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1.
EMBO Rep ; 24(12): e57849, 2023 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-37877678

RESUMO

Oxygen is essential for viability in mammalian organisms. However, cells are often exposed to changes in oxygen availability, due to either increased demand or reduced oxygen supply, herein called hypoxia. To be able to survive and/or adapt to hypoxia, cells activate a variety of signalling cascades resulting in changes to chromatin, gene expression, metabolism and viability. Cellular signalling is often mediated via post-translational modifications (PTMs), and this is no different in response to hypoxia. Many enzymes require oxygen for their activity and oxygen can directly influence several PTMS. Here, we review the direct impact of changes in oxygen availability on PTMs such as proline, asparagine, histidine and lysine hydroxylation, lysine and arginine methylation and cysteine dioxygenation, with a focus on mammalian systems. In addition, indirect hypoxia-dependent effects on phosphorylation, ubiquitination and sumoylation will also be discussed. Direct and indirect oxygen-regulated changes to PTMs are coordinated to achieve the cell's ultimate response to hypoxia. However, specific oxygen sensitivity and the functional relevance of some of the identified PTMs still require significant research.


Assuntos
Lisina , Oxigênio , Animais , Humanos , Oxigênio/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Cromatina , Hipóxia/metabolismo , Mamíferos/metabolismo
2.
Cell ; 133(1): 78-89, 2008 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-18394991

RESUMO

Characteristics of transformed and tumor cells include increased levels of protein synthesis and elevated expression of RNA polymerase (pol) III products, such as tRNAs and 5S rRNA. However, whether deregulated pol III transcription contributes to transformation has been unclear. Generating cell lines expressing an inducible pol III-specific transcription factor, Brf1, allowed us to raise tRNA and 5S rRNA levels specifically. Brf1 induction caused an increase in cell proliferation and oncogenic transformation, whereas depletion of Brf1 impeded transformation. Among the gene products induced by Brf1 is the tRNA(iMet) that initiates polypeptide synthesis. Overexpression of tRNA(iMet) is sufficient to stimulate cell proliferation and allow immortalized fibroblasts to form foci in culture and tumors in mice. The data indicate that elevated tRNA synthesis can promote cellular transformation.


Assuntos
Proliferação de Células , Transformação Celular Neoplásica , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo , Células 3T3 , Animais , Células CHO , Ciclo Celular , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Camundongos , Biossíntese de Proteínas , Interferência de RNA , RNA Polimerase III/metabolismo , Fator de Transcrição TFIIIB/genética , Fator de Transcrição TFIIIB/metabolismo , Transcrição Gênica
3.
Biochem J ; 479(3): 245-257, 2022 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-35119457

RESUMO

Hypoxia is a common denominator in the pathophysiology of a variety of human disease states. Insight into how cells detect, and respond to low oxygen is crucial to understanding the role of hypoxia in disease. Central to the hypoxic response is rapid changes in the expression of genes essential to carry out a wide range of functions to adapt the cell/tissue to decreased oxygen availability. These changes in gene expression are co-ordinated by specialised transcription factors, changes to chromatin architecture and intricate balances between protein synthesis and destruction that together establish changes to the cellular proteome. In this article, we will discuss the advances of our understanding of the cellular oxygen sensing machinery achieved through the application of 'omics-based experimental approaches.


Assuntos
Hipóxia Celular/genética , Regulação da Expressão Gênica , Oxigênio/metabolismo , Transdução de Sinais/genética , Transcriptoma/genética , Animais , Cromatina/genética , Cromatina/metabolismo , Humanos , Metaboloma/genética , Proteoma/genética , Proteoma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Mol Cell ; 45(4): 541-52, 2012 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-22281053

RESUMO

Polo-like kinase Plk1 controls numerous aspects of cell-cycle progression. We show that it associates with tRNA and 5S rRNA genes and regulates their transcription by RNA polymerase III (pol III) through direct binding and phosphorylation of transcription factor Brf1. During interphase, Plk1 promotes tRNA and 5S rRNA expression by phosphorylating Brf1 directly on serine 450. However, this stimulatory modification is overridden at mitosis, when elevated Plk1 activity causes Brf1 phosphorylation on threonine 270 (T270), which prevents pol III recruitment. Thus, although Plk1 enhances net tRNA and 5S rRNA production, consistent with its proliferation-stimulating function, it also suppresses untimely transcription when cells divide. Genomic instability is apparent in cells with Brf1 T270 mutated to alanine to resist Plk1-directed inactivation, suggesting that chromosome segregation is vulnerable to inappropriate pol III activity.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Regulação da Expressão Gênica , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , RNA Ribossômico 5S/genética , RNA de Transferência/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/genética , Instabilidade Genômica , Células HeLa , Humanos , Mitose , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Polimerase III/metabolismo , RNA Polimerase III/fisiologia , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIIIB/metabolismo , Quinase 1 Polo-Like
5.
Nucleic Acids Res ; 46(8): 3878-3890, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29529249

RESUMO

HIF1α (hypoxia inducible factor 1α) is the central regulator of the cellular response to low oxygen and its activity is deregulated in multiple human pathologies. Consequently, given the importance of HIF signaling in disease, there is considerable interest in developing strategies to modulate HIF1α activity and down-stream signaling events. In the present study we find that under hypoxic conditions, activation of the PERK branch of the unfolded protein response (UPR) can suppress the levels and activity of HIF1α by preventing efficient HIF1α translation. Activation of PERK inhibits de novo HIF1α protein synthesis by preventing the RNA-binding protein, YB-1, from interacting with the HIF1α mRNA 5'UTR. Our data indicate that activation of the UPR can sensitise tumor cells to hypoxic stress, indicating that chemical activation of the UPR could be a strategy to target hypoxic malignant cancer cells.


Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteína 1 de Ligação a Y-Box/metabolismo , eIF-2 Quinase/metabolismo , Regiões 5' não Traduzidas , Regulação para Baixo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células PC-3 , Biossíntese de Proteínas , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Tapsigargina/farmacologia , Hipóxia Tumoral/genética , Resposta a Proteínas não Dobradas , eIF-2 Quinase/antagonistas & inibidores
6.
Nucleic Acids Res ; 45(16): 9336-9347, 2017 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-28666324

RESUMO

The cellular response to hypoxia is characterised by a switch in the transcriptional program, mediated predominantly by the hypoxia inducible factor family of transcription factors (HIF). Regulation of HIF1 is primarily controlled by post-translational modification of the HIF1α subunit, which can alter its stability and/or activity. This study identifies an unanticipated role for the X-linked inhibitor of apoptosis (XIAP) protein as a regulator of Lys63-linked polyubiquitination of HIF1α. Lys63-linked ubiquitination of HIF1α by XIAP is dependent on the activity of E2 ubiquitin conjugating enzyme Ubc13. We find that XIAP and Ubc13 dependent Lys63-linked polyubiquitination promotes HIF1α nuclear retention leading to an increase in the expression of HIF1 responsive genes. Inhibition of the Lys63-linked polyubiquitination pathway leads to reduced levels of nuclear HIF1α, promoter occupancy, HIF-dependent gene expression and cell viability. Our data reveals an additional and significant level of control of the HIF1 by XIAP, with important implications in understanding the role of HIF1 and XIAP in human disease.


Assuntos
Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lisina/metabolismo , Ubiquitinação , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Hipóxia Celular , Linhagem Celular , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Regiões Promotoras Genéticas , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/metabolismo , Regulação para Cima
8.
Biochem J ; 449(1): 275-84, 2013 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23016877

RESUMO

The IKK [inhibitor of NF-κB (nuclear factor κB) kinase] complex has an essential role in the activation of the family of NF-κB transcription factors in response to a variety of stimuli. To identify novel IKK-interacting proteins, we performed an unbiased proteomics screen where we identified TfR1 (transferrin receptor 1). TfR1 is required for transferrin binding and internalization and ultimately for iron homoeostasis. TfR1 depletion does not lead to changes in IKK subunit protein levels; however, it does reduce the formation of the IKK complex, and inhibits TNFα (tumour necrosis factor α)-induced NF-κB-dependent transcription. We find that, in the absence of TfR1, NF-κB does not translocate to the nucleus efficiently, and there is a reduction in the binding to target gene promoters and consequentially less target gene activation. Significantly, depletion of TfR1 results in an increase in apoptosis in response to TNFα treatment, which is rescued by elevating the levels of RelA/NF-κB. Taken together, these results indicate a new function for TfR1 in the control of IKK and NF-κB. Our data indicate that IKK-NF-κB responds to changes in iron within the cell.


Assuntos
Antígenos CD/metabolismo , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Receptores da Transferrina/metabolismo , Transdução de Sinais/fisiologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Quinase I-kappa B/fisiologia , Ferro/química , Ferro/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/fisiologia , Ligação Proteica/fisiologia , Receptores da Transferrina/deficiência
9.
PLoS Genet ; 7(1): e1001285, 2011 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-21298084

RESUMO

Hypoxia Inducible Factor-1 (HIF-1) is essential for mammalian development and is the principal transcription factor activated by low oxygen tensions. HIF-α subunit quantities and their associated activity are regulated in a post-translational manner, through the concerted action of a class of enzymes called Prolyl Hydroxylases (PHDs) and Factor Inhibiting HIF (FIH) respectively. However, alternative modes of HIF-α regulation such as translation or transcription are under-investigated, and their importance has not been firmly established. Here, we demonstrate that NF-κB regulates the HIF pathway in a significant and evolutionary conserved manner. We demonstrate that NF-κB directly regulates HIF-1ß mRNA and protein. In addition, we found that NF-κB-mediated changes in HIF-1ß result in modulation of HIF-2α protein. HIF-1ß overexpression can rescue HIF-2α protein levels following NF-κB depletion. Significantly, NF-κB regulates HIF-1ß (tango) and HIF-α (sima) levels and activity (Hph/fatiga, ImpL3/ldha) in Drosophila, both in normoxia and hypoxia, indicating an evolutionary conserved mode of regulation. These results reveal a novel mechanism of HIF regulation, with impact in the development of novel therapeutic strategies for HIF-related pathologies including ageing, ischemia, and cancer.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Regulação da Expressão Gênica , NF-kappa B/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Fibroblastos/efeitos dos fármacos , Redes Reguladoras de Genes/genética , Células HEK293 , Células HeLa , Humanos , Hipóxia/genética , Camundongos , NF-kappa B/genética , RNA Interferente Pequeno/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia
10.
Biosci Rep ; 44(6)2024 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-38808772

RESUMO

Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in most types of chronic liver disease. At the cellular level, liver fibrosis is associated with the activation of hepatic stellate cells (HSCs) which transdifferentiate into a myofibroblast-like phenotype that is contractile, proliferative and profibrogenic. HSC transdifferentiation induces genome-wide changes in gene expression that enable the cell to adopt its profibrogenic functions. We have previously identified that the deubiquitinase ubiquitin C-terminal hydrolase 1 (UCHL1) is highly induced following HSC activation; however, the cellular targets of its deubiquitinating activity are poorly defined. Here, we describe a role for UCHL1 in regulating the levels and activity of hypoxia-inducible factor 1 (HIF1), an oxygen-sensitive transcription factor, during HSC activation and liver fibrosis. HIF1 is elevated during HSC activation and promotes the expression of profibrotic mediator HIF target genes. Increased HIF1α expression correlated with induction of UCHL1 mRNA and protein with HSC activation. Genetic deletion or chemical inhibition of UCHL1 impaired HIF activity through reduction of HIF1α levels. Furthermore, our mechanistic studies have shown that UCHL1 elevates HIF activity through specific cleavage of degradative ubiquitin chains, elevates levels of pro-fibrotic gene expression and increases proliferation rates. As we also show that UCHL1 inhibition blunts fibrogenesis in a pre-clinical 3D human liver slice model of fibrosis, these results demonstrate how small molecule inhibitors of DUBs can exert therapeutic effects through modulation of HIF transcription factors in liver disease. Furthermore, inhibition of HIF activity using UCHL1 inhibitors may represent a therapeutic opportunity with other HIF-related pathologies.


Assuntos
Células Estreladas do Fígado , Subunidade alfa do Fator 1 Induzível por Hipóxia , Cirrose Hepática , Ubiquitina Tiolesterase , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Cirrose Hepática/genética , Cirrose Hepática/patologia , Cirrose Hepática/metabolismo , Animais , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Humanos , Regulação da Expressão Gênica , Transdiferenciação Celular/genética
11.
Biochem J ; 443(2): 355-9, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22332634

RESUMO

A recent study revealed that ES (embryonic stem) cell lines derived from the 129 murine strain carry an inactivating mutation within the caspase 11 gene (Casp4) locus [Kayagaki, Warming, Lamkanfi, Vande Walle, Louie, Dong, Newton, Qu, Liu, Heldens, Zhang, Lee, Roose-Girma and Dixit (2011) Nature 479, 117-121]. Thus, if 129 ES cells are used to target genes closely linked to caspase 11, the resulting mice might also carry the caspase 11 deficiency as a passenger mutation. In the present study, we examined the genetic loci of mice targeted for the closely linked c-IAP (cellular inhibitor of apoptosis) genes, which were generated in 129 ES cells, and found that, despite extensive backcrossing into a C57BL/6 background, c-IAP1(-/-) animals are also deficient in caspase 11. Consequently, data obtained from these mice should be re-evaluated in this new context.


Assuntos
Caspases/genética , Proteínas Inibidoras de Apoptose/metabolismo , Mutação , Animais , Caspases/metabolismo , Caspases Iniciadoras , Linhagem Celular , Ativação Enzimática , Proteínas Inibidoras de Apoptose/deficiência , Camundongos , Camundongos da Linhagem 129
12.
Sci Rep ; 12(1): 7943, 2022 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-35562367

RESUMO

The product encoded by the X-linked inhibitor of apoptosis (XIAP) gene is a multi-functional protein which not only controls caspase-dependent cell death, but also participates in inflammatory signalling, copper homeostasis, response to hypoxia and control of cell migration. Deregulation of XIAP, either by elevated expression or inherited genetic deletion, is associated with several human disease states. Reconciling XIAP-dependent signalling pathways with its role in disease progression is essential to understand how XIAP promotes the progression of human pathologies. In this study we have created a panel of genetically modified XIAP-null cell lines using TALENs and CRISPR/Cas9 to investigate the functional outcome of XIAP deletion. Surprisingly, in our genetically modified cells XIAP deletion had no effect on programmed cell death, but instead the primary phenotype we observed was a profound increase in cell migration rates. Furthermore, we found that XIAP-dependent suppression of cell migration was dependent on XIAPdependent control of C-RAF levels, a protein kinase which controls cell signalling pathways that regulate the cytoskeleton. These results suggest that XIAP is not necessary for control of the apoptotic signalling cascade, however it does have a critical role in controlling cell migration and motility that cannot be compensated for in XIAP-knockout cells.


Assuntos
Linfócitos Nulos , Proteínas Proto-Oncogênicas c-raf , Apoptose , Caspases/metabolismo , Linfócitos Nulos/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Transdução de Sinais , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
13.
Mol Neurobiol ; 59(10): 5987-6008, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35831557

RESUMO

The neuroblastoma cell line SH-SY5Y is widely used to study retinoic acid (RA)-induced gene expression and differentiation and as a tool to study neurodegenerative disorders. SH-SY5Y cells predominantly exhibit adrenergic neuronal properties, but they can also exist in an epigenetically interconvertible alternative state with more mesenchymal characteristics; as a result, these cells can be used to study gene regulation circuitry controlling neuroblastoma phenotype. Using a combination of pharmacological inhibition and targeted gene inactivation, we have probed the requirement for DNA topoisomerase IIB (TOP2B) in RA-induced gene expression and differentiation and in the balance between adrenergic neuronal versus mesenchymal transcription programmes. We found that expression of many, but not all genes that are rapidly induced by ATRA in SH-SY5Y cells was significantly reduced in the TOP2B null cells; these genes include BCL2, CYP26A1, CRABP2, and NTRK2. Comparing gene expression profiles in wild-type versus TOP2B null cells, we found that long genes and genes expressed at a high level in WT SH-SY5Y cells were disproportionately dependent on TOP2B. Notably, TOP2B null SH-SY5Y cells upregulated mesenchymal markers vimentin (VIM) and fibronectin (FN1) and components of the NOTCH signalling pathway. Enrichment analysis and comparison with the transcription profiles of other neuroblastoma-derived cell lines supported the conclusion that TOP2B is required to fully maintain the adrenergic neural-like transcriptional signature of SH-SY5Y cells and to suppress the alternative mesenchymal epithelial-like epigenetic state.


Assuntos
DNA Topoisomerases Tipo II , Neuroblastoma , Proteínas de Ligação a Poli-ADP-Ribose , Adrenérgicos , Diferenciação Celular , Linhagem Celular Tumoral , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Humanos , Neuroblastoma/metabolismo , Fenótipo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Tretinoína/metabolismo , Tretinoína/farmacologia
14.
Cells ; 10(6)2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34200988

RESUMO

PBRM1, a component of the chromatin remodeller SWI/SNF, is often deleted or mutated in human cancers, most prominently in renal cancers. Core components of the SWI/SNF complex have been shown to be important for the cellular response to hypoxia. Here, we investigated how PBRM1 controls HIF-1α activity. We found that PBRM1 is required for HIF-1α transcriptional activity and protein levels. Mechanistically, PBRM1 is important for HIF-1α mRNA translation, as absence of PBRM1 results in reduced actively translating HIF-1α mRNA. Interestingly, we found that PBRM1, but not BRG1, interacts with the m6A reader protein YTHDF2. HIF-1α mRNA is m6A-modified, bound by PBRM1 and YTHDF2. PBRM1 is necessary for YTHDF2 binding to HIF-1α mRNA and reduction of YTHDF2 results in reduced HIF-1α protein expression in cells. Our results identify a SWI/SNF-independent function for PBRM1, interacting with HIF-1α mRNA and the epitranscriptome machinery. Furthermore, our results suggest that the epitranscriptome-associated proteins play a role in the control of hypoxia signalling pathways.


Assuntos
Hipóxia Celular , Proteínas de Ligação a DNA/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/fisiologia , Células A549 , Células HeLa , Humanos , Biossíntese de Proteínas , Transdução de Sinais
15.
Nucleic Acids Res ; 36(11): 3757-64, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18487626

RESUMO

RNA polymerase (pol) III contains a dissociable subcomplex that is required for initiation, but not for elongation or termination of transcription. This subcomplex is composed of subunits RPC3, RPC6 and RPC7, and interacts with TFIIIB, a factor that is necessary and sufficient to support accurate pol III transcription in vitro. Direct binding of TFIIIB to RPC6 is believed to recruit pol III to its genetic templates. However, this has never been tested in vivo. Here we combine chromatin immunoprecipitation with RNA interference to demonstrate that the RPC3/6/7 subcomplex is required for pol III recruitment in mammalian cells. Specific knockdown of RPC6 by RNAi results in post-transcriptional depletion of the other components of the subcomplex, RPC3 and RPC7, without destabilizing core pol III subunits or TFIIIB. The resultant core enzyme is defective in associating with TFIIIB and target genes in vivo. Promoter occupancy by pol II is unaffected, despite sharing five subunits with the pol III core. These observations provide evidence for the validity in vivo of the model for pol III recruitment that was built on biochemical data.


Assuntos
Proteínas de Transporte/metabolismo , Subunidades Proteicas/metabolismo , RNA Polimerase III/metabolismo , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Linhagem Celular , Camundongos , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Interferência de RNA , RNA Polimerase III/antagonistas & inibidores , Fator de Transcrição TFIIIB/metabolismo , Transcrição Gênica
16.
Biochem J ; 412(3): 477-84, 2008 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-18393939

RESUMO

HIF (hypoxia-inducible factor) is the main transcription factor activated by low oxygen tensions. HIF-1alpha (and other alpha subunits) is tightly controlled mostly at the protein level, through the concerted action of a class of enzymes called PHDs (prolyl hydroxylases) 1, 2 and 3. Most of the knowledge of HIF derives from studies following hypoxic stress; however, HIF-1alpha stabilization is also found in non-hypoxic conditions through an unknown mechanism. In the present study, we demonstrate that NF-kappaB (nuclear factor kappaB) is a direct modulator of HIF-1alpha expression. The HIF-1alpha promoter is responsive to selective NF-kappaB subunits. siRNA (small interfering RNA) studies for individual NF-kappaB members revealed differential effects on HIF-1alpha mRNA levels, indicating that NF-kappaB can regulate basal HIF-1alpha expression. Finally, when endogenous NF-kappaB is induced by TNFalpha (tumour necrosis factor alpha) treatment, HIF-1alpha levels also change in an NF-kappaB-dependent manner. In conclusion, we find that NF-kappaB can regulate basal TNFalpha and, in certain circumstances, the hypoxia-induced HIF-1alpha.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , NF-kappa B/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
17.
Cells ; 8(2)2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30717305

RESUMO

Hypoxia-Inducible Factors (HIFs) play essential roles in the physiological response to low oxygen in all multicellular organisms, while their deregulation is associated with human diseases. HIF levels and activity are primarily controlled by the availability of the oxygen-sensitive HIFα subunits, which is mediated by rapid alterations to the rates of HIFα protein production and degradation. While the pathways that control HIFα degradation are understood in great detail, much less is known about the targeted control of HIFα protein synthesis and what role this has in controlling HIF activity during the hypoxic response. This review will focus on the signalling pathways and RNA binding proteins that modulate HIFα mRNA half-life and/or translation rate, and their contribution to hypoxia-associated diseases.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Oxigênio/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Hipóxia Celular/efeitos dos fármacos , Humanos , Proteínas de Ligação a RNA/metabolismo , Ubiquitina/metabolismo
18.
Nucleic Acids Res ; 34(1): 286-94, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16407335

RESUMO

RNA polymerase (pol) III transcription decreases when primary cultures of rat neonatal cardiomyocytes are exposed to low oxygen tension. Previous studies in fibroblasts have shown that the pol III-specific transcription factor IIIB (TFIIIB) is bound and regulated by the proto-oncogene product c-Myc, the mitogen-activated protein kinase ERK and the retinoblastoma tumour suppressor protein, RB. The principal function of TFIIIB is to recruit pol III to its cognate gene template, an activity that is known to be inhibited by RB and stimulated by ERK. We demonstrate by chromatin immunoprecipitation (ChIP) that c-Myc also stimulates pol III recruitment by TFIIIB. However, hypoxic conditions cause TFIIIB dissociation from c-Myc and ERK, at the same time as increasing its interaction with RB. Consistent with this, ChIP assays indicate that the occupancy of tRNA genes by pol III is significantly reduced, whereas promoter binding by TFIIIB is undiminished. The data suggest that hypoxia can inhibit pol III transcription by altering the interactions between TFIIIB and its regulators and thus compromising its ability to recruit the polymerase. These effects are independent of cell cycle changes.


Assuntos
Regulação da Expressão Gênica , Miócitos Cardíacos/metabolismo , RNA Polimerase III/antagonistas & inibidores , RNA de Transferência/genética , Transcrição Gênica , Animais , Hipóxia Celular , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Polimerase III/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína do Retinoblastoma/metabolismo
19.
Biochem Soc Symp ; (73): 141-54, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16626295

RESUMO

The proto-oncogene product c-Myc can induce cell growth and proliferation. It regulates a large number of RNA polymerase II-transcribed genes, many of which encode ribosomal proteins, translation factors and other components of the biosynthetic apparatus. We have found that c-Myc can also activate transcription by RNA polymerases I and III, thereby stimulating production of rRNA and tRNA. As such, c-Myc may possess the unprecedented capacity to induce expression of all ribosomal components. This may explain its potent ability to drive cell growth, which depends on the accumulation of ribosomes. The activation of RNA polymerase II transcription by c-Myc is often inefficient, but its induction of rRNA and tRNA genes can be very strong in comparison. We will describe what is known about the mechanisms used by c-Myc to activate transcription by RNA polymerases I and II.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ativação Transcricional , Animais , RNA Polimerases Dirigidas por DNA/genética , Humanos , Camundongos , Modelos Biológicos , Proto-Oncogene Mas , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , RNA Ribossômico/genética , RNA de Transferência/genética
20.
Cell Cycle ; 2(3): 181-4, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12734418

RESUMO

The synthesis of tRNA and 5S rRNA by RNA polymerase (pol) III is cell cycle regulated in higher organisms. Overexpression of pol III products is a general feature of transformed cells. These observations may be explained by the fact that a pol III-specific transcription factor, TFIIIB, is strongly regulated by the tumor suppressors RB and p53, as well as the proto-oncogene product c-Myc. RB and p53 repress TFIIIB, but this restraint can be lost in tumors through a variety of mechanisms. In contrast, c-Myc binds and activates TFIIIB, causing potent induction of pol III transcription. Using chromatin immunoprecipitation and RNA interference, we show that c-Myc interacts with tRNA and 5S rRNA genes in transformed cervical cells, stimulating their expression. Availability of pol III products may be an important determinant of a cell's capacity to grow. The ability to regulate pol III output may therefore be integral to the growth control functions of RB, p53 and c-Myc.


Assuntos
Divisão Celular/genética , Transformação Celular Neoplásica/metabolismo , DNA Polimerase III/metabolismo , Células Eucarióticas/enzimologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína do Retinoblastoma/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Transformação Celular Neoplásica/genética , DNA Polimerase III/genética , Humanos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myc/genética , RNA/genética , Proteína do Retinoblastoma/genética , Transcrição Gênica/genética , Proteína Supressora de Tumor p53/genética
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