RESUMO
The importance of epigenetic changes in the development of hepatic steatosis is largely unknown. The histone variant macroH2A1 under alternative splicing gives rise to macroH2A1.1 and macroH2A1.2. In this study, we show that the macroH2A1 isoforms play an important role in the regulation of lipid accumulation in hepatocytes. Hepatoma cell line and immortalized human hepatocytes transiently transfected or knocked down with macroH2A1 isoforms were used as in vitro model of fat-induced steatosis. Gene expressions were analyzed by quantitative PCR array and Western blot. Chromatin immunoprecipitation analysis was performed to check the association of histone H3 lysine 27 trimethylation (H3K27me3) and histone H3 lysine 4 trimethylation (H3K4me3) with the promoter of lipogenic genes. Livers from knockout mice that are resistant to lipid deposition despite a high-fat diet were used for histopathology. We found that macroH2A1.2 is regulated by fat uptake and that its overexpression caused an increase in lipid uptake, triglycerides, and lipogenic genes compared with macroH2A1.1. This suggests that macroH2A1.2 is important for lipid uptake, whereas macroH2A1.1 was found to be protective. The result was supported by a high positivity for macroH2A1.1 in knockout mice for genes targeted by macroH2A1 (Atp5a1 and Fam73b), that under a high-fat diet presented minimal lipidosis. Moreover, macroH2A1 isoforms differentially regulate the expression of lipogenic genes by modulating the association of the active (H3K4me3) and repressive (H3K27me3) histone marks on their promoters. This study underlines the importance of the replacement of noncanonical histones in the regulation of genes involved in lipid metabolism in the progression of steatosis.
Assuntos
Biomarcadores/metabolismo , Carcinoma Hepatocelular/patologia , Dieta Hiperlipídica/efeitos adversos , Epigenômica , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Hepatócitos/patologia , Histonas/metabolismo , Animais , Western Blotting , Carcinoma Hepatocelular/metabolismo , Células Cultivadas , Imunoprecipitação da Cromatina , Fígado Gorduroso/etiologia , Feminino , Imunofluorescência , Perfilação da Expressão Gênica , Hepatócitos/metabolismo , Histonas/genética , Humanos , Técnicas Imunoenzimáticas , Peroxidação de Lipídeos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , ATPases Mitocondriais Próton-Translocadoras/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Aberrant Hedgehog (Hh) signalling has been reported in a number of malignancies, particularly basal cell carcinoma (BCC) of the skin. Clinical trials of Hh inhibitors are underway in many cancers, and these have produced significant clinical benefit in BCC patients, although regrowth of new, or clinically aggressive, variants, as well as development of secondary malignancies, has been reported. αvß6 integrin is expressed in many cancers, where it has been shown to correlate with an aggressive tumour phenotype and poor prognosis. We have previously reported αvß6 up-regulation in aggressive, morphoeic BCC variants, where it modulates the stromal response and induces invasion. To examine a possible link between Hh and αvß6 function, we generated BCC models, overexpressing Gli1 in immortalized keratinocytes (NTert1, HaCaT). Unexpectedly, we found that suppressing Gli1 significantly increased αvß6 expression. This promoted tumour cell motility and also stromal myofibroblast differentiation through integrin-dependent TGF-ß1 activation. Gli1 inhibited αvß6 expression by suppressing TGF-ß1-induced Smad2/3 activation, blocking a positive feedback loop maintaining high αvß6 levels. A similar mechanism was observed in AsPC1 pancreatic cancer cells expressing endogenous Gli1, suggesting a common mechanism across tumour types. In vitro findings were supported using human clinical samples, where we showed an inverse correlation between αvß6 and Gli1 expression in different BCC subtypes and pancreatic cancers. In summary, we show that expression of Gli1 and αvß6 inversely correlates in tumours in vivo, and Hh targeting up-regulates TGF-ß1/Smad2/3-dependent αvß6 expression, promoting pro-tumourigenic cell functions in vitro. These results have potential clinical significance, given the reported recurrence of BCC variants and secondary malignancies in patients treated by Hh targeting.
Assuntos
Antígenos de Neoplasias/metabolismo , Carcinoma Basocelular/metabolismo , Transformação Celular Neoplásica/metabolismo , Proteínas Hedgehog/metabolismo , Integrinas/metabolismo , Neoplasias Pancreáticas/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Fatores de Transcrição/metabolismo , Antígenos de Neoplasias/genética , Carcinoma Basocelular/genética , Carcinoma Basocelular/patologia , Linhagem Celular , Movimento Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Técnicas de Cocultura , Regulação para Baixo , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Integrinas/genética , Queratinócitos/metabolismo , Queratinócitos/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Interferência de RNA , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fatores de Transcrição/genética , Transfecção , Fator de Crescimento Transformador beta1/metabolismo , Proteína GLI1 em Dedos de ZincoRESUMO
Polycomb group proteins are essential regulators of stem cell function during embryonic development and in adult tissue homeostasis. Bmi1, a key component of the Polycomb Repressive Complex 1, is highly expressed in undifferentiated neural stem cells (NSC) as well as in several human cancers including high-grade gliomas--highly aggressive brain tumors. Using a conditional gene activation approach in mice, we show that overexpression of Bmi1 induces repressive epigenetic regulation of the promoter of Survivin, a well-characterized antiapoptotic protein. This phenomenon is cell type-specific and it leads to apoptotic death of progenitor cells exclusively upon commitment toward a neuronal fate. Moreover, we show that this is triggered by increased oxidative stress-induced DNA damage. In contrast, undifferentiated NSC as well as glioma-initiating cells display an open chromatin configuration at the Survivin promoter and do not undergo apoptotic death. These findings raise the possibility that normal and neoplastic stem cells depend on the same mechanism for surviving the hyperproliferative state induced by increased Bmi1 expression.
Assuntos
Glioma/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Células-Tronco Neurais/fisiologia , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Animais , Neoplasias Encefálicas/metabolismo , Diferenciação Celular , Proliferação de Células , Córtex Cerebral/citologia , Cromatina/metabolismo , Dano ao DNA , Metilação de DNA , Epigênese Genética , Perfilação da Expressão Gênica , Glioma/genética , Proteínas Inibidoras de Apoptose/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Camundongos , Neurogênese , Estresse Oxidativo , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Survivina , UbiquitinaçãoRESUMO
The developmental protein Numb is a major determinant of binary cell fates. It is also required for the differentiation of cerebellar granule cell progenitors (GCPs) at a stage of development responsive to the morphogenic glycoprotein Hedehog. Hedgehog signalling is crucial for the physiological maintenance and self-renewal of neural stem cells and its deregulation is responsible for their progression towards tumorigenesis. The mechanisms that inhibit this pathway during the differentiation stage are poorly understood. Here, we identify Numb as a Hedgehog-pathway inhibitor that is downregulated in early GCPs and GCP-derived cancer cells. We demonstrate that the Hedgehog transcription factor Gli1 is targeted by Numb for Itch-dependent ubiquitination, which suppresses Hedgehog signals, thus arresting growth and promoting cell differentiation. This novel Numb-dependent regulatory loop may limit the extent and duration of Hedgehog signalling during neural-progenitor differentiation, and its subversion may be a relevant event in brain tumorigenesis.
Assuntos
Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/metabolismo , Animais , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Cerebelo/citologia , Cerebelo/patologia , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Fatores de Transcrição Kruppel-Like/genética , Meduloblastoma/genética , Camundongos , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Fatores de Transcrição/genética , Proteína GLI1 em Dedos de ZincoRESUMO
Histone deacetylases (HDACs) play a crucial role in several physiological and pathological cell functions, including cell development and cancer, by deacetylating both histones and others proteins. HDACs belong to a large family of enzymes including Class I, II and IV as well as Class III or sirtuins subfamilies, that undergo a complex transcriptional and post-translational regulation. In current years, antitumor therapy is attempting to exploit several chemical classes of inhibitors that target HDACs, frequently reported to be misregulated in cancer. Nevertheless, the identity of gene products directly involved in tumorigenesis and preventing HDAC misregulation in cancer is still poorly understood. Recent evidence has demonstrated that the tumor suppressors HIC1 and DBC1 induce direct repression of Sirt1 function, whereas Chfr and REN(KCTD11/KASH family) downregulate HDAC1, by inducing its ubiquitin-dependent degradation. Loss of these gene products leads to imbalanced enhancement of HDAC activity and subsequently to oncogenesis. All these genes are frequently deleted or silenced in human cancers, highlighting the role of endogenous HDAC inhibitors to counteracts HDAC-mediated tumorigenesis. Thus, endogenous HDAC inhibitors represent a promising class of "antitumor agents" thanks to which oncogenic addiction pathways may be selectively therapeutically targeted.
Assuntos
Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Animais , Humanos , Neoplasias/patologiaRESUMO
During the early development of the cerebellum, a burst of granule cell progenitor (GCP) proliferation occurs in the outer external granule layer (EGL), which is sustained mainly by Purkinje cell-derived Sonic Hedgehog (Shh). Shh response is interrupted once GCPs move into the inner EGL, where granule progenitors withdraw proliferation and start differentiating and migrating toward the internal granule layer (IGL). Failure to interrupt Shh signals results in uncoordinated proliferation and differentiation of GCPs and eventually leads to malignancy (i.e., medulloblastoma). The Shh inhibitory mechanisms that are responsible for GCP growth arrest and differentiation remain unclear. Here we report that REN, a putative tumor suppressor frequently deleted in human medulloblastoma, is expressed to a higher extent in nonproliferating inner EGL and IGL granule cells than in highly proliferating outer EGL cells. Accordingly, upregulated REN expression occurs along GCP differentiation in vitro, and, in turn, REN overexpression promotes growth arrest and increases the proportion of p27/Kip1+ GCPs. REN also impairs both Gli2-dependent gene transcription and Shh-enhanced expression of the target Gli1 mRNA, thus antagonizing the Shh-induced effects on the proliferation and differentiation of cultured GCPs. Conversely, REN functional knock-down impairs Hedgehog antagonism and differentiation and sustains the proliferation of GCPs. Finally, REN enhances caspase-3 activation and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling apoptotic GCP numbers; therefore, the pattern of REN expression, its activity, and its antagonism on the Hedgehog pathway suggest that this gene may represent a restraint of Shh signaling at the outer to inner EGL GCP transitions. Medulloblastoma-associated REN loss of function might withdraw such a limiting signal for immature cell expansion, thus favoring tumorigenesis.
Assuntos
Apoptose/fisiologia , Divisão Celular/fisiologia , Cerebelo/fisiologia , Bulbo/fisiologia , Proteínas do Tecido Nervoso/genética , Transativadores/antagonistas & inibidores , Animais , Caspase 3 , Caspases/metabolismo , Técnicas de Cultura de Células , Proteínas de Ciclo Celular , Diferenciação Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p27/genética , Ativação Enzimática , Regulação da Expressão Gênica/fisiologia , Proteínas Hedgehog , Camundongos , Proteínas Recombinantes/metabolismo , TransferasesRESUMO
Non-alcoholic fatty liver disease (NAFLD), an accumulation of intra-hepatic triglycerides that is often considered the hepatic manifestation of insulin resistance, is the most common cause of chronic liver disease in the Western countries with up to one third of the population affected. NAFLD is a spectrum of disturbances that encompasses various degrees of liver damage ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). NASH is characterized by hepatocellular injury/inflammation with or without fibrosis. The individuals with NAFLD develop NASH in 10% of the cases, and are also at risk of developing hepatocellular carcinoma (HCC). Epigenetic mechanisms of nuclear chromatin remodeling, such as DNA methylation, post-translational modifications of histones, and incorporation of histone variants into the chromatin are increasingly recognized as crucial factors in the pathophysiology of NAFLD. NAFLD is often accompanied by oxidative stress: reactive oxygen species (ROS) are implicated in altered reduction/oxidation (redox) reactions that attack cellular macromolecules and are detected in the liver of patients and animal models of NAFLD. In this review, we summarize recent knowledge advancements in the hepatic epigenetic and redox mechanisms, and their possible links, involved in the pathogenesis and treatment of NAFLD.
Assuntos
Epigênese Genética , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Homeostase , Doenças Cardiovasculares/complicações , Doenças Cardiovasculares/genética , Fígado Gorduroso/complicações , Humanos , Hepatopatia Gordurosa não Alcoólica , Oxirredução , Estresse OxidativoRESUMO
BACKGROUND: Hepatitis C virus (HCV) infects approximately 3% of the world population and is the leading cause of liver disease, impacting hepatocyte metabolism, depending on virus genotype. Hepatic metabolic functions show rhythmic fluctuations with 24-h periodicity (circadian), driven by molecular clockworks ticking through translational-transcriptional feedback loops, operated by a set of genes, called clock genes, encoding circadian proteins. Disruption of biologic clocks is implicated in a variety of disorders including fatty liver disease, obesity and diabetes. The relation between HCV replication and the circadian clock is unknown. METHODS: We investigated the relationship between HCV core infection and viral replication and the expression of clock genes (Rev-Erbα, Rorα, ARNTL, ARNTL2, CLOCK, PER1, PER2, PER3, CRY1 and CRY2) in two cellular models, the Huh-7 cells transiently expressing the HCV core protein genotypes 1b or 3a, and the OR6 cells stably harboring the full-length hepatitis C genotype 1b replicon, and in human liver biopsies, using qRT-PCR, immunoblotting, luciferase assays and immunohistochemistry. RESULTS: In Huh-7 cells expressing the HCV core protein genotype 1b, but not 3a, and in OR6 cells, transcript and protein levels of PER2 and CRY2 were downregulated. Overexpression of PER2 led to a consistent decrease in HCV RNA replicating levels and restoration of altered expression pattern of a subset of interferon stimulated genes (ISGs) in OR6 cells. Furthermore, in liver biopsies from HCV genotype 1b infected patients, PER2 was markedly localized to the nucleus, consistent with an auto-inhibitory transcriptional feedback loop. CONCLUSIONS: HCV can modulate hepatic clock gene machinery, and the circadian protein PER2 counteracts viral replication. Further understanding of circadian regulation of HCV replication and rhythmic patterns of host-hosted relationship may improve the effectiveness of HCV antiviral therapy. This would extend to hepatic viral infections the current spectrum of chronotherapies, implemented to treat metabolic, immune related and neoplastic disease.
Assuntos
Hepacivirus/fisiologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Proteínas Circadianas Period/antagonistas & inibidores , Proteínas Circadianas Period/metabolismo , Replicação Viral , Adulto , Idoso , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Genoma Viral/genética , Genótipo , Hepacivirus/genética , Hepatite C/genética , Hepatite C/virologia , Humanos , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , Masculino , Pessoa de Meia-Idade , Proteínas Circadianas Period/genética , RNA Viral/biossíntese , Proteínas do Core Viral/genéticaRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Prevention and risk reduction are important and the identification of specific biomarkers for early diagnosis of HCC represents an active field of research. Increasing evidence indicates that fat accumulation in the liver, defined as hepatosteatosis, is an independent and strong risk factor for developing an HCC. MacroH2A1, a histone protein generally associated with the repressed regions of chromosomes, is involved in hepatic lipid metabolism and is present in two alternative spliced isoforms, macroH2A1.1 and macroH2A1.2. These isoforms have been shown to predict lung and colon cancer recurrence but to our knowledge, their role in fatty-liver associated HCC has not been investigated previously. METHODS: We examined macroH2A1.1 and macroH2A1.2 protein expression levels in the liver of two murine models of fat-associated HCC, the high fat diet/diethylnistrosamine (DEN) and the phosphatase and tensin homolog (PTEN) liver specific knock-out (KO) mouse, and in human liver samples of subjects with steatosis or HCC, using immunoblotting and immunohistochemistry. RESULTS: Protein levels for both macroH2A1 isoforms were massively upregulated in HCC, whereas macroH2A1.2 was specifically upregulated in steatosis. In addition, examination of human liver samples showed a significant difference (p<0.01) in number of positive nuclei in HCC (100% of tumor cells positive for either macroH2A1.1 or macroH2A1.2), when compared to steatosis (<2% of hepatocytes positive for either isoform). The steatotic areas flanking the tumors were highly immunopositive for macroH2A1.1 and macroH2A1.2. CONCLUSIONS: These data obtained in mice and humans suggest that both macroH2A1 isoforms may play a role in HCC pathogenesis and moreover may be considered as novel diagnostic markers for human HCC.
Assuntos
Carcinoma Hepatocelular/genética , Fígado Gorduroso/genética , Histonas/genética , Neoplasias Hepáticas/genética , Animais , Biomarcadores/metabolismo , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Dieta Hiperlipídica , Dietilnitrosamina , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/complicações , Fígado Gorduroso/metabolismo , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Hepatócitos/patologia , Histonas/metabolismo , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Microambiente Tumoral/genéticaRESUMO
The family of human proteins containing a potassium channel tetramerization domain (KCTD) includes 21 members whose function is largely unknown. Recent reports have however suggested that these proteins are implicated in very important biological processes. KCTD11/REN, the best-characterized member of the family to date, plays a crucial role in the ubiquitination of HDAC1 by acting, in complex with Cullin3, as an E3 ubiquitin ligase. By combining bioinformatics and mutagenesis analyses, here we show that the protein is expressed in two alternative variants: a short previously characterized form (sKCTD11) composed by 232 amino acids and a longer variant (lKCTD11) which contains an N-terminal extension of 39 residues. Interestingly, we demonstrate that lKCTD11 starts with a non-canonical AUU codon. Although both sKCTD11 and lKCTD11 bear a POZ/BTB domain in their N-terminal region, this domain is complete only in the long form. Indeed, sKCTD11 presents an incomplete POZ/BTB domain. Nonetheless, sKCTD11 is still able to bind Cul3, although to much lesser extent than lKCTD11, and to perform its biological activity. The heterologous expression of sKCTD11 and lKCTD11 and their individual domains in Escherichia coli yielded soluble products as fusion proteins only for the longer form. In contrast to the closely related KCTD5 which is pentameric, the characterization of both lKCTD11 and its POZ/BTB domain by gel filtration and light scattering indicates that the protein likely forms stable tetramers. In line with this result, experiments conducted in cells show that the active protein is not monomeric. Based on these findings, homology-based models were built for lKCTD11 BTB and for its complex with Cul3. These analyses indicate that a stable lKCTD11 BTB-Cul3 three-dimensional model with a 4:4 stoichiometry can be generated. Moreover, these models provide insights into the determinants of the tetramer stability and into the regions involved in lKCTD11-Cul3 recognition.
Assuntos
Canais de Potássio/química , Canais de Potássio/genética , Ubiquitina-Proteína Ligases/química , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Ciclo Celular , Proteínas Culina/química , Proteínas Culina/genética , Células HEK293 , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/métodos , Ligação Proteica/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transferases , Ubiquitina/químicaRESUMO
Medulloblastoma is the most common pediatric malignant brain tumor, arising from aberrant cerebellar precursors' development, a process mainly controlled by Hedgehog (Hh) signaling pathway. Histone deacetylase HDAC1 has been recently shown to modulate Hh signaling, deacetylating its effectors Gli1/2 and enhancing their transcriptional activity. Therefore, HDAC may represent a potential therapeutic target for Hh-dependent tumors, but still little information is available on the physiological mechanisms of HDAC regulation. The putative tumor suppressor REN(KCTD11) acts through ubiquitination-dependent degradation of HDAC1, thereby affecting Hh activity and medulloblastoma growth. We identify and characterize here two REN(KCTD11) homologues, defining a new family of proteins named KCASH, as "KCTD containing, Cullin3 adaptor, suppressor of Hedgehog." Indeed, the novel genes (KCASH2(KCTD21) and KCASH3(KCTD6)) share with REN(KCTD11) a number of features, such as a BTB domain required for the formation of a Cullin3 ubiquitin ligase complex and HDAC1 ubiquitination and degradation capability, suppressing the acetylation-dependent Hh/Gli signaling. Expression of KCASH2 and -3 is observed in cerebellum, whereas epigenetic silencing and allelic deletion are observed in human medulloblastoma. Rescuing KCASHs expression reduces the Hedgehog-dependent medulloblastoma growth, suggesting that loss of members of this novel family of native HDAC inhibitors is crucial in sustaining Hh pathway-mediated tumorigenesis. Accordingly, they might represent a promising class of endogenous "agents" through which this pathway may be targeted.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Neoplasias Cerebelares/genética , Proteínas Hedgehog/antagonistas & inibidores , Meduloblastoma/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Proteínas de Ciclo Celular , Células Cultivadas , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Clonagem Molecular , Proteínas Culina/metabolismo , Feminino , Idade Gestacional , Proteínas Hedgehog/metabolismo , Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/metabolismo , Humanos , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Modelos Biológicos , Canais de Potássio/química , Gravidez , Homologia de Sequência de Aminoácidos , Transferases , Adulto JovemRESUMO
Hedgehog signalling is crucial for development and is deregulated in several tumours, including medulloblastoma. Regulation of the transcriptional activity of Gli (glioma-associated oncogene) proteins, effectors of the Hedgehog pathway, is poorly understood. We show here that Gli1 and Gli2 are acetylated proteins and that their HDAC-mediated deacetylation promotes transcriptional activation and sustains a positive autoregulatory loop through Hedgehog-induced upregulation of HDAC1. This mechanism is turned off by HDAC1 degradation through an E3 ubiquitin ligase complex formed by Cullin3 and REN, a Gli antagonist lost in human medulloblastoma. Whereas high HDAC1 and low REN expression in neural progenitors and medulloblastomas correlates with active Hedgehog signalling, loss of HDAC activity suppresses Hedgehog-dependent growth of neural progenitors and tumour cells. Consistent with this, abrogation of Gli1 acetylation enhances cellular proliferation and transformation. These data identify an integrated HDAC- and ubiquitin-mediated circuitry, where acetylation of Gli proteins functions as an unexpected key transcriptional checkpoint of Hedgehog signalling.
Assuntos
Proteínas Culina/metabolismo , Proteínas Hedgehog/metabolismo , Histona Desacetilases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Acetilação , Animais , Proteínas de Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Imunoprecipitação da Cromatina , Proteínas Culina/genética , Eletroforese em Gel de Poliacrilamida , Proteínas Hedgehog/genética , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Histona Desacetilases/genética , Humanos , Immunoblotting , Imuno-Histoquímica , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Meduloblastoma/genética , Meduloblastoma/metabolismo , Camundongos , Células NIH 3T3 , Proteínas do Tecido Nervoso/genética , Proteínas Oncogênicas/genética , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Espectrometria de Massas por Ionização por Electrospray , Transativadores/genética , Transferases , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de ZincoRESUMO
Medulloblastoma (MB) is the most common malignant brain tumor of childhood arising from deregulated cerebellar development. Sonic Hedgehog (Shh) pathway plays a critical role in cerebellar development and its aberrant expression has been identified in MB. Gene expression profiling of cerebella from 1- to 14-day-old mice unveiled a cluster of genes whose expression correlates with the levels of Hedgehog (HH) activity. From this cluster, we identified Insm1 and Nhlh1/NSCL1 as novel HH targets induced by Shh treatment in cultured cerebellar granule cell progenitors. Nhlh1 promoter was found to be bound and activated by Gli1 transcription factor. Remarkably, the expression of these genes is also upregulated in mouse and human HH-dependent MBs, suggesting that they may be either a part of the HH-induced tumorigenic process or a specific trait of HH-dependent tumor cells.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Neoplasias Cerebelares/genética , Cerebelo/crescimento & desenvolvimento , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/metabolismo , Meduloblastoma/genética , Organogênese/genética , Fatores de Transcrição/genética , Animais , Sequência de Bases , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/farmacologia , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Oncogênicas/metabolismo , Proteínas Repressoras , Transativadores/metabolismo , Células Tumorais Cultivadas , Proteína GLI1 em Dedos de ZincoRESUMO
Hedgehog pathway is crucial for the maintenance and self-renewal of neural stem cells and for tumorigenesis. Hedgehog signaling is limited by multiple E3 ubiquitin ligases that process the downstream transcription factors Gli. Cullin family-based ubiquitination results in either Cullin1-Slimb/betaTrCP- or Cullin3-HIB/Roadkill/SPOP-dependent proteolytic processing or degradation of Drosophila Cubitus interruptus or mammalian Gli proteins. We have recently identified Itch as an additional HECT family E3 ligase, able to ubiquitinate and degrade Gli1. A functional link with the influence of Hedgehog signaling on cell development and tumorigenesis is suggested by the identification of Numb as a promoter of such an Itch-dependent ubiquitination process that leads to Gli1 degradation, thus suppressing its transcriptional function. Numb is an evolutionary conserved developmental protein that, during progenitor division, asymmetrically segregates to daughter cells thereby determining distinct binary cell fates. Numb is downregulated in cerebellar progenitors and their malignant derivatives (i.e. medulloblastoma cells). Furthermore, Numb has anti-proliferative and pro-differentiation effects on both cerebellar progenitors and medulloblastoma cells, due to its suppression of functional Gli1. These findings unveil a novel Numb/Itch-dependent regulatory loop that limits the extent and duration of Hedgehog signaling during neural progenitor differentiation. Its subversion emerges as a relevant event in brain tumorigenesis.