RESUMEN
There is growing evidence suggesting that urban pollution has adverse effects on lung health. However, how urban pollution affects alveolar mesenchymal and epithelial stem cell niches remains unknown. This study aimed to determine how complex representative urban atmospheres alter alveolar stem cell niche properties. Mice were placed in an innovative chamber realistically simulating the atmosphere of a megalopolis, or "clean air," for 7 days. Lungs were collected, and fibroblasts and epithelial cells (EpCAM+) were isolated. Proliferative capacities of fibroblasts were tested by population doubling levels (PDL), and microarray analyses were performed. Fibroblasts and EpCAM+ cells from exposed, nonexposed, or naive mice were cocultured in organoid assays to assess the stem cell properties. Collagen deposition (Sirius red), lipofibroblasts (ADRP, COL1A1), myofibroblasts (αSMA), alveolar type 2 cells (AT2, SFTPC+), and alveolar differentiation intermediate cell [ADI, keratin-8-positive (KRT8+)/claudin-4-positive (CLDN4+)] markers were quantified in the lungs. Fibroblasts obtained from mice exposed to urban atmosphere had lower PDL and survival and produced fewer and smaller organoids. Microarray analysis showed a decrease of adipogenesis and an increase of genes associated with fibrosis, suggesting a lipofibroblast to myofibroblast transition. Collagen deposition and myofibroblast number increased in the lungs of urban atmosphere-exposed mice. AT2 number was reduced and associated with an increase in ADI cells KRT8+/CLDN4+. Furthermore, EpCAM+ cells from exposed mice also produced fewer and smaller organoids. In conclusion, urban atmosphere alters alveolar mesenchymal stem cell niche properties by inducing a lipofibroblast to myofibroblast shift. It also results in alveolar epithelial dysfunction and a fibrotic-like phenotype.NEW & NOTEWORTHY Urban pollution is known to have major adverse effects on lung health. To assess the effect of pollution on alveolar regeneration, we exposed adult mice to a simulated high-pollution urban atmosphere, using an innovative CESAM simulation chamber (Multiphase Atmospheric Experimental Simulation Chamber, https://cesam.cnrs.fr/). We demonstrated that urban atmosphere alters alveolar mesenchymal stem cell niche properties by inducing a lipofibroblast to myofibroblast shift and induces alveolar epithelial dysfunction.
Asunto(s)
Fibrosis Pulmonar , Ratones , Animales , Fibrosis Pulmonar/patología , Molécula de Adhesión Celular Epitelial/metabolismo , Células Epiteliales Alveolares/metabolismo , Pulmón/metabolismo , Diferenciación Celular , Células Madre , Colágeno/metabolismoRESUMEN
The chromosome translocations generating PAX3-FOXO1 and PAX7-FOXO1 chimeric proteins are the primary hallmarks of the paediatric fusion-positive alveolar subtype of Rhabdomyosarcoma (FP-RMS). Despite the ability of these transcription factors to remodel chromatin landscapes and promote the expression of tumour driver genes, they only inefficiently promote malignant transformation in vivo. The reason for this is unclear. To address this, we developed an in ovo model to follow the response of spinal cord progenitors to PAX-FOXO1s. Our data demonstrate that PAX-FOXO1s, but not wild-type PAX3 or PAX7, trigger the trans-differentiation of neural cells into FP-RMS-like cells with myogenic characteristics. In parallel, PAX-FOXO1s remodel the neural pseudo-stratified epithelium into a cohesive mesenchyme capable of tissue invasion. Surprisingly, expression of PAX-FOXO1s, similar to wild-type PAX3/7, reduce the levels of CDK-CYCLIN activity and increase the fraction of cells in G1. Introduction of CYCLIN D1 or MYCN overcomes this PAX-FOXO1-mediated cell cycle inhibition and promotes tumour growth. Together, our findings reveal a mechanism that can explain the apparent limited oncogenicity of PAX-FOXO1 fusion transcription factors. They are also consistent with certain clinical reports indicative of a neural origin of FP-RMS.
Asunto(s)
Transdiferenciación Celular/genética , Transformación Celular Neoplásica/genética , Proteínas de Fusión Oncogénica/metabolismo , Factores de Transcripción Paired Box/metabolismo , Rabdomiosarcoma Alveolar/genética , Animales , Biopsia , Embrión de Pollo , Niño , Ciclina D1/genética , Conjuntos de Datos como Asunto , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Proteína Proto-Oncogénica N-Myc/genética , Invasividad Neoplásica/genética , Células-Madre Neurales/patología , Tubo Neural/citología , Proteínas de Fusión Oncogénica/genética , Factor de Transcripción PAX3/genética , Factor de Transcripción PAX3/metabolismo , Factor de Transcripción PAX7/genética , Factor de Transcripción PAX7/metabolismo , Factores de Transcripción Paired Box/genética , Rabdomiosarcoma Alveolar/patología , Fase S/genéticaRESUMEN
Past work has shown that the protein O-fucosyltransferase 1 (POFUT1) is involved in mammal myogenic differentiation program. Pofut1 knockdown (Po -) in murine C2C12 cells leads to numerous elongated and thin myotubes, suggesting significant defects in secondary fusion. Among the few pathways involved in this process, NFATc2/IL-4 is described as the major one. To unravel the impact of POFUT1 on secondary fusion, we used wild-type (WT) C2C12 and Po - cell lines to follow Myf6, Nfatc2, Il-4 and Il-4rα expressions during a 120 h myogenic differentiation time course. Secreted IL-4 was quantified by ELISA. IL-4Rα expression and its labeling on myogenic cell types were investigated by Western blot and immunofluorescence, respectively. Phenotypic observations of cells treated with IL-4Rα blocking antibody were performed. In Po -, we found a decrease in nuclei number per myotube and a downexpression of Myf6. The observed downregulation of Nfatc2 is correlated to a diminution of secreted IL-4 and to the low level of IL-4Rα for reserve cells. Neutralization of IL-4Rα on WT C2C12 promotes myonuclear accretion defects, similarly to those identified in Po -. Thus, POFUT1 could be a new controller of myotube growth during myogenesis, especially through NFATc2/IL-4 signaling pathway.
Asunto(s)
Fucosiltransferasas/genética , Regulación de la Expresión Génica , Interleucina-4/metabolismo , Desarrollo de Músculos/genética , Fibras Musculares Esqueléticas/metabolismo , Factores de Transcripción NFATC/metabolismo , Transducción de Señal , Animales , Diferenciación Celular/genética , Línea Celular , Técnicas de Silenciamiento del Gen , Ratones , Factores de Transcripción NFATC/genética , Receptores Notch/metabolismoRESUMEN
BACKGROUND: Obesity is associated with oxidative stress, a major factor in carcinogenesis, and with high leptin concentration. The aim of this study was to determine the effects of leptin on the antioxidant response in three human mammary epithelial cells each presenting a different neoplastic status: healthy human mammary epithelial cells (HMEC), oestrogen-receptor positive MCF-7 cells and triple-negative MDA-MB-231 cells. METHODS: This in vitro kinetic study characterized the cell antioxidant response after 1, 6 and 24 h in the presence of leptin (10 or 100 ng/ml).The antioxidant response was defined in terms of cell glutathione content, gene expression and catalytic activity of antioxidant enzymes (i.e. glutathione peroxidase 1 (Gpx1), glutathione reductase (GR), glutathione S transferase (GST), heme-oxygenase 1 (HO-1) and cyclooxygenase-2 (COX-2)). Oxidative stress occurrence was assessed by lipid hydro peroxide (HPLIP) and isoprostane concentrations in culture media at 24 h. RESULTS: At both concentrations used, leptin induced ROS production in all cell models, contributing to various antioxidant responses linked to neoplastic cell status. HMEC developed a highly inducible antioxidant response based on antioxidant enzyme activation and an increase in cell GSH content at 10 ng/ml of leptin. However, at 100 ng/ml of leptin, activation of antioxidant response was lower. Conversely, in tumour cells, MCF-7 and MDA-MB-231, leptin did not induce an efficient antioxidant response, at either concentration, resulting in an increase of lipid peroxidation products. CONCLUSIONS: Leptin can modulate the oxidative status of mammary epithelial cells differently according to their neoplastic state. These novel results shed light on oxidative status changes in mammary cells in the presence of leptin.
Asunto(s)
Antioxidantes/administración & dosificación , Leptina/administración & dosificación , Glándulas Mamarias Humanas/metabolismo , Obesidad/metabolismo , Antioxidantes/metabolismo , Carcinogénesis/efectos de los fármacos , Ciclooxigenasa 2/metabolismo , Femenino , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Glutatión Transferasa/metabolismo , Hemo-Oxigenasa 1/metabolismo , Humanos , Leptina/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Células MCF-7 , Glándulas Mamarias Humanas/efectos de los fármacos , Obesidad/tratamiento farmacológico , Obesidad/enzimología , Obesidad/patología , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo , Glutatión Peroxidasa GPX1RESUMEN
Muscle satellite cells (MuSCs) are the quiescent muscle stem cells required for adult skeletal muscle repair. The impact of environmental stress such as pollution on MuSC behavior remains unexplored. We evaluated the impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure, a ubiquitous and highly toxic pollutant, on MuSCs by combining in vivo mouse molecular genetic models with ex vivo studies. While all MuSCs express the transcription factor PAX7, we show that a subset also express PAX3 and exhibit resistance to environmental stress. Upon systemic TCDD treatment, PAX3-negative MuSCs display impaired survival, atypical activation, and sporadic differentiation through xenobiotic aryl hydrocarbon receptor signaling. We further show that PAX3-positive MuSCs become sensitized to environmental stress when PAX3 function is impaired and that PAX3-mediated induction of mTORC1 is required for protection. Our study, therefore, identifies a functional heterogeneity of MuSCs in response to environmental stress controlled by PAX3.
Asunto(s)
Células Madre Adultas/fisiología , Contaminación Ambiental/efectos adversos , Factor de Transcripción PAX3/metabolismo , Factor de Transcripción PAX7/metabolismo , Dibenzodioxinas Policloradas/efectos adversos , Células Satélite del Músculo Esquelético/fisiología , Animales , Diferenciación Celular , Supervivencia Celular , Células Cultivadas , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Transcripción PAX3/genética , Factor de Transcripción PAX7/genética , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de SeñalRESUMEN
The protein O-glucosyltransferase 1 (Poglut1) links O-glucose to epidermal growth factor-like repeats harboring the C1XSX(P/A)C2 consensus sequence. Poglut1 is a ubiquitous endoplasmic reticulum-resident protein largely found in metazoans, but only about 50 proteins possess this consensus sequence. Among them, Notch receptors have multiple O-glucosylation sites and their activation depends on this status. In adult skeletal muscle, Notch signaling contributes to the maintenance of satellite cell (SC) quiescence and the proliferation of myoblasts after SC activation. To address the role of Poglut1 in myogenesis, we created two stable C2C12 cell lines where Poglut1 was downexpressed by 42% and 81%, and assessed their ability to differentiate. We showed that Poglut1 knockdown reduced Notch signaling and largely affected the key regulators of myogenic differentiation, with PAX7 decrease and MYOD increase. This perturbed Pax7/MyoD expression balance led to a premature myogenic differentiation and an increase in myotube size, accentuated in case of strong Poglut1 downexpression. Differences observed between myotubes of the two Poglut1 knockdown cell lines could reflect dissimilar fusion defects. We concluded that Poglut1 contributes to myogenesis by regulating Notch signaling and defining, directly or indirectly, the proportion of cells that commit differentiation.
Asunto(s)
Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Fibras Musculares Esqueléticas/fisiología , Animales , Diferenciación Celular/fisiología , Línea Celular , Factor de Crecimiento Epidérmico , Ratones , Desarrollo de Músculos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Proteína MioD/metabolismo , Mioblastos/citología , Receptores Notch/metabolismo , Transducción de SeñalRESUMEN
NADPH oxidase (NOX) complexes (a family of seven isoforms) drive cellular ROS production in patho-logical processes such as cancer. NOX-driven ROS production is involved in cell mechanisms from signalling to oxidative stress. Leptin, an adipokine overexpressed in obese patients, has been investigated in studies on breast carcinogenesis, but its effects on oxidative stress remain largely unexplored, especially in breast cancer. The study used three human mammary epithelial cell models presenting different neoplastic status (healthy primary HMECs, neoplastic MCF-7 cells and neoplastic MDA-MB-231 cells) to determine the effects of leptin on short-term ROS production and to characterize the enzymes involved. All three cell models significantly expressed NADPH oxidase isoform 5 (NOX5) in our culture conditions. All models showed induced ROS production regardless of leptin concentration (10 ng/ml mimicking good health, 100 ng/ml mimicking obesity). Cell treatment with either siRNA against NOX5, NOX inhibitor DPI or a calcium channel blocker (verapamil) confirmed the putative involvement of the NOX5 isoenzyme in ROS production. Moreover, cell treatments suppressed ROS production under leptin at both concentrations. Neoplastic cells appeared unable to downregulate NOX5 mRNA expression under leptin. Leptin emerged as a potential activator of ROS production in human epithelial mammary cells, where the ROS production was apparently linked to NOX5 activation. This novel finding could shed light on the potential role of obesity-associated hyperleptinemia in mammary cells via the activation of NOX enzymes.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Leptina/genética , NADPH Oxidasa 5/genética , Estrés Oxidativo/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Células Epiteliales/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Glándulas Mamarias Humanas/metabolismo , Glándulas Mamarias Humanas/patología , NADPH Oxidasa 5/antagonistas & inhibidores , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno/metabolismo , Verapamilo/administración & dosificaciónRESUMEN
The Notch signaling pathway plays a crucial role in skeletal muscle regeneration in mammals by controlling the transition of satellite cells from quiescence to an activated state, their proliferation, and their commitment toward myotubes or self-renewal. O-fucosylation on Notch receptor epidermal growth factor (EGF)-like repeats is catalyzed by the protein O-fucosyltransferase 1 (Pofut1) and primarily controls Notch interaction with its ligands. To approach the role of O-fucosylation in myogenesis, we analyzed a murine myoblastic C2C12 cell line downregulated for Pofut1 expression by short hairpin RNA (shRNA) inhibition during the time course of differentiation. Knockdown of Pofut1 affected the signaling pathway activation by a reduction of the amount of cleaved Notch intracellular domain and a decrease in downstream Notch target gene expression. Depletion in Pax7(+)/MyoD(-) cells and earlier myogenic program entrance were observed, leading to an increase in myotube quantity with a small number of nuclei, reflecting fusion defects. The rescue of Pofut1 expression in knockdown cells restored Notch signaling activation and a normal course in C2C12 differentiation. Our results establish the critical role of Pofut1 on Notch pathway activation during myogenic differentiation.