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JCI Insight ; 52019 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-31393855


It has been hypothesized that interleukin-1alpha (IL-1α) is released from damaged cardiomyocytes following myocardial infarction (MI) and activates cardiac fibroblasts via its receptor (IL-1R1) to drive the early stages of cardiac remodeling. This study aimed to definitively test this hypothesis using cell type-specific IL-1α and IL-1R1 knockout (KO) mouse models. A floxed Il1α mouse was created and used to generate a cardiomyocyte-specific IL-1α KO mouse line (MIL1AKO). A tamoxifen-inducible fibroblast-specific IL-1R1 hemizygous KO mouse line (FIL1R1KO) was also generated. Mice underwent experimental MI (permanent left anterior descending coronary artery ligation) and cardiac function was determined 4 weeks later by conductance pressure-volume catheter analysis. Molecular markers of remodeling were evaluated at various time points by real-time RT-PCR and histology. MIL1AKO mice showed no difference in cardiac function or molecular markers of remodeling post-MI compared with littermate controls. In contrast, FIL1R1KO mice showed improved cardiac function and reduced remodeling markers post-MI compared with littermate controls. In conclusion, these data highlight a key role for the IL-1R1/cardiac fibroblast signaling axis in regulating post-MI remodeling and provide support for the continued development of anti-IL-1 therapies for improving cardiac function after MI. Cardiomyocyte-derived IL-1α was not an important contributor to post-MI remodeling in this model.

Nature ; 515(7526): 279-282, 2014 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-25119035


The mechanisms by which physical forces regulate endothelial cells to determine the complexities of vascular structure and function are enigmatic. Studies of sensory neurons have suggested Piezo proteins as subunits of Ca(2+)-permeable non-selective cationic channels for detection of noxious mechanical impact. Here we show Piezo1 (Fam38a) channels as sensors of frictional force (shear stress) and determinants of vascular structure in both development and adult physiology. Global or endothelial-specific disruption of mouse Piezo1 profoundly disturbed the developing vasculature and was embryonic lethal within days of the heart beating. Haploinsufficiency was not lethal but endothelial abnormality was detected in mature vessels. The importance of Piezo1 channels as sensors of blood flow was shown by Piezo1 dependence of shear-stress-evoked ionic current and calcium influx in endothelial cells and the ability of exogenous Piezo1 to confer sensitivity to shear stress on otherwise resistant cells. Downstream of this calcium influx there was protease activation and spatial reorganization of endothelial cells to the polarity of the applied force. The data suggest that Piezo1 channels function as pivotal integrators in vascular biology.

Células Endoteliais/citologia , Células Endoteliais/fisiologia , Fricção , Canais Iônicos/metabolismo , Estresse Mecânico , Animais , Embrião de Mamíferos/irrigação sanguínea , Embrião de Mamíferos/metabolismo , Feminino , Hemorreologia , Masculino , Camundongos
Nucleic Acids Res ; 39(7): 2671-7, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21109536


Cyclin E supports pre-replication complex (pre-RC) assembly, while cyclin A-associated kinase activates DNA synthesis. We show that cyclin E, but not A, is mounted upon the nuclear matrix in sub-nuclear foci in differentiated vertebrate cells, but not in undifferentiated cells or cancer cells. In murine embryonic stem cells, Xenopus embryos and human urothelial cells, cyclin E is recruited to the nuclear matrix as cells differentiate and this can be manipulated in vitro. This suggests that pre-RC assembly becomes spatially restricted as template usage is defined. Furthermore, failure to become restricted may contribute to the plasticity of cancer cells.

Ciclina E/metabolismo , Neoplasias/metabolismo , Matriz Nuclear/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Camundongos , Transporte Proteico , Xenopus laevis
Hum Mutat ; 28(10): 993-1004, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17508423


Cip1-interacting zinc finger protein 1 (CIZ1, also known as CDKN1A-interacting zinc finger protein 1) stimulates initiation of mammalian DNA replication and is normally tethered to the nuclear matrix within DNA replication foci. Here, we show that an alternatively spliced human CIZ1 variant, lacking exon 4 (Delta E4), is misexpressed as a consequence of intronic mutation in Ewing tumor (ET) cell lines. In all ET lines tested, exon 4 is skipped and an upstream mononucleotide repeat element is expanded to contain up to 28 thymidines, compared to 16 in controls. In exon-trap experiments, a 24T variant produced three-fold more exon skipping than a 16T variant, demonstrating a direct effect on splicing. In functional assays, Delta E4 protein retains replication activity, but fails to form subnuclear foci. Furthermore, coexpression of mouse Delta E4 with Ciz1 prevents Ciz1 from localizing appropriately, having a dominant negative effect on foci formation. The data show that conditional exclusion of exon 4 influences the spatial distribution of the Ciz1 protein within the nucleus, and raise the possibility that CIZ1 alternative splicing could influence organized patterns of DNA replication.

Processamento Alternativo , Replicação do DNA , Neoplasias/genética , Proteínas Nucleares/genética , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Éxons , Variação Genética , Humanos , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos
J Cell Sci ; 120(Pt 1): 115-24, 2007 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17182902


Cip1-interacting zinc finger protein 1 (Ciz1) stimulates DNA replication in vitro and is required for mammalian cells to enter S phase. Here, we show that a significant proportion of Ciz1 is retained in nuclear foci following extraction with nuclease and high salt. This suggests that Ciz1 is normally immobilized by interaction with non-chromatin nuclear structures, consistent with the nuclear matrix. Furthermore, matrix-associated Ciz1 foci strikingly colocalize with sites of newly synthesized DNA in S phase nuclei, suggesting that Ciz1 is present in DNA replication factories. Analysis of green fluorescent protein-tagged fragments indicates that nuclear immobilization of Ciz1 is mediated by sequences in its C-terminal third, encoded within amino acids 708-830. Immobilization occurs in a cell-cycle-dependent manner, most probably during late G1 or early S phase, to coincide with its reported point of action. Although C-terminal domains are sufficient for immobilization, N-terminal domains are also required to specify focal organization. Combined with previous work, which showed that the DNA replication activity of Ciz1 is encoded by N-terminal sequences, we suggest that Ciz1 is composed of two functionally distinct domains: an N-terminal replication domain and a C-terminal nuclear matrix anchor. This could contribute to the formation or function of DNA replication factories in mammalian cells.

Replicação do DNA/fisiologia , Matriz Nuclear/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Dedos de Zinco/fisiologia , Animais , Desoxirribonucleases , Proteínas de Fluorescência Verde/genética , Camundongos , Células NIH 3T3 , Matriz Nuclear/genética , Estrutura Terciária de Proteína , Fase S/fisiologia , Sais
Dev Genes Evol ; 213(7): 336-44, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12750886


A subset of autosomal genes undergo genomic imprinting which results in expression from only the paternal or maternal chromosome. While this phenomenon is restricted to mammals and angiosperms, the underlying silencing mechanisms appear to be evolutionarily conserved. A biallelically unmethylated DNaseI hypersensitive region (A6-A4) between the imprinted Igf2 and H19 genes is conserved in humans and mice and functions as a tissue-specific maintenance element for the imprinted growth factor IGF2. In order to analyse A6-A4 for potentially conserved transcriptional maintenance properties, we have generated transgenic Drosophila harbouring the element in a reporter construct. These flies depicted silencing of the reporter genes lacZ and mini -white. The silenced state of the mini -white gene showed variegation and sensitivity to temperature changes. In addition, two members of the conserved Polycomb group, Enhancer of zeste and Posterior sex combs, were needed for repression. Polycomb group proteins are essential for gene silencing during development. Our results indicate that Polycomb group proteins may also be involved in the regulation of mammalian imprinted genes.

Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Inativação Gênica , Impressão Genômica , Fator de Crescimento Insulin-Like II/genética , RNA não Traduzido/genética , Animais , Animais Geneticamente Modificados , Sequência Conservada , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Elementos Facilitadores Genéticos , Genes Reporter , Camundongos , Camundongos Mutantes , Proteínas Musculares/genética , Proteínas Nucleares/metabolismo , Plasmídeos , Complexo Repressor Polycomb 1 , Complexo Repressor Polycomb 2 , RNA Longo não Codificante , Proteínas Repressoras/metabolismo , Temperatura Ambiente , Transcrição Genética , Transgenes