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1.
Nature ; 557(7705): 439-445, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29743679

RESUMO

In vertebrate hearts, the ventricular trabecular myocardium develops as a sponge-like network of cardiomyocytes that is critical for contraction and conduction, ventricular septation, papillary muscle formation and wall thickening through the process of compaction 1 . Defective trabeculation leads to embryonic lethality2-4 or non-compaction cardiomyopathy (NCC) 5 . There are divergent views on when and how trabeculation is initiated in different species. In zebrafish, trabecular cardiomyocytes extrude from compact myocardium 6 , whereas in chicks, chamber wall thickening occurs before overt trabeculation 7 . In mice, the onset of trabeculation has not been described, but is proposed to begin at embryonic day 9.0, when cardiomyocytes form radially oriented ribs 2 . Endocardium-myocardium communication is essential for trabeculation, and numerous signalling pathways have been identified, including Notch2,8 and Neuregulin (NRG) 4 . Late disruption of the Notch pathway causes NCC 5 . Whereas it has been shown that mutations in the extracellular matrix (ECM) genes Has2 and Vcan prevent the formation of trabeculae in mice9,10 and the matrix metalloprotease ADAMTS1 promotes trabecular termination 3 , the pathways involved in ECM dynamics and the molecular regulation of trabeculation during its early phases remain unexplored. Here we present a model of trabeculation in mice that integrates dynamic endocardial and myocardial cell behaviours and ECM remodelling, and reveal new epistatic relationships between the involved signalling pathways. NOTCH1 signalling promotes ECM degradation during the formation of endocardial projections that are critical for individualization of trabecular units, whereas NRG1 promotes myocardial ECM synthesis, which is necessary for trabecular rearrangement and growth. These systems interconnect through NRG1 control of Vegfa, but act antagonistically to establish trabecular architecture. These insights enabled the prediction of persistent ECM and cardiomyocyte growth in a mouse NCC model, providing new insights into the pathophysiology of congenital heart disease.


Assuntos
Coração/embriologia , Miocárdio/citologia , Miocárdio/metabolismo , Neuregulina-1/metabolismo , Organogênese , Receptor Notch1/metabolismo , Animais , Modelos Animais de Doenças , Endocárdio/citologia , Endocárdio/metabolismo , Matriz Extracelular/metabolismo , Cardiopatias/congênito , Cardiopatias/metabolismo , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Neuregulina-1/genética , Receptor Notch1/genética , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo
2.
Mol Oncol ; 11(5): 470-490, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28173629

RESUMO

Activating KRAS mutations drive colorectal cancer tumorigenesis and influence response to anti-EGFR-targeted therapy. Despite recent advances in understanding Ras signaling biology and the revolution in therapies for melanoma using BRAF inhibitors, no targeted agents have been effective in KRAS-mutant cancers, mainly due to activation of compensatory pathways. Here, by leveraging the largest synthetic lethal genetic interactome in yeast, we identify that KRAS-mutated colorectal cancer cells have augmented homologous recombination repair (HRR) signaling. We found that KRAS mutation resulted in slowing and stalling of the replication fork and accumulation of DNA damage. Moreover, we found that KRAS-mutant HCT116 cells have an increase in MYC-mediated RAD51 expression with a corresponding increase in RAD51 recruitment to irradiation-induced DNA double-strand breaks (DSBs) compared to genetically complemented isogenic cells. MYC depletion using RNA interference significantly reduced IR-induced RAD51 foci formation and HRR. On the contrary, overexpression of either HA-tagged wild-type (WT) MYC or phospho-mutant S62A increased RAD51 protein levels and hence IR-induced RAD51 foci. Likewise, depletion of RAD51 selectively induced apoptosis in HCT116-mutant cells by increasing DSBs. Pharmacological inhibition targeting HRR signaling combined with PARP inhibition selectivity killed KRAS-mutant cells. Interestingly, these differences were not seen in a second isogenic pair of KRAS WT and mutant cells (DLD-1), likely due to their nondependency on the KRAS mutation for survival. Our data thus highlight a possible mechanism by which KRAS-mutant-dependent cells drive HRR in vitro by upregulating MYC-RAD51 expression. These data may offer a promising therapeutic vulnerability in colorectal cancer cells harboring otherwise nondruggable KRAS mutations, which warrants further investigation in vivo.


Assuntos
Neoplasias Colorretais/genética , Recombinação Homóloga , Proteínas Proto-Oncogênicas p21(ras)/genética , Rad51 Recombinase/genética , Saccharomyces cerevisiae/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Proteínas de Ligação a DNA/genética , Relação Dose-Resposta a Droga , Receptores ErbB/genética , Células HCT116 , Humanos , Mutação , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , RNA Interferente Pequeno/genética , Rad51 Recombinase/metabolismo , Fatores de Transcrição/genética
3.
Sci Rep ; 7: 39873, 2017 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-28051153

RESUMO

In breast cancer metastasis, the dynamic continuum involving pro- and anti-inflammatory regulators can become compromised. Over 600 genes have been implicated in metastasis to bone, lung or brain but how these genes might contribute to perturbation of immune function is poorly understood. To gain insight, we adopted a gene co-expression network approach that draws on the functional parallels between naturally occurring bone marrow-derived mesenchymal stem cells (BM-MSCs) and cancer stem cells (CSCs). Our network analyses indicate a key role for metastasis suppressor RARRES3, including potential to regulate the immunoproteasome (IP), a specialized proteasome induced under inflammatory conditions. Knockdown of RARRES3 in near-normal mammary epithelial and breast cancer cell lines increases overall transcript and protein levels of the IP subunits, but not of their constitutively expressed counterparts. RARRES3 mRNA expression is controlled by interferon regulatory factor IRF1, an inducer of the IP, and is sensitive to depletion of the retinoid-related receptor RORA that regulates various physiological processes including immunity through modulation of gene expression. Collectively, these findings identify a novel regulatory role for RARRES3 as an endogenous inhibitor of IP expression, and contribute to our evolving understanding of potential pathways underlying breast cancer driven immune modulation.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores do Ácido Retinoico/metabolismo , Células da Medula Óssea/citologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Metástase Neoplásica/genética , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/imunologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptores do Ácido Retinoico/genética
4.
BMC Biol ; 13: 21, 2015 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-25857663

RESUMO

BACKGROUND: We recently identified a novel protein, Rearranged L-myc fusion (Rlf), that is required for DNA hypomethylation and transcriptional activity at two specific regions of the genome known to be sensitive to epigenetic gene silencing. To identify other loci affected by the absence of Rlf, we have now analysed 12 whole genome bisulphite sequencing datasets across three different embryonic tissues/stages from mice wild-type or null for Rlf. RESULTS: Here we show that the absence of Rlf results in an increase in DNA methylation at thousands of elements involved in transcriptional regulation and many of the changes occur at enhancers and CpG island shores. ChIP-seq for H3K4me1, a mark generally found at regulatory elements, revealed associated changes at many of the regions that are differentially methylated in the Rlf mutants. RNA-seq showed that the numerous effects of the absence of Rlf on the epigenome are associated with relatively subtle effects on the mRNA population. In vitro studies suggest that Rlf's zinc fingers have the capacity to bind DNA and that the protein interacts with other known epigenetic modifiers. CONCLUSION: This study provides the first evidence that the epigenetic modifier Rlf is involved in the maintenance of DNA methylation at enhancers and CGI shores across the genome.


Assuntos
Alelos , Ilhas de CpG/genética , Elementos Facilitadores Genéticos/genética , Epigênese Genética , Genes Modificadores , Fatores de Transcrição/genética , Animais , Cromatina/metabolismo , DNA/metabolismo , Metilação de DNA/genética , Replicação do DNA/genética , Éxons/genética , Regulação da Expressão Gênica no Desenvolvimento , Loci Gênicos , Fatores de Troca do Nucleotídeo Guanina , Células HEK293 , Histonas/metabolismo , Homozigoto , Humanos , Fígado/embriologia , Fígado/metabolismo , Lisina/metabolismo , Camundongos , Mutação/genética , Especificidade de Órgãos/genética , Ligação Proteica , Fatores de Transcrição/metabolismo , Transcrição Gênica
5.
Genomics ; 105(2): 116-22, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25451176

RESUMO

Position-effect variegation of transgene expression is sensitive to the chromatin state. We previously reported a forward genetic screen in mice carrying a variegated α-globin GFP transgene to find novel genes encoding epigenetic regulators. We named the phenovariant strains "Mommes" for modifiers of murine metastable epialleles. Here we report positional cloning of mutations in two Momme strains which result in suppression of variegation. Both strains harbour point mutations in the erythroid transcription factor, Klf1. One (D11) generates a stop codon in the zinc finger domain and a homozygous null phenotype. The other (D45) generates an amino acid transversion (H350R) within a conserved linker between zinc fingers two and three. Homozygous MommeD45 mice have chronic microcytic anaemia which models the phenotype in a recently described family. This is the first genetic evidence that the linkers between the zinc fingers of transcription factors have a function beyond that of a simple spacer.


Assuntos
Efeitos da Posição Cromossômica , Fatores de Transcrição Kruppel-Like/genética , Mutação , alfa-Globinas/genética , Anemia/genética , Animais , Testes Genéticos/métodos , Camundongos , Camundongos Transgênicos/embriologia , Camundongos Transgênicos/genética , Esplenomegalia/genética , Dedos de Zinco/genética
6.
Mamm Genome ; 25(7-8): 293-303, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24781204

RESUMO

An ENU mutagenesis screen to identify novel epigenetic modifiers was established in mice carrying a multi-copy GFP transgene, which is expressed in a variegated manner in erythrocytes and is highly sensitive to epigenetic silencing. The screen has produced mouse mutants of both known modifiers of epigenetic state, such as Dnmt1 and Smarca5, and novel modifiers, such as Smchd1 and Rlf. Here we report two mouse lines generated from the screen, MommeD6 and MommeD20, with point mutations in D14Abb1e. These are the first mouse mutants of D14Abb1e (also known as Fam208a), a gene about which little is known. Heterozygous intercrosses show that homozygous mutants from both the MommeD6 and MommeD20 lines are not viable beyond gastrulation, demonstrating an important role for D14Abb1e in development. We demonstrate that haploinsufficiency for D14Abb1e effects transgene expression at the RNA level. Analysis of the predicted D14Abb1e protein sequence reveals that it contains putative nuclear localisation signals and a domain of unknown function, DUF3715. Our studies reveal that D14Abb1e is localised to the nucleus and is expressed in skin and testes.


Assuntos
Desenvolvimento Embrionário , Proteínas Nucleares/genética , Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Núcleo Celular/metabolismo , Eritrócitos/metabolismo , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Dados de Sequência Molecular , Mutação/genética , Proteínas Nucleares/química , Transporte Proteico , Proteínas/química , Pele/metabolismo , Testículo/metabolismo , Transgenes
7.
Genome Biol ; 14(9): R96, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24025402

RESUMO

BACKGROUND: We have used a sensitized ENU mutagenesis screen to produce mouse lines that carry mutations in genes required for epigenetic regulation. We call these lines Modifiers of murine metastable epialleles (Mommes). RESULTS: We report a basic molecular and phenotypic characterization for twenty of the Momme mouse lines, and in each case we also identify the causative mutation. Three of the lines carry a mutation in a novel epigenetic modifier, Rearranged L-myc fusion (Rlf), and one gene, Rap-interacting factor 1 (Rif1), has not previously been reported to be involved in transcriptional regulation in mammals. Many of the other lines are novel alleles of known epigenetic regulators. For two genes, Rlf and Widely-interspaced zinc finger (Wiz), we describe the first mouse mutants. All of the Momme mutants show some degree of homozygous embryonic lethality, emphasizing the importance of epigenetic processes. The penetrance of lethality is incomplete in a number of cases. Similarly ,abnormalities in phenotype seen in the heterozygous individuals of some lines occur with incomplete penetrance. CONCLUSIONS: Recent advances in sequencing enhance the power of sensitized mutagenesis screens to identify the function of previously uncharacterized factors and to discover additional functions for previously characterized proteins. The observation of incomplete penetrance of phenotypes in these inbred mutant mice, at various stages of development, is of interest. Overall, the Momme collection of mouse mutants provides a valuable resource for researchers across many disciplines.


Assuntos
Epigênese Genética , Etilnitrosoureia/farmacologia , Genes Letais , Mutagênese , Mutagênicos/farmacologia , Mutação/efeitos dos fármacos , Alelos , Animais , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Genótipo , Fatores de Troca do Nucleotídeo Guanina , Heterozigoto , Homozigoto , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Fenótipo , Proteínas de Ligação a Telômeros/genética , Fatores de Transcrição/genética
8.
Mamm Genome ; 24(5-6): 206-17, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23636699

RESUMO

Observations of inherited phenotypes that cannot be explained solely through genetic inheritance are increasing. Evidence points to transmission of non-DNA molecules in the gamete as mediators of the phenotypes. However, in most cases it is unclear what the molecules are, with DNA methylation, chromatin proteins, and small RNAs being the most prominent candidates. From a screen to generate novel mouse mutants of genes involved in epigenetic reprogramming, we produced a DNA methyltransferase 3b allele that is missing exon 13. Mice that are homozygous for the mutant allele have smaller stature and reduced viability, with particularly high levels of female post-natal death. Reduced DNA methylation was also detected at telocentric repeats and the X-linked Hprt gene. However, none of the abnormal phenotypes or DNA methylation changes worsened with multiple generations of homozygous mutant inbreeding. This suggests that in our model the abnormalities are reset each generation and the processes of transgenerational epigenetic reprogramming are effective in preventing their inheritance.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Camundongos/genética , Alelos , Animais , Sequência de Bases , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Epigênese Genética , Éxons , Feminino , Homozigoto , Masculino , Camundongos/crescimento & desenvolvimento , Camundongos/metabolismo , Camundongos Transgênicos , Dados de Sequência Molecular , Linhagem , DNA Metiltransferase 3B
9.
Circ Res ; 112(12): 1583-91, 2013 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-23564640

RESUMO

RATIONALE: Hypoxia followed by reoxygenation promotes inflammation by activating nuclear factor κB transcription factors in endothelial cells (ECs). This process involves modification of the signaling intermediary tumor necrosis factor receptor-associated factor 6 with polyubiquitin chains. Thus, cellular mechanisms that suppress tumor necrosis factor receptor-associated factor 6 ubiquitination are potential therapeutic targets to reduce inflammation in hypoxic tissues. OBJECTIVE: In this study, we tested the hypothesis that endothelial activation in response to hypoxia-reoxygenation can be influenced by Cezanne, a deubiquitinating enzyme that cleaves ubiquitin from specific modified proteins. METHODS AND RESULTS: Studies of cultured ECs demonstrated that hypoxia (1% oxygen) induced Cezanne via p38 mitogen-activated protein kinase-dependent transcriptional and post-transcriptional mechanisms. Hypoxia-reoxygenation had minimal effects on proinflammatory signaling in unmanipulated ECs but significantly enhanced Lys63 polyubiquitination of tumor necrosis factor receptor-associated factor 6, activation of nuclear factor κB, and expression of inflammatory genes after silencing of Cezanne. Thus, although hypoxia primed cells for inflammatory activation, it simultaneously induced Cezanne, which impeded signaling to nuclear factor κB by suppressing tumor necrosis factor receptor-associated factor 6 ubiquitination. Similarly, ischemia induced Cezanne in the murine kidney in vascular ECs, glomerular ECs, podocytes, and epithelial cells, and genetic deletion of Cezanne enhanced renal inflammation and injury in murine kidneys exposed to ischemia followed by reperfusion. CONCLUSIONS: We conclude that inflammatory responses to ischemia are controlled by a balance between ubiquitination and deubiquitination, and that Cezanne is a key regulator of this process. Our observations have important implications for therapeutic targeting of inflammation and injury during ischemia-reperfusion.


Assuntos
Endopeptidases/metabolismo , Células Endoteliais/enzimologia , Inflamação/prevenção & controle , Rim/irrigação sanguínea , Traumatismo por Reperfusão/enzimologia , Fator 6 Associado a Receptor de TNF/metabolismo , Animais , Hipóxia Celular , Células Cultivadas , Modelos Animais de Doenças , Endopeptidases/deficiência , Endopeptidases/genética , Células Endoteliais/imunologia , Humanos , Inflamação/enzimologia , Inflamação/genética , Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Oxigênio/metabolismo , Interferência de RNA , Ratos , Ratos Endogâmicos F344 , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/imunologia , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/genética , Fatores de Tempo , Transcrição Gênica , Transfecção , Ubiquitinação , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
10.
Lab Invest ; 91(12): 1684-94, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21876537

RESUMO

Dimethyloxalylglycine (DMOG) is an inhibitor of prolyl-4-hydroxylase domain enzymes. Its potential value and mechanism of actions in preventing/treating gastrointestinal injury are, however, poorly understood. We, therefore, examined the effect of DMOG on influencing gut injury and repair using a variety of in vitro and in vivo models. We performed in vitro studies utilising pro-migratory (wounded monolayer) and proliferation (using DNA quantitation) assays of human stomach (AGS) and colonic (HT29) carcinoma cells. Time course studies examined changes in hypoxia-inducible factor (HIF) and vascular endothelial growth factor (VEGF) levels, a growth factor known to be regulated via HIF. In vivo studies utilised a rat gastric (indomethacin, 20 mg/kg and 3 h restraint) damage model. DMOG stimulated migration in a dose-dependent manner, increasing migration twofold when added at 25µM (P<0.01). Additive effects were seen when DMOG was added to cells in hypoxic conditions. DMOG stimulated proliferation dose dependently, increasing proliferation threefold when added at 70 µM (P<0.01). DMOG caused upregulation of both HIF and VEGF within 4 h of administration. Addition of VEGF neutralising antibody truncated migratory and proliferative activity of DMOG by about 70%. Both oral and subcutaneous administration of DMOG decreased gastric injury without influencing intragastric pH (50% reduction in injury when 1 ml gavaged at 0.57 mM, P < 0.01). Indomethacin reduced tissue HIF and VEGF levels but this was prevented if DMOG was present. In conclusion, DMOG stimulates the early phases of gut repair and VEGF-dependent processes appear relevant. Non-peptide factors such as this may be useful to stabilise or repair gut mucosa.


Assuntos
Aminoácidos Dicarboxílicos/uso terapêutico , Gastroenteropatias/tratamento farmacológico , Fator 1 Induzível por Hipóxia/metabolismo , Regeneração/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Aminoácidos Dicarboxílicos/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Células HT29 , Humanos , Masculino , Ratos , Ratos Sprague-Dawley
11.
Mol Cancer ; 10: 89, 2011 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-21791076

RESUMO

BACKGROUND: 209 000 new cases of renal carcinoma are diagnosed each year worldwide and new therapeutic targets are urgently required. The great majority of clear cell renal cancer involves inactivation of VHL, which acts as a gatekeeper tumour suppressor gene in renal epithelial cells. However how VHL exerts its tumour suppressor function remains unclear. A gene expression microarray comparing RCC10 renal cancer cells expressing either VHL or an empty vector was used to identify novel VHL regulated genes. FINDINGS: NMU (Neuromedin U) is a neuropeptide that has been implicated in energy homeostasis and tumour progression. Here we show for the first time that VHL loss-of-function results in dramatic upregulation of NMU expression in renal cancer cells. The effect of VHL inactivation was found to be mediated via activation of Hypoxia Inducible Factor (HIF). Exposure of VHL expressing RCC cells to either hypoxia or dimethyloxalylglycine resulted in HIF activation and increased NMU expression. Conversely, suppression of HIF in VHL defective RCC cells via siRNA of HIF-α subunits or expression of Type 2C mutant VHLs reduced NMU expression levels. We also show that renal cancer cells express a functional NMU receptor (NMUR1), and that NMU stimulates migration of renal cancer cells. CONCLUSIONS: These findings suggest that NMU may act in an autocrine fashion, promoting progression of kidney cancer. Hypoxia and HIF expression are frequently observed in many non-renal cancers and are associated with a poor prognosis. Our study raises the possibility that HIF may also drive NMU expression in non-renal tumours.


Assuntos
Carcinoma de Células Renais/genética , Inativação Gênica/fisiologia , Neoplasias Renais/genética , Neuropeptídeos/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor/fisiologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Neoplasias Renais/patologia , Análise em Microsséries , Neuropeptídeos/metabolismo , Regulação para Cima , Proteína Supressora de Tumor Von Hippel-Lindau/antagonistas & inibidores
12.
J Am Soc Nephrol ; 21(12): 2041-6, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20864688

RESUMO

The ciliary hypothesis for cystic renal diseases postulates that most of these conditions result from abnormalities in the primary cilium, a microtubule-based structure that acts as a sensor for extracellular cues. Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene predisposes to renal cysts and clear cell renal cell carcinoma. VHL plays a critical role in the formation of primary cilia in kidney epithelium, but the underlying mechanisms are poorly understood. Here, we demonstrate that VHL inactivation induces HEF1/Cas-L/NEDD9 and Aurora kinase A via the stabilization of hypoxia-inducible factors 1 and 2. Aurora kinase A is a mitotic kinase commonly upregulated in cancer that causes regression of the primary cilium by promoting histone deacetylase-dependent tubulin depolymerization of the ciliary axoneme. HEF1/Cas-L/NEDD9 is a component of focal adhesions that has a prominent role in inducing metastasis and that colocalizes with Aurora kinase A at the centrosome, thereby enhancing the harmful effect of Aurora kinase A on the cilium. Suppression of this pathway improved the formation of primary cilia and reduced cell motility in VHL-defective renal cancer cells. Our results highlight the gatekeeper role of VHL in the kidney epithelium.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinoma de Células Renais/genética , Doenças Renais Císticas/genética , Neoplasias Renais/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Aurora Quinase A , Aurora Quinases , Carcinoma de Células Renais/fisiopatologia , Linhagem Celular Tumoral , Células Cultivadas , Cílios/metabolismo , Cílios/fisiologia , Feminino , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Doenças Renais Císticas/fisiopatologia , Neoplasias Renais/fisiopatologia , Masculino , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/análise , Sensibilidade e Especificidade , Proteína Supressora de Tumor Von Hippel-Lindau/efeitos dos fármacos , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
13.
PLoS One ; 5(6): e11103, 2010 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-20559438

RESUMO

BACKGROUND: Hypoxia plays a key role in ischaemic and neovascular disorders of the retina. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs) that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the tissue distribution of retinal hypoxia during oxygen-induced retinopathy (OIR) in mice using the bio-reductive drug pimonidazole. We measured the levels of HIF-1alpha and HIF-2alpha proteins by Western blotting and determined their cellular distribution by immunohistochemistry during the development of OIR. We measured the temporal expression profiles of two downstream mediators, vascular endothelial growth factor (VEGF) and erythropoietin (Epo) by ELISA. Pimonidazole labelling was evident specifically in the inner retina. Labelling peaked at 2 hours after the onset of hypoxia and gradually declined thereafter. Marked binding to Müller glia was evident during the early hypoxic stages of OIR. Both HIF-1alpha and HIF-2alpha protein levels were significantly increased during retinal hypoxia but were evident in distinct cellular distributions; HIF-1alpha stabilisation was evident in neuronal cells throughout the inner retinal layers whereas HIF-2alpha was restricted to Müller glia and astrocytes. Hypoxia and HIF-alpha stabilisation in the retina were closely followed by upregulated expression of the downstream mediators VEGF and EPO. CONCLUSIONS/SIGNIFICANCE: Both HIF-1alpha and HIF-2alpha are activated in close correlation with retinal hypoxia but have contrasting cell specificities, consistent with differential roles in retinal ischaemia. Our findings suggest that HIF-2alpha activation plays a key role in regulating the response of Müller glia to hypoxia.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/metabolismo , Vasos Retinianos/metabolismo , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Eritropoetina/metabolismo , Imuno-Histoquímica , Camundongos , Fator A de Crescimento do Endotélio Vascular/metabolismo
14.
Antioxid Redox Signal ; 12(4): 459-80, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19737089

RESUMO

Abstract Ischemic stroke is a major cause of death worldwide, and current therapeutic options are very limited. Preconditioning with an ischemic or hypoxic insult is beneficial in experimental models of ischemic stroke. Ischemia/hypoxia results in activation of numerous transcription factors, including hypoxia inducible factor (HIF), which is a master regulator of oxygen homeostasis. HIF activation induces a diverse range of target genes, encompassing a wide variety of cellular processes; including angiogenesis, energy metabolism, cell survival, radical production/scavenging, iron metabolism, stem cell homing, and differentiation. Inhibition of HIF prolyl hydroxylase domain (PHD) enzymes results in activation of HIF and is likely to mimic, at least in part, the effects of hypoxia preconditioning. A caveat is that not all consequences of HIF activation will be beneficial and some could even be deleterious. Nevertheless, PHD inhibitors may be therapeutically useful in the treatment of stroke. Prototype PHD inhibitors have shown promising results in preclinical models.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Citoproteção , Inibidores Enzimáticos/farmacologia , Fator 1 Induzível por Hipóxia/agonistas , Fármacos Neuroprotetores/farmacologia , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Barreira Hematoencefálica/enzimologia , Encéfalo/irrigação sanguínea , Isquemia Encefálica/fisiopatologia , Bloqueadores dos Canais de Cálcio/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Sequestradores de Radicais Livres/farmacologia , Humanos , Precondicionamento Isquêmico , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Ratos , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Acidente Vascular Cerebral/enzimologia , Acidente Vascular Cerebral/patologia
16.
Mol Biol Cell ; 20(3): 1089-101, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19073886

RESUMO

Epithelial-to-mesenchymal transitions (EMT) are important in renal development, fibrosis, and cancer. Loss of function of the tumor suppressor VHL leads to many features of EMT, and it has been hypothesized that the pivotal mediator is down-regulation of the adherens junction (AJ) protein E-cadherin. Here we show that VHL loss-of-function also has striking effects on the expression of the tight junction (TJ) components occludin and claudin 1 in vitro in VHL-defective clear cell renal cell carcinoma (CCRCC) cells and in vivo in VHL-defective sporadic CCRCCs (compared with normal kidney). Occludin is also down-regulated in premalignant foci in kidneys from patients with germline VHL mutations, consistent with a contribution to CCRCC initiation. Reexpression of E-cadherin was sufficient to restore AJ but not TJ assembly, indicating that the TJ defect is independent of E-cadherin down-regulation. Additional experiments show that activation of hypoxia inducible factor (HIF) contributes to both TJ and AJ abnormalities, thus the VHL/HIF pathway contributes to multiple aspects of the EMT phenotype that are not interdependent. Despite the independent nature of the defects, we show that treatment with the histone deacetylase inhibitor sodium butyrate, which suppresses HIF activation, provides a method for reversing EMT in the context of VHL inactivation.


Assuntos
Células Epiteliais/metabolismo , Rim/metabolismo , Proteínas de Membrana/metabolismo , Junções Íntimas/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/metabolismo , Butiratos/farmacologia , Caderinas/metabolismo , Linhagem Celular Tumoral , Claudina-1 , Regulação para Baixo/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Epitélio/patologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Rim/patologia , Mesoderma/efeitos dos fármacos , Mesoderma/patologia , Mutação/genética , Ocludina , Fenótipo , Fatores de Transcrição da Família Snail , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/patologia , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
17.
J Clin Invest ; 119(1): 125-35, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19065050

RESUMO

Defective insulin secretion in response to glucose is an important component of the beta cell dysfunction seen in type 2 diabetes. As mitochondrial oxidative phosphorylation plays a key role in glucose-stimulated insulin secretion (GSIS), oxygen-sensing pathways may modulate insulin release. The von Hippel-Lindau (VHL) protein controls the degradation of hypoxia-inducible factor (HIF) to coordinate cellular and organismal responses to altered oxygenation. To determine the role of this pathway in controlling glucose-stimulated insulin release from pancreatic beta cells, we generated mice lacking Vhl in pancreatic beta cells (betaVhlKO mice) and mice lacking Vhl in the pancreas (PVhlKO mice). Both mouse strains developed glucose intolerance with impaired insulin secretion. Furthermore, deletion of Vhl in beta cells or the pancreas altered expression of genes involved in beta cell function, including those involved in glucose transport and glycolysis, and isolated betaVhlKO and PVhlKO islets displayed impaired glucose uptake and defective glucose metabolism. The abnormal glucose homeostasis was dependent on upregulation of Hif-1alpha expression, and deletion of Hif1a in Vhl-deficient beta cells restored GSIS. Consistent with this, expression of activated Hif-1alpha in a mouse beta cell line impaired GSIS. These data suggest that VHL/HIF oxygen-sensing mechanisms play a critical role in glucose homeostasis and that activation of this pathway in response to decreased islet oxygenation may contribute to beta cell dysfunction.


Assuntos
Glucose/metabolismo , Homeostase , Células Secretoras de Insulina/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau , Animais , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 2/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxigênio/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
18.
J Mol Med (Berl) ; 86(12): 1329-39, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18813897

RESUMO

Ischaemia followed by reperfusion (I/R) can induce inflammation and injury and is a risk factor for delayed graft function and rejection of transplanted kidneys. Inflammation is regulated by NF-kappaB transcription factors which induce pro-inflammatory molecules in endothelial cells (EC). We examined whether A20, a negative regulator of NF-kappaB, can protect kidneys from I/R injury. To mimic the fluctuations in endothelial oxygenation that occur during I/R we exposed cultured human umbilical vein EC (HUVEC) to hypoxia (1% O(2) for 4 h) followed by re-oxygenation (21% O(2) for 1 h-24 h). We observed transient expression of pro-inflammatory molecules (E-selectin, VCAM-1 and IL-8) and sustained expression of A20 in HUVEC exposed to hypoxia/re-oxygenation. The effect of A20 on endothelial responses to hypoxia/re-oxygenation was assessed. We observed that pre-treatment of HUVEC with an adenovirus containing A20 (Ad-A20) suppressed activation of NF-kappaB and induction of pro-inflammatory molecules by hypoxia/re-oxygenation, whereas a control adenovirus had little or no effect. Thus the induction of A20 may form a negative feedback loop in pro-inflammatory signalling in cells exposed to hypoxia/re-oxygenation. To validate our cell culture experiments we examined the role of A20 in renal responses to I/R. We observed that A20 was induced in rat kidneys exposed to I/R. Moreover, pre-treatment of animals with Ad-A20 significantly reduced acute tubular necrosis, renal expression of VCAM-1 and NF-kappaB activation in response to I/R, whereas pre-treatment with control adenovirus did not. Our observations suggest that A20 maintains physiological homeostasis in kidneys exposed to I/R by protecting them from inflammation and injury.


Assuntos
Proteínas de Ligação a DNA/imunologia , Células Endoteliais/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Rim/imunologia , Proteínas Nucleares/imunologia , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Rim/efeitos dos fármacos , Masculino , NF-kappa B/genética , NF-kappa B/imunologia , Proteínas Nucleares/genética , Ratos , Ratos Endogâmicos F344 , Transfecção , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Fator de Necrose Tumoral alfa/imunologia
20.
Nat Genet ; 40(2): 170-80, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18176562

RESUMO

HIF prolyl hydroxylases (PHD1-3) are oxygen sensors that regulate the stability of the hypoxia-inducible factors (HIFs) in an oxygen-dependent manner. Here, we show that loss of Phd1 lowers oxygen consumption in skeletal muscle by reprogramming glucose metabolism from oxidative to more anaerobic ATP production through activation of a Pparalpha pathway. This metabolic adaptation to oxygen conservation impairs oxidative muscle performance in healthy conditions, but it provides acute protection of myofibers against lethal ischemia. Hypoxia tolerance is not due to HIF-dependent angiogenesis, erythropoiesis or vasodilation, but rather to reduced generation of oxidative stress, which allows Phd1-deficient myofibers to preserve mitochondrial respiration. Hypoxia tolerance relies primarily on Hif-2alpha and was not observed in heterozygous Phd2-deficient or homozygous Phd3-deficient mice. Of medical importance, conditional knockdown of Phd1 also rapidly induces hypoxia tolerance. These findings delineate a new role of Phd1 in hypoxia tolerance and offer new treatment perspectives for disorders characterized by oxidative stress.


Assuntos
Metabolismo Basal , Glucose/metabolismo , Hipóxia/metabolismo , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Pró-Colágeno-Prolina Dioxigenase/deficiência , Animais , Dióxido de Carbono/metabolismo , Isótopos de Carbono/metabolismo , Radioisótopos de Carbono/metabolismo , Embrião de Mamíferos , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Fibroblastos/metabolismo , Glutamatos/metabolismo , Homozigoto , Imuno-Histoquímica , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Modelos Biológicos , Músculo Esquelético/metabolismo , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Consumo de Oxigênio/genética , Consumo de Oxigênio/fisiologia , Pró-Colágeno-Prolina Dioxigenase/genética , Tomografia Computadorizada por Raios X
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