RESUMO
In a study in the December 15, 2011, issue of Genes & Development, Valenta and colleagues (pp. 2631-2643) constructed a series of ß-catenin mutants that allowed them to separate ß-catenin's activity as a mediator of Wnt signaling from its activity as cell adhesion component. In doing so, they uncovered some surprising properties of Wnt signaling.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , beta Catenina/genética , beta Catenina/metabolismo , AnimaisRESUMO
Danon disease is a familial cardiomyopathy associated with impaired autophagy due to mutations in the gene encoding lysosomal-associated membrane protein type 2 (LAMP-2). Emerging evidence has highlighted the importance of autophagy in regulating cardiomyocyte bioenergetics, function, and survival. However, the mechanisms responsible for cellular dysfunction and death in cardiomyocytes with impaired autophagic flux remain unclear. To investigate the molecular mechanisms responsible for Danon disease, we created induced pluripotent stem cells (iPSCs) from two patients with different LAMP-2 mutations. Danon iPSC-derived cardiomyocytes (iPSC-CMs) exhibited impaired autophagic flux and key features of heart failure such as increased cell size, increased expression of natriuretic peptides, and abnormal calcium handling compared to control iPSC-CMs. Additionally, Danon iPSC-CMs demonstrated excessive amounts of mitochondrial oxidative stress and apoptosis. Using the sulfhydryl antioxidant N-acetylcysteine to scavenge free radicals resulted in a significant reduction in apoptotic cell death in Danon iPSC-CMs. In summary, we have modeled Danon disease using human iPSC-CMs from patients with mutations in LAMP-2, allowing us to gain mechanistic insight into the pathogenesis of this disease. We demonstrate that LAMP-2 deficiency leads to an impairment in autophagic flux, which results in excessive oxidative stress, and subsequent cardiomyocyte apoptosis. Scavenging excessive free radicals with antioxidants may be beneficial for patients with Danon disease. In vivo studies will be necessary to validate this new treatment strategy.
Assuntos
Doença de Depósito de Glicogênio Tipo IIb/genética , Insuficiência Cardíaca/genética , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/genética , Apoptose , Autofagia , Doença de Depósito de Glicogênio Tipo IIb/patologia , Insuficiência Cardíaca/patologia , Humanos , Células-Tronco Pluripotentes InduzidasRESUMO
MYCN activation, mainly by gene amplification, is one of the most frequent molecular events in neuroblastoma (NB) oncogenesis, and is associated with increased malignancy and decreased neuronal differentiation propensity. The frequency of concomitant loss of heterozygosity at the 1p36.3 locus, which harbours the p53 anti-oncogene homologue TP73, indicates that MYCN and p73 alterations may cooperate in the pathogenesis of NB. We have previously shown that p73 isoforms are deregulated in NB tumours and that TAp73 co-operates synergistically with p53 for apoptosis of NB cells, whereas DeltaNp73 activates the expression of neuronal differentiation genes such as BTG2. Herein, using both ectopic expression and RNA interference-mediated silencing of p73 in MYCN amplified NB cells, we show that p73alpha isoforms inhibit MYCN expression at both transcript and protein levels, in spite of transactivator effects on MYCN promoter. To explain this paradox, we found that TAp73alpha exerts negative post-transcriptional effects leading to reduced MYCN mRNA stability. RNA immunoprecipitation experiments suggest that this dominant inhibitory post-transcriptional effect could be due to an interaction between the p73 protein and MYCN mRNA, a hypothesis also raised for the regulation of certain genes by the p53 protein.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/genética , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular Tumoral , Genes myc , Humanos , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/metabolismo , Proteínas Nucleares/biossíntese , Proteínas Oncogênicas/biossíntese , Proteínas Oncogênicas/metabolismo , Regiões Promotoras Genéticas , Isoformas de Proteínas/metabolismo , Ativação Transcricional , Proteína Tumoral p73RESUMO
WNT5A, a member of the WNT family of secreted lipid-modified glycoproteins, is a critical regulator of a host of developmental processes, including limb formation, lung morphogenesis, intestinal elongation and mammary gland development. Altered WNT5A expression has been associated with a number of cancers. Interestingly, in certain types of cancers, such as hematological malignancies and colorectal carcinoma, WNT5A is inactivated and exerts a tumor suppressive function, while in other cancers, such as melanoma and gastric carcinoma, WNT5A is overexpressed and promotes tumor progression. The mechanism by which WNT5A achieves these distinct activities in cancers is poorly understood. Here, we provide evidence that the WNT5A gene produces two protein isoforms, WNT5A-long (WNT5A-L) and WNT5A-short (WNT5A-S). Amino-terminal sequencing and a WNT5A-L specific antibody demonstrate that the mature and secreted isoforms are distinct, with WNT5A-L carrying an additional 18 N-terminal amino acids. Biochemical analysis indicates that both purified proteins are similar with respect to their stability, hydrophobicity and WNT/ß-catenin signaling activity. Nonetheless, modulation of these two WNT5A isoforms, either through ectopic expression or knockdown, demonstrates that they exert distinct activities in cancer cell lines: while WNT5A-L inhibits proliferation of tumor cell lines, WNT5A-S promotes their growth. Finally, we show that expression of these two WNT5A isoforms is altered in breast and cervix carcinomas, as well as in the most aggressive neuroblastoma tumors. In these cancers, WNT5A-L is frequently down-regulated, whereas WNT5A-S is found overexpressed in a significant fraction of tumors. Altogether, our study provides evidence that the distinct activities of WNT5A in cancer can be attributed to the production of two WNT5A isoforms.
Assuntos
Proteínas Proto-Oncogênicas/genética , Proteínas Wnt/genética , Processamento Alternativo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Proliferação de Células , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Ordem dos Genes , Humanos , Dados de Sequência Molecular , Isoformas de Proteínas , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Proteínas Wnt/química , Proteínas Wnt/metabolismo , Proteína Wnt-5a , beta Catenina/metabolismoRESUMO
Functional and mechanistic studies of Wnt signaling have been severely hindered by the inaccessibility of bioactive proteins. To overcome this long-standing barrier, we engineered and characterized a panel of Chinese hamster ovary (CHO) cell lines with inducible transgenes encoding tagged and un-tagged human WNT1, WNT3A, WNT5A, WNT7A, WNT11, WNT16 or the soluble Wnt antagonist Fzd8CRD, all integrated into an identical genomic locus. Using a quantitative real-time bioluminescence assay, we show that cells expressing WNT1, 3A and 7A stimulate Wnt/beta-catenin reporter activity, while the other WNT expressing cell lines interfere with this activation. Additionally, in contrast to WNT3A, WNT1 only exhibits activity when cell-associated, and thus only signals to neighboring cells. The reporter assay also revealed a rapid decline of Wnt activity at 37°C, indicating that Wnt activity is highly labile. These engineered cell lines will reduce the cost of making and purifying Wnt proteins and serve as a continuous, reliable and regulatable source of Wnts to research laboratories around the world.
Assuntos
Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Animais , Células CHO , Cricetinae , Expressão Gênica , Regulação da Expressão Gênica , Genes Reporter , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Wnt/isolamento & purificaçãoRESUMO
The developmental and oncogenic roles of MYC proteins are well established, but the transcriptional targets mediating their functions remain elusive. Using small interfering RNA-mediated knockdown in breast and cervix carcinoma cell lines, which overexpress c-MYC, we show that c-MYC independently controls metabolism and cell proliferation, and can, depending on the cells, promote or inhibit migration. We identified new c-MYC target genes in these cell lines, and show that selective regulation of some targets correlates with the phenotypic responses of these different cell lines to c-MYC depletion. Notably, we show that a positive regulation of the WNT signalling pathway contributes to c-MYC pro-mitogenic effects in breast and cervix carcinoma cells. We also show that repression of CCL5/RANTES accounts for c-MYC anti-migratory effects in specific breast cancer cells. Our combined genomic and phenotypic analysis indicates that c-MYC functions are cellular-context-dependent and that selectively regulated genes are responsible for its differential properties.