RESUMO
Iodide is captured by thyrocytes through the Na(+)/I(-) symporter (NIS) before being released into the follicular lumen, where it is oxidized and incorporated into thyroglobulin for the production of thyroid hormones. Several reports point to pendrin as a candidate protein for iodide export from thyroid cells into the follicular lumen. Here, we show that a recently discovered Ca(2+)-activated anion channel, TMEM16A or anoctamin-1 (ANO1), also exports iodide from rat thyroid cell lines and from HEK 293T cells expressing human NIS and ANO1. The Ano1 mRNA is expressed in PCCl3 and FRTL-5 rat thyroid cell lines, and this expression is stimulated by thyrotropin (TSH) in rat in vivo, leading to the accumulation of the ANO1 protein at the apical membrane of thyroid follicles. Moreover, ANO1 properties, i.e., activation by intracellular calcium (i.e., by ionomycin or by ATP), low but positive affinity for pertechnetate, and nonrequirement for chloride, better fit with the iodide release characteristics of PCCl3 and FRTL-5 rat thyroid cell lines than the dissimilar properties of pendrin. Most importantly, iodide release by PCCl3 and FRTL-5 cells is efficiently blocked by T16Ainh-A01, an ANO1-specific inhibitor, and upon ANO1 knockdown by RNA interference. Finally, we show that the T16Ainh-A01 inhibitor efficiently blocks ATP-induced iodide efflux from in vitro-cultured human thyrocytes. In conclusion, our data strongly suggest that ANO1 is responsible for most of the iodide efflux across the apical membrane of thyroid cells.
Assuntos
Polaridade Celular , Canais de Cloreto/metabolismo , Iodetos/metabolismo , Proteínas de Neoplasias/metabolismo , Glândula Tireoide/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Anoctamina-1 , Transporte Biológico , Cálcio/metabolismo , Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/genética , Células HEK293 , Humanos , Moduladores de Transporte de Membrana/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Interferência de RNA , Ratos , Glândula Tireoide/citologia , Glândula Tireoide/efeitos dos fármacos , Tireotropina/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
Human T cell leukemia virus type-1 (HTLV-1) is the causative agent of a fatal adult T-cell leukemia. Through deregulation of multiple cellular signaling pathways the viral Tax protein has a pivotal role in T-cell transformation. In response to stressful stimuli, cells mount a cellular stress response to limit the damage that environmental forces inflict on DNA or proteins. During stress response, cells postpone the translation of most cellular mRNAs, which are gathered into cytoplasmic mRNA-silencing foci called stress granules (SGs) and allocate their available resources towards the production of dedicated stress-management proteins. Here we demonstrate that Tax controls the formation of SGs and interferes with the cellular stress response pathway. In agreement with previous reports, we observed that Tax relocates from the nucleus to the cytoplasm in response to environmental stress. We found that the presence of Tax in the cytoplasm of stressed cells prevents the formation of SGs and counteracts the shutoff of specific host proteins. Unexpectedly, nuclear localization of Tax promotes spontaneous aggregation of SGs, even in the absence of stress. Mutant analysis revealed that the SG inhibitory capacity of Tax is independent of its transcriptional abilities but relies on its interaction with histone deacetylase 6, a critical component of SGs. Importantly, the stress-protective effect of Tax was also observed in the context of HTLV-1 infected cells, which were shown to be less prone to form SGs and undergo apoptosis under arsenite exposure. These observations identify Tax as the first virally encoded inhibitory component of SGs and unravel a new strategy developed by HTLV-1 to deregulate normal cell processes. We postulate that inhibition of the stress response pathway by Tax would favor cell survival under stressful conditions and may have an important role in HTLV-1-induced cellular transformation.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Produtos do Gene tax/fisiologia , Histona Desacetilases/fisiologia , RNA Mensageiro/metabolismo , Estresse Fisiológico , Células HeLa , Desacetilase 6 de Histona , HumanosRESUMO
Tumour necrosis factor (TNF)-alpha mRNA contains an AU-rich element (ARE) in its 3' untranslated region (3'UTR), which determines its half-life and translational efficiency. In unstimulated macrophages, TNF-alpha mRNA is repressed translationally, and becomes efficiently translated upon cell activation. Gel retardation experiments and screening of a macrophage cDNA expression library with the TNF-alpha ARE allowed the identification of TIA-1-related protein (TIAR), T-cell intracellular antigen-1 (TIA-1) and tristetraprolin (TTP) as TNF-alpha ARE-binding proteins. Whereas TIAR and TIA-1 bind the TNF-alpha ARE independently of the activation state of macrophages, the TTP-ARE complex is detectable upon stimulation with lipopolysaccharide (LPS). Moreover, treatment of LPS-induced macrophage extracts with phosphatase significantly abrogates TTP binding to the TNF-alpha ARE, indicating that TTP phosphorylation is required for ARE binding. Carballo, Lai and Blackshear [(1998) Science 281, 1001-1005] showed that TTP was a TNF-alpha mRNA destabilizer. In contrast, TIA-1, and most probably TIAR, acts as a TNF-alpha mRNA translational silencer. A two-hybrid screening with TIAR and TIA-1 revealed the capacity of these proteins to interact with other RNA-binding proteins. Interestingly, TIAR and TIA-1 are not engaged in the same interaction, indicating for the first time that TIAR and TIA-1 can be functionally distinct. These findings also suggest that ARE-binding proteins interact with RNA as multimeric complexes, which might define their function and their sequence specificity.
Assuntos
Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Ativação Transcricional , Regiões 3' não Traduzidas , Animais , Proteínas Imediatamente Precoces/metabolismo , Lipopolissacarídeos/metabolismo , Camundongos , Modelos Biológicos , Ligação Proteica , Biossíntese de Proteínas , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Tristetraprolina , Fator de Necrose Tumoral alfa/metabolismoAssuntos
Células Eucarióticas/metabolismo , Biossíntese de Proteínas/fisiologia , Animais , Artefatos , Sítios de Ligação/fisiologia , Genes , Vetores Genéticos , Humanos , Fatores de Iniciação de Peptídeos/metabolismo , Processamento Pós-Transcricional do RNA/fisiologia , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , RNA Viral/metabolismo , Reprodutibilidade dos Testes , Projetos de Pesquisa , Ribossomos/metabolismoRESUMO
In vitro studies have indicated that the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression is regulated at the posttranscriptional level by the AU-rich element (ARE) sequence present in its 3' untranslated region (UTR). This study investigated the importance of the ARE in the control of GM-CSF gene expression in vivo. For this purpose, transgenic mice bearing GM-CSF gene constructs containing or lacking the ARE (GM-CSF AU(+) or GM-CSF AU(-), respectively) were generated. Both transgenes were under the transcriptional control of the immediate early promoter of the cytomegalovirus (CMV) to ensure their early, widespread, and constitutive expression. The regulation imposed by the ARE was revealed by comparing transgene expression at day 14 of embryonic development (E14); only the ARE-deleted but not the ARE-containing construct was expressed. Although GM-CSF AU(+) embryos were phenotypically normal, overexpression of GM-CSF in E14 GM-CSF AU(-) embryos led to severe hematopoietic alterations such as abnormal proliferation of granulocytes and macrophages accompanied by an increased number of peroxidase-expressing cells, their putative progenitor cells. These abnormalities compromise development because no viable GM-CSF AU(-) transgenic pups could be obtained. Surprisingly, by E18, significant accumulation of transgene messenger RNA was also observed in GM-CSF AU(+) embryos leading to similar phenotypic abnormalities. Altogether, these observations reveal that GM-CSF ARE is a developmentally controlled regulatory element and highlight the consequences of GM-CSF overexpression on myeloid cell proliferation and differentiation.
Assuntos
Regiões 3' não Traduzidas/genética , Desenvolvimento Embrionário e Fetal/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Sistema Hematopoético/embriologia , Animais , Citomegalovirus/genética , Doenças Fetais/genética , Genes Letais , Genes Sintéticos , Idade Gestacional , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Células HeLa , Humanos , Células L , Leucocitose/embriologia , Leucocitose/genética , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Deleção de Sequência , Transcrição Gênica , TransgenesRESUMO
Here we have investigated the regulation of TNF-alpha expression in macrophages during HSV-2 infection. Despite a low basal level of TNF-alpha mRNA present in resting macrophages, no TNF-alpha protein is detectable. HSV-2 infection marginally increases the level of TNF-alpha mRNA and protein in resting macrophages, whereas a strong increase is observed in IFN-gamma-activated cells infected with the virus. By reporter gene assay it was found that HSV infection augments TNF-alpha promoter activity. Moreover, treatment of the cells with actinomycin D, which totally blocked mRNA synthesis, only partially prevented accumulation of TNF-alpha protein, indicating that the infection lifts a block on translation of TNF-alpha mRNA. EMSA analysis showed that specific binding to the kappaB#3 site of the murine TNF-alpha promoter was induced within 1 h after infection and persisted beyond 5 h where TNF-alpha expression is down-modulated. Binding to the cAMP responsive element site was also induced but more transiently with kinetics closely following activation of the TNF-alpha promoter. Inhibitors against either NF-kappaB activation or the activating transcription factor 2 kinase p38 abrogated TNF-alpha expression, showing a requirement for both signals for activation of the promoter. This observation was corroborated by reporter gene assays. As to the translational regulation of TNF-alpha, the AU-rich sequence in the 3' untranslated region of the mRNA was found to be responsible for this control because deletion of this region renders mRNA constitutively translationable. These results show that TNF-alpha production is induced by HSV-2 in macrophages through both transcriptional and translational regulation.
Assuntos
Regiões 3' não Traduzidas/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Herpesvirus Humano 2/imunologia , Proteínas I-kappa B , Macrófagos Peritoneais/imunologia , NF-kappa B/fisiologia , Proteínas Proto-Oncogênicas c-jun/fisiologia , Fatores de Transcrição/fisiologia , Ativação Transcricional/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , Fator 2 Ativador da Transcrição , Animais , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Repetições de Dinucleotídeos/fisiologia , Feminino , Interferon gama/farmacologia , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/virologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Regiões Promotoras Genéticas/imunologia , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The human c-myc proto-oncogene is transcribed from four alternative promoters (P0, P1, P2, and P3) giving rise to mRNAs having 5' leader sequences of various length. The c-myc P0 mRNA contains three open reading frames (ORFs), the last one encoding c-Myc1 and c-Myc2 proteins generated by alternative translation initiated at CUG and AUG codons. The middle ORF (MYCHEX1) and the 5' ORF (ORF1) code for proteins 188 and 114 amino acids in length, respectively. We and others previously identified an internal ribosome entry site (IRES) in P0 and P2 c-myc mRNAs, promoting the cap-independent translation of c-Myc1 and c-Myc2. Here, we report the presence of a second IRES (named IRES1) promoting the cap-independent translation of MYCHEX1 in c-myc P0 mRNA. Using deletion analysis, we mapped an 80-nt region essential for IRES1 activity. c-myc P0 mRNA is thus the first eukaryotic polycistronic mRNA described for which translation initiation of two different open reading frames (MYCHEX1 and c-Myc1/c-Myc2) involves internal ribosome entry.
Assuntos
Genes myc , Biossíntese de Proteínas , RNA Mensageiro/genética , Ribossomos/genética , Regiões 5' não Traduzidas , Sequência de Bases , Códon , Primers do DNA , Células HeLa , Humanos , Fases de Leitura Aberta , Proto-Oncogene MasRESUMO
TNF-alpha gene expression is regulated at transcriptional and post-transcriptional levels in mouse macrophages. The post-transcriptional regulation is mediated by the AU-rich element (ARE) located in the TNF-alpha mRNA 3' untranslated region (UTR), which controls its translation and stability. In resting macrophages, the ARE represses TNF-alpha mRNA translation. Activation of macrophages with various agents [for example lipopolysaccharide (LPS), viruses] results in translational derepression, leading to the production of high levels of TNF-alpha. TNF-alpha ARE has also been shown to confer mRNA instability as its deletion from the mouse genome leads to an increase in the TNF-alpha mRNA half-life [Kontoyiannis, D., Pasparakis, M., Pizzaro, T., Cominelli, F. & Kollias, G. (1999) Immunity 10, 387-398]. In this study, we measured the half-life as well as the poly(A) tail length of TNF-alpha mRNA in the course of macrophage activation by LPS. We report that TNF-alpha mRNA is short lived even in conditions of maximal TNF-alpha synthesis. Moreover, TNF-alpha mRNA is hypoadenylated in a constitutive manner. These results reveal that TNF-alpha mRNA rapid turnover does not constitute a regulatory step of TNF-alpha biosynthesis in macrophages and that TNF-alpha mRNA translational activation upon LPS stimulation is not accompanied by a change of poly(A) tail length.
Assuntos
Proteínas de Ligação a DNA , Proteínas Imediatamente Precoces , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/genética , Regiões 3' não Traduzidas/genética , Animais , Linhagem Celular , Citosol/metabolismo , Retroalimentação , Regulação da Expressão Gênica/efeitos dos fármacos , Meia-Vida , Células HeLa/efeitos dos fármacos , Células HeLa/metabolismo , Humanos , Ativação de Macrófagos/genética , Camundongos , Biossíntese de Proteínas , Proteínas/metabolismo , Tristetraprolina , Dedos de ZincoRESUMO
TIA-1 and TIAR are related proteins that bind to an AU-rich element (ARE) in the 3' untranslated region of tumor necrosis factor alpha (TNF-alpha) transcripts. To determine the functional significance of this interaction, we used homologous recombination to produce mutant mice lacking TIA-1. Although lipopolysaccharide (LPS)-stimulated macrophages derived from wild-type and TIA-1(-/-) mice express similar amounts of TNF-alpha transcripts, macrophages lacking TIA-1 produce significantly more TNF-alpha protein than wild-type controls. The half-life of TNF-alpha transcripts is similar in wild-type and TIA-1(-/-) macrophages, indicating that TIA-1 does not regulate transcript stability. Rather, the absence of TIA-1 significantly increases the proportion of TNF-alpha transcripts that associate with polysomes, suggesting that TIA-1 normally functions as a translational silencer. TIA-1 does not appear to regulate the production of interleukin 1 beta, granulocyte-macrophage colony-stimulating factor or interferon gamma, indicating that its effects are, at least partially, transcript specific. Mice lacking TIA-1 are hypersensitive to the toxic effects of LPS, indicating that this translational control pathway may regulate the organismal response to microbial stress.
Assuntos
Proteínas de Membrana/metabolismo , Biossíntese de Proteínas , Proteínas , Proteínas de Ligação a RNA/metabolismo , Fator de Necrose Tumoral alfa/genética , Regiões 3' não Traduzidas , Animais , Citocinas/biossíntese , Regulação da Expressão Gênica , Macrófagos Peritoneais/imunologia , Proteínas de Membrana/genética , Camundongos , Camundongos Mutantes , Proteínas de Ligação a RNA/genética , Choque Séptico/mortalidade , Antígeno-1 Intracelular de Células TRESUMO
In monocyte/macrophages, the translation of tumor necrosis factor alpha (TNF-alpha) mRNA is tightly regulated. In unstimulated cells, translation of TNF-alpha mRNA is blocked. Upon stimulation with lipopolysaccharides, this repression is overcome, and the mRNA becomes efficiently translated. The key element in this regulation is the AU-rich element (ARE). We have previously reported the binding of two cytosolic protein complexes to the TNF-alpha mRNA ARE. One of these complexes (complex 1) forms with extracts of both unstimulated and lipopolysaccharide-stimulated macrophages and requires a large fragment of the ARE containing clustered AUUUA pentamers. The other complex (complex 2) is only detected after cell activation, binds to a minimal UUAUUUAUU nonamer, and is composed of a 55-kDa protein. Here, we report the identification of the RNA-binding protein TIAR as a protein involved in complex 1. The RNA sequence bound by TIAR and the cytoplasmic localization of this protein in macrophages argue for an involvement of TIAR in TNF mRNA posttranscriptional regulation.
Assuntos
Biossíntese de Proteínas , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Macrófagos/metabolismo , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Frações Subcelulares/metabolismo , Fator de Necrose Tumoral alfa/genéticaAssuntos
Sequência Conservada/genética , Ligação Genética/genética , Genoma Humano , Integrina beta1/genética , Mapeamento Físico do Cromossomo , Proteínas de Ligação a RNA/genética , Animais , Cromossomos Humanos Par 10/genética , Evolução Molecular , Humanos , Células Híbridas , Hibridização in Situ Fluorescente , Camundongos , Ratos , Homologia de Sequência do Ácido NucleicoRESUMO
In monocyte/macrophage cells, the translation of tumor necrosis factor-alpha (TNF-alpha) mRNA is tightly controlled. In unstimulated cells, TNF-alpha mRNA is translationally repressed. However, upon stimulation of the cells with various agents (e.g. lipopolysaccharides (LPS) and viruses), this repression is overcome and translation occurs. The key element in this regulation is the AU-rich sequence present in the 3'-untranslated region of TNF-alpha mRNA. Several groups have described the binding of proteins on AU-rich elements (AREs). We have previously reported the binding of two cytosolic protein complexes (1 and 2) to the TNF-alpha mRNA ARE, one of which (complex 2) is observed only following induction of TNF-alpha production by LPS. In this report, we have demonstrated that complex 1 involves a long fragment of the ARE, whereas the formation of the LPS-inducible complex 2 requires a minimal sequence which corresponds to the nonanucleotide UUAUUUAUU. Furthermore, we show that the RNA-binding protein involved in complex 2 has an apparent molecular mass of 55 kDa. Finally, we tested other AREs for their ability to form complex 2. We observed that the ARE derived from granulocyte/macrophage colony-stimulating factor mRNA, which does contain the nonanucleotide, is able to sustain the LPS-induced binding of the 55-kDa protein. However, c-myc mRNA, which does not contain the nonanucleotide, is unable to promote the formation of any LPS-induced complex.
Assuntos
RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator de Necrose Tumoral alfa/genética , Animais , Sequência de Bases , Linhagem Celular , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genéticaRESUMO
The production of tumor necrosis factor-alpha (TNF-alpha) by lipopolysaccharide (LPS)-stimulated macrophages can be markedly inhibited by the two closely related cytokines, interleukin (IL)-4 and IL-13. To investigate the molecular mechanism of this inhibition, we analyzed the effect of the two cytokines on TNF-alpha production and TNF-alpha mRNA accumulation in the mouse macrophage cell lines RAW 264.7 and J774 stimulated by LPS. Whereas LPS-induced TNF-alpha production is strongly suppressed by both cytokines, TNF-alpha mRNA accumulation is not significantly affected, indicating that IL-4 and IL-13 induce a translational repression of TNF-alpha mRNA. Transfection of reporter gene constructs containing different regions of the TNF-alpha gene revealed that the inhibitory action of IL-4 and IL-13 is mediated by the UA-rich sequence present in the TNF-alpha mRNA 3'-untranslated region.
Assuntos
Interleucina-13/farmacologia , Interleucina-4/farmacologia , Macrófagos/metabolismo , Ativação Transcricional/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossíntese , Animais , Linhagem Celular , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Camundongos , RNA Mensageiro/biossíntese , Fator de Necrose Tumoral alfa/genéticaRESUMO
BACKGROUND: Tumor necrosis factor (TNF) production by macrophages plays an important role in the host response to infection. TNF-alpha gene expression in RAW 264.7 macrophages is predominantly regulated at the translational level. A key element in this regulation is an AU-rich (AUR) sequence located in the 3' untranslated region (UTR) of TNF mRNA. In unstimulated macrophages, the translation of TNF mRNA is inhibited via this AUR sequence. Upon stimulation with LPS, this repression is overcome and translation occurs. In this study, we attempted to identify cellular proteins that interact with the AUR sequence and thereby regulate TNF mRNA translation. MATERIALS AND METHODS: RNA probes corresponding to portions of TNF mRNA 3' UTR were synthesized. These labeled RNAs were incubated with cytoplasmic extracts of either unstimulated or lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophages. The RNA/protein complexes formed were analyzed by gel retardation. Ultraviolet (UV) cross-linking experiments were performed to determine the molecular weight of the proteins involved in the complexes. RESULTS: TNF mRNA AUR sequence formed two complexes (1 and 2) of distinct electrophoretic mobilities. While the formation of complex 1 was independent of the activation state of the macrophages from which the extracts were obtained, complex 2 was detected only using cytoplasmic extracts from LPS-stimulated macrophages. Upon UV cross-linking, two proteins, of 50 and 80 kD, respectively, were capable of binding the UAR sequence. The 50-kD protein is likely to be part of the LPS-inducible complex 2, since its binding ability was enhanced upon LPS stimulation. Interestingly, complex 2 formation was also triggered by Sendaï virus infection, another potent activator of TNF mRNA translation in RAW 264.7 macrophages. In contrast, complex 2 was not detected with cytoplasmic extracts obtained from B and T cell lines which are unable to produce TNF in response to LPS. Protein tyrosine phosphorylation is required for LPS-induced TNF mRNA translation. Remarkably, the protein tyrosine phosphorylation inhibitor herbimycin A abolished LPS-induced complex 2 formation. Complex 2 was already detectable after 0.5 hr of LPS treatment and was triggered by a minimal LPS dose of 10 pg/ml. CONCLUSIONS: The tight correlation between TNF production and the formation of an LPS-inducible cytoplasmic complex suggests that this complex plays a role in the translational regulation of TNF mRNA.
Assuntos
Proteínas/metabolismo , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/genética , Animais , Sequência de Bases , Benzoquinonas , Citoplasma/química , Eletroforese em Gel de Poliacrilamida , Cinética , Lactamas Macrocíclicas , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas Tirosina Quinases/antagonistas & inibidores , Quinonas/farmacologia , Rifabutina/análogos & derivados , Raios UltravioletaRESUMO
Macrophage activation by lipopolysaccharide (LPS) results in the translational activation of tumor necrosis factor (TNF) mRNA. The initial phase of macrophage activation is followed by a refractory state called LPS tolerance characterized by an impaired TNF production in response to a secondary LPS challenge. LPS-tolerant macrophages contain high amounts of TNF mRNA, suggesting a translational regulation of TNF biosynthesis. The induction of LPS tolerance was studied in RAW 264.7 macrophages stably transfected with a chloramphenicol acetyl-transferase (CAT) reporter gene construct driven by a constitutive cytomegalovirus promoter and containing the 3' untranslated region of the murine TNF gene. We found that primary stimulation of transfected cells by LPS (1 ng/ml, 12 hr) resulted in a marked suppression (80%) of CAT accumulation in response to a secondary LPS challenge (1 microgram/ml, 6 hr). In contrast, the accumulation of CAT mRNA was not influenced by LPS tolerance. Using the same CAT reporter, we observed that the serine/threonine phosphatases 1 and 2A inhibitor okadaic acid induced TNF mRNA translation and that this activation was not inhibited by LPS-tolerance. In conclusion, these data indicate that deficient production of TNF in LPS-tolerant macrophages in response to a second LPS challenge is characterized by a defective translation of TNF mRNA. However, this hyporesponsiveness to LPS is specific, since translation of TNF mRNA induced by okadaic acid is not inhibited in LPS-tolerant macrophages.
Assuntos
Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , Fator de Necrose Tumoral alfa/genética , Animais , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Citomegalovirus/genética , Tolerância a Medicamentos , Inibidores Enzimáticos/farmacologia , Éteres Cíclicos/farmacologia , Regulação da Expressão Gênica , Genes Reporter , Cinética , Ativação de Macrófagos , Camundongos , Ácido Okadáico , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/biossínteseRESUMO
In recent years, the chain of events that connects introduction of bacterial endotoxin (lipopolysaccharide; LPS) into a mammalian host, and the syndrome of organ damage and vascular collapse that ensues, have come into sharper focus. Several of the molecules that engage LPS, and a rough outline of the signaling cascade that leads to cytokine release from mononuclear cells, have been elucidated. The principal cytokines that mediate the untoward effects of LPS have also been identified. The most important of these is tumor necrosis factor (TNF), which elicits biologic responses from virtually every type of cell to which it binds. Two distinct receptors transduce the TNF signal. Mechanisms of TNF receptor action are becoming increasing clear, and there is reason to hope that, through intervention at many distinct levels, the devastating effects of LPS might be attenuated or averted.
Assuntos
Lipopolissacarídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/fisiologia , Animais , Humanos , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
Tumor necrosis factor (TNF)-a gene expression can be induced primarily in cells of the monocyte-macrophage lineage by a variety of inducers, including lipopolysaccharides (LPS), phorbol esters, ultraviolet (UV) light, and viruses. In this paper, we analyzed the regulatory mechanisms of TNF-alpha production induced by infection with the Sendai" virus in RAW 264.7 macrophages. We show that in these cells TNF-a synthesis results mainly from TNF-alpha mRNA translational activation. Using CAT reporter genes, we identified the UA- rich (UAR) sequences localized in the TNF-alpha mRNA 3' untranslated region (UTR) as the main sequence involved in this regulation. This sequence has been previously shown to be the essential regulatory element involved in LPS- induced translational activation of TNF mRNA. Activation of TNF gene expression by viral infection presents other similarities with those induced by LPS. First, TNF production in response to viral infection is inhibited by the protein-tyrosine kinase inhibitor herbimycin A as it is in response to LPS. More specifically, we show here that TNF mRNA translational activation induced by viral infection or by LPS is inhibited by pretreating the cells with herbimycin A. Second, TNF production in response to viruses is tissue-specific and is abrogated in RAW 264.7x NIH3T3 hybrid cells, which lack the ability to produce TNF in response to LPS, as a consequence of a defect in the LPS signaling pathway. However, viral infection induces TNF production in LPS- unresponsive C3H/HeJ mouse-derived peritoneal macro phages indicating that viruses and LPS signaling pathways differ for at least one intermediate which is the product of the Lps gene. Finally, we show that this regulatory mechanism can be triggered by different classes of viruses.
Assuntos
Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Macrófagos/virologia , Respirovirus/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , Células 3T3 , Animais , Benzoquinonas , Cloranfenicol O-Acetiltransferase/genética , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Genes Reporter , Lactamas Macrocíclicas , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Biossíntese de Proteínas , Proteínas Tirosina Quinases/antagonistas & inibidores , Quinonas/farmacologia , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico , Rifabutina/análogos & derivados , Transdução de Sinais , Fator de Necrose Tumoral alfa/genéticaRESUMO
The early embryonic development of Xenopus is mainly governed by post-transcriptional regulations until the mid-blastula transition. In this report, we present evidence demonstrating that fertilization of Xenopus eggs triggers a complete translational arrest of mRNAs containing UA-rich elements in their 3'-untranslated region. This control is maintained at least until the mid-blastula transition. Neither maturation nor pseudo-fertilization of the egg is sufficient for triggering this control, suggesting that components originating from the male gamete are involved in the mechanism. Moreover, this control is exerted whether the mRNA is polyadenylated or not.
Assuntos
Fertilização , Oócitos/fisiologia , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Animais , Sequência de Bases , Cloranfenicol O-Acetiltransferase/biossíntese , Cloranfenicol O-Acetiltransferase/metabolismo , Feminino , Técnicas In Vitro , Cinética , Masculino , Dados de Sequência Molecular , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Plasmídeos , Progesterona/farmacologia , RNA Mensageiro/química , Proteínas Recombinantes de Fusão/biossíntese , Sequências Repetitivas de Ácido Nucleico , Espermatozoides/fisiologia , Fatores de Tempo , Transcrição Gênica , Xenopus laevisRESUMO
Several messenger RNAs which are transiently expressed contain a conserved uridine-adenosine-rich sequence in their 3' untranslated region. Many of these mRNas encode cytokines, growth factors or oncoproteins. This UA-rich sequence is composed of several interpsersed repeats of the octanucleotide UUAUUUAU and plays a key role in the post-transcriptional regulation of these mRNAs. Known as instability determinants, these UA-rich elements can also strongly affect mRNA translational efficiency. In this report, we review the data which illustrate this translational regulation and give insight the underlying mechanism.
Assuntos
Sequência de Bases/genética , Citocinas/biossíntese , Regulação da Expressão Gênica , Biossíntese de Proteínas , Adenosina/genética , Animais , Sequência de Bases/fisiologia , Citocinas/fisiologia , Humanos , Sequências Reguladoras de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico , Uridina/genéticaRESUMO
Ultraviolet (UV) light induces the biosynthesis of chloramphenicol acetyltransferase (CAT) in the skin of mice bearing the CATTNF reporter transgene. Moreover, nuclear run-on assays indicate that UV light induces transcription of the TNF gene in RAW 264.7 macrophages. These observations suggest that the TNF gene (and/or its mRNA product) responds to signals elicited by UV light. To identify transcriptional UV response elements within the TNF promoter, and to determine whether a posttranscriptional response might also exist, a series of reporter constructs using a CAT coding sequence attached to various portions of the TNF promoter and 3' untranslated region were devised and transfected into several cultured cell lines. All cells tested were found to be UV responsive, and in NIH 3T3 cells, induction was found to depend upon two general regions of the promoter. The more distal region encompassed nucleotides (nt) -1059 through -451 with respect to the cap site, while the more proximal region spanned nt -403 through -261. A negative element, blocking the UV response, was interposed (nt -451 through -403). As with the response to LPS, the response to UV irradiation appears to involve translational activation in macrophages. However, the UV and LPS signaling pathways have little in common with one another, as indicated by three observations. First, no difference in responsiveness was observed on comparison of TNF gene induction in macrophages derived from C3H/HeN as opposed to C3H/HeJ mice. Second, cell fusion studies showed that while the LPS signaling pathway is extinguished by fusion of RAW 264.7 cells with NIH 3T3 cells, the UV signaling pathway remained intact. Finally, induction did not depend upon the NF-kappa B binding sites that are known to be required for LPS response in macrophages, since mutation of these sites did not impair the UV response.