RESUMO
The JunB gene is activated by many stimuli including transforming growth factor beta (TGFbeta) family members and interleukin-6 (IL-6). Here the effect of TGFbeta activated kinase 1 (TAK1), a mitogen activated protein kinase kinase kinase (MAPKKK) implicated in TGFbeta, bone morphogenetic protein (BMP) and interleukin-1 (IL-1) signaling, on JunB promoter activity was investigated. Promoter analysis led to the identification of a CCAAT motif in the JunB gene, essential for activation by TAK1. Transfer of this CCAAT element to a heterologous minimal promoter conferred TAK1-responsiveness. The CCAAT-binding transcription factor, nuclear factor Y (NF-Y), activated the JunB promoter and a dominant negative NF-YA construct inhibited TAK1 activation of JunB. Our results demonstrate that JunB gene activation by TAK1 is mediated by the CCAAT-binding factor NF-Y.
Assuntos
Fator de Ligação a CCAAT/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-jun/genética , Animais , Sequência de Bases , Linhagem Celular , DNA , Primers do DNA , Regulação da Expressão Gênica/genética , Genes Reporter , Humanos , Interleucina-1/fisiologia , Luciferases/genética , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional , Fator de Crescimento Transformador beta/fisiologiaRESUMO
In the present study, signal transducer and activator of transcription 3 (STAT3) Ser(727) phosphorylation and transactivation was investigated in relation to activation of mitogen-activated protein (MAP) kinase family members including extracellular-signal-regulated protein kinase (ERK)-1, c-Jun N-terminal kinase (JNK)-1 and p38 ('reactivating kinase') in response to interleukin (IL)-6 stimulation. Although IL-6 can activate ERK-1 in HepG2 cells, STAT3 transactivation and Ser(727) phosphorylation were not reduced by using the MAP kinase/ERK kinase (MEK) inhibitor PD98059 or by overexpression of dominant-negative Raf. IL-6 did not activate JNK-1 in HepG2 cells and STAT3 was a poor substrate for JNK-1 activated by anisomycin, excluding a role for JNK1 in IL-6-induced STAT3 activation. However, SEK-1/MKK-4 [where SEK-1 stands for stress-activated protein kinase (SAPK)/ERK kinase 1, and MKK-4 stands for MAP kinase kinase 4] was activated in response to IL-6 and overexpression of dominant-negative SEK-1/MKK-4(A-L) reduced both IL-6-induced STAT3 Ser(727) phosphorylation as well as STAT3 transactivation. Subsequently, the SEK-1/MKK-4 upstream components Vav, Rac-1 and MEKK were identified as components of a signal transduction cascade that leads to STAT3 transactivation in response to IL-6 stimulation. Furthermore, inhibition of p38 kinase activity with the inhibitor SB203580 did not block STAT3 Ser(727) phosphorylation but rather increased both basal as well as IL-6-induced STAT3 transactivation, indicating that p38 may act as a negative regulator of IL-6-induced STAT3 transactivation through a presently unknown mechanism. In conclusion, these data indicate that IL-6-induced STAT3 transactivation and Ser(727) phosphorylation is independent of ERK-1 or JNK-1 activity, but involves a gp130 receptor-signalling cascade that includes Vav, Rac-1, MEKK and SEK-1/MKK-4 as signal transduction components.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , Interleucina-6/farmacologia , MAP Quinase Quinase 4 , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Serina , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Substituição de Aminoácidos , Carcinoma Hepatocelular , Proteínas de Ligação a DNA/química , Inibidores Enzimáticos/farmacologia , Genes Reporter , Humanos , Imidazóis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Neoplasias Hepáticas , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Proto-Oncogênicas c-vav , Piridinas/farmacologia , Proteínas Recombinantes/metabolismo , Fator de Transcrição STAT3 , Transativadores/química , Ativação Transcricional , Transfecção , Células Tumorais CultivadasRESUMO
In the present study we examined in more detail the dual role of the c-JUN N-terminal kinase (JNK) and p38 stress-activated protein kinase pathways in mediating apoptosis or cellular activation in hematopoietic cells. Growth factor deprivation of the erythroleukemic cell line TF-1 led to apoptosis which was associated with an enhanced activity of JNK and p38 and immediate dephosphorylation of the extracellular signal-regulated kinases (ERKs). Enhanced activity of p38 and JNK was not only observed during apoptosis but also in TF-1 cells stimulated with IL-1. IL-1 rescued TF-1 cells from apoptosis. In this case, the upregulation of p38 and JNK was associated with an enhanced activity of ERK. By using SB203580, a specific inhibitor of the p38 signaling pathway, it was demonstrated that p38 plays a pivotal role in the apoptotic process. SB203580 repressed the apoptotic process to a large extent. In contrast, PD98059, a specific inhibitor of the ERK pathway, counteracted the suppressive effects of SB203580 and IL-1 on the apoptotic process indicating that the protective effect of SB203580 and IL-1 might be the result of a shift in the balance between the ERK1/2 and p38/JNK route. This was also supported by experiments with TF-1 cells overexpressing the Shc protein that demonstrated a significantly lower percentage of apoptotic cells, which coincided with higher ERK activity. Finally, the IL-1 and SB203580-mediated effects were associated with an enhanced nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) binding activity, which could also be blocked by PD98059. These data demonstrate a dual function of the p38 pathway whereby other factors, such as ERK kinases, AP-1 and NF-kappaB, might determine the final cellular response.
Assuntos
Apoptose/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células-Tronco Hematopoéticas/enzimologia , Proteínas Quinases Ativadas por Mitógeno , Proteínas Quinases Dependentes de Cálcio-Calmodulina/biossíntese , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Ativação Enzimática , Indução Enzimática , Substâncias de Crescimento/deficiência , Humanos , Interleucina-1/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Leucemia Eritroblástica Aguda/enzimologia , Leucemia Eritroblástica Aguda/patologia , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Smad proteins have been identified as mediators of intracellular signal transduction by members of the transforming growth factor-beta (TGF-beta) superfamily, which affect cell proliferation, differentiation, as well as pattern formation during early vertebrate development. Following receptor activation, Smads are assembled into heteromeric complexes consisting of a pathway-restricted Smad and the common Smad4 that are subsequently translocated into the nucleus where they are thought to play an important role in gene transcription. Here we report the identification of Smad Binding Elements (SBEs) composed of the sequence CAGACA in the promoter of the JunB gene, an immediate early gene that is potently induced by TGF-beta, activin, and bone morphogenetic protein (BMP) 2. Two JunB SBEs are arranged as an inverted repeat that is transactivated in response to Smad3 and Smad4 co-overexpression and shows inducible binding of a Smad3- and Smad4-containing complex in nuclear extracts from TGF-beta-treated cells. Bacterial-expressed Smad proteins bind directly to the SBE. Multimerization of the SBE creates a powerful TGF-beta-inducible enhancer that is also responsive to activin and BMPs. The identification of the sequence CAGACA as a direct binding site for Smad proteins will facilitate the identification of regulatory elements in genes that are activated by members of the TGF-beta superfamily.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transativadores/metabolismo , Ativinas , Sequência de Bases , Proteínas Morfogenéticas Ósseas/genética , DNA , Inibinas/genética , Dados de Sequência Molecular , Ligação Proteica , Proteína Smad3 , Ativação Transcricional , Fator de Crescimento Transformador beta/genética , Células Tumorais CultivadasRESUMO
In humans, at least three types of inositol (1,4,5)-trisphosphate receptor (IP3R) are present. The gene encoding type 1 IP3R (IP3R-I) is expressed in all cell types, although expression predominates in Purkinje cells. To study the regulation of the human IP3R-I gene, we isolated and characterized a 2.1-kb 5' flanking region. In transient expression assays using a rat cell line, analysis of various deletion mutants demonstrated that a fragment of only 86 bp 5' of the putative tsp displayed a promoter activity similar to that of the 2.1-kb fragment. Also, we compared the sequence of the human IP3R-I promoter with the sequence of the mouse IP3R-I promoter. Considerable sequence homology is present in four distinct domains, which include several conserved putative binding sites for transcription factors. Further, we demonstrate a decrease in the activity of the isolated human IP3R-I promoter and of the endogenous IP3R-I promoter after 48 h of treatment with retinoic acid. Analysis of deletion constructs of the human promoter indicates that the decreased promoter activity in response to retinoic acid is likely to be mediated by a conserved AP-2 binding site.
Assuntos
Canais de Cálcio/genética , Inositol 1,4,5-Trifosfato , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/genética , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA , Regulação da Expressão Gênica , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ratos , Homologia de Sequência do Ácido NucleicoRESUMO
The chicken ovalbumin upstream promoter transcription factor, COUP-TF I, and the protein ARP-1 (COUP-TF II) are two highly homologous orphan receptors of the nuclear hormone receptor family. In this study we investigated their expression patterns in the adult nervous system of the mouse. In situ hybridizations were performed on brain sections with 35S-labeled cRNA probes derived from the 3'-non-coding regions of the mARP-1 and mCOUP-TF I mRNAs. Both COUP-TF I and ARP-1 were shown to be expressed in the adult brain and they displayed restricted and distinct expression patterns. COUP-TF I transcripts were predominantly found in the rostral and caudal parts of the adult mouse brain, whereas ARP-1 transcripts prevaled in the middle part of the brain. High expression of COUP-TF I was detected in the olfactory nucleus, in neocortex layers I/II and V/VL, in the dentate gyrus and in areas CA1/CA3/CA4 of the hippocampus, and in the granular layer of the cerebellum. Only low amounts of COUP-TF I mRNA were detected in the ventral, the laterodorsal and in the interanteromedial thalamic nuclei. Small amounts of COUP-TF I transcripts were also found in the epithelial layer of the ventricle and in arachnoid membranes. High expression of ARP-I was detected in the reticular, the ventral lateral and the gelatinosus thalamic nuclei. Other hot spots of ARP-1 mRNA expression were the amygdaloid nucleus and the arachnoid membranes. Lower amounts of ARP-1 transcripts were found in the anterior and lateral hypothalamic areas, in the suprachiasmatic nucleus, and in the choroid plexus.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Encéfalo/metabolismo , Proteínas de Ligação a DNA/biossíntese , Receptores de Glucocorticoides/biossíntese , Receptores de Esteroides , Fatores de Transcrição/biossíntese , Animais , Autorradiografia , Fator I de Transcrição COUP , Fator II de Transcrição COUP , Fatores de Transcrição COUP , Proteínas de Ligação a DNA/genética , Hipocampo/metabolismo , Hibridização In Situ , Camundongos , Camundongos Endogâmicos , RNA Mensageiro/metabolismo , Receptores de Glucocorticoides/genética , Telencéfalo/metabolismo , Fatores de Transcrição/genéticaRESUMO
We have isolated the murine homologs of the members of the COUP-family of steroid hormone receptors, COUP-TF1, ARP-1 and EAR2. The proteins encoded by the murine genes appeared to be highly conserved when compared to their human counterparts. The expression of COUP-TF1 and ARP-1 was induced during differentiation of P19 embryonal carcinoma (EC) cells into derivatives of all three germ layers. Retinoic acid (RA) treatment rapidly induced expression of both genes, while other methods of differentiation were less effective. Undifferentiated P19 cells were found to express EAR2 mRNA and the expression level was only slightly elevated by RA-treatment. In addition, we analyzed the expression in P19 cells of three members of the retinoid X receptor (RXR) family, which have been shown to heterodimerize with members of the COUP-family. During RA mediated differentiation of P19 cells, RXR alpha expression was induced while RXR beta expression was not modulated and RXR gamma expression was down regulated. Gel shift analysis revealed that in P19 cells the members of the COUP-family comprise the major portion of proteins binding to a RA-responsive direct repeat of the consensus steroid hormone receptor binding half site (AGGTCA) spaced by one nucleotide (DR + 1). The members of the COUP-family appeared to down regulate RA-induced activation of RA-response element-containing reporter constructs in a promoter context-dependent manner. The expression patterns of COUP-TF1, ARP-1 and EAR2 during development were investigated by in situ hybridization. In agreement with the results obtained in vitro, the three genes appeared to be expressed in tissues derived from all three germ layers. However, COUP-TF1 and ARP-1 were found to be expressed predominantly in the developing central nervous system in mutually exclusive domains. Furthermore, strong ARP-1 expression was detected in lung and kidney. Our data strongly suggest an important role for the members of the COUP-family in the hormonal control of gene expression regulating embryogenesis.
Assuntos
Proteínas de Ligação a DNA/genética , Receptores de Esteroides/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Fator I de Transcrição COUP , Fator II de Transcrição COUP , Fatores de Transcrição COUP , Carcinoma Embrionário/química , Carcinoma Embrionário/genética , Carcinoma Embrionário/patologia , Clonagem Molecular , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/análise , Regulação Neoplásica da Expressão Gênica , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores do Ácido Retinoico/análise , Receptores do Ácido Retinoico/genética , Receptores de Esteroides/análise , Receptores X de Retinoides , Fatores de Transcrição/análise , Tretinoína/farmacologia , Células Tumorais CultivadasRESUMO
The vitamin A derivative retinoic acid (RA) is involved in vertebrate anteroposterior axis formation and cellular differentiation and has been shown to modulate the expression of a number of genes implicated in the control of early embryonal development. Under defined culture conditions, all-trans-RA induces differentiation of P19 embryonal carcinoma cells into neural derivatives. In this report, we describe the isolation and partial characterization of 14 RA-regulated genes from P19 cells committed to differentiate along the neural pathway. In addition to the previously recognized genes encoding vimentin, heat stable antigen, neural cadherin, Hox-B2, F52/MacMARCKS, thymosin beta 4b, and the murine homolog of COUP-TF1, we identified 7 novel genes, 5 of which are predominantly expressed in developing neural tissues as shown by in situ hybridization. The results confirm the usefulness of the P19 system in the isolation and study of the regulation of developmentally expressed genes.
Assuntos
Desenvolvimento Embrionário e Fetal , Regulação da Expressão Gênica/efeitos dos fármacos , Tretinoína/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Diferenciação Celular , Clonagem Molecular , Citoesqueleto/fisiologia , DNA Complementar/química , DNA Complementar/isolamento & purificação , Células-Tronco de Carcinoma Embrionário , Dados de Sequência Molecular , Células-Tronco Neoplásicas/metabolismo , RNA Mensageiro/análise , Receptores Citoplasmáticos e Nucleares/biossíntese , Receptores do Ácido Retinoico/biossíntese , Receptores X de Retinoides , Fatores de Transcrição/genéticaRESUMO
Protein phosphorylation on tyrosine residues is one of the major mechanisms of cell signal transduction and is regulated by protein tyrosine kinases and protein tyrosine phosphatases. Here we report the molecular cloning of an additional member of the protein tyrosine phosphatase-family from differentiated murine P19 embryonal carcinoma cells. This non-receptor protein tyrosine phosphatase, P19-PTP, does not contain regulatory sequences, homologous to the ones found in other non-receptor PTPases. P19-PTP is differentially expressed during in vitro differentiation of P19 EC cells, in that P19-PTP mRNA could only be detected in embryoid bodies, derived from P19 cells.
Assuntos
Diferenciação Celular , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas Tirosina Fosfatases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Biblioteca Gênica , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais , TeratomaRESUMO
The very lysine-rich replacement histone variant H1(0) is found to be present in different murine (C1003, PC13, P19) and human (Tera-2) embryonal carcinoma cell lines. The proportion of H1(0) increases upon induction of differentiation of the different cell lines by various treatments. In undifferentiated PC13 EC cells H1(0) mRNA is present at a low level. During retinoic acid induced differentiation of mitotically synchronized PC13 EC cells, accumulation of H1(0) mRNA starts in the first cell cycle. The H1(0) protein level starts to increase in the second synchronous cycle preceding changes in the cycle parameters that become apparent in the third cycle. The results provide further support for an important role of H1(0) in the control of cellular differentiation in early mammalian development.
Assuntos
Divisão Celular/efeitos dos fármacos , Histonas/genética , Células-Tronco Neoplásicas/metabolismo , RNA Mensageiro/genética , Tretinoína/farmacologia , Animais , Anticorpos Monoclonais , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/genética , Células-Tronco de Carcinoma Embrionário , Histonas/biossíntese , Humanos , Immunoblotting , Camundongos , Células-Tronco Neoplásicas/citologia , Hibridização de Ácido Nucleico , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
Differentiation of P19 EC cells along different pathways into derivatives resembling cells of the three embryonic germ layers is accompanied by characteristic differences in modulation of expression of each of the three retinoic acid receptor genes, RAR alpha, -beta and -gamma. Differentiation induced by addition of RA to P19 EC cells cultured in monolayer is accompanied by a rapid increase in expression of both RAR alpha and -beta. Induction of RAR beta occurs in a characteristic biphasic manner, suggesting that multiple factors and/or different mechanisms are involved in controlling its expression. RAR beta mRNA is induced to a far higher level during early aggregation in the presence of RA than during early differentiation in monolayer, suggesting that the direction of differentiation depends on the number and/or ratio of alpha and beta type of RA receptors. Aggregation of P19 EC cells in the presence of RA, but not DMSO, is accompanied by repression of RAR gamma, suggesting that the expression of RAR beta and RAR gamma during neuroectodermal differentiation is mutually exclusive. The effects of RA on RAR expression are significantly greater in G1 than in S-phase of the cell cycle. These results extend previous observations that commitment to differentiation is cell cycle dependent and indicates that critical target gene regulation in response to RA has to take place in G1 for differentiation to occur.
Assuntos
Proteínas de Transporte/genética , Diferenciação Celular/genética , Agregação Celular , Ciclo Celular , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células-Tronco de Carcinoma Embrionário , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Células-Tronco Neoplásicas , RNA Mensageiro/análise , Receptores do Ácido Retinoico , Tretinoína/farmacologiaRESUMO
P19 embryonal carcinoma (EC) cells differentiate when treated with retinoic acid (RA). The P19 EC-derived mutant cell line RAC65 is resistant to the differentiation-inducing activity of RA. We show that these cells express a truncated retinoic acid receptor alpha(mRAR alpha-RAC65), probably due to the integration of a transposon-like element in the RAR alpha gene. This receptor lacks 71 C-terminal amino acids and terminates in the ligand-binding domain. In CAT assays in RAC65 cells, mRAR alpha-RAC65 fails to trans-activate the RAR beta promoter, which contains a RA-response element. In wild-type P19 EC cells mRAR alpha-RAC65 functions as a dominant-negative repressor of RA-induced RAR beta activation. Gel retardation assays demonstrate that mRAR alpha-RAC65 is still able to bind to the RA-response element of the RAR beta promoter, indicating that competition with functional RARs for the same binding site leads to the observed dominant-negative effect. In addition, in two RAC65 clones in which wild-type hRAR alpha was stably transfected RA-sensitivity was restored and in one RAR beta expression could be induced by RA. Taken together, these data show that the primary cause of RA-resistance of RAC65 cells is the expression of a defective RAR alpha, which prevents the trans-activation of RA-responsive genes and results in a loss of the ability to differentiate.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Neoplasias/fisiologia , Teratoma/genética , Tretinoína/farmacologia , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Clonagem Molecular , DNA/análise , Resistência a Medicamentos , Regulação da Expressão Gênica , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Receptores do Ácido Retinoico , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , TransfecçãoRESUMO
Balb/c mice were immunized with affinity purified Ro(SS-A) from human origin in order to allow the preparation of monoclonal anti-Ro(SS-A) antibodies. After fusion of mouse myeloma cells (line Sp2/0 A914) with spleen cells from one of these mice, anti-Ro(SS-A) monoclonals were not obtained, but, instead, two IgM producing hybridomas reactive with histone H1 and one with histone H2B. The specificity of the anti-H1 monoclonals was investigated by means of immunoblotting of very lysine-rich histone variants from mouse which were separated by two-dimensional gelelectrophoresis. One of them (CLB-ANA 105) has H1(0) specificity with respect to the histone variants of mouse and man, but recognizes H5 as well as H1 from Xenopus laevis. Another monoclonal (CLB-ANA 108) reacts with the variant H1c from mouse, exclusively. From the way these monoclonals were produced, we postulate that they were not the result of immunization, but comprise specificities of naturally occurring autoantibodies.