RESUMEN
In 1993, we described an English family with beta-thalassaemia that was not linked to the beta-globin locus. Whole genome sequence analyses revealed potential causative mutations in 15 different genes, of which 4 were consistently and uniquely associated with the phenotype in all 7 affected family members, also confirmed by genetic linkage analysis. Of the 4 genes, which are present in a centromeric region of chromosome 1, ASH1L was proposed as causative through functional mRNA knock-down and chromatin-immunoprecipitation studies in human erythroid progenitor cells. Our data suggest a putative role for ASH1L (Trithorax protein) in the regulation of globin genes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Factores de Transcripción/metabolismo , Globinas beta/genética , Talasemia beta/genética , Talasemia beta/metabolismo , Línea Celular , Mapeo Cromosómico , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/metabolismo , Silenciador del Gen , Ligamiento Genético , Variación Genética , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento , N-Metiltransferasa de Histona-Lisina , Histonas/metabolismo , Humanos , Fenotipo , Interferencia de ARN , Talasemia beta/diagnósticoRESUMEN
Induced pluripotent stem cells (iPSCs) have revolutionized human biomedicine through their use in disease modeling and therapy. In comparison, little progress has been made toward the application of iPSCs in veterinary species. In that regard, skeletal myocytes from iPSCs would have great potential for understanding muscle function and disease in the equine athlete. In this study, we generated skeletal myotubes by transducing equine iPSC-derived mesenchymal derivatives with an inducible lentiviral vector coding for the human sequence of the myogenic factor, MyoD. Myosin heavy chain-positive myotubes generated from two different iPSC lines were compared to myotubes from adult equine skeletal muscle progenitor cells (MPCs). iPSC myotubes had a smaller mean area than MPC myotubes (≤2-fold). In addition, quantitative polymerase chain reaction analyses showed that iPSC myotubes expressed MYH2 and MYH3 isoforms (at similar or lower levels than MPC myotubes), but they did not express the mature muscle isoform, MYH1. Compared to MPC myotubes, iPSC myotubes expressed reduced levels of the myogenic factors, MYOD1 and MYF6, but did not express MYF5. Finally, iPSC myotubes responded to KCl-induced membrane depolarization by releasing calcium and did so in a manner similar to MPC myotubes. In conclusion, this is the first study to report the generation of functional myocytes from equine iPSCs.
Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas/citología , Fibras Musculares Esqueléticas/citología , Músculo Esquelético/citología , Animales , Células Cultivadas , CaballosRESUMEN
CONTEXT: Levels of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), which regenerates active glucocorticoids, are selectively elevated in adipose tissue in human obesity and metabolic syndrome, both conditions associated with chronic low-grade inflammation. 11ß-HSD1 expression is induced by proinflammatory cytokines in a variety of cell types, including in human adipocytes differentiated in vitro. OBJECTIVE: Our objective was to determine the mechanisms by which proinflammatory cytokines induce 11ß-HSD1 in human adipocytes. RESULTS: The proinflammatory cytokines IL-1α (10 ng/mL) and TNFα (20 ng/mL) increased 11ß-HSD1 mRNA levels in human primary adipocyte fractions and Simpson-Golabi-Behmel syndrome (SGBS) adipocytes (P<.001). Inhibition of the MAPK/ERK kinase (MEK) attenuated CCAAT/enhancer binding protein (C/EBP) ß phosphorylation at Thr235 and IL-1α/TNFα induction of 11ß-HSD1 (P≤.007). The small interfering RNA-mediated knockdown of C/EBPß and nuclear factor (NF)-κB/RelA or inhibition of NF-κB/RelA also attenuated cytokine induction of 11ß-HSD1 (P≤.001). Moreover, induction of 11ß-HSD1 by IL-1α in SGBS cells was associated with nuclear localization of C/EBPß and NF-κB/RelA. Chromatin immunoprecipitation experiments showed C/EBPß and NF-κB/RelA located to the 11ß-HSD1 promoter in human adipose tissue. Treatment of adipocyte fractions or SGBS adipocytes with metformin or acetylsalicylic acid, which target C/EBPß and NF-κB/RelA signaling, attenuated the IL-1α induction of 11ß-HSD1 (P≤.002). CONCLUSIONS: Increased proinflammatory signaling in inflamed adipose tissue may mediate elevated 11ß-HSD1 expression at this site via MEK, C/EBPß, and NF-κB/RelA. These molecules/signaling pathways are, therefore, potential targets for drugs, including metformin and acetylsalicylic acid, to prevent/decreased up-regulation of 11ß-HSD1 in human obese/metabolic syndrome adipose tissue.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/biosíntesis , Adipocitos/efectos de los fármacos , Proteína beta Potenciadora de Unión a CCAAT/fisiología , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Mediadores de Inflamación/farmacología , FN-kappa B/fisiología , Adipocitos/metabolismo , Adulto , Células Cultivadas , Citocinas/farmacología , Inducción Enzimática/efectos de los fármacos , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Factor de Transcripción ReIA/fisiología , Adulto JovenRESUMEN
11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) converts inert glucocorticoids into active forms, thereby increasing intracellular glucocorticoid levels, important to restrain acute inflammation. 11ß-HSD1 is induced by pro-inflammatory cytokines in a variety of cells. Here, we show 11ß-HSD1 expression in human A549 epithelial cells is increased by pro-inflammatory cytokines (IL-1α/TNFα) via the P2 promoter of the HSD11B1 gene. Inhibition of p38 MAPK attenuated the pro-inflammatory cytokine induction of mRNA encoding 11ß-HSD1 as well as that encoding C/EBPß. IL-1α/TNFα-induced phosphorylation of C/EBPß at Thr235 was also attenuated by p38 MAPK inhibition suggesting involvement of a p38 MAPK-C/EBPß pathway. siRNA-mediated knock-down of C/EBPß and NF-κB/RelA implicated both transcription factors in the IL-1α/TNFα induction of HSD11B1 mRNA. Transient transfections of HSD11B1 promoter-reporter constructs identified the proximal region of the P2 promoter of HSD11B1 as essential for this induction. IL-1α increased binding of C/EBPß to the HSD11B1 P2 promoter, but this was not observed for NF-κB/RelA, suggesting indirect regulation by NF-κB/RelA. Ectopic expression of mutant chicken C/EBPß constructs unable to undergo phosphorylation at the threonine equivalent to Thr235 attenuated the IL-1α-induction of HSD11B1, whereas mimicking constitutive phosphorylation of Thr235 (by mutation to aspartate) increased basal expression of HSD11B1 mRNA without affecting IL-1α-induced levels. These data clearly demonstrate a role for both C/EBPß and NF-κB/RelA in the pro-inflammatory cytokine induction of HSD11B1 in human epithelial cells and show that p38 MAPK-induced phosphorylation of C/EBPß at Thr235 is critical in this.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasas/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Citocinas/metabolismo , Regulación de la Expresión Génica/inmunología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , Animales , Proteína beta Potenciadora de Unión a CCAAT , Línea Celular Tumoral , Pollos , Glucocorticoides/metabolismo , Humanos , Ligasas/metabolismo , FN-kappa B/metabolismo , Fosforilación , Regiones Promotoras Genéticas/genéticaRESUMEN
Pluripotent stem cells offer unprecedented potential not only for human medicine but also for veterinary medicine, particularly in relation to the horse. Induced pluripotent stem cells (iPSCs) are particularly promising, as they are functionally similar to embryonic stem cells and can be generated in vitro in a patient-specific manner. In this study, we report the generation of equine iPSCs from skin fibroblasts obtained from a foal and reprogrammed using viral vectors coding for murine Oct4, Sox2, c-Myc, and Klf4 sequences. The reprogrammed cell lines were morphologically similar to iPSCs reported from other species and could be stably maintained over more than 30 passages. Immunostaining and polymerase chain reaction analyses revealed that these cell lines expressed an array of endogenous markers associated with pluripotency, including OCT4, SOX2, NANOG, REX1, LIN28, SSEA1, SSEA4, and TRA1-60. Furthermore, under the appropriate conditions, the equine iPSCs readily formed embryoid bodies and differentiated in vitro into cells expressing markers of ectoderm, mesoderm, and endoderm, and when injected into immunodeficient mice, gave raise to tumors containing differentiated derivatives of the 3 germ layers. Finally, we also reprogrammed fibroblasts from a 2-year-old horse. The reprogrammed cells were similar to iPSCs derived from neonatal fibroblasts in terms of morphology, expression of pluripotency markers, and differentiation ability. The generation of these novel cell lines constitutes an important step toward the understanding of pluripotency in the horse, and paves the way for iPSC technology to potentially become a powerful research and clinical tool in veterinary biomedicine.
Asunto(s)
Antígenos de Diferenciación , Desdiferenciación Celular , Cuerpos Embrioides , Fibroblastos , Células Madre Pluripotentes Inducidas , Factores de Transcripción , Animales , Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/genética , Células Cultivadas , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Caballos , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Ratones , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
Histone H3 trimethylation on lysine 27 is one of the histone modifications associated with chromatin of silenced regions. H3K27me3 labeling is initially asymmetrical between pronuclei in mammalian embryos, and then it is remodeled during early development. However, in mouse embryos obtained after somatic cell nuclear transfer (SCNT), H3K27me3 histones inherited from the somatic female cell and associated with X chromosome inactivation have been reported to escape remodeling. Using immunostaining, we investigated the remodeling of H3K27me3 in Bos taurus embryos obtained after in vitro fertilization (IVF) and SCNT. In this species, transfer-induced chromatin remodeling can be clearly separated from embryonic genome activation (EGA), which occurs at the 8-16-cell stage, and cloning by SCNT is 10 times more successful than in the mouse. In early IVF bovine embryos, dense H3K27me3 labeling was localized in the pericentric heterochromatin as recently described in the mouse. Labeling was however unevenly distributed up to the 8-cell stage, suggesting that the parental genomes partitioned before EGA. In female IVF blastocysts, a somatic-like female profile appeared in 21% of the trophoblast cells. This profile, which had one major nuclear H3K27me3 patch, the putative inactive X chromosome (Xi), was absent in male blastocysts. In contrast, the somatic-like female H3K27me3 profile was observed in the majority of the nuclei of female bovine SCNT embryos before EGA. At the 8-16-cell stage, this profile was transiently replaced by pericentric-like labeling in most nuclei. Immunostaining of mitotic chromosomes suggested that the ratio of H3K27me3 labeling in pericentric heterochromatin vs. euchromatin was then rapidly altered. Finally, Xi-like H3K27me3 staining appeared again in trophoblast cells in female SCNT blastocysts. These results suggest a role for EGA in H3K27me3 remodeling, which affects the heterochromatin inherited from the donor cell or produced during development.
Asunto(s)
Bovinos/embriología , Núcleo Celular/metabolismo , Fertilización In Vitro , Histonas/metabolismo , Técnicas de Transferencia Nuclear , Animales , Bovinos/metabolismo , Células Cultivadas , Ensamble y Desensamble de Cromatina/fisiología , Embrión de Mamíferos , Desarrollo Embrionario/fisiología , Femenino , Fertilización In Vitro/métodos , Heterocromatina/metabolismo , Histonas/química , Células Híbridas/metabolismo , Células Híbridas/fisiología , Lisina/metabolismo , Masculino , Metaboloma , Metilación , Ratones , EmbarazoRESUMEN
BACKGROUND: Expression of several copies of the heat-inducible Hsp70.1Luciferase (LUC) transgene inserted at a single X chromosome locus of a bull (Bos taurus) was assessed in females after X-chromosome inactivation (XCI). Furthermore, impact of the chromosomal environment on the spontaneous expression of these transgene copies before XCI was studied during early development in embryos obtained after in vitro fertilization (IVF), when the locus was carried by the X chromosome inherited from the bull, and after somatic cell nuclear transfer (SCNT) cloning, when the locus could be carried by the inactive Xi or the active Xa chromosome in a female donor cell, or by the (active) X in a male donor cell. FINDINGS: Transgene copies were mapped to bovine Xp22. In XXLUC female fibroblasts, i.e. after random XCI, the proportions of late-replicating inactive and early-replicating active XLUC chromosomes were not biased and the proportion of cells displaying an increase in the level of immunostained luciferase protein after heat-shock induction was similar to that in male fibroblasts. Spontaneous transgene expression occurred at the 8-16-cell stage both in transgenic (female) embryos obtained after IVF and in male and female embryos obtained after SCNT. CONCLUSIONS: The XLUC chromosome is normally inactivated but at least part of the inactivated X-linked Hsp70.1Luciferase transgene copies remains heat-inducible after random XCI in somatic cells. Before XCI, the profile of the transgenes' spontaneous expression is independent of the epigenetic origin of the XLUC chromosome since it is similar in IVF female, SCNT male and SCNT female embryos.