RESUMEN
Promoter-bound steroid receptors activate gene expression by recruiting members of the p160 family of coactivators. Many steroid receptors, most notably the progesterone and estrogen receptors, are regulated both by cognate hormone and independently by growth factors. Here we show that epidermal growth factor regulates the activities of the p160 GRIP1 through the extracellular signal-regulated kinase (ERK) family of mitogen-activated protein kinases. ERKs phosphorylate GRIP1 at a specific site, Ser-736, the integrity of which is required for full growth factor induction of GRIP1 transcriptional activation and coactivator function. We propose that growth factors signal to nuclear receptors in part by targeting the p160 coactivators.
Asunto(s)
Factor de Crecimiento Epidérmico/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores de Esteroides/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Células HeLa , Humanos , Coactivador 2 del Receptor Nuclear , FosforilaciónRESUMEN
Regulation of nuclear receptor gene expression involves dynamic and coordinated interactions with histone acetyl transferase (HAT) and deacetylase complexes. The estrogen receptor (ERalpha) contains two transactivation domains regulating ligand-independent and -dependent gene transcription (AF-1 and AF-2 (activation functions 1 and 2)). ERalpha-regulated gene expression involves interactions with cointegrators (e.g. p300/CBP, P/CAF) that have the capacity to modify core histone acetyl groups. Here we show that the ERalpha is acetylated in vivo. p300, but not P/CAF, selectively and directly acetylated the ERalpha at lysine residues within the ERalpha hinge/ligand binding domain. Substitution of these residues with charged or polar residues dramatically enhanced ERalpha hormone sensitivity without affecting induction by MAPK signaling, suggesting that direct ERalpha acetylation normally suppresses ligand sensitivity. These ERalpha lysine residues also regulated transcriptional activation by histone deacetylase inhibitors and p300. The conservation of the ERalpha acetylation motif in a phylogenetic subset of nuclear receptors suggests that direct acetylation of nuclear receptors may contribute to additional signaling pathways involved in metabolism and development.
Asunto(s)
Estrógenos/metabolismo , Receptores de Estrógenos/genética , Transducción de Señal , Activación Transcripcional , Acetilación , Animales , Receptor alfa de Estrógeno , Receptores de Estrógenos/metabolismoRESUMEN
Estrogen receptors (ERs alpha and beta) enhance transcription in response to estrogens by binding to estrogen response elements (EREs) within target genes and utilizing transactivation functions (AF-1 and AF-2) to recruit p160 coactivator proteins. The ERs also enhance transcription in response to estrogens and antiestrogens by modulating the activity of the AP-1 protein complex. Here, we examine the role of AF-1 and AF-2 in ER action at AP-1 sites. Estrogen responses at AP-1 sites require the integrity of the ERalpha AF-1 and AF-2 activation surfaces and the complementary surfaces on the p160 coactivator GRIP1 (glucocorticoid receptor interacting protein 1), the NID/AF-1 region, and NR boxes. Thus, estrogen-liganded ERalpha utilizes the same protein-protein contacts to transactivate at EREs and AP-1 sites. In contrast, antiestrogen responses are strongly inhibited by ERalpha AF-1 and weakly inhibited by AF-2. Indeed, ERalpha truncations that lack AF-1 enhance AP-1 activity in the presence of antiestrogens, but not estrogens. This phenotype resembles ERbeta, which naturally lacks constitutive AF-1 activity. We conclude that the ERs enhance AP-1 responsive transcription by distinct mechanisms with different requirements for ER transactivation functions. We suggest that estrogen-liganded ER enhances AP-1 activity via interactions with p160s and speculate that antiestrogen-liganded ER enhances AP-1 activity via interactions with corepressors.
Asunto(s)
Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Factor de Transcripción AP-1/metabolismo , Sitios de Unión , Estradiol/metabolismo , Estradiol/farmacología , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Células HeLa/efectos de los fármacos , Humanos , Fenotipo , Receptores de Estrógenos/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Elementos de Respuesta , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Eliminación de Secuencia , Serina , Tamoxifeno/farmacología , Factor de Transcripción AP-1/genética , Activación TranscripcionalRESUMEN
Cross-interference (squelching) among nuclear receptors has been proposed to reflect the titration of coactivators that bind the receptors in a hormone-dependent manner. We have tested whether the coactivators are the only target titrated during squelching of one receptor by another, or whether proteins needed for coactivator function are titrated as well. That the coactivators are indeed one target of squelching is apparent. The isolated ligand-binding domain of the estrogen receptor (ER-LBD) squelches transcriptional activation by the thyroid hormone receptor (TR) only when the LBD is bound to ligands that promote coactivator interactions and only when regions of the LBD that promote coactivator interactions are undisturbed. Furthermore, the ER-LBD and the TR compete in vitro for the related p160 coactivators, SRC1a and GRIP1 (glucocorticoid receptor interacting protein 1), or the putative corepressor, RIP140. Finally TR action becomes more potent when coactivator levels are raised. Nonetheless, supplying excess SRC1a or GRIP1 does not abolish squelching by the ER. In fact, squelching becomes even more severe when coactivators are abundant. Supplying combinations of coactivators from the p160 class and the CREB-binding protein (CBP)/p300 class makes squelching most severe. Elevated RIP140 inhibits TR action, but also protects the residual TR action from squelching by the ER-LBD. We conclude that ER-LBD squelches TR both by titrating p160-CBP coactivators and additionally by cooperating with the coactivators to titrate a second factor. The second factor would be needed by the TR for coactivator-mediated transcriptional stimulation.
Asunto(s)
Receptores de Estrógenos/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Unión Competitiva , Proteína de Unión a CREB , Proteínas Portadoras/metabolismo , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Proteínas de Unión al ADN , Estradiol/metabolismo , Estradiol/farmacología , Regulación de la Expresión Génica , Genes Reporteros , Histona Acetiltransferasas , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Coactivador 1 de Receptor Nuclear , Coactivador 2 del Receptor Nuclear , Proteína de Interacción con Receptores Nucleares 1 , Proteínas de Transporte Nucleocitoplasmático , Proteínas de Unión al ARN , Receptores de Estrógenos/efectos de los fármacos , Receptores de Estrógenos/genética , Receptores de Glucocorticoides/metabolismo , Receptores de Progesterona/metabolismo , Receptores de Hormona Tiroidea/efectos de los fármacos , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tamoxifeno/metabolismo , Tamoxifeno/farmacología , Volumetría , Transactivadores/genética , Factor de Transcripción TFIIB , Factores de Transcripción/genética , Transcripción Genética , Triyodotironina/metabolismo , Triyodotironina/farmacologíaRESUMEN
We find that tamoxifen is a potent activator of estrogen receptor (ER)- mediated induction of promoters regulated by AP-1 sites including the human collagenase gene promoter and constructs in which an AP-1 site is fused to the herpes thymidine kinase promoter. This contrasts with the inability of tamoxifen to activate otherwise identical promoters bearing classical estrogen response elements. Tamoxifen agonism at AP-1 sites is cell type specific, occurring in cell lines of uterine, but not of breast, origin. It thus parallels tamoxifen agonism in vivo. AP-1 proteins such as Jun or Jun/Fos are needed for tamoxifen stimulation, and tamoxifen increases the transcriptional efficiency of these proteins even when they are provided at optimal amounts. The DNA binding domain (DBD) of ER is required for tamoxifen activation at AP-1 sites. In contrast, estrogen activation is partially independent of this domain. This suggests the existence of two pathways of ER action at AP-1: an alpha (DBD-dependent) pathway activated by tamoxifen, and a beta (DBD-independent) pathway activated by estrogen. Fusing VP16 transcriptional activation functions to ER potentiates the beta, but not the alpha, pathway. We discuss models for the two pathways and the possibility that the AP-1 pathway is a major route by which ER affects target tissue growth and differentiation in vivo.
Asunto(s)
Mama/efectos de los fármacos , Endometrio/efectos de los fármacos , Antagonistas de Estrógenos/farmacología , Receptores de Estrógenos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Tamoxifeno/farmacología , Factor de Transcripción AP-1/metabolismo , Células 3T3/efectos de los fármacos , Animales , Secuencia de Bases , Sitios de Unión , Neoplasias de la Mama/patología , Células CHO/efectos de los fármacos , Colagenasas/biosíntesis , Colagenasas/genética , Secuencia de Consenso , Cricetinae , Cricetulus , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Células HeLa/efectos de los fármacos , Humanos , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Regiones Promotoras Genéticas/efectos de los fármacos , Receptores de Estrógenos/antagonistas & inhibidores , Receptores de Estrógenos/química , Proteínas Recombinantes de Fusión/biosíntesis , Transcripción Genética/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Células Tumorales CultivadasAsunto(s)
Artefactos , Clonación Molecular , ADN Bacteriano/genética , Células Eucariotas/metabolismo , Vectores Genéticos/genética , Plásmidos/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Sitios de Unión , ADN Bacteriano/metabolismo , Sustancias de Crecimiento/fisiología , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-fos/metabolismo , Receptores de Esteroides/fisiología , Transcripción GenéticaRESUMEN
While steroid response is generally restricted by the availability of steroid receptors, the theoretical limits of the response are not known. We have constructed a series of cell lines that stably express the estrogen receptor (ER) at levels up to 5,000,000 ERs per cell and employed these cells to explore the limits of the estrogen response. Several reporter genes with estrogen response elements upstream of the herpes thymidine kinase promoter showed hyperbolic saturation kinetics with increasing ER. Maximum response was 10 times that seen in cell lines with receptor titers comparable to physiological levels. Half-maximal responses required 500,000 receptors per cell, and cells with 5,000,000 ERs showed greater than 90% maximum induction. Estradiol dose-response studies indicated that the receptors are limiting below 500,000 ERs per cell, but at higher ER titers there are spare receptors. In contrast to most reporters, the widely used reporter pA2-CAT, which has 200 base pairs of Xenopus vitellogenin DNA between the response element and the promoter, showed squelching at ER levels beyond 500,000 per cell. Cell lines that expressed ER above this level activated pA2-CAT with a distorted hormone dependence, where saturating ligand concentrations were inhibitory. All reporters displayed squelching when the ER was provided by transient transfection at a level that we judge is 20,000,000 per cell by extrapolation from the behavior of stable cell lines. These findings suggest that saturation of the cellular capacity to mediate an estrogen response and ER-dependent squelching occur at receptor titers well above those encountered in nature. If current models of steroid hormone action are correct, the findings also imply that estrogen response elements are occupied to very small extents under normal conditions.