RESUMO
Hutchinson-Gilford progeria (HGPS) is a premature ageing syndrome caused by a mutation in LMNA, resulting in a truncated form of lamin A called progerin. Progerin triggers loss of the heterochromatic marker H3K27me3, and premature senescence, which is prevented by telomerase. However, the mechanism how progerin causes disease remains unclear. Here, we describe an inducible cellular system to model HGPS and find that LAP2α (lamina-associated polypeptide-α) interacts with lamin A, while its interaction with progerin is significantly reduced. Super-resolution microscopy revealed that over 50% of telomeres localize to the lamina and that LAP2α association with telomeres is impaired in HGPS. This impaired interaction is central to HGPS since increasing LAP2α levels rescues progerin-induced proliferation defects and loss of H3K27me3, whereas lowering LAP2 levels exacerbates progerin-induced defects. These findings provide novel insights into the pathophysiology underlying HGPS, and how the nuclear lamina regulates proliferation and chromatin organization.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Lamina Tipo A/metabolismo , Proteínas de Membrana/metabolismo , Progéria/patologia , Telômero/metabolismo , Humanos , Microscopia , Ligação ProteicaRESUMO
The establishment of a receptive uterus is the prime requirement for embryo implantation. In mice, the E2-induced cytokine leukemia inhibitory factor (LIF) is essential in switching the uterine luminal epithelium (LE) from a nonreceptive to a receptive state. Here we define the LIF-mediated switch using array analysis and informatics to identify LIF-induced changes in gene expression and annotated signaling pathways specific to the LE. We compare gene expression profiles at 0, 1, 3, and 6 h, following LIF treatment. During the first hour, the JAK-STAT signaling pathway is activated and the expression of 54 genes declines, primarily affecting LE cytoskeletal and chromatin organization as well as a transient reduction in the progesterone, TGFbetaR1, and ACVR1 receptors. Simultaneously 256 genes increase expression, of which 42 are transcription factors, including Sox, Kfl, Hes, Hey, and Hox families. Within 3 h, the expression of 3987 genes belonging to more than 25 biological process pathways was altered. We confirmed the mRNA and protein distribution of key genes from 10 pathways, including the Igf-1, Vegf, Toll-like receptors, actin cytoskeleton, ephrin, integrins, TGFbeta, Wnt, and Notch pathways. These data identify novel LIF-activated pathways in the LE and define the molecular basis between the refractory and receptive uterine phases. More broadly, these findings highlight the staggering capacity of a single cytokine to induce a dynamic and complex network of changes in a simple epithelium essential to mammalian reproduction and provide a basis for identifying new routes to regulating female reproduction.
Assuntos
Implantação do Embrião , Endométrio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fator Inibidor de Leucemia/metabolismo , Transdução de Sinais , Animais , Western Blotting , Montagem e Desmontagem da Cromatina , Biologia Computacional , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Dinoprostona/administração & dosagem , Dinoprostona/metabolismo , Endométrio/citologia , Endométrio/enzimologia , Feminino , Perfilação da Expressão Gênica , Injeções Intraperitoneais , Fator Inibidor de Leucemia/administração & dosagem , Fator Inibidor de Leucemia/genética , Camundongos Endogâmicos C3H , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Human LMNA gene mutations result in laminopathies that include Emery-Dreifuss muscular dystrophy (AD-EDMD) and Hutchinson-Gilford progeria, the premature aging syndrome (HGPS). The Lmna null (Lmna(-/-)) and progeroid LmnaΔ9 mutant mice are models for AD-EDMD and HGPS, respectively. Both animals develop severe tissue pathologies with abbreviated life spans. Like HGPS cells, Lmna(-/-) and LmnaΔ9 fibroblasts have typically misshapen nuclei. Unexpectedly, Lmna(-/-) or LmnaΔ9 mice that are also deficient for the inner nuclear membrane protein Sun1 show markedly reduced tissue pathologies and enhanced longevity. Concordantly, reduction of SUN1 overaccumulation in LMNA mutant fibroblasts and in cells derived from HGPS patients corrected nuclear defects and cellular senescence. Collectively, these findings implicate Sun1 protein accumulation as a common pathogenic event in Lmna(-/-), LmnaΔ9, and HGPS disorders.
Assuntos
Lamina Tipo A/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Distrofia Muscular de Emery-Dreifuss/metabolismo , Distrofia Muscular de Emery-Dreifuss/patologia , Proteínas Nucleares/metabolismo , Progéria/metabolismo , Animais , Linhagem Celular , Senescência Celular , Modelos Animais de Doenças , Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Progéria/patologiaRESUMO
The segmental premature aging disease Hutchinson-Gilford Progeria syndrome (HGPS) is caused by a truncated and farnesylated form of Lamin A called progerin. HGPS affects mesenchymal lineages, including the skeletal system, dermis, and vascular smooth muscle (VSMC). To understand the underlying molecular pathology of HGPS, we derived induced pluripotent stem cells (iPSCs) from HGPS dermal fibroblasts. The iPSCs were differentiated into neural progenitors, endothelial cells, fibroblasts, VSMCs, and mesenchymal stem cells (MSCs). Progerin levels were highest in MSCs, VSMCs, and fibroblasts, in that order, with these lineages displaying increased DNA damage, nuclear abnormalities, and HGPS-VSMC accumulating numerous calponin-staining inclusion bodies. Both HGPS-MSC and -VSMC viability was compromised by stress and hypoxia in vitro and in vivo (MSC). Because MSCs reside in low oxygen niches in vivo, we propose that, in HGPS, this causes additional depletion of the MSC pool responsible for replacing differentiated cells lost to progerin toxicity.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Mesenquimais/patologia , Músculo Liso Vascular/patologia , Senilidade Prematura/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Fibroblastos/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos , Músculo Liso Vascular/citologia , Oxigênio/metabolismo , Progéria/patologia , Progéria/terapiaRESUMO
The segmental premature aging disease Hutchinson-Gilford Progeria (HGPS) is caused by a truncated and farnesylated form of Lamin A. In a mouse model for HGPS, a similar Lamin A variant causes the proliferative arrest and death of postnatal, but not embryonic, fibroblasts. Arrest is due to an inability to produce a functional extracellular matrix (ECM), because growth on normal ECM rescues proliferation. The defects are associated with inhibition of canonical Wnt signaling, due to reduced nuclear localization and transcriptional activity of Lef1, but not Tcf4, in both mouse and human progeric cells. Defective Wnt signaling, affecting ECM synthesis, may be critical to the etiology of HGPS because mice exhibit skeletal defects and apoptosis in major blood vessels proximal to the heart. These results establish a functional link between the nuclear envelope/lamina and the cell surface/ECM and may provide insights into the role of Wnt signaling and the ECM in aging.
Assuntos
Matriz Extracelular/metabolismo , Lâmina Nuclear/metabolismo , Progéria/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Xenopus laevis/embriologia , Animais , Apoptose , Biomarcadores/metabolismo , Western Blotting , Proliferação de Células , Células Cultivadas , Imunoprecipitação da Cromatina , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Ensaio de Imunoadsorção Enzimática , Fibroblastos/citologia , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Humanos , Lamina Tipo A/fisiologia , Luciferases/metabolismo , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Progéria/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SíndromeRESUMO
Embryonic stem (ES) cells were first derived from inner cell mass (ICM) explants of preimplantation stage mouse blastocysts some 30 years ago. ES cells are of primary interest as they are used to genetically modify the genome of mice via gene targeting. Although many founder ES lines have been established, there is still a need to obtain new ES lines or their derivatives, often from new mutant mouse lines, to study the function of a mutated gene in different cell types. Existing methods for isolating ES cell lines are inefficient. Here, we describe a reproducible, efficient, and economical method to derive ES cells from different mouse strains using a defined serum-free, serum replacement (KO-SR) media, with 50-85% efficiency. We have derived over 100 ES lines, which when karyotyped>70% were euploid. Two of these lines, when tested, produced germ-line chimeras. We also present procedures for the routine maintenance and karyotyping of the ES cells.