RESUMO
Steroid receptor coactivator 2 (SRC-2) is a coactivator that regulates nuclear receptor activity. We previously reported that SRC-2 protein is degraded through the action of cAMP-dependent protein kinase A (PKA) and cAMP response element binding protein (CREB). In the study presented here, we aimed to identify proteins that interact with and thereby regulate SRC-2. We isolated cyclin C (CCNC) as an interacting partner with the SRC-2 degradation domain aa 347-758 in a yeast two-hybrid assay and confirmed direct interaction in an in vitro assay. The protein level of SRC-2 was increased with CCNC overexpression in COS-1 cells and decreased with CCNC silencing in COS-1 and MCF-7 cells. In a pulse-chase assay, we further show that silencing of CCNC resulted in a different SRC-2 degradation pattern during the first 6 h after the pulse. Finally, we provide evidence that CCNC regulates expression of cell cycle genes upregulated by SRC-2. In conclusion, our results suggest that CCNC temporarily protects SRC-2 against degradation and this event is involved in the transcriptional regulation of SRC-2 cell cycle target genes.
Assuntos
Ciclo Celular/fisiologia , Ciclina C/biossíntese , Coativador 2 de Receptor Nuclear/metabolismo , Proteólise , Transcrição Gênica/fisiologia , Regulação para Cima/fisiologia , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ciclina C/genética , Humanos , Coativador 2 de Receptor Nuclear/genética , Estrutura Terciária de ProteínaRESUMO
Bone remodeling is a continuous process regulated by several hormones such as estrogens and parathyroid hormone (PTH). Here we investigated the influence of PTH on estrogen receptor alpha (ERα)-dependent transcriptional activity in MC3T3-E1 osteoblasts. Cells that were transfected with an ER-responsive reporter plasmid and treated with PTH showed increased luciferase activity. However, in the presence of 17ß-estradiol, we observed that PTH inhibited ERα-mediated transcription. cAMP mimicked the effects by PTH, and the findings were confirmed in COS-1 cells transfected with expression vector encoding the catalytic subunit of cAMP-dependent protein kinase (PKA). Furthermore, PTH exhibited specific effects on the mRNA expression of the decoy receptor osteoprotegerin (OPG) and the receptor activator of NF kappa-B ligand (RANKL) in MC3T3-E1 osteoblasts. In the absence of 17ß-estradiol, PTH and cAMP enhanced the OPG/RANKL ratio, whereas, OPG/RANKL was suppressed when estradiol was present. In conclusion, our results indicate that the presence of estradiol determines whether PTH and cAMP stimulates or inhibits ERα-dependent activity and the OPG/RANKL mRNA expression in an osteoblastic cell line.
Assuntos
Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Hormônio Paratireóideo/administração & dosagem , Ativação Transcricional/fisiologia , Células 3T3 , Animais , Células COS , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Chlorocebus aethiops , Camundongos , Osteoblastos/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacosRESUMO
Steroid receptor coactivator 2 (SRC-2) is a nuclear receptor coactivator, important for the regulation of estrogen receptor alpha (ERα)-mediated transcriptional activity in breast cancer cells. However, the transcriptional role of SRC-2 in breast cancer is still ambiguous. Here we aimed to unravel a more precise transcriptional role of SRC-2 and uncover unique target genes in MCF-7 breast cancer cells, as opposed to the known oncogene SRC-3. Gene expression analyses of cells depleted of either SRC-2 or SRC-3 showed that they transcriptionally regulate mostly separate gene sets. However, individual unique gene sets were implicated in some of the same major gene ontology biological processes, such as cellular structure and development. This finding was supported by three-dimensional cell cultures, demonstrating that depletion of SRC-2 and SRC-3 changed the morphology of the cells into epithelial-like hollow acinar structures, indicating that both SRC proteins are involved in maintaining the hybrid E/M phenotype. In clinical ER-positive, HER2-negative breast cancer samples the expression of SRC-2 was negatively correlated with the expression of MCF-7-related luminal, cell cycle and cellular morphogenesis genes. Finally, elucidating SRC-2 unique transcriptional effects, we identified Lyn kinase (an EMT biomarker) to be upregulated exclusively after SRC-2 depletion. In conclusion, we show that both SRC-2 and SRC-3 are essential for the EMT in breast cancer cells, controlling different transcriptional niches.
Assuntos
Neoplasias da Mama/patologia , Transição Epitelial-Mesenquimal/fisiologia , Coativador 2 de Receptor Nuclear/metabolismo , Coativador 3 de Receptor Nuclear/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células MCF-7 , Coativador 2 de Receptor Nuclear/genética , Coativador 3 de Receptor Nuclear/genética , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Esferoides Celulares/citologia , Transcrição Gênica/genética , Células Tumorais Cultivadas , Quinases da Família src/biossíntese , Quinases da Família src/genéticaRESUMO
Steroid receptor coactivators (SRCs), such as glucocorticoid receptor interacting protein 1 (GRIP1) are recruited to the DNA-bound nuclear receptors (NRs) and are also shown to enhance the gene transactivation by other transcription factors. In contrast to the two other members of the SRC family, SRC-1 and SRC-3/amplified in breast cancer 1, SRC-2/GRIP1 is regulated by the cAMP-dependent protein kinase [protein kinase A (PKA)] that stimulates its ubiquitination and degradation. In this report we demonstrate that COS-1 and MCF-7 cells treated with cAMP-elevating agents and 8-para-chlorophenylthio-cAMP for short periods of time showed an increase in GRIP1 coactivator function, whereas prolonged stimulation of the cAMP/PKA pathway led to a decline in GRIP1-mediated activation and protein levels. Furthermore, MCF-7 breast cancer cells were subjected to chromatin immunoprecipitation assays after stimulation of the cAMP/PKA pathway. cAMP/PKA initiated a rapid recruitment of GRIP1 to the endogenous estrogen receptor (ER)-alpha target pS2 gene promoter. In contrast to the estradiol-induced recruitment of GRIP1 to pS2, we observed an additional increase in GRIP1 recruitment on inhibition of the proteasome, suggesting that inhibition of GRIP1 degradation leads to accumulation at the pS2. Real-time PCR experiments confirmed that cAMP/PKA enhanced the expression of pS2. Moreover, confocal imaging of COS-1 cells transfected with yellow fluorescent protein-GRIP1 and cyan fluorescent protein-ERalpha revealed that PKA led to redistribution and colocalization of yellow fluorescent protein-GRIP1 and cyan fluorescent protein-ERalpha in subnuclear foci. In conclusion, these results suggest that activation of the cAMP/PKA pathway stimulates recruitment of GRIP1 to an ER-responsive gene promoter. The initial stimulation of GRIP1 coactivator function is followed by an increased turnover and subsequent degradation of GRIP1 protein.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Receptor alfa de Estrogênio/metabolismo , Coativador 2 de Receptor Nuclear/metabolismo , Animais , Células COS , Chlorocebus aethiops , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/fisiologia , Coativador 2 de Receptor Nuclear/genética , Regiões Promotoras Genéticas , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
INTRODUCTION: Tamoxifen is an anti-estrogen drug used in treatment of Estrogen Receptor (ER) positive breast cancer. Effects and side effects of tamoxifen is the sum of tamoxifen and all its metabolites. 4-Hydroxytamoxifen (4OHtam) and 4-hydroxy-N-demethyltamoxifen (4OHNDtam, endoxifen) both have ER affinity exceeding that of the parent drug tamoxifen. 4OHNDtam is considered the main active metabolite of tamoxifen. Ndesmethyltamoxifen (NDtam) is the major tamoxifen metabolite. It has low affinity to the ER and is not believed to influence tumor growth. However, NDtam might mediate adverse effects of tamoxifen treatment. In this study we investigated the gene regulatory effects of the three metabolites of tamoxifen in MCF-7 breast cancer cells. MATERIAL AND METHODS: Using concentrations that mimic the clinical situation we examined effects of 4OHtam, 4OHNDtam and NDtam on global gene expression in 17ß-estradiol (E2) treated MCF-7 cells. Transcriptomic responses were assessed by correspondence analysis, differential expression, gene ontology analysis and quantitative real time PCR (Q-rt-PCR). E2 deprivation and knockdown of Steroid Receptor Coactivator-3 (SRC-3)/Amplified in Breast Cancer 1 (AIB1) mRNA in MCF-7 cells were performed to further characterize specific effects on gene expression. RESULTS: 4OHNDtam and 4OHtam caused major changes in gene expression compared to treatment with E2 alone, with a stronger effect of 4OHNDtam. NDtam had nearly no effect on the global gene expression profile. Treatment of MCF-7 cells with 4OHNDtam led to a strong down-regulation of the CytoKeratin 6 isoforms (KRT6A, KRT6B and KRT6C). The CytoKeratin 6 mRNAs were also down-regulated in MCF-7 cells after E2 deprivation and after SRC-3/AIB1 knockdown. CONCLUSION: Using concentrations that mimic the clinical situation we report global gene expression changes that were most pronounced with 4OHNDtam and minimal with NDtam. Genes encoding CytoKeratin 6, were highly down-regulated by 4OHNDtam, as well as after E2 deprivation and knockdown of SRC-3/AIB1, indicating an estrogen receptor-dependent regulation.
Assuntos
Neoplasias da Mama/metabolismo , Regulação para Baixo/efeitos dos fármacos , Queratina-6/metabolismo , Tamoxifeno/análogos & derivados , Neoplasias da Mama/patologia , Feminino , Humanos , Células MCF-7 , Tamoxifeno/farmacologiaRESUMO
The p160/Steroid Receptor Coactivators SRC-1, SRC-2/GRIP1, and SRC-3/AIB1 are important regulators of Estrogen Receptor alpha (ERα) activity. However, whereas the functions of SRC-1 and SRC-3 in breast tumourigenesis have been extensively studied, little is known about the role of SRC-2. Previously, we reported that activation of the cAMP-dependent protein kinase, PKA, facilitates ubiquitination and proteasomal degradation of SRC-2 which in turn leads to inhibition of SRC-2-coactivation of ERα and changed expression of the ERα target gene, pS2. Here we have characterized the global program of transcription in SRC-2-depleted MCF-7 breast cancer cells using short-hairpin RNA technology, and in MCF-7 cells exposed to PKA activating agents. In order to identify genes that may be regulated through PKA-induced downregulation of SRC-2, overlapping transcriptional targets in response to the respective treatments were characterized. Interestingly, we observed decreased expression of several breast cancer tumour suppressor genes (e.g., TAGLN, EGR1, BCL11b, CAV1) in response to both SRC-2 knockdown and PKA activation, whereas the expression of a number of other genes implicated in cancer progression (e.g., RET, BCAS1, TFF3, CXCR4, ADM) was increased. In line with this, knockdown of SRC-2 also stimulated proliferation of MCF-7 cells. Together, these results suggest that SRC-2 may have an antiproliferative function in breast cancer cells.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica , Coativador 2 de Receptor Nuclear/genética , Coativador 2 de Receptor Nuclear/metabolismo , Proliferação de Células , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Células MCF-7 , Oncogenes/genética , Proteólise , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
The glucocorticoid receptor interacting protein (GRIP1) belongs to the p160 steroid receptor coactivator family that plays essential roles in nuclear receptor-dependent transcriptional regulation. Previously, we reported that the cAMP-dependent protein kinase (PKA) induces ubiquitination leading to degradation of GRIP1. Here we show that the cAMP response element-binding protein (CREB) downregulates GRIP1 and is necessary for the PKA-stimulated degradation of GRIP1, which leads to changes in the expression of a subset of genes regulated by estrogen receptor-α in MCF-7 breast cancer cells. Our data of domain-mapping and ubiquitination analyses suggest that CREB promotes the proteasomal breakdown of ubiquitinated GRIP1 through 2 functionally independent protein domains containing amino acids 347 to 758 and 1121 to 1462. We provide evidence that CREB interacts directly with GRIP1 and that CREB Ser-133 phosphorylation or transcriptional activity is not required for GRIP1 interaction and degradation. The basic leucine zipper domain (bZIP) of CREB is important for the interaction with GRIP1, and deletion of this domain led to an inability to downregulate GRIP1. We propose that CREB mediates the PKA-stimulated degradation of GRIP1 through protein-protein interaction and stimulation of proteasomal degradation of ubiquitinated GRIP1.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Coativador 2 de Receptor Nuclear/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação para Baixo , Receptor alfa de Estrogênio/metabolismo , Humanos , Células MCF-7 , Plasmídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional , Transfecção , Ubiquitinação/fisiologiaRESUMO
Nuclear receptors and their coactivators are key regulators of numerous physiological functions. GRIP1 (glucocorticoid receptor-interacting protein) is a member of the steroid receptor coactivator family. Here, we show that GRIP1 is regulated by cAMP-dependent protein kinase (PKA) that induces its degradation through the ubiquitin-proteasome pathway. GRIP1 was down-regulated in transiently transfected COS-1 cells after treatment with 8-para-chlorophenylthio-cAMP or forskolin and 3-isobutyl-1-methylxanthine and in adrenocortical Y1 cells after incubation with adrenocorticotropic hormone. Pulse-chase experiments with transiently transfected COS-1 cells demonstrated that the half-life of GRIP1 was markedly reduced in cells overexpressing the PKA catalytic subunit, suggesting that activation of PKA increases the turnover of GRIP1 protein. The proteasome inhibitors MG132 and lactacystin abolished the PKA-mediated degradation of GRIP1. Using ts20 cells, a temperature-sensitive cell line that contains a thermolabile ubiquitin-activating E1 enzyme, it was confirmed that PKA-mediated degradation of GRIP1 is dependent upon the ubiquitin-proteasome pathway. Coimmunoprecipitation studies of COS-1 cells transfected with expression vectors encoding GRIP1 and ubiquitin using anti-GRIP1 and anti-ubiquitin antibodies showed that the ubiquitination of GRIP1 was increased by overexpression of PKA. Finally, we show that PKA regulates the intracellular distribution pattern of green fluorescent protein-GRIP1 and stimulates recruitment of GRIP1 to subnuclear foci that are colocalized with the proteasome. Taken together, these data demonstrate that GRIP1 is ubiquitinated and degraded through activation of the PKA pathway. This may represent a novel regulatory mechanism whereby hormones down-regulate a nuclear receptor coactivator.