RESUMO
The PTEN tumour suppressor gene is induced by the early growth response 1 (EGR1) transcription factor, which also transactivates p53, p73, and p300/CBP as well as other proapoptotic and anti-cancer genes. Here, we describe a novel Akt-EGR1-alternate reading frame (ARF)-PTEN axis, in which PTEN activation in vivo requires p14ARF-mediated sumoylation of EGR1. This modification is dependent on the phosphorylation of EGR1 at S350 and T309 by Akt, which promotes interaction of EGR1 with ARF at K272 in its repressor domain by the ARF/Ubc9/SUMO system. EGR1 sumoylation is decreased by ARF reduction, and no EGR1 sumoylation is detected in ARF(-/-) mice, which also exhibit reduced amounts of PTEN. Our model predicts that perturbation of any of the clinically important tumour suppressors, PTEN, EGR1, and ARF, will cause some degree of dysfunction of the others. These results also explain the known negative feedback regulation by PTEN on its own synthesis through PI3 kinase inhibition.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Regulação da Expressão Gênica , Proteína Oncogênica v-akt/metabolismo , PTEN Fosfo-Hidrolase/biossíntese , Animais , Linhagem Celular , Humanos , Camundongos , Camundongos Knockout , Modelos Biológicos , FosforilaçãoRESUMO
The molecular mechanisms controlling inductive events leading to the specification and terminal differentiation of cardiomyocytes are still largely unknown. We have investigated the role of Cripto, an EGF-CFC factor, in the earliest stages of cardiomyogenesis. We find that both the timing of initiation and the duration of Cripto signaling are crucial for priming differentiation of embryonic stem (ES) cells into cardiomyocytes, indicating that Cripto acts early to determine the cardiac fate. Furthermore, we show that failure to activate Cripto signaling in this early window of time results in a direct conversion of ES cells into a neural fate. Moreover, the induction of Cripto activates the Smad2 pathway, and overexpression of activated forms of type I receptor ActRIB compensates for the lack of Cripto signaling in promoting cardiomyogenesis. Finally, we show that Nodal antagonists inhibit Cripto-regulated cardiomyocyte induction and differentiation in ES cells. All together our findings provide evidence for a novel role of the Nodal/Cripto/Alk4 pathway in this process.
Assuntos
Diferenciação Celular/genética , Fator de Crescimento Epidérmico , Glicoproteínas de Membrana , Miocárdio/metabolismo , Proteínas de Neoplasias/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Receptores de Ativinas Tipo I/metabolismo , Receptores de Activinas Tipo II/metabolismo , Sequência de Aminoácidos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Indução Embrionária , Proteínas Ligadas por GPI , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Dados de Sequência Molecular , Mutação , Miocárdio/citologia , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/farmacologia , Proteína Nodal , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/farmacologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/genética , Proteína Smad2 , Células-Tronco/citologia , Transativadores/metabolismo , Proteínas de XenopusRESUMO
Cells lacking vinculin are highly metastatic and motile. The reasons for this finding have remained unclear. Both enhanced survival and motility are critical to metastasis. Here, we show that vinculin null (vin-/-) cells and cells expressing a vinculin Y822F mutant have increased survival due to up-regulated activity of extracellular signal-regulated kinase (ERK). This increase is shown to result from vinculin's modulation of paxillin-FAK interactions. A vinculin fragment (amino acids 811-1066) containing the paxillin binding site restored apoptosis and suppressed ERK activity in vin-/- cells. Both vinY822F and vin-/- cells exhibit increased interaction between paxillin and focal adhesion kinase (FAK) and increased paxillin and FAK phosphorylation. Transfection with paxillin Y31FY118F dominant-negative mutant in these cells inhibits ERK activation and restores apoptosis. The enhanced motility of vin-/- and vinY822F cells is also shown to be due to a similar mechanism. Thus, vinculin regulates survival and motility via ERK by controlling the accessibility of paxillin for FAK interaction.
Assuntos
Movimento Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Vinculina/fisiologia , Animais , Apoptose/genética , Sítios de Ligação/genética , Adesão Celular/genética , Sobrevivência Celular/genética , Proteínas do Citoesqueleto/genética , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Camundongos , Mutação/genética , Metástase Neoplásica/genética , Células-Tronco Neoplásicas , Paxilina , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/genética , Fosforilação , Transfecção , Regulação para Cima/genética , Vinculina/genética , Vinculina/metabolismoRESUMO
The early growth response 1 (Egr1) gene is a transcription factor that acts as both a tumor suppressor and a tumor promoter. Egr1-null mouse embryo fibroblasts bypass replicative senescence and exhibit a loss of DNA damage response and an apparent immortal growth, suggesting loss of p53 functions. Stringent expression analysis revealed 266 transcripts with >2-fold differential expression in Egr1-null mouse embryo fibroblasts, including 143 known genes. Of the 143 genes, program-assisted searching revealed 66 informative genes linked to Egr1. All 66 genes could be placed on a single regulatory network consisting of three branch points of known Egr1 target genes: TGFbeta1, IL6, and IGFI. Moreover, 19 additional genes that are known targets of p53 were identified, indicating that p53 is a fourth branch point. Electrophoretic mobility shift assay as well as chromatin immunoprecipitation confirmed that p53 is a direct target of Egr1. Because deficient p53 expression causes tumors in mice, we tested the role of Egr1 in a two-step skin carcinogenesis study (144 mice) that revealed a uniformly accelerated development of skin tumors in Egr1-null mice (P < 0.005). These studies reveal a new role for Egr1 as an in vivo tumor suppressor.
Assuntos
Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/fisiologia , Genes Supressores de Tumor , Proteínas Imediatamente Precoces/fisiologia , Neoplasias Cutâneas/genética , Fatores de Transcrição/fisiologia , Proteína Supressora de Tumor p53/fisiologia , 9,10-Dimetil-1,2-benzantraceno , Animais , Processos de Crescimento Celular/genética , Mapeamento Cromossômico , Dano ao DNA , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce , Feminino , Fibroblastos/fisiologia , Perfilação da Expressão Gênica , Proteínas Imediatamente Precoces/deficiência , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Neoplasias Cutâneas/induzido quimicamente , Acetato de Tetradecanoilforbol , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Regulação para CimaRESUMO
Although vinculin (-/-) mouse embryo fibroblasts assemble focal adhesions (FAs), they spread more slowly, less extensively, and close a wound more rapidly than vinculin (+/+) cells. To investigate the structure and dynamics of FAs in these cells, we used real-time interference reflection microscopy (IRM) thus avoiding the need to express exogenous GFP-tagged FA proteins which may be misregulated. This showed that the FAs were smaller, less abundant and turned over more rapidly in vinculin null compared to wild-type cells. Expression of vinculin rescued the spreading defect and resulted in larger and more stable FAs. Phosphatidylinositol 4,5-bisphosphate (PIP2) is thought to play a role in vinculin activation by relieving an intramolecular association between the vinculin head (Vh) and tail (Vt) that masks the ligand binding sites in Vh and Vt. To investigate the role of the vinculin/PIP2 interaction in FA dynamics, we used a vinculin mutant lacking the C-terminal arm (residues 1053-1066) and referred to as the deltaC mutation. This mutation reduced PIP2 binding to a Vt deltaC polypeptide by >90% compared to wild type without affecting binding to Vh or F-actin. Interestingly, cells expressing the vinculin deltaC mutant assembled remarkably stable FAs. The results suggest that vinculin inhibits cell migration by stabilising FAs, and that binding of inositol phospholipids to Vt plays an important role in FA turnover.
Assuntos
Adesões Focais/metabolismo , Vinculina/metabolismo , Animais , Varredura Diferencial de Calorimetria , Movimento Celular/fisiologia , Fibroblastos/citologia , Camundongos , Modelos Moleculares , Mutação/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Ligação Proteica , Conformação Proteica , Vinculina/química , Vinculina/deficiênciaRESUMO
DNA methylation and copy number in the genomes of three immortalized prostate epithelial and five cancer cell lines (LNCaP, PC3, PC3M, PC3M-Pro4, and PC3M-LN4) were compared using a microarray-based technique. Genomic DNA is cut with a methylation-sensitive enzyme HpaII, followed by linker ligation, polymerase chain reaction (PCR) amplification, labeling, and hybridization to an array of promoter sequences. Only those parts of the genomic DNA that have unmethylated restriction sites within a few hundred base pairs generate PCR products detectable on an array. Of 2732 promoter sequences on a test array, 504 (18.5%) showed differential hybridization between immortalized prostate epithelial and cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, there were eight (CD44, CDKN1A, ESR1, PLAU, RARB, SFN, TNFRSF6, and TSPY) previously observed in prostate cancer and 13 previously known methylation targets in other cancers (ARHI, bcl-2, BRCA1, CDKN2C, GADD45A, MTAP, PGR, SLC26A4, SPARC, SYK, TJP2, UCHL1, and WIT-1). The majority of genes that appear to be both differentially methylated and differentially regulated between prostate epithelial and cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, and GG2-1, representing a rich new source of candidate genes used to study the role of DNA methylation in prostate tumors.
Assuntos
Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Regiões Promotoras Genéticas , Neoplasias da Próstata/genética , Linhagem Celular Tumoral , DNA de Neoplasias , DNA-Citosina Metilases , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/metabolismo , RNA Neoplásico/metabolismoRESUMO
DNA microarrays of promoter sequences have been developed in order to identify the profile of genes bound and activated by DNA regulatory proteins such as the transcription factors c-Jun and ATF2 as well as DNA-modifying methylases. The arrays contain 3083 unique human promoter sequences from +500 to -1000 nts from the transcription start site. Cisplatin-induced DNA damage rapidly leads to specific activation of the Jun kinase pathway leading to increased phosphorylation of c-Jun and ATF2-DNA complexes at hundreds of sites within 3 hours. Using three statistical criteria, approximately 269 most commonly phosphorylated c-Jun/ATF2-DNA complexes were identified and representative cases were verified by qPCR measurement of ChIP-captured DNA. Expression was correlated at the mRNA and protein levels. The largest functional cohort was 24 genes of known DNA repair function, most of which exhibited increased protein expression indicated coordinate gene regulation. In addition, cell lines of prostate cancer exhibit stable methylation or copy number changes that reflect the alterations of the corresponding primary tumors. 504 (18.5%) promoters showed differential hybridization between immortalized control prostate epithelial and cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, eight had previously been observed in prostate cancer, and 13 were previously determined methylation targets in other cancers. The vast majority of genes that appear to be both differentially methylated and differentially regulated between prostate epithelial and cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, GG2-1, representing a rich new source of candidate genes to study the role of DNA methylation in prostate tumors. Earlier studies using prototype promoter arrays examine approximately 7% of the proximal regulatory sequences while the current gene regulatory events surveyed here occur on a large scale and may rapidly effect the coordinated expression of a large number of genes.
Assuntos
Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica , Humanos , Modelos Biológicos , Neoplasias/genética , Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , FosforilaçãoRESUMO
The Early Growth Response protein (Egr-1) is a C(2)H(2)-zinc finger-containing transcriptional regulator involved in the control of cell proliferation and apoptosis. Its DNA-binding activity is redox regulated in vitro through the oxidation-reduction of Cys residues within its DNA-binding domain. APE/Ref-1 is a DNA-repair enzyme with redox modulating activities on several transcription factors. In this study, by evaluating the effects of different stimuli, we found a similar timing of activation being suggestive for a common and co-linear regulation for the two proteins. Indeed, we show that APE/Ref-1 increases the Egr-1 DNA-binding activity in unstimulated osteoblastic HOBIT cells. H(2)O(2) stimulation induces a strong interaction between Egr-1 and APE/Ref-1 at early times upon activation, as assayed by immunoprecipitation experiments. By using a cell transfection approach, we demonstrated the functional role of this interaction showing that two specific Egr-1 target genes, the PTEN phosphatase and the thymidine kinase (TK) genes promoters, are activated by contransfection of APE/Ref-1. Interestingly, by using a cell transfection approach and Chromatin immunoprecipitation assays, we were able to demonstrate that Egr-1 stimulates the transcriptional activity of APE/Ref-1 gene promoter by a direct interaction with specific DNA-binding site on its promoter. Taken together, our data delineate a new molecular mechanism of Egr-1 activation occurring soon after H(2)O(2) stimulation in osteoblastic cells and suggest a model for a positive loop between APE/Ref-1 and Egr-1 that could explain the early transcriptional activation of APE/Ref-1 gene expression.
Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteínas Imediatamente Precoces/metabolismo , Osteoblastos , Estresse Oxidativo , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Proliferação de Células , Imunoprecipitação da Cromatina , DNA/metabolismo , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Peróxido de Hidrogênio/farmacologia , Proteínas Imediatamente Precoces/genética , Dados de Sequência Molecular , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Oxidantes/farmacologia , Oxirredução , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Regiões Promotoras Genéticas/genética , Timidina Quinase/genética , Timidina Quinase/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Dedos de ZincoRESUMO
The lipid phosphatase, phosphatase and tensin homolog (PTEN), is a key element in controlling cell growth and survival and has a well established role as tumor suppressor protein in many neoplasia. Several data indicate that silencing of PTEN gene expression may be relevant in follicular thyroid cell transformation. Thus, in the present study regulation of PTEN gene expression in thyroid cells was investigated. Cotransfection experiments indicated that in normal FRTL-5 rat thyroid cells, PTEN promoter activity was increased by overexpression of the transcription factor early growth response protein-1 (Egr-1). Moreover, Western blot experiments indicated that when Egr-1 expression was up-regulated by treating FRTL-5 cells with H2O2, an increase in PTEN expression was also observed. TSH induced opposite modifications on PTEN and Egr-1 protein levels. Moreover, acute or chronic TSH stimulation determined distinct effects. In fact, acute TSH stimulation (30 and 60 min) induced a decrease in PTEN, but an increase in Egr-1 protein levels. These effects were cAMP dependent; in fact, they were mimicked by forskolin. A chronic TSH treatment (5 d) stimulated PTEN protein expression, whereas Egr-1 protein was down-regulated. In contrast to normal thyroid cells, when the thyroid tumor cell lines ARO and BCPAP were exposed to H2O2, neither Egr-1 nor PTEN protein levels were increased. Acute stimulation of ARO and BCPAP cells with forskolin increased Egr-1, but not PTEN, protein levels. Therefore, thyroid tumor cell lines show alteration of PTEN gene expression regulation. RT-PCR experiments performed on human thyroid tumors showed that the absence of Egr-1 mRNA is always paralleled by the absence of PTEN mRNA. Thus, modification of the Egr-1-dependent mechanisms may play a role in the silencing of PTEN gene expression occurring during thyroid cell transformation.
Assuntos
Monoéster Fosfórico Hidrolases/metabolismo , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular , AMP Cíclico/farmacologia , Regulação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Peróxido de Hidrogênio/farmacologia , Oxidantes/farmacologia , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/genética , Regiões Promotoras Genéticas , Ratos , Glândula Tireoide/citologia , Neoplasias da Glândula Tireoide/patologia , Tireotropina/farmacologia , Transfecção , Proteínas Supressoras de Tumor/genéticaRESUMO
Egr1 is a multifunctional transcription factor regulating a remarkable spectrum of cellular responses from survival to apoptosis, growth to growth arrest, differentiation to transformation, senescence as well as memory and learning effects. In prostate cancer, Egr1 levels are constitutively high and closely linked to cancer development and progression. This zinc-finger protein is a short-lived, immediate early growth response gene known to be induced by a large number of extracellular stimuli such as irradiation (all wavelengths tested), hypoxia, hyperoxia, chemotherapy agents, and more. Therefore the target genes that Egr1 regulates in prostate cancer cells play an important role in generating many of the cellular responses that characterize these cells. After Egr1 binds to its binding sites on gene promoters, specificity of response is determined by whether Egr1 transcriptionally up- or downregulates the target genes. Expression microarray analyses combined with binding data promise new ways to identify stage specific cancer markers, to aid in patient risk assessment and in therapeutic choices.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Neoplasias da Próstata/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Proteína 1 de Resposta de Crescimento Precoce , Previsões , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Imediatamente Precoces/genética , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Neoplasias da Próstata/patologia , Transdução de Sinais/genética , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: UV irradiation activates the epidermal growth factor receptor, induces Egr1 expression and promotes apoptosis in a variety of cell types. We examined the hypothesis that Egr1 regulates genes that mediate this process by use of a chip-on-chip protocol in human tumorigenic prostate M12 cells. RESULTS: UV irradiation led to significant binding of 288 gene promoters by Egr1. A major functional subgroup consisted of apoptosis related genes. The largest subgroup of 24 genes belongs to the epidermal growth factor receptor-signal transduction pathway. Egr1 promoter binding had a significant impact on gene expression of target genes. Conventional chromatin immunoprecipitation and quantitative real time PCR were used to validate promoter binding and expression changes. Small interfering RNA experiments were used to demonstrate the specific role of Egr1 in gene regulation. UV stimulation promotes growth arrest and apoptosis of M12 cells and our data clearly show that a downstream target of the epidermal growth factor receptor, namely Egr1, mediates this apoptotic response. Our study also identified numerous previously unknown targets of Egr1. These include FasL, MAX and RRAS2, which may play a role in the apoptotic response/growth arrest. CONCLUSIONS: Our results indicate that M12 cells undergo Egr1-dependent apoptotic response upon UV stimulation and led to the identification of downstream targets of Egr1, which mediate epidermal growth factor receptor function.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , Apoptose/efeitos da radiação , Linhagem Celular Transformada , Imunoprecipitação da Cromatina , Perfilação da Expressão Gênica , Humanos , Masculino , Regiões Promotoras Genéticas , Ativação Transcricional , Raios UltravioletaRESUMO
p300 and CBP are large (300 kDa) nuclear scaffold proteins that act as co-activators of transcription in most cell types and are over-expressed in prostate cancer. Recently, the Egr1 transcription factor was shown to up- or down-regulate p300 and CBP transcription based on the nature of its post-translational modification. Notably, interactions of the three proteins provide fine tuning for Egr1-induced growth or cell death responses.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas Imediatamente Precoces/fisiologia , Proteínas Nucleares/fisiologia , Próstata/fisiologia , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologia , Proteína 1 de Resposta de Crescimento Precoce , Humanos , MasculinoRESUMO
PTEN is a frequently mutated tumor suppressor in malignancies. Interestingly, some malignancies exhibit undetectable PTEN protein without mutations or loss of PTEN mRNA. The cause(s) for this reduction in PTEN is unknown. Cancer cells frequently exhibit loss of cadherin, beta-catenin, alpha-catenin and/or vinculin, key elements of adherens junctions. Here we show that F9 vinculin-null (vin(-/-)) cells lack PTEN protein despite normal PTEN mRNA levels. Their PTEN protein expression was restored by transfection with vinculin or by inhibition of PTEN degradation. F9 vin(-/-) cells express PTEN protein upon transfection with a vinculin fragment (amino acids 243-1066) that is capable of interacting with alpha-catenin but unable to target into focal adhesions. On the other hand, disruption of adherens junctions with an E-cadherin blocking antibody reduced PTEN protein to undetectable levels in wild-type F9 cells. PTEN protein levels were restored in F9 vin(-/-) cells upon transfection with an E-cadherin-alpha-catenin fusion protein, which targets into adherens junctions and interacts with beta-catenin in F9 vin(-/-) cells. beta-Catenin is known to interact with MAGI-2. MAGI-2 interaction with PTEN in the cell membrane is known to prevent PTEN protein degradation. Thus, MAGI-2 overexpression in F9 vin(-/-) cells restored PTEN protein levels. Moreover, expression of vinculin mutants that reinstated the disrupted interactions of beta-catenin with MAGI-2 in F9 vin(-/-) cells also restored PTEN protein levels. These studies indicate that PTEN protein levels are dependent on the maintenance of beta-catenin-MAGI-2 interaction, in which vinculin plays a critical role.
Assuntos
Junções Aderentes/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transativadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Vinculina/metabolismo , Animais , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/genética , Proteínas de Membrana/genética , Camundongos , Mutação/genética , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Proteínas Supressoras de Tumor/genética , Vinculina/genética , alfa Catenina , beta CateninaRESUMO
The transcription factor, Egr1, so-called because it is encoded by the immediate early growth response gene, Egr1, is rapidly induced by growth factors to transduce the proliferative signal. The induction of Egr1 by external stimuli is generally transient but appears to be sustained in some prostate tumor cell lines and tumors, suggesting that Egr1 stimulates tumor cell growth. In contradiction, in breast, lung and brain tumors, Egr1 expression is often absent or reduced and when re-expressed, results in growth suppression. Re-expression of Egr1 in tumor cells also leads to antiapoptotic activity, which would encourage tumor cell survival. Egr1 is also required for, or stimulates, the differentiation of several cell types. Another contradiction is that after stress stimuli to some cell types, Egr1 is required for programmed cell death or apoptosis in both normal and tumor cells. Egr1 also plays a role in tumor progression, through the hypoxic signal generated in growing tumors. Egr1 is highly induced under these conditions and its activities stimulate angiogenesis and improved survival of tumor cells. How this large agenda can be achieved lies in the choice of Egr1 target genes, and varying patterns of coordinated expression have been described, but the mechanisms for this choice are not clear. This review points to areas where research should be focussed.
Assuntos
Adenocarcinoma/metabolismo , Proteínas de Ligação a DNA/fisiologia , Regulação Neoplásica da Expressão Gênica , Proteínas Imediatamente Precoces , Proteínas de Neoplasias/fisiologia , Neoplasias da Próstata/metabolismo , Fatores de Transcrição/fisiologia , Adenocarcinoma/irrigação sanguínea , Adenocarcinoma/patologia , Animais , Apoptose , Diferenciação Celular , Divisão Celular , Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce , Genes Supressores de Tumor , Substâncias de Crescimento/fisiologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Família Multigênica , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neovascularização Patológica/metabolismo , Especificidade de Órgãos , Neoplasias da Próstata/irrigação sanguínea , Neoplasias da Próstata/patologia , Receptores de Fatores de Crescimento/fisiologia , Fatores de Transcrição/genética , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologiaRESUMO
Cripto, a growth factor with an EGF-like domain, and the first member of the EGF-CFC family of genes to be sequenced and characterized, contributes to deregulated growth of cancer cells. A role for Cripto in tumor development has been described in the human and the mouse. Members of the EGF-CFC family are found only in vertebrates: CFC proteins in zebrafish, Xenopus, chick, mouse and human have been characterized and indicate some common general functions in development. Cripto expression was first found in human and mouse embryonal carcinoma cells and male teratocarcinomas, and was demonstrated to be over-expressed in breast, cervical, ovarian, gastric, lung, colon, and pancreatic carcinomas in contrast to normal tissues where Cripto expression was invariably low or absent. Cripto may play a role in mammary tumorigenesis, since in vitro, Cripto induces mammary cell proliferation, reduces apoptosis, increases cell migration, and inhibits milk protein expression. This prediction is strengthened by observations of Cripto expression in 80% of human and mouse mammary tumors. At least three important roles for Cripto in development have created considerable interest, and each activity may be distinct in its mechanism of receptor signaling. One role is in the patterning of the anterior-posterior axis of the early embryo, a second is a crucial role in the development of the heart, and a third is in potentiating branching morphogenesis and modulating differentiation in the developing mammary gland. Whether these properties are functions of different forms of Cripto, different Cripto receptors or the distinct domains within this 15-38 kDa glycoprotein are examined here, but much remains to be revealed about this evolutionarily conserved gene product. Since all Cripto receptors have not yet been determined with certainty, future possible uses as therapeutic targets remain to be developed. Cripto is released or shed from expressing cells and may serve as an accessible marker gene in the early to mid-progressive stages of breast and other cancers. Meanwhile some speculations on possible receptor complexes for Cripto signaling in mammary cells are offered here as a spur to further discoveries.
Assuntos
Fator de Crescimento Epidérmico , Proteínas de Homeodomínio , Glicoproteínas de Membrana , Proteínas de Neoplasias/fisiologia , Fatores de Transcrição , Proteínas de Xenopus , Proteínas de Peixe-Zebra , Animais , Comunicação Autócrina/fisiologia , Fatores de Crescimento de Fibroblastos/fisiologia , Proteínas Ligadas por GPI , Expressão Gênica , Substâncias de Crescimento/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas de Membrana , Família Multigênica , Neoplasias/fisiopatologia , Receptores de Superfície Celular/fisiologia , Transdução de Sinais/fisiologiaRESUMO
The proliferation of most primary cells in culture is limited by replicative senescence and crisis, p53-dependent events. However, the regulation of p53 itself has not been defined. We find that deletion of the early growth response 1 (EGR1) transcription factor leads to a striking phenotype, including complete bypass of senescence and apparent immortal growth consistent with loss of a suppressor gene. EGR1-null mouse embryo fibroblasts (MEFs) exhibit decreased expression of p53, p21(Cip1/Waf1), and other p53 "marker" proteins. Precrisis WT but not EGR1-null cells exhibit irradiation-induced arrest. WT MEFs that emerge from crisis exhibit a mutated p53 (sequence confirmed), colony formation, and tumorigenicity. In contrast, high-passage EGR1-null MEFs retain the WT p53 sequence but with much reduced expression, remain untransformed, and grow continuously. An EGR1-expressing retrovirus restores p53 expression and sencescence to EGR1-null but not p53-null MEFs or postcrisis WT cells. Taken together, the results establish EGR1 as a major regulator of cell senescence and previously undescribed upstream "gatekeeper" of the p53 tumor suppressor pathway.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas Imediatamente Precoces , Fatores de Transcrição/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Divisão Celular , Células Cultivadas , Senescência Celular , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Dano ao DNA , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce , Feminino , Expressão Gênica , Camundongos , Camundongos Knockout , Camundongos Nus , Retroviridae/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Ensaio Tumoral de Célula-TroncoRESUMO
Related coactivators p300 and CBP affect the transcriptional activities of many transcription factors (TF), producing multiple downstream effects. Here we show that immediate early response TF, Egr1, acts upstream of p300/CBP to induce or to repress transcription, depending on the stimulus. Cells induced with serum to increase endogenous Egr1 increase the transcription of p300/CBP only when Egr1 binding sites in the promoter are not mutated, causing the expression of downstream targets of Egr1 which leads to survival and growth. Induction of p300/CBP by Egr1 results in acetylation and stabilization of Egr1 and transactivation of survival genes but repression of Egr1 and p300/CBP in negative feedback loops. In contrast, induction of Egr1 by UV-C irradiation leads to repression of p300/CBP transcription: Egr1 is preferentially phosphorylated, leading to regulation of target genes that cause cell death. This complex balance of opposing effects appears to finely modulate important cellular life and death responses.
Assuntos
Carcinoma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Proteínas Nucleares/metabolismo , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Acetilação/efeitos da radiação , Animais , Sítios de Ligação/genética , Proteína de Ligação a CREB , Carcinoma/genética , Morte Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Proteínas de Ligação a DNA/genética , Regulação para Baixo/genética , Regulação para Baixo/efeitos da radiação , Proteína p300 Associada a E1A , Proteína 1 de Resposta de Crescimento Precoce , Retroalimentação Fisiológica/genética , Retroalimentação Fisiológica/efeitos da radiação , Feto , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica/genética , Genes Reguladores/genética , Genes Reguladores/efeitos da radiação , Humanos , Proteínas Imediatamente Precoces/genética , Masculino , Camundongos , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/efeitos da radiação , Próstata/citologia , Próstata/crescimento & desenvolvimento , Neoplasias da Próstata/genética , Transativadores/genética , Fatores de Transcrição/genética , Ativação Transcricional/genética , Ativação Transcricional/efeitos da radiação , Regulação para Cima/genéticaRESUMO
The PTEN tumor suppressor phosphatase directly counteracts the multiple functions of phosphatidylinositol 3-kinase by removing phosphate from the D3 position of inositol phospholipids. Like many lymphomas and leukemias, the Jurkat T cell line lacks PTEN protein due to frame-shift mutations in both PTEN alleles and therefore survives in long-term cell culture. We report that PTEN reintroduced into Jurkat was highly phosphorylated on serines 380 and 385 in its C terminus, particularly the former site. Phosphate was also detected at Ser(380) in PTEN in untransformed human T cells. Treatments that reduced the levels of D3-phospholipids in the cells resulted in reduced phosphorylation and accelerated degradation of PTEN. In contrast, expression of inactive PTEN-C124G or coexpression of a constitutively active protein kinase B led to increased phosphorylation and slower degradation of PTEN. These results suggest that PTEN normally is subjected to a feedback mechanism of regulation aimed at maintaining homeostatic levels of D3-phosphoinositides, which are crucial for T cell survival and activation.
Assuntos
Regulação para Baixo/imunologia , Fosfatidilinositóis/fisiologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Serina/metabolismo , Linfócitos T/enzimologia , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismo , Alanina/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Células Cultivadas , Regulação para Baixo/genética , Retroalimentação , Meia-Vida , Humanos , Células Jurkat/enzimologia , Células Jurkat/metabolismo , Dados de Sequência Molecular , PTEN Fosfo-Hidrolase , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatos de Fosfatidilinositol/fisiologia , Fosfatidilinositóis/metabolismo , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Serina/genética , Linfócitos T/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Egr1, an immediate early transcription factor, responds to diverse stimuli and affects gene transcription to accomplish its biological effects. One important effect of Egr1 expression is to decrease the growth and tumorigenic potential of several tumor cell types. To identify important Egr1 target genes, we have adapted a methodology involving formaldehyde-induced protein-DNA cross-linking, chromatin immunoprecipitation, and multiplex PCR. Using this approach, we report the cloning of a new Egr1 target gene that is able to account, at least in part, for the growth inhibitory activity of Egr1. We have named this new protein TOE1 for target of Egr1.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Imediatamente Precoces , Proteínas/química , Proteínas/genética , Fatores de Transcrição/metabolismo , Northern Blotting , Cromatina/metabolismo , Clonagem Molecular , Reagentes de Ligações Cruzadas/farmacologia , DNA/metabolismo , DNA Complementar/metabolismo , Proteína 1 de Resposta de Crescimento Precoce , Citometria de Fluxo , Formaldeído/farmacologia , Biblioteca Gênica , Humanos , Luciferases/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Proteínas Nucleares , Reação em Cadeia da Polimerase , Testes de Precipitina , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Ativação Transcricional , Transfecção , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1RESUMO
The relation between the role of the organizer at the gastrula stage and the activity of earlier signals in the specification, maintenance, and regionalization of the developing brain anlage is still controversial. Mouse embryos homozygous for null mutation in the cripto gene die at about 9.0 days postcoitum (d.p.c.) and fail to gastrulate and to form the node (the primary organizer). Here, we study the presence and the distribution of anterior neural plate molecular domains in cripto null mutants. We demonstrate that, in cripto(-/-) embryos, the main prosencephalic and mesencephalic regions are present and that they assume the correct topological organization. The identity of the anterior neural domains is maintained in mutant embryos at 8.5 d.p.c., as well as in mutant explants dissected at 8.5 d.p.c. and cultured in vitro for 24 h. Our data imply the existence of a stable neural regionalization of anterior character inside the cripto(-/-) embryos, despite the failure in both the gastrulation process and node formation. These results suggest that, in mouse embryos, the specification of the anterior neural identities can be maintained without an absolute requirement for the embryonic mesoderm and the node.