

RESUMEN

The mineralocorticoid receptor (MR) is a tightly regulated nuclear hormone receptor that selectively transmits corticosteroid signals. Steroid treatment transforms MR from a transcriptionally inert state, in which it is distributed equally between the nucleus and cytoplasm, to an active completely nuclear transcription factor. We report here that MR is an atypical nuclear hormone receptor that moves unidirectionally from the cytoplasm to the nucleus. We show that nuclear import of MR is controlled through three nuclear localization signals (NLSs) of distinct types. Nuclear localization of naive MR was mediated primarily through a novel serine/threonine-rich NLS (NL0) in the receptor N terminus. Specific amino acid substitutions that mimicked phosphorylation selectively enhanced or repressed NL0 activity, highlighting the potential for active regulation of this new type of NLS. The second NLS (NL2) within the ligand-binding domain also lacks a recognizable basic motif. Nuclear transfer through this signal was strictly dependent on steroid agonist, but was independent of the interaction of MR with coactivator proteins. The third MR NLS (NL1) is a bipartite basic motif localized to the C terminus of the MR DNA-binding domain with properties distinct from those of NL1 of the closely related glucocorticoid receptor. NL1 acted in concert with NL0 and NL2 to stimulate nuclear uptake of the agonist-treated receptor, but also directed the complete nuclear localization of MR in response to treatment with steroid antagonist. These results present MR as a nuclear hormone receptor whose unidirectional transfer to the nucleus may be regulated through multiple pathways.


Asunto(s)
Núcleo Celular/metabolismo , Señales de Localización Nuclear , Receptores de Mineralocorticoides/metabolismo , Serina/química , Treonina/química , Transporte Activo de Núcleo Celular , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Transporte Biológico , Western Blotting , Células COS , Citoplasma/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Técnica del Anticuerpo Fluorescente Indirecta , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Ligandos , Datos de Secuencia Molecular , Matriz Nuclear/metabolismo , Fosforilación , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Receptores de Glucocorticoides/metabolismo , Esteroides/metabolismo , Factores de Tiempo , Transfección
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