RESUMEN
The gastrointestinal tract consists of an enormous surface area that is optimized to efficiently absorb nutrients, water, and electrolytes from food. At the same time, it needs to provide a tight barrier against the ingress of harmful substances, and protect against a reaction to omnipresent harmless compounds. A dysfunctional intestinal barrier is associated with various diseases and disorders. In this review, the role of intestinal permeability in common disorders such as infections with intestinal pathogens, inflammatory bowel disease, irritable bowel syndrome, obesity, celiac disease, non-celiac gluten sensitivity, and food allergies will be discussed. In addition, the effect of the frequently prescribed drugs proton pump inhibitors and non-steroidal anti-inflammatory drugs on intestinal permeability, as well as commonly used methods to assess barrier function will be reviewed.
RESUMEN
Threshold of Toxicological Concern (TTC) aids assessment of human health risks from exposure to low levels of chemicals when toxicity data are limited. The objective here was to explore the potential refinement of exposure for applying the oral TTC to chemicals found in cosmetic products, for which there are limited dermal absorption data. A decision tree was constructed to estimate the dermally absorbed amount of chemical, based on typical skin exposure scenarios. Dermal absorption was calculated using an established predictive algorithm to derive the maximum skin flux adjusted to the actual 'dose' applied. The predicted systemic availability (assuming no local metabolism), can then be ranked against the oral TTC for the relevant structural class. The predictive approach has been evaluated by deriving the experimental/prediction ratio for systemic availability for 22 cosmetic chemical exposure scenarios. These emphasise that estimation of skin penetration may be challenging for penetration enhancing formulations, short application times with incomplete rinse-off, or significant metabolism. While there were a few exceptions, the experiment-to-prediction ratios mostly fell within a factor of 10 of the ideal value of 1. It can be concluded therefore, that the approach is fit-for-purpose when used as a screening and prioritisation tool.
Asunto(s)
Cosméticos/toxicidad , Árboles de Decisión , Absorción Intestinal , Modelos Biológicos , Absorción Cutánea , Piel/metabolismo , Pruebas de Toxicidad/métodos , Administración Cutánea , Administración Oral , Algoritmos , Animales , Disponibilidad Biológica , Seguridad de Productos para el Consumidor , Cosméticos/administración & dosificación , Cosméticos/farmacocinética , Relación Dosis-Respuesta a Droga , Humanos , Nivel sin Efectos Adversos Observados , Medición de RiesgoRESUMEN
Primary murine mammary epithelial cells cultured on a laminin-rich-extracellular matrix (ECM) require c-Jun N-terminal kinase (JNK) activity for acinus formation. Inhibition of JNK (using SP600125) or small interfering RNA-mediated knockdown of JNK1 blocked acinus formation, impaired cell polarisation and lumen clearance and allowed sustained extracellular signal-regulated kinase (ERK) phosphorylation, cell proliferation, adhesion-independent cell survival and expression of epithelial-mesenchymal transition markers. ERK inhibition abolished the effects of JNK blockade. Interestingly, inhibition of JNK from the time of cell seeding blocked cell polarisation and lumen clearance; later inhibition (≥ 6 h) only affected lumen clearance. ERK inhibition effectively protected cell polarisation but less so, lumen clearance. SP600125-treatment similarly affected acinus formation by the 'normal' human mammary epithelial MCF10A cell line. Expression of dominant-negative JNK1 in MCF10A cells also undermined acinus formation, generating large 'multi-acinar spheres' whose formation is probably driven by excessive luminal cell proliferation and cell survival. As JNK activity must be suppressed from the time of cell seeding to block cell polarisation, we studied the behaviour of MCF10A cells immediately after seeding in laminin rich matrix: we detected engagement of cells with the matrix, early polarisation, movement of cells into clusters and 'epithelial-cell- like' behaviour of clustered cells. Inhibition of JNK activity or expression of dominant-negative JNK1 allowed cell engagement to the matrix, but blocked cell polarisation and all subsequent 'behaviours'. While integrin activation occurred, tyrosine-phosphorylation of paxillin, Fak and Src was significantly damped by JNK inhibition. These results emphasise the multi-phase dependency of the organisation of mammary cells in 3D on JNK activity and suggest a 'permissive' support of ECM-integrin 'outside-in' signalling and a 'damping' of growth-factor ERK signalling as its two key cell physiological effects.
Asunto(s)
Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/enzimología , Esferoides Celulares/citología , Esferoides Celulares/enzimología , Animales , Secuencia de Bases , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Polaridad Celular , Células Epiteliales/citología , Células Epiteliales/enzimología , Transición Epitelial-Mesenquimal , Matriz Extracelular/enzimología , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Proteína Quinasa 8 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 8 Activada por Mitógenos/genética , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , ARN Interferente Pequeño/genética , Transducción de SeñalRESUMEN
Mammary epithelial cells organize in three dimensions and generate acini when supported on laminin-rich extracellular matrix. Acinus formation begins with the apicobasal polarisation of the outer cells of the assembly and the withdrawal of these cells from the cell cycle. Internal cells then clear out to form a hollow lumen. Here, we show that PKCζ is phosphorylated (at T410) and activated in the early stages of acinus formation in both primary cells and MCF10A cells, and during mammary tree maturation in vivo. Phospho-PKCζ colocalised with tight junction components and bound to the Par polarising complex in developing acini. To further investigate the importance of PKCζ phosphorylation in this context, acinus formation was studied in MCF10A cells overexpressing non-phosphorylatable (T410A) or 'constitutively phosphorylated' (T410E) PKCζ. In both cell types, acinus-associated cell polarisation and lumen clearance were compromised, emphasising the importance of regulated phosphorylation of PKCζ at T410 for successful acinus formation. PKCζ can be activated in a phosphorylation (at T410)-dependent and a phosphorylation-independent manner. Cells overexpressing a complete kinase-deficient PKCζ (K281W) displayed a cell polarising deficit, but also generated large 'multi-acinar' structures with associated early lumenal cell hyperproliferation. Therefore our data shows, for the first time, that two separable PKCζ activities (one phosphorylation-dependent, the other not) are required to support the cell polarisation and proliferation restriction that underpins successful acinus formation. Paralleling these contributions, we found that low levels of PKCζ mRNA expression are associated with more 'poorly differentiated' tumours and a poor outcome in a cohort of 295 breast cancer patients.
Asunto(s)
Neoplasias de la Mama/metabolismo , Células Epiteliales/metabolismo , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Humanas/metabolismo , Proteína Quinasa C/metabolismo , Adulto , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Neoplasias de la Mama/fisiopatología , Diferenciación Celular/genética , Línea Celular Tumoral , Polaridad Celular/genética , Transformación Celular Neoplásica , Progresión de la Enfermedad , Células Epiteliales/patología , Femenino , Humanos , Hiperplasia/genética , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/patología , Glándulas Mamarias Humanas/patología , Ratones , Ratones Endogámicos , Análisis por Micromatrices , Proteínas Mutantes/genética , Organogénesis/genética , Fosforilación , Embarazo , Proteína Quinasa C/genética , Análisis de Supervivencia , Transgenes/genéticaRESUMEN
The amyloid precursor protein (APP) is critically involved in the pathogenesis of Alzheimer's disease, and is strongly up-regulated in response to traumatic, metabolic, or toxic insults to the nervous system. The processing of APP by gamma/epsilon-secretase activity results in the generation of the APP intracellular domain (AICD). Previously, we have shown that AICD induces the expression of genes (transgelin, alpha2-actin) with functional roles in actin organization and dynamics and demonstrated that the induction of AICD and its co-activator Fe65 (AICD/Fe65) resulted in a loss of organized filamentous actin structures within the cell. As mitochondrial function is thought to be reliant on ordered actin dynamics, we examined mitochondrial function in human SHEP neuroblastoma cells inducibly expressing AICD/Fe65. Confocal analysis of the mitochondrial membrane potential (DeltaPsim) identified a significant decrease in the DeltaPsim in the AICD50/Fe65 over-expressing cells. This was paralleled by significantly reduced ATP levels and decreased basal superoxide production. Overexpression of the proposed AICD target gene transgelin in SHEP-SF parental cells and primary neurons was sufficient to destabilize actin filaments, depolarize DeltaPsim, and significantly alter mitochondrial distribution and morphology. Our data demonstrate that the induction of AICD/Fe65 or transgelin significantly alters actin dynamics and mitochondrial function in neuronal cells.
Asunto(s)
Actinas/metabolismo , Precursor de Proteína beta-Amiloide/química , Metabolismo Energético/genética , Regulación de la Expresión Génica/genética , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Adenosina Trifosfato/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Línea Celular Tumoral , Doxiciclina/farmacología , Metabolismo Energético/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/genética , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Rodaminas/metabolismo , Estadísticas no Paramétricas , Superóxidos/metabolismo , Transfección/métodosRESUMEN
Seven classes of mitogen-activated protein kinase (MAPK) intracellular signalling cascades exist, four of which are implicated in breast disease and function in mammary epithelial cells. These are the extracellular regulated kinase (ERK)1/2 pathway, the ERK5 pathway, the p38 pathway and the c-Jun N-terminal kinase (JNK) pathway. In some forms of human breast cancer and in many experimental models of breast cancer progression, signalling through the ERK1/2 pathway, in particular, has been implicated as being important. We review the influence of ERK1/2 activity on the organised three-dimensional association of mammary epithelial cells, and in models of breast cancer cell invasion. We assess the importance of epidermal growth factor receptor family signalling through ERK1/2 in models of breast cancer progression and the influence of ERK1/2 on its substrate, the oestrogen receptor, in this context. In parallel, we consider the importance of these MAPK-centred signalling cascades during the cycle of mammary gland development. Although less extensively studied, we highlight the instances of signalling through the p38, JNK and ERK5 pathways involved in breast cancer progression and mammary gland development.
Asunto(s)
Neoplasias de la Mama/enzimología , Sistema de Señalización de MAP Quinasas , Neoplasias Mamarias Experimentales/enzimología , Animales , Neoplasias de la Mama/patología , Progresión de la Enfermedad , Femenino , Humanos , Glándulas Mamarias Animales/enzimología , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Humanas/enzimología , Glándulas Mamarias Humanas/crecimiento & desarrollo , Neoplasias Mamarias Experimentales/patología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismoRESUMEN
Cytoplasmic dynein functions at several sites during mitosis; however, the basis of targeting to each site remains unclear. Tandem mass spectrometry analysis of mitotic dynein revealed a phosphorylation site in the dynein intermediate chains (ICs) that mediates binding to kinetochores. IC phosphorylation directs binding to zw10 rather than dynactin, and this interaction is needed for kinetochore dynein localization. Phosphodynein associates with kinetochores from nuclear envelope breakdown to metaphase, but bioriented microtubule (MT) attachment and chromosome alignment induce IC dephosphorylation. IC dephosphorylation stimulates binding to dynactin and poleward streaming. MT depolymerization, release of kinetochore tension, and a PP1-gamma mutant each inhibited IC dephosphorylation, leading to the retention of phosphodynein at kinetochores and reduced poleward streaming. The depletion of kinetochore dynactin by moderate levels of p50(dynamitin) expression disrupted the ability of dynein to remove checkpoint proteins by streaming at metaphase but not other aspects of kinetochore dynein activity. Together, these results suggest a new model for localization of kinetochore dynein and the contribution of kinetochore dynactin.