RESUMO
A previous study reported the use of a biosensing technique based on surface plasmon resonance (SPR) for the ligand binding detection of peroxisome proliferator activator receptor gamma (PPARγ). This detection was designed based on the structural properties of PPARγ. Because of cross-linked protein inactivation and the low molecular weight of conventional ligands, direct ligand binding detection based on SPR has low stability and repeatability. In this study, we report an indirect response methodology based on SPR technology in which anti-His CM5 chip binds fresh PPARγ every cycle, resulting in more stable detection. We developed a remarkable improvement in ligand-protein binding detectability in vitro by introducing two coregulator-related polypeptides into this system. In parallel, a systematic indirect response methodology can reflect the interaction relationship between ligands and proteins to some extent by detecting the changes in SA-SRC1 and GST-NCOR2 binding to PPARγ. Rosiglitazone, a PPARγ agonist with strong affinity, is a potent insulin-sensitizing agent. Some ligands may be competitively exerted at the same sites of PPARγ (binding rosiglitazone). We demonstrated using indirect response methodology that selective PPARγ modulator (SPPARM) candidates of PPARγ can be found by competing for the binding of the rosiglitazone site on PPARγ, although they may have no effect on polypeptides and PPARγ binding.
Assuntos
Coativador 1 de Receptor Nuclear , PPAR gama , Ligação Proteica , Ressonância de Plasmônio de Superfície , Ressonância de Plasmônio de Superfície/métodos , PPAR gama/metabolismo , PPAR gama/química , Ligantes , Coativador 1 de Receptor Nuclear/metabolismo , Coativador 1 de Receptor Nuclear/química , Peptídeos/química , Peptídeos/metabolismo , Humanos , Rosiglitazona/farmacologia , Correpressor 2 de Receptor NuclearRESUMO
SRC-1 functions as a transcriptional coactivator for steroid receptors and various transcriptional factors. Notably, SRC-1 has been implicated in oncogenic roles in multiple cancers, including breast cancer and prostate cancer. Previous investigations from our laboratory have established the high expression of SRC-1 in human HCC specimens, where it accelerates HCC progression by enhancing Wnt/beta-catenin signalling. In this study, we uncover a previously unknown role of SRC-1 in HCC metastasis. Our findings reveal that SRC-1 promotes HCC metastasis through the augmentation of MMP-9 expression. The knockdown of SRC-1 effectively mitigated HCC cell metastasis both in vitro and in vivo by suppressing MMP-9 expression. Furthermore, we observed a positive correlation between SRC-1 mRNA levels and MMP-9 mRNA levels in limited and larger cohorts of HCC specimens from GEO database. Mechanistically, SRC-1 operates as a coactivator for NF-κB and AP-1, enhancing MMP-9 promoter activity in HCC cells. Higher levels of SRC-1 and MMP-9 expression are associated with worse overall survival in HCC patients. Treatment with Bufalin, known to inhibit SRC-1 expression, significantly decreased MMP-9 expression and inhibited HCC metastasis in both in vitro and in vivo settings. Our results demonstrated the pivotal role of SRC-1 as a critical modulator in HCC metastasis, presenting a potential therapeutic target for HCC intervention.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Masculino , Humanos , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Coativador 1 de Receptor Nuclear/genética , Coativador 1 de Receptor Nuclear/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , RNA Mensageiro , Invasividade Neoplásica/genética , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão GênicaRESUMO
BACKGROUND: Corticosteroid is commonly used before surgery to control cerebral oedema in brain tumours and is frequently continued throughout treatment. Its long-term effect of on the recurrence of WHO-Grade 4 astrocytoma remains controversial. The interaction between corticosteroid, SRC-1 gene and cytotoxic T-cells has never been investigated. METHODS: A retrospective cohort of 36 patients with WHO-Grade 4 astrocytoma were examined for CD8 + T-cell and SRC-1 gene expressions through IHC and qRT-PCR. The impact of corticosteroid on CD8+T-cells infiltration, SRC-1 expression, and tumour recurrence was analyzed. RESULTS: The mean patients age was 47-years, with a male to female ratio 1.2. About 78% [n = 28] of the cases showed reduced or no CD8+T-cell expression while 22% [n = 8] of cases have showed medium to high CD8+T-cell expression. SRC-1 gene was upregulated in 5 cases [14%] and 31 cases [86%] showed SRC-1 downregulation. The average of total days and doses of administered corticosteroid from the preoperative period to the postoperative period was at range of 14-106 days and 41-5028 mg, respectively. There was no significant statistical difference in RFI among tumours expressing high or low CD8+T-cells when corticosteroid was administered in recommended or exceeded doses [p-value = 0.640]. There was a significant statistical difference in RFI between CD8+T-Cell expression and SRC-1 gene dysregulation [p-value = 002]. Tumours with high CD8+T T-cell expression and SRC-1 gene downregulation had late recurrence. CONCLUSIONS: Corticosteroid treatment can directly affect the SRC-1 gene regulation but does not directly influence cytotoxic T-cells infiltration or tumor progression. However, SRC-1 gene downregulation can facilitate late tumor recurrence.
Assuntos
Astrocitoma , Glioblastoma , Coativador 1 de Receptor Nuclear , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Corticosteroides/uso terapêutico , Astrocitoma/tratamento farmacológico , Astrocitoma/genética , Astrocitoma/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Estudos Retrospectivos , Organização Mundial da Saúde , Coativador 1 de Receptor Nuclear/genética , Coativador 1 de Receptor Nuclear/metabolismoRESUMO
Steroid receptors (SRs) are ligand-dependent transcription factors (TFs) relevant to key cellular processes in both physiology and pathology, including some types of cancer. SOX2 is a master TF of pluripotency and self-renewal of embryonic stem cells, and its dysregulation is also associated with various types of human cancers. A potential crosstalk between these TFs could be relevant in malignant cells yet, to the best of our knowledge, no formal study has been performed thus far. Here we show, by quantitative live-cell imaging microscopy, that ectopic expression of SOX2 disrupts the formation of hormone-dependent intranuclear condensates of many steroid receptors (SRs), including those formed by the glucocorticoid receptor (GR). SOX2 also reduces GR's binding to specific DNA targets and modulates its transcriptional activity. SOX2-driven effects on GR condensates do not require the intrinsically disordered N-terminal domain of the receptor and, surprisingly, neither relies on GR/SOX2 interactions. SOX2 also alters the intranuclear dynamics and compartmentalization of the SR coactivator NCoA-2 and impairs GR/NCoA-2 interactions. These results suggest an indirect mechanism underlying SOX2-driven effects on SRs involving this coactivator. Together, these results highlight that the transcriptional program elicited by GR relies on its nuclear organization and is intimately linked to the distribution of other GR partners, such as the NCoA-2 coactivator. Abnormal expression of SOX2, commonly observed in many tumors, may alter the biological action of GR and, probably, other SRs as well. Understanding this crosstalk may help to improve steroid hormone-based therapies in cancers with elevated SOX2 expression.
Assuntos
Receptores de Glucocorticoides , Fatores de Transcrição SOXB1 , Ativação Transcricional , Humanos , Coativador 1 de Receptor Nuclear/genética , Coativador 1 de Receptor Nuclear/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
Germline mutations to the breast cancer 2 (BRCA2) gene have been associated with hereditary breast cancer. In addition to estrogen uptake, BRCA2 expression increases in the S phase of the cell cycle and largely contributes to DNA damage repair associated with DNA replication. However, the role of BRCA2 in estrogen induction remains unclear. An expression plasmid was created to induce BRCA2 activation upon the addition of estradiol by introducing mutations to the binding sequences for the transcription factors USF1, E2F1, and NF-κB within the promoter region of BRCA2. Then, the estrogen receptor (ER) sites of the proteins that interact with BRCA2 upon the addition of estradiol were identified. Both proteins were bound by the helical domain of BRCA2 and activation function-2 of the ER, suggesting that this binding may regulate the transcriptional activity of pS2, a target gene of the estradiol-ER, by suppressing the binding of SRC-1, a coactivator required for activation of the transcription factor.
Assuntos
Proteína BRCA2/metabolismo , Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Proteínas/genética , Transcrição Gênica , Fator Trefoil-1/genética , Proteína BRCA2/química , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Coativador 1 de Receptor Nuclear/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Domínios Proteicos , Proteínas/metabolismo , Fatores de Transcrição/metabolismo , Fator Trefoil-1/metabolismoRESUMO
Octocrylene (OC) is an extensively prescribed organic ultraviolet B filter used in sunscreen products. Due to its extensive use, a significant level of OC is detected in marine and freshwater environments. Notably, the bioaccumulation of OC in aquatic biota may affect human health. In this study, the effect of OC on metabolism was investigated using the adipogenesis model of human bone marrow mesenchymal stem cells (hBM-MSCs). OC promoted adiponectin production during adipogenesis in hBM-MSCs compared to the vehicle-treated control (EC50, 29.6 µM). In target identification, OC directly bound to peroxisome proliferator-activated receptor (PPAR) γ (Ki, 37.8 µM). OC-bound PPARγ also significantly recruited nuclear receptor coactivator proteins SRC-1 (EC50, 54.1 µM) and SRC-2 (EC50, 58.6 µM). In the molecular docking simulation study, the optimal ligand-binding mode of OC suggested that OC is a PPARγ partial agonist. A competitive analysis with a PPARγ full agonist pioglitazone revealed that OC acted as a PPARγ partial agonist. OC altered the gene transcription profile of lipid-metabolism associated enzymes in normal human keratinocytes, primarily exposed human cells after the application of sunscreens. In conclusion, OC is a potential metabolic disrupting obesogen.
Assuntos
Acrilatos/toxicidade , Adipócitos/fisiologia , Células da Medula Óssea/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Obesidade/induzido quimicamente , PPAR gama/agonistas , Adipócitos/efeitos dos fármacos , Células da Medula Óssea/fisiologia , Domínio Catalítico , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Queratinócitos/efeitos dos fármacos , Metabolismo dos Lipídeos , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Coativador 1 de Receptor Nuclear/genética , Coativador 1 de Receptor Nuclear/metabolismo , Coativador 2 de Receptor Nuclear/genética , Coativador 2 de Receptor Nuclear/metabolismo , Conformação ProteicaRESUMO
SRY-box 2 (Sox2) is a transcription factor with critical roles in maintaining embryonic stem (ES) cell and adult stem cell functions and in tumorigenesis. However, how Sox2 exerts its transcriptional function remains unclear. Here, we used an in vitro protein-protein interaction assay to discover transcriptional regulators for ES cell core transcription factors (Oct4, Sox2, Klf4, and c-Myc) and identified members of the steroid receptor coactivators (SRCs) as Sox2-specific interacting proteins. The SRC family coactivators have broad roles in transcriptional regulation, but it is unknown whether they also serve as Sox2 coactivators. We demonstrated that these proteins facilitate Sox2 transcriptional activity and act synergistically with p300. Furthermore, we uncovered an acetylation-enhanced interaction between Sox2 and SRC-2/3, but not SRC-1, demonstrating it is Sox2 acetylation that promotes the interaction. We identified putative Sox2 acetylation sites required for acetylation-enhanced interaction between Sox2 and SRC-3 and demonstrated that acetylation on these sites contributes to Sox2 transcriptional activity and recruitment of SRC-3. We showed that activation domains 1 and 2 of SRC-3 both display a preferential binding to acetylated Sox2. Finally, functional analyses in mouse ES cells demonstrated that knockdown of SRC-2/3 but not SRC-1 in mouse ES cells significantly downregulates the transcriptional activities of various Sox2 target genes and impairs ES cell stemness. Taken together, we identify specific SRC family proteins as novel Sox2 coactivators and uncover the role of Sox2 acetylation in promoting coactivator recruitment and Sox2 transcriptional function.
Assuntos
Coativador 1 de Receptor Nuclear/metabolismo , Coativador 2 de Receptor Nuclear/metabolismo , Coativador 3 de Receptor Nuclear/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transcrição Gênica , Acetilação , Animais , Células HEK293 , Células HeLa , Humanos , Camundongos , Coativador 1 de Receptor Nuclear/genética , Coativador 2 de Receptor Nuclear/genética , Coativador 3 de Receptor Nuclear/genética , Fatores de Transcrição SOXB1/genéticaRESUMO
Steroid receptor coactivator-1 (SRC-1) is a transcription coactivator playing a pivotal role in mediating a wide range of signaling pathways by interacting with related transcription factors and nuclear receptors. Aberrantly elevated SRC-1 activity is associated with cancer metastasis and progression, and therefore, suppression of SRC-1 is emerging as a promising therapeutic strategy. In this study, we developed a novel SRC-1 degrader for targeted degradation of cellular SRC-1. This molecule consists of a selective ligand for SRC-1 and a bulky hydrophobic group. Since the hydrophobic moiety on the protein surface could mimic a partially denatured hydrophobic region of a protein, SRC-1 could be recognized as an unfolded protein and experience the chaperone-mediated degradation in the cells through the ubiquitin-proteasome system (UPS). Our results demonstrate that a hydrophobic-tagged chimeric molecule is shown to significantly reduce cellular levels of SRC-1 and suppress cancer cell migration and invasion. Together, these results highlight that our SRC-1 degrader represents a novel class of therapeutic candidates for targeting cancer metastasis. Moreover, we believe that the hydrophobic tagging strategy would be widely applicable to develop peptide-based protein degraders with enhanced cellular activity.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , Coativador 1 de Receptor Nuclear/metabolismo , Proteólise , Transativadores/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Movimento Celular , Humanos , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Chaperonas Moleculares/metabolismo , Invasividade Neoplásica , Peptídeos/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Necroptosis is an alternative form of programmed cell death that generally occurs under apoptosis-deficient conditions. Our previous work showed that connexin32 (Cx32) promotes the malignant progress of hepatocellular carcinoma (HCC) by enhancing the ability of resisting apoptosis in vivo and in vitro. Whether triggering necroptosis is a promising strategy to eliminate the apoptosis-resistant HCC cells with high Cx32 expression remains unknown. In this study, we found that Cx32 expression was positively correlated with the expression of necroptosis protein biomarkers in human HCC specimens, cell lines, and a xenograft model. Treatment with shikonin, a well-used necroptosis inducer, markedly caused necroptosis in HCC cells. Interestingly, overexpressed Cx32 exacerbated shikonin-induced necroptosis, but downregulation of Cx32 alleviated necroptosis in vitro and in vivo. Mechanistically, Cx32 was found to bind to Src and promote Src-mediated caspase 8 phosphorylation and inactivation, which ultimately reduced the activated caspase 8-mediated proteolysis of receptor-interacting serine-threonine protein kinase 1/3, the key molecule for necroptosis activation. In conclusion, we showed that Cx32 contributed to the activation of necroptosis in HCC cells through binding to Src and then mediating the inactivation of caspase 8. The present study suggested that necroptosis inducers could be more favorable than apoptosis inducers to eliminate HCC cells with high expression of Cx32.
Assuntos
Carcinoma Hepatocelular/metabolismo , Caspase 8/metabolismo , Conexinas/metabolismo , Neoplasias Hepáticas/metabolismo , Necroptose/genética , Coativador 1 de Receptor Nuclear/metabolismo , Transdução de Sinais/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Conexinas/genética , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Naftoquinonas/administração & dosagem , Necroptose/efeitos dos fármacos , Coativador 1 de Receptor Nuclear/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Transdução de Sinais/efeitos dos fármacos , Transfecção , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Proteína beta-1 de Junções ComunicantesRESUMO
Recurrent fusion genes involving C11orf95, C11orf95-RELA, have been identified only in supratentorial ependymomas among primary CNS tumors. Here, we report hitherto histopathologically unclassifiable high-grade tumors, under the tentative label of "ependymoma-like tumors with mesenchymal differentiation (ELTMDs)," harboring C11orf95-NCOA1/2 or -RELA fusion. We examined the clinicopathological and molecular features in five cases of ELTMDs. Except for one adult case (50 years old), all cases were in children ranging from 1 to 2.5 years old. All patients presented with a mass lesion in the cerebral hemisphere. Histologically, all cases demonstrated a similar histology with a mixture of components. The major components were embryonal-appearing components forming well-delineated tumor cell nests composed of small uniform cells with high proliferative activity, and spindle-cell mesenchymal components with a low- to high-grade sarcoma-like appearance. The embryonal-appearing components exhibited minimal ependymal differentiation including a characteristic EMA positivity and tubular structures, but histologically did not fit with ependymoma because they lacked perivascular pseudorosettes, a histological hallmark of ependymoma, formed well-delineated nests, and had diffuse and strong staining for CAM5.2. Molecular analysis identified C11orf95-NCOA1, -NCOA2, and -RELA in two, one, and two cases, respectively. t-distributed stochastic neighbor embedding analysis of DNA methylation data from two cases with C11orf95-NCOA1 or -NCOA2 and a reference set of 380 CNS tumors revealed that these two cases were clustered together and were distinct from all subgroups of ependymomas. In conclusion, although ELTMDs exhibited morphological and genetic associations with supratentorial ependymoma with C11orf95-RELA, they cannot be regarded as ependymoma. Further analyses of more cases are needed to clarify their differences and similarities.
Assuntos
Ependimoma/genética , Ependimoma/patologia , Coativador 1 de Receptor Nuclear/metabolismo , Proteínas/metabolismo , Fator de Transcrição RelA/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias do Sistema Nervoso Central/genética , Pré-Escolar , Metilação de DNA/genética , Fusão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Coativador 1 de Receptor Nuclear/genética , Proteínas/genética , Neoplasias Supratentoriais/genética , Neoplasias Supratentoriais/patologia , Fator de Transcrição RelA/genéticaRESUMO
Glioblastoma (GBM) recurrence is attributed to the presence of therapy-resistant glioblastoma stem cells. Steroid receptor coactivator-1 (SRC-1) acts as an oncogenic regulator in many human tumors. The relationship between SRC-1 and GBM has not yet been studied. Herein, we investigate the role of SRC-1 in GBM. In this study, we found that SRC-1 expression is positively correlated with grades of glioma and inversely correlated with glioma patient's prognosis. Steroid receptor coactivator-1 promotes the proliferation, migration, and tumor growth of GBM cells. Notably, SRC-1 knockdown suppresses the stemness of GBM cells. Mechanistically, long noncoding RNA X-inactive specific transcript (XIST) is regulated by SRC-1 at the posttranscriptional level and mediates the function of SRC-1 in promoting stemness-like properties of GBM. Steroid receptor coactivator-1 can promote the expression of Kruppel-like factor 4 (KLF4) through the XIST/microRNA (miR)-152 axis. Additionally, arenobufagin and bufalin, SRC small molecule inhibitors, can reduce the proliferation and stemness of GBM cells. This study reveals SRC-1 promotes the stemness of GBM by activating the long noncoding RNA XIST/miR-152/KLF4 pathway and provides novel markers for diagnosis and therapy of GBM.
Assuntos
Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Glioblastoma/patologia , Coativador 1 de Receptor Nuclear/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Proliferação de Células/genética , Glioblastoma/genética , Glioblastoma/metabolismo , Xenoenxertos , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/fisiologiaRESUMO
The retinoid X receptor (RXR) is a ligand-sensing transcription factor acting mainly as a universal heterodimer partner for other nuclear receptors. Despite presenting as a potential therapeutic target for cancer and neurodegeneration, adverse effects typically observed for RXR agonists, likely due to the lack of isoform selectivity, limit chemotherapeutic application of currently available RXR ligands. The three human RXR isoforms exhibit different expression patterns; however, they share high sequence similarity, presenting a major obstacle toward the development of subtype-selective ligands. Here, we report the discovery of the saturated fatty acid, palmitic acid, as an RXR ligand and disclose a uniform set of crystal structures of all three RXR isoforms in an active conformation induced by palmitic acid. A structural comparison revealed subtle differences among the RXR subtypes. We also observed an ability of palmitic acid as well as myristic acid and stearic acid to induce recruitment of steroid receptor co-activator 1 to the RXR ligand-binding domain with low micromolar potencies. With the high, millimolar endogenous concentrations of these highly abundant lipids, our results suggest their potential involvement in RXR signaling.
Assuntos
Ácido Palmítico/metabolismo , Isoformas de Proteínas/metabolismo , Receptores X de Retinoides/metabolismo , Linhagem Celular , Dimerização , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Ligantes , Ácido Mirístico/metabolismo , Coativador 1 de Receptor Nuclear/metabolismo , Transdução de Sinais/fisiologia , Ácidos Esteáricos/metabolismoRESUMO
Aberrantly elevated steroid receptor coactivator-1 (SRC-1) expression and activity are strongly correlated with cancer progression and metastasis. Here we report, for the first time, the development of a proteolysis targeting chimera (PROTAC) that is composed of a selective SRC-1 binder linked to a specific ligand for UBR box, a unique class of E3 ligases recognizing N-degrons. We showed that the bifunctional molecule efficiently and selectively induced the degradation of SRC-1 in cells through the N-degron pathway. Importantly, given the ubiquitous expression of the UBR protein in most cells, PROTACs targeting the UBR box could degrade a protein of interest regardless of cell types. We also showed that the SRC-1 degrader significantly suppressed cancer cell invasion and migration in vitro and in vivo. Together, these results demonstrate that the SRC-1 degrader can be an invaluable chemical tool in the studies of SRC-1 functions. Moreover, our findings suggest PROTACs based on the N-degron pathway as a widely useful strategy to degrade disease-relevant proteins.
Assuntos
Coativador 1 de Receptor Nuclear/antagonistas & inibidores , Peptídeos/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biocatálise , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Humanos , Fator Estimulador de Colônias de Macrófagos/metabolismo , Camundongos Endogâmicos BALB C , Invasividade Neoplásica/prevenção & controle , Neoplasias/tratamento farmacológico , Coativador 1 de Receptor Nuclear/metabolismo , Peptídeos/metabolismo , Peptídeos/uso terapêutico , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
A homozygous missense mutation in the gene encoding the estrogen receptor α (ERα) was previously identified in a female patient with estrogen insensitivity syndrome. We investigated the molecular features underlying the impaired transcriptional response of this mutant (ERα-Q375H) and four other missense mutations at this position designed to query alternative mechanisms. The identity of residue 375 greatly affected the sensitivity of the receptor to agonists without changing the ligand binding affinity. Instead, the mutations caused changes in the affinity of coactivator binding and alterations in the balance of coactivator and corepressor recruitment. Comparisons among the transcriptional regulatory responses of these six ERα genotypes to a set of ER agonists showed that both steric and electrostatic factors contributed to the functional deficits in gene regulatory activity of the mutant ERα proteins. ERα-coregulator peptide binding in vitro and RIME (rapid immunoprecipitation mass spectrometry of endogenous) analysis in cells showed that the degree of functional impairment paralleled changes in receptor-coregulator binding interactions. These findings uncover coupling between ligand binding and coregulator recruitment that affects the potency rather than the efficacy of the receptor response without substantially altering ligand binding affinity. This highlights a molecular mechanism for estrogen insensitivity syndrome involving mutations that perturb a bidirectional allosteric coupling between ligand binding and coregulator binding that determines receptor transcriptional output.
Assuntos
Receptor alfa de Estrogênio/genética , Estrogênios/metabolismo , Mutação de Sentido Incorreto , Coativador 1 de Receptor Nuclear/genética , Coativador 3 de Receptor Nuclear/genética , Sítios de Ligação/genética , Resistência a Medicamentos/genética , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Regulação da Expressão Gênica , Células HEK293 , Células Hep G2 , Humanos , Cinética , Ligantes , Simulação de Dinâmica Molecular , Coativador 1 de Receptor Nuclear/metabolismo , Coativador 3 de Receptor Nuclear/metabolismo , Ligação Proteica , Domínios ProteicosRESUMO
The endometrium, composed of epithelial and stromal cell compartments, is tightly regulated by the ovarian steroid hormones estrogen (E2) and progesterone (P4) during early pregnancy. Through the progesterone receptor (PGR), steroid receptor coactivators, and other transcriptional coregulators, progesterone inhibits E2-induced cell proliferation and induces the differentiation of stromal cells in a process called decidualization to promote endometrial receptivity. Although interleukin-13 receptor subunit alpha-2 (Il13ra2) is expressed in the human and mouse endometrium, its potential role in the steroid hormone regulation of the endometrium has not been thoroughly examined. In this study, we employed PGR knockout mice and steroid receptor coactivator-1 knockout mice (SRC-1-/-) to profile the expression of Il13ra2 in the murine endometrium and determine the role of these transcriptional regulators in the hormone-responsiveness of Il13ra2 expression. Furthermore, we utilized a well-established decidualization-inducing steroidogenic cocktail and a siRNA-based knockdown of IL13RA2 to determine the importance of IL13RA2 in the decidualization of primary human endometrial stromal cells. Our findings demonstrate that Il13ra2 is expressed in the subepithelial stroma of the murine endometrium in response to ovarian steroid hormones and during early pregnancy in a PGR- and SRC-1-dependent manner. Furthermore, we show that knockdown of IL13RA2 before in vitro decidualization of primary human endometrial stromal cells partially compromises the full decidualization response. We conclude that Il13ra2 is a downstream target of progesterone through PGR and SRC-1 and plays a role in mediating the stromal action of ovarian steroid hormones.
Assuntos
Regulação da Expressão Gênica/fisiologia , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Subunidade alfa2 de Receptor de Interleucina-13/metabolismo , Coativador 1 de Receptor Nuclear/metabolismo , Útero/metabolismo , Animais , Feminino , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Subunidade alfa2 de Receptor de Interleucina-13/genética , Camundongos , Camundongos Knockout , Coativador 1 de Receptor Nuclear/genética , Interferência de RNA , RNA Mensageiro , RNA Interferente PequenoRESUMO
Recently, the action of steroid receptor coactivators (SRCs) has been recognized to be an important molecular initiating event (MIE) in estrogenic adverse outcome pathways (AOPs). However, the role of SRCs in the molecular mechanisms of many highly concerned environmental estrogens remains poorly understood. In this study, the widely studied environmental estrogen, 4-n-nonylphenol (4-n-NP), was used as a typical pollutant to study SRCs recruitment in its estrogenic effects. In MCF7 cell proliferation (E-SCREEN) assay and MVLN cell assay, 4-n-NP showed significant estrogenic potency that involved an increase in estrogen receptor α (ERα), SRC1 and SRC3 transcript levels. Moreover, 4-n-NP was found to induce estrogen response element (ERE)-mediated activity via ERα in MVLN cells. To investigate the mechanism by which SRCs recruitment is induced by 4-n-NP-ERα, a coactivators recruitment assay was performed, and the results showed that 4-n-NP-ERα recruited both SRC1 and SRC3, whereas it failed to recruit SRC2. Similarly, it had no interaction with SRC2 in the ERα-SRC2 two-hybrid yeast assay. This is the first report to investigate the novel MIE of SRCs recruitment in 4-n-NP-ERα-induced estrogenicity. Overall, our results suggest that the action of 4-n-NP on estrogenic effects involves the following MIEs: the activation of ERα, the recruitment of SRC1 and SRC3, and the induction of ERE-mediated activity. The findings also provide valuable insights into the MIE associated with the different SRCs that are recruited in the adverse outcome pathways of environmental estrogens.
Assuntos
Poluentes Ambientais/farmacologia , Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Coativador 1 de Receptor Nuclear/metabolismo , Coativador 3 de Receptor Nuclear/metabolismo , Fenóis/farmacologia , Proliferação de Células/efeitos dos fármacos , Humanos , Células MCF-7RESUMO
Pheochromocytoma is a relatively uncommon tumor, and the histomorphologic and biochemical features that may portend malignant behavior have poor overall consensus across various proposed classification systems. Steroid receptor coactivator-1 (SRC-1) is a nuclear protein that mediates transcriptional activity. Current diagnostic applications of SRC-1 are limited, and include distinguishing adrenocortical carcinoma (ACC) from renal cell carcinoma, and other mimickers. SRC-1 expression in pheochromocytoma has not been previously studied. Pheochromocytoma cases were retrieved from our Urological Pathology database and expert consultation files of the senior author, from 2015 to 2019. Clinicopathological data were obtained. SRC-1 expression was scored systematically. Thirty-eight cases were included, with a female predominance, and a mean age of 52 years (range, 16 to 75 y). Seven patients had heritable mutations including RET (n=3), VHL (2), SDHB (1), and ATM and PDGFRA (1). Two patients developed clinical metastasis, who individually had ATM and PDGFRA mutations, and SDHB p.V140F mutation. All heritable tumors were positive for SRC-1, including diffuse/strong staining and intensity in the VHL cases, and diffuse staining with variable intensity in RET cases. Diffuse positivity was seen in most of our heritable cases, providing evidence for a putative link between RET and downstream SRC-1 signaling. An inverse relationship was observed between SRC-1 expression and Pheochromocytoma of the Adrenal Gland Scaled Score/tumor size, suggesting that SRC-1 phenotype may become muted in pheochromocytomas that have malignant potential. SRC-1 expression in aggressive pheochromocytomas, may also be a potential diagnostic pitfall in view of the fact that these tumors may be misinterpreted as ACC in the primary or metastatic setting.
Assuntos
Neoplasias das Glândulas Suprarrenais/metabolismo , Carcinoma Adrenocortical/diagnóstico , Coativador 1 de Receptor Nuclear/metabolismo , Feocromocitoma/metabolismo , Adolescente , Neoplasias das Glândulas Suprarrenais/diagnóstico , Adulto , Idoso , Diagnóstico Diferencial , Erros de Diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Metástase Neoplásica , Feocromocitoma/diagnóstico , Proteínas Proto-Oncogênicas c-ret/genética , Adulto JovemRESUMO
OBJECTIVE: Roux-en-Y gastric bypass surgery (RYGB) improves the first phase of glucose-stimulated insulin secretion (GSIS) in patients with type 2 diabetes. How it does so remains unclear. Farnesoid X receptor (FXR), the nuclear receptor of bile acids (BAs), is implicated in bariatric surgery. Moreover, the transient receptor potential ankyrin 1 (TRPA1) channel is expressed in pancreatic ß-cells and involved in insulin secretion. We aimed to explore the role of BAs/FXR and TRPA1 in improved GSIS in diabetic rats after RYGB. METHODS: RYGB or sham surgery was conducted in spontaneous diabetic Goto-Kakizaki (GK) rats, or FXR or TRPA1 transgenic mice. Gene and protein expression of islets were assessed by qPCR and western blotting. Electrophysiological properties of single ß-cells were studied using patch-clamp technique. Binding of FXR and histone acetyltransferase steroid receptor coactivator-1 (SRC1) to the TRPA1 promoter, acetylated histone H3 (ACH3) levels at the TRPA1 promoter were determined using ChIP assays. GSIS was measured using enzyme-linked immunosorbent assays or intravenous glucose tolerance test (IVGTT). RESULTS: RYGB increases GSIS, particularly the first-phase of GSIS in both intact islets and GK rats in vivo, and ameliorates hyperglycemia of GK rats. Importantly, the effects of RYGB were attenuated in TRPA1-deficient mice. Moreover, GK ß-cells displayed significantly decreased TRPA1 expression and current. Patch-clamp recording revealed that TRPA1-/- ß-cells displayed a marked hyperpolarization and decreased glucose-evoked action potential firing, which was associated with impaired GSIS. RYGB restored TRPA1 expression and current in GK ß-cells. This was accompanied by improved glucose-evoked electrical activity and insulin secretion. Additionally, RYGB-induced TRPA1 expression involved BAs/FXR-mediated recruitment of SRC1, promoting ACH3 at the promoter of TRPA1. CONCLUSIONS: The BAs/FXR/SRC1 axis-mediated restoration of TRPA1 expression plays a critical role in the enhanced GSIS and remission of diabetes in GK rats after RYGB.
Assuntos
Diabetes Mellitus Tipo 2/patologia , Secreção de Insulina , Receptores Citoplasmáticos e Nucleares/metabolismo , Canal de Cátion TRPA1/metabolismo , Animais , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/cirurgia , Diabetes Mellitus Tipo 2/veterinária , Potenciais Evocados , Derivação Gástrica , Histonas/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Coativador 1 de Receptor Nuclear/antagonistas & inibidores , Coativador 1 de Receptor Nuclear/genética , Coativador 1 de Receptor Nuclear/metabolismo , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Ratos Zucker , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Canal de Cátion TRPA1/genéticaRESUMO
Hypothalamic neurons expressing the anorectic peptide Pro-opiomelanocortin (Pomc) regulate food intake and body weight. Here, we show that Steroid Receptor Coactivator-1 (SRC-1) interacts with a target of leptin receptor activation, phosphorylated STAT3, to potentiate Pomc transcription. Deletion of SRC-1 in Pomc neurons in mice attenuates their depolarization by leptin, decreases Pomc expression and increases food intake leading to high-fat diet-induced obesity. In humans, fifteen rare heterozygous variants in SRC-1 found in severely obese individuals impair leptin-mediated Pomc reporter activity in cells, whilst four variants found in non-obese controls do not. In a knock-in mouse model of a loss of function human variant (SRC-1L1376P), leptin-induced depolarization of Pomc neurons and Pomc expression are significantly reduced, and food intake and body weight are increased. In summary, we demonstrate that SRC-1 modulates the function of hypothalamic Pomc neurons, and suggest that targeting SRC-1 may represent a useful therapeutic strategy for weight loss.