RESUMO
Elk-1 is a transcription factor that binds together with a dimer of the serum response factor (SRF) to the serum-response element (SRE), a genetic element that connects cellular stimulation with gene transcription. Elk-1 plays an important role in the regulation of cellular proliferation and apoptosis, thymocyte development, glucose homeostasis and brain function. The biological function of Elk-1 relies essentially on the interaction with other proteins. Elk-1 binds to SRF and generates a functional ternary complex that is required to activate SRE-mediated gene transcription. Elk-1 is kept in an inactive state under basal conditions via binding of a SUMO-histone deacetylase complex. Phosphorylation by extracellular signal-regulated protein kinase, c-Jun N-terminal protein kinase or p38 upregulates the transcriptional activity of Elk-1, mediated by binding to the mediator of RNA polymerase II transcription (Mediator) and the transcriptional coactivator p300. Strong and extended phosphorylation of Elk-1 attenuates Mediator and p300 recruitment and allows the binding of the mSin3A-histone deacetylase corepressor complex. The subsequent dephosphorylation of Elk-1, catalyzed by the protein phosphatase calcineurin, facilitates the re-SUMOylation of Elk-1, transforming Elk-1 back to a transcriptionally inactive state. Thus, numerous protein-protein interactions control the activation cycle of Elk-1 and are essential for its biological function.
Assuntos
Proteínas Elk-1 do Domínio ets/metabolismo , Proteínas Elk-1 do Domínio ets/fisiologia , Animais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Camundongos , Proteínas Nucleares/metabolismo , Fosforilação , Domínios e Motivos de Interação entre Proteínas/fisiologia , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Fator de Resposta Sérica/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Ativação Transcricional/genética , Proteínas Elk-1 do Domínio ets/genéticaRESUMO
BACKGROUND: Although FOXE1 was initially recognized for its role in thyroid organogenesis, more recently a strong association has been identified between the FOXE1 locus and thyroid cancer. The role of FOXE1 in adult thyroid, and in particular regarding cancer risk, has not been well established. We hypothesised that discovering key FOXE1 transcriptional partners would in turn identify regulatory pathways relevant to its role in oncogenesis. RESULTS: In a transcription factor-binding array, ELK1 was identified to bind FOXE1. We confirmed this physical association in heterologously transfected cells by IP and mammalian two-hybrid assays. In thyroid tissue, endogenous FOXE1 was shown to bind ELK1, and using ChIP assays these factors bound thyroid-relevant gene promoters TPO and TERT in close proximity to each other. Using a combination of electromobility shift assays, TERT promoter assays and siRNA-silencing, we found that FOXE1 positively regulated TERT expression in a manner dependent upon its association with ELK1. Treating heterologously transfected thyroid cells with MEK inhibitor U0126 inhibited FOXE1-ELK1 interaction, and reduced TERT and TPO promoter activity. METHODOLOGY: We investigated FOXE1 interactions within in vitro thyroid cell models and human thyroid tissue using a combination of immunoprecipitation (IP), chromatin IP (ChIP) and gene reporter assays. CONCLUSIONS: FOXE1 interacts with ELK1 on thyroid relevant gene promoters, establishing a new regulatory pathway for its role in adult thyroid function. Co-regulation of TERT suggests a mechanism by which allelic variants in/near FOXE1 are associated with thyroid cancer risk.
Assuntos
Fatores de Transcrição Forkhead/fisiologia , Regiões Promotoras Genéticas , Telomerase/genética , Neoplasias da Glândula Tireoide/etiologia , Proteínas Elk-1 do Domínio ets/fisiologia , Butadienos/farmacologia , Células HEK293 , Humanos , Iodeto Peroxidase/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Nitrilas/farmacologia , Neoplasias da Glândula Tireoide/tratamento farmacológicoRESUMO
B-Raf inhibitors have been used for the treatment of some B-Raf-mutated cancers. They effectively inhibit B-Raf/MEK/ERK signaling in cancers harboring mutant B-Raf, but paradoxically activates MEK/ERK in Ras-mutated cancers. Death receptor 5 (DR5), a cell surface pro-apoptotic protein, triggers apoptosis upon ligation with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or aggregation. This study focused on determining the effects of B-Raf inhibition on DR5 expression and DR5 activation-induced apoptosis in Ras-mutant cancer cells. Using chemical and genetic approaches, we have demonstrated that the B-Raf inhibitor PLX4032 induces DR5 upregulation exclusively in Ras-mutant cancer cells; this effect is dependent on Ras/c-Raf/MEK/ERK signaling activation. PLX4032 induces DR5 expression at transcriptional levels, largely due to enhancing CHOP/Elk1-mediated DR5 transcription. Pre-exposure of Ras-mutated cancer cells to PLX4032 sensitizes them to TRAIL-induced apoptosis; this is also a c-Raf/MEK/ERK-dependent event. Collectively, our findings highlight a previously undiscovered effect of B-Raf inhibition on the induction of DR5 expression and the enhancement of DR5 activation-induced apoptosis in Ras-mutant cancer cells and hence may suggest a novel therapeutic strategy against Ras-mutated cancer cells by driving their death due to DR5-dependent apoptosis through B-Raf inhibition.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes ras , Indóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas de Neoplasias/biossíntese , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Sulfonamidas/farmacologia , Apoptose/efeitos dos fármacos , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Proteínas de Neoplasias/genética , Oximas/farmacologia , Proteínas Proto-Oncogênicas B-raf/fisiologia , Proteínas Proto-Oncogênicas c-raf/fisiologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Fator de Transcrição CHOP/fisiologia , Transcrição Gênica/efeitos dos fármacos , Vemurafenib , Proteínas Elk-1 do Domínio ets/fisiologiaRESUMO
Inflammation plays an important role in the pathophysiological process after carotid artery stenting (CAS). Monocyte is a significant source of inflammatory cytokines in vascular remodeling. Telmisartan could reduce inflammation. In our study, we first found that, after CAS, the serum IL-1ß, IL-6, TGF-ß, and MMP-9 levels were significantly increased, but only MMP-9 level was elevated no less than 3 months. Second, we established a new in vitro model, where THP-1 monocytes were treated with the supernatants of human umbilical vein endothelial cells (HUVECs) that were scratched by pipette tips, which mimics monocytes activated by mechanical injury of stenting. The treatment enhanced THP-1 cell adhesion, migration and invasion ability, and the phosphorylation of ERK1/2 and Elk-1 and MMP-9 expression were significantly increased. THP-1 cells pretreated with PD98095 (ERK1/2 inhibitor) attenuated the phosphorylation of ERK1/2 and Elk-1 and upregulation of MMP-9, while pretreatment with telmisartan merely decreased the phosphorylation of Elk-1 and MMP-9 expression. These results suggested that IL-1ß, IL-6, TGF-ß, and MMP-9 participate in the pathophysiological process after CAS. Our new in vitro model mimics monocytes activated by stenting. MMP-9 expression could be regulated through ERK1/2/Elk-1 pathway, and the protective effects of telmisartan after stenting are partly attributed to its MMP-9 inhibition effects via suppression of Elk-1.
Assuntos
Estenose das Carótidas/terapia , Interleucina-1beta/sangue , Interleucina-6/sangue , Metaloproteinase 9 da Matriz/sangue , Stents , Fator de Crescimento Transformador beta/sangue , Idoso , Benzimidazóis/uso terapêutico , Benzoatos/uso terapêutico , Estenose das Carótidas/sangue , Células Cultivadas , Quimiocina CCL2/análise , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Pessoa de Meia-Idade , PPAR gama/fisiologia , Telmisartan , Molécula 1 de Adesão de Célula Vascular/análise , Proteínas Elk-1 do Domínio ets/fisiologiaRESUMO
Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Estilbenos/farmacologia , Proteínas Elk-1 do Domínio ets/fisiologia , Sequência de Bases , Fosfatase 1 de Especificidade Dupla/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Genes Reporter , Células HEK293 , Humanos , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Elementos de Resposta , Resveratrol , Ativação Transcricional/efeitos dos fármacos , Regulação para CimaRESUMO
Mutations in neurokinin B (NKB) and its receptor, NK3R, were identified in human patients with hypogonadotropic hypogonadism, a disorder characterized by lack of puberty and infertility. Further studies have suggested that NKB acts at the level of the hypothalamus to control GnRH neuron activity, either directly or indirectly. We recently reported that treatment with senktide, a NK3R agonist, induced GnRH secretion and expression of c-fos mRNA in GT1-7 cells. Here, we map the responsive region in the murine c-fos promoter to between -400 and -200 bp, identify the signal transducer and activator of transcription (STAT) (-345) and serum response element (-310) sites as required for induction, a modulatory role for the Ets site (-318), and show that induction is protein kinase C dependent. Using gel shift and Gal4 assays, we further show that phosphorylation of Elk-1 leads to binding to DNA in complex with serum response factor at serum response element and Ets sites within the c-fos promoter. Thus, we determine molecular mechanisms involved in NKB regulation of c-fos induction, which may play a role in modulation of GnRH neuron activation.
Assuntos
Hormônio Liberador de Gonadotropina/metabolismo , Neurocinina B/fisiologia , Neurônios/efeitos dos fármacos , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Fator de Resposta Sérica/metabolismo , Transcrição Gênica/fisiologia , Proteínas Elk-1 do Domínio ets/fisiologia , Animais , Linhagem Celular Transformada , Camundongos , Neurocinina B/farmacologia , Neurônios/metabolismo , Ratos , Transdução de Sinais , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
CD133 is a cellular surface protein that has been reported to be a cancer stem cell marker, and thus it is considered to be a potential target for cancer treatment. However, the mechanism regulating CD133 expression is not yet understood. In this study, we analyzed the activity of five putative promoters (P1-P5) of CD133 in human embryonic kidney (HEK) 293 cells and colon cancer cell line WiDr, and found that the activity of promoters, particularly of P5, is elevated by overexpression of hypoxia-inducible factors (HIF-1α and HIF-2α). Deletion and mutation analysis identified one of the two E-twenty six (ETS) binding sites (EBSs) in the P5 region as being essential for its promoter activity induced by HIF-1α and HIF-2α. In addition, a chromatin imunoprecipitation assay demonstrated that HIF-1α and HIF-2α bind to the proximal P5 promoter at the EBSs. The immunoprecipitation assay showed that HIF-1α physically interacts with Elk1; however, HIF-2α did not bind to Elk1 or ETS1. Furthermore, knockdown of both HIF-1α and HIF-2α resulted in a reduction of CD133 expression in WiDr. Taken together, our results revealed that HIF-1α and HIF-2α activate CD133 promoter through ETS proteins.
Assuntos
Antígenos CD/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Glicoproteínas/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Peptídeos/genética , Proteína Proto-Oncogênica c-ets-1/fisiologia , Ativação Transcricional , Proteínas Elk-1 do Domínio ets/fisiologia , Antígeno AC133 , Antígenos CD/metabolismo , Sítios de Ligação , Hipóxia Celular , Linhagem Celular Tumoral , Expressão Gênica , Técnicas de Silenciamento de Genes , Glicoproteínas/metabolismo , Células HEK293 , Humanos , Peptídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Regulação para CimaRESUMO
Elk-1 belongs to the ternary complex factors (TCFs) subfamily of the ETS domain proteins, and plays a critical role in the expression of immediate-early genes (IEGs) upon mitogen stimulation and activation of the mitogen-activated protein kinase (MAPK) cascade. The association of TCFs with serum response elements (SREs) on IEG promoters has been widely studied and a role for Elk-1 in promoting cell cycle entry has been determined. However, the presence of the ETS domain transcription factor Elk-1 in axons and dendrites of post-mitotic adult brain neurons has implications for an alternative function for Elk-1 in neurons other than controlling proliferation. In this study, possible alternative roles for Elk-1 in neurons were investigated, and it was demonstrated that blocking TCF-mediated transactivation in neuronal cells leads to apoptosis through a caspase-dependent mechanism. Indeed RNAi-mediated depletion of endogenous Elk-1 results in increased caspase activity. Conversely, overexpression of either Elk-1 or Elk-VP16 fusion proteins was shown to rescue PC12 cells from chemically-induced apoptosis, and that higher levels of endogenous Elk-1 correlated with longer survival of DRGs in culture. It was shown that Elk-1 regulated the Mcl-1 gene expression required for survival, and that RNAi-mediated degradation of endogenous Elk-1 resulted in elimination of the mcl-1 message. We have further identified the survival-of-motor neuron-1 (SMN1) gene as a novel target of Elk-1, and show that the ets motifs in the SMN1 promoter are involved in this regulation.
Assuntos
Neurônios/fisiologia , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteínas Elk-1 do Domínio ets/fisiologia , Animais , Apoptose , Caspases/fisiologia , Sobrevivência Celular , Proteína de Sequência 1 de Leucemia de Células Mieloides , Neurônios/citologia , Células PC12 , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2/genética , RatosRESUMO
We investigated whether altering caveolin-2 (cav-2) expression affects the proliferation of cancer cells. Cav-2 was not detected in HepG2, SH-SY5Y and LN-CaP cells, and the loss of cav-2 expression was not restored by 5-aza-2'-deoxycytidine treatment. In contrast, C6, HeLa, A549, MCF7 and PC3M cells expressed cav-2. Effects of re-expression of exogenous cav-2 in HepG2, SH-SY5Y and LN-CaP cells, and siRNA-mediated down-regulation of endogenous cav-2 in C6, HeLa, A549, MCF7 and PC3M cells on cancer proliferation were examined by MTT assay, colony formation assay and flow cytometric analysis. Cav-2 transfection in HepG2 hepatocellular carcinoma cells and knockdown in C6 glioma cells caused reduction in cell proliferation and growth with retarded entry into the S phase. Cav-2 re-expression in SH-SY5Y neuroblastoma cells and depletion in HeLa epithelial cervical cancer and A549 lung adenocarcinoma cells promoted cancer cell proliferation. Luciferase reporter assay showed that transcriptional activation of Elk-1 and STAT3 was significantly decreased in cav-2-transfected HepG2 hepatocellular carcinoma and down-regulated C6 glioma cells. Our data suggest that cav-2 acts as a modulator of cancer progression.
Assuntos
Caveolina 2/fisiologia , Proliferação de Células , Neoplasias/patologia , Animais , Caveolina 2/antagonistas & inibidores , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Glioma/patologia , Células Hep G2 , Humanos , Neuroblastoma/patologia , Ratos , Fator de Transcrição STAT3/fisiologia , Proteínas Elk-1 do Domínio ets/fisiologiaRESUMO
PAI-1 (plasminogen activator inhibitor-1) in breast cancer cells is involved in tumour development and metastasis of breast cancer cells. PAI-1 function and the regulation of its expression have been precisely investigated. Here we report that EGF, which promotes breast cancer tumour growth and survival, rapidly induces PAI-1 expression in the breast adenocarcinoma cell line MCF-7 through the activation of the transcription factor Elk-1. We have found that the PAI-1 promoter fragment (-140 to +173) containing the Ets consensus binding site is activated by Elk-1. Chromatin immunoprecipitation analysis confirms in vivo binding of Elk-1 to the PAI-1 promoter and demonstrates that Elk-1 phosphorylation on the Ets binding site is EGF-dependent.
Assuntos
Fator de Crescimento Epidérmico/fisiologia , Inibidor 1 de Ativador de Plasminogênio/genética , Proteínas Elk-1 do Domínio ets/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Sítios de Ligação/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Fator de Crescimento Epidérmico/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Fosforilação/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteína Proto-Oncogênica c-ets-1/metabolismo , Ativação Transcricional/efeitos dos fármacos , Transfecção , Células Tumorais Cultivadas , Proteínas Elk-1 do Domínio ets/fisiologiaRESUMO
MDMX is an important regulator of p53 transcriptional activity and stress response. MDMX overexpression and gene amplification are implicated in p53 inactivation and tumor development. Unlike MDM2, MDMX is not inducible by p53, and little is known about its regulation at the transcriptional level. We found that MDMX levels in tumor cell lines closely correlate with promoter activity and mRNA level. Activated K-Ras and insulin-like growth factor 1 induce MDMX expression at the transcriptional level through mechanisms that involve the mitogen-activated protein kinase and c-Ets-1 transcription factors. Pharmacological inhibition of MEK results in down-regulation of MDMX in tumor cell lines. MDMX overexpression was detected in approximately 50% of human colon tumors and showed strong correlation with increased extracellular signal-regulated kinase phosphorylation. Therefore, MDMX expression is regulated by mitogenic signaling pathways. This mechanism may protect normal proliferating cells from p53 but also hamper p53 response during tumor development.
Assuntos
Regulação Neoplásica da Expressão Gênica/fisiologia , Mitose/genética , Proteínas de Neoplasias/biossíntese , Proteínas Nucleares/biossíntese , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/biossíntese , Fatores de Transcrição/fisiologia , Sítios de Ligação , Proteínas de Ciclo Celular , Linhagem Celular Tumoral/citologia , Linhagem Celular Tumoral/metabolismo , Cicloeximida/farmacologia , Feminino , Genes ras , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Proteína Proto-Oncogênica c-ets-1/fisiologia , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Proteínas Recombinantes/farmacologia , Proteína Supressora de Tumor p53/fisiologia , Proteínas Elk-1 do Domínio ets/fisiologiaRESUMO
Astrocytes play important roles in guiding the construction of the nervous system, controlling extracellular ions and neurotransmitters, and regulating CNS synaptogenesis. Egr-1 is a transcription factor involved in neuronal differentiation and astrocyte cell proliferation. In this study, we investigated whether the tricyclic antidepressant (TCA) amitriptyline induces Egr-1 expression in astrocytes using rat C6 glioma cells as a model. We found that amitriptyline increased the expression of Egr-1 in a dose- and time-dependent manner. The amitriptyline-induced Egr-1 expression was mediated through serum response elements (SREs) in the Egr-1 promoter. SREs were activated by the Ets-domain transcription factor Elk-1 through the ERK and JNK mitogen-activated protein (MAP) kinase pathways. The inhibition of the ERK and JNK MAP kinase signals attenuated amitriptyline-induced transactivation of Gal4-Elk-1 and Egr-1 promoter activity. Our findings suggest that the induction of Egr-1 expression in astrocytes may be required to attain the therapeutic effects of antidepressant drugs.
Assuntos
Amitriptilina/farmacologia , Antidepressivos Tricíclicos/farmacologia , Proteína 1 de Resposta de Crescimento Precoce/biossíntese , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Neuroglia/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Proteína 1 de Resposta de Crescimento Precoce/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ratos , Ativação Transcricional/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Proteínas Elk-1 do Domínio ets/genética , Proteínas Elk-1 do Domínio ets/fisiologiaRESUMO
Cyclooxygenase-2 (COX-2) expression is associated with many aspects of physiological and pathological conditions, including pancreatic beta-cell dysfunction. Prostaglandin E2 (PGE2) production, as a consequence of COX-2 gene induction, has been reported to impair beta-cell function. The molecular mechanisms involved in the regulation of COX-2 gene expression are not fully understood. In this report, we used pancreatic beta-cells (RINm5F) to explore the potential transcription factors regulating COX-2 promoter activity. Using promoter screening method, we selected several transcription factors in our study. Through luciferase reporter studies, we found that these factors can regulate COX-2 promoter activity in RINm5F cells. Among these factors, cyclic AMP response-element binding protein (CREB), Ets family members Ets-1 and Elk-1 can positively regulate COX-2 promoter activity. On the contrary, signal transducer and activator of transcription 1 (STAT1) plays a negative role on COX-2 promoter. Our findings will be helpful for better understanding the transcriptional regulation of COX-2 in pancreatic beta-cells. Moreover, these transcriptional regulators of COX-2 expression will be potential targets for the prevention of beta-cell damage mediated by PGE2.
Assuntos
Ciclo-Oxigenase 2/genética , Regulação Enzimológica da Expressão Gênica , Células Secretoras de Insulina/enzimologia , Fatores de Transcrição/fisiologia , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Células Secretoras de Insulina/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteína Proto-Oncogênica c-ets-1/fisiologia , Ratos , Fator de Transcrição STAT1/fisiologia , Fatores de Transcrição/metabolismo , Ativação Transcricional , Células Tumorais Cultivadas , Proteínas Elk-1 do Domínio ets/fisiologiaRESUMO
Expression of homeobox A1 (HOXA1) results in oncogenic transformation of immortalized human mammary epithelial cells with aggressive tumor formation in vivo. However, the mechanisms by which HOXA1 mediates oncogenic transformation is not well defined. To identify molecules that could potentially be involved in HOXA1-mediated oncogenic transformation, microarray analysis was utilized to characterize and compare the gene expression pattern in response to forced expression or depletion of HOXA1 in human mammary carcinoma cells. Gene expression profiling identified that genes involved in the p44/42 mitogen-activated protein (MAP) kinase activation pathway (GRB2, MAP kinase kinase (MEK1) and SDFR1) or p44/42 MAP kinase-regulated genes (IER3, EPAS1, PCNA and catalase) are downstream expression targets of HOXA1. Forced expression of HOXA1 increased GRB2 and MEK1 mRNA and protein expression and increased p44/42 MAP kinase phosphorylation, activity and Elk-1-mediated transcription. Use of a MEK1 inhibitor demonstrated that increased p44/42 MAP kinase activity is required for the HOXA1-mediated increase in cell proliferation, survival, oncogenicity and oncogenic transformation. Thus, modulation of the p44/42 MAP kinase pathway is one mechanism by which HOXA1 mediates oncogenic transformation of the human mammary epithelial cell.
Assuntos
Proteínas de Homeodomínio/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Fatores de Transcrição/fisiologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Transfecção , Proteínas Elk-1 do Domínio ets/genética , Proteínas Elk-1 do Domínio ets/fisiologiaRESUMO
Emerging evidence supports the idea that the c-Jun N-terminal kinases (JNKs) possess overlapping but distinct functions. The potential roles of the ubiquitously expressed JNK1 and JNK2 in regulating expression of the central transcription initiation factor, TATA-binding protein (TBP), were examined. Relative to wild-type fibroblasts, TBP was decreased in Jnk1(-/-) cells and increased in Jnk2(-/-) cells. Similarly, reduction of JNK1 in human hepatoma cells decreased TBP expression, whereas reduction of JNK2 enhanced it. JNK-mediated regulation of TBP expression occurs at the transcriptional level through their ability to target Elk-1, which directly regulates the TBP promoter in response to epidermal growth factor stimulation. JNK1 increases, whereas JNK2 decreases, the phosphorylation state of Elk-1, which differentially affects Elk-1 occupancy at a defined site within the TBP promoter. These JNK-mediated alterations in TBP expression, alone, serve to regulate c-Jun expression and fibroblast proliferation rates. These studies uncovered several new molecular events that distinguish the functions of JNK1 and JNK2 that are critical for their regulation of cellular proliferation.
Assuntos
Regulação da Expressão Gênica , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Proteína Quinase 9 Ativada por Mitógeno/fisiologia , Proteínas Proto-Oncogênicas c-jun/biossíntese , Proteína de Ligação a TATA-Box/biossíntese , Proteína de Ligação a TATA-Box/fisiologia , Proteínas Elk-1 do Domínio ets/fisiologia , Animais , Apoptose , Proliferação de Células , Fibroblastos/metabolismo , Humanos , Camundongos , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-ets-1/metabolismo , Transcrição GênicaRESUMO
Glioblastoma multiforme (GBM) is the highest grade of astrocytoma. GBM pathogenesis has been linked to receptor tyrosine kinases and kinases further down signal-transduction pathways - in particular, members of the protein kinase C (PKC) family. The expression and activity of various PKC isoforms are increased in malignant astrocytomas, but not in non-neoplastic astrocytes. This suggests that PKC activity contributes to tumor progression. The level of PKC-eta expressed correlates with the degree of phorbol-12-myristate-13-acetate (PMA)-induced proliferation of two glioblastoma cell lines, U-1242 MG and U-251 MG. Normally, U-1242 cells do not express PKC-eta, and PMA inhibits their proliferation. Conversely, PMA increases proliferation of U-1242 cells that are stably transfected with PKC-eta (U-1242-PKC-eta). PMA treatment also stimulates proliferation of U-251 cells, which express PKC-eta. Here, we determined that extracellular signal-regulated kinase (ERK) and Elk-1 are downstream targets of PKC-eta. Elk-1-mediated transcriptional activity correlates with the PKC-eta-mediated mitogenic response. Pretreatment of U-1242-PKC-eta cells with inhibitors of PKC or MAPK/ERK kinase (MEK) (bisindolyl maleimide (BIM) or U0126, respectively) blocked both PMA-induced Elk-1 transcriptional activity and PMA-stimulated proliferation. An overexpressed dominant-negative PKC-eta reduced the mitogenic response in U-251 cells, as did reduction of Elk-1 by small interfering RNA. Taken together, these results strongly suggest that PKC-eta-mediated glioblastoma proliferation involves MEK/mitogen-activated protein (MAP) kinase phosphorylation, activation of ERK and subsequently of Elk-1. Elk-1 target genes involved in GBM proliferative responses have yet to be identified.
Assuntos
Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Glioblastoma/patologia , Proteína Quinase C/fisiologia , Proteínas Elk-1 do Domínio ets/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genes fos/fisiologia , Genes jun/fisiologia , Humanos , Isoenzimas/fisiologia , Modelos Biológicos , Fosforilação , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição AP-1/genética , Transcrição Gênica/genética , TransfecçãoRESUMO
Inactivating mutations in the adenomatous polyposis coli gene (APC), and activating mutations in RAS, occur in a majority of colorectal carcinomas. However, the relationship between these changes and tumorigenesis is poorly understood. RAS-induced activation of the ERK pathway was reduced by overexpressing APC in DLD-1 colorectal cancer cells. ERK activity was increased by Cre-virus-induced Apc knockout in primary Apc(flox/flox) mouse embryonic fibroblasts, indicating that APC inhibits ERK activity. ERK activity was increased by overexpression and decreased by knock down of beta-catenin. The activation of Raf1, MEK and ERK kinases by beta-catenin was reduced by co-expression of APC. These results indicate that APC inhibits the ERK pathway by an action on beta-catenin. RAS-induced activation of the ERK pathway was reduced by the dominant negative form of TCF4, indicating that the ERK pathway regulation by APC/beta-catenin signaling is, at least, partly caused by effects on beta-catenin/TCF4-mediated gene expression. The GTP loading and the protein level of mutated RAS were decreased in cells with reduced ERK activity as a result of APC overexpression, indicating that APC regulates RAS-induced ERK activation at least partly by reduction of the RAS protein level. APC regulates cellular proliferation and transformation induced by activation of both RAS and beta-catenin signaling.
Assuntos
Proteína da Polipose Adenomatosa do Colo/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas ras/fisiologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Regulação da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Células NIH 3T3 , Transdução de Sinais/fisiologia , Fatores de Transcrição TCF/genética , Fatores de Transcrição TCF/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição , Fator de Transcrição AP-1/fisiologia , beta Catenina/genética , beta Catenina/metabolismo , Proteínas Elk-1 do Domínio ets/fisiologiaRESUMO
OBJECTIVE: To investigate the role of extracellular signal-regulated kinase (ERK) 1/2 and Elk1 in pulmonary disease induced by crocidolite asbestos fiber. METHOD: Western blotting and Immunoprecipitation were used to detect the expression of phosphorylated ERK1/2 and Elk1 in human bronchial airway A549 cell line stimulated by crocidolite. RESULTS: The expression of phosphorylated ERK1/2 and Elk1 were striking higher than those of the control, the differences were significant (P < 0.05). CONCLUSION: Phosphorylated ERK1/2 and Elk1 probably involved in the process of the diseases induced by crocidolite.