RESUMEN
We have examined the role of phosphoinositide 3-kinases (PI3K) in interleukin (IL)-3-dependent cell cycle progression and compared the effects of LY294002 with expression of a dominant negative form of p85, termed Deltap85, which more specifically inhibits class I(A) PI3Ks. Inhibition of PI3Ks in BaF/3 led to accumulation of cells in G1 and extension of cell cycle transit times. Biochemically, both LY294002 and Deltap85 decreased levels of p107 and cyclins D2, D3 and E and reduced retinoblastoma protein (pRb) phosphorylation. Significantly, only LY294002 treatment increased expression of p27(Kip1). Interestingly, LY294002 decreased IL-3-induced proliferation of primary bone marrow-derived mast cells (BMMC) derived from both wild-type and p27(Kip1)-deficient mice and importantly, LY294002 treatment failed to upregulate p27(Kip1) in wild-type BMMC. These data support a role for class I(A) PI3K in regulating optimal cell cycle progression in response to IL-3 and demonstrate that upregulation of p27(Kip1) is not essential for attenuation of the cell cycle resulting from PI3K inhibition.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Interleucina-3/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Secuencia de Bases , Ciclo Celular , Proliferación Celular , Cromonas/farmacología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Eliminación de Gen , Mastocitos/citología , Ratones , Datos de Secuencia Molecular , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de las Quinasa Fosfoinosítidos-3RESUMEN
To investigate the function of the Grb10 adapter protein, we have generated mice in which the Grb10 gene was disrupted by a gene-trap insertion. Our experiments confirm that Grb10 is subject to genomic imprinting with the majority of Grb10 expression arising from the maternally inherited allele. Consistent with this, disruption of the maternal allele results in overgrowth of both the embryo and placenta such that mutant mice are at birth approximately 30% larger than normal. This observation establishes that Grb10 is a potent growth inhibitor. In humans, GRB10 is located at chromosome 7p11.2-p12 and has been associated with Silver-Russell syndrome, in which approximately 10% of those affected inherit both copies of chromosome 7 from their mother. Our results indicate that changes in GRB10 dosage could, in at least some cases, account for the severe growth retardation that is characteristic of Silver-Russell syndrome. Because Grb10 is a signaling protein capable of interacting with tyrosine kinase receptors, we tested genetically whether Grb10 might act downstream of insulin-like growth factor 2, a paternally expressed growth-promoting gene. The result indicates that Grb10 action is essentially independent of insulin-like growth factor 2, providing evidence that imprinting acts on at least two major fetal growth axes in a manner consistent with parent-offspring conflict theory.
Asunto(s)
Desarrollo Embrionario y Fetal/genética , Macrosomía Fetal/genética , Impresión Genómica , Inhibidores de Crecimiento/fisiología , Placenta/anomalías , Proteínas/fisiología , Alelos , Empalme Alternativo , Animales , Línea Celular , Quimera , Cruzamientos Genéticos , Femenino , Proteína Adaptadora GRB10 , Dosificación de Gen , Marcación de Gen , Genes Reporteros , Genes Sintéticos , Inhibidores de Crecimiento/química , Inhibidores de Crecimiento/deficiencia , Inhibidores de Crecimiento/genética , Factor II del Crecimiento Similar a la Insulina/fisiología , Operón Lac , Hígado/embriología , Hígado/patología , Pulmón/anomalías , Pulmón/embriología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Modelos Biológicos , Especificidad de Órganos , Proteínas/química , Proteínas/genética , ARN Mensajero/biosíntesis , Eliminación de Secuencia , Transducción de Señal , Células Madre/citologíaRESUMEN
Many epithelial renewal tissues in vertebrates are organised into structural-proliferative units. We have examined the effect of IGF2 dose on the structure of structural-proliferative units in skin and colon. The mouse strains used were the Igf2 knockout, wild type and K:Igf2, a transgenic in which Igf2 is overexpressed under control of a keratin promoter. For both skin and colon, the histological organisation of structural-proliferative units was unaltered with increasing IGF2 dose, although there was a higher fraction of dividing cells in the proliferative compartment. In the colon an increase in IGF2 dose increases the overall area of the epithelium. This is due to an increase in the number of crypts with no change of cell size or of crypt area. Growth stimulation appears to be due to a reduction in the duration of crypt fission. The conclusion is that the IGF2 pathway can stimulate the multiplication of colonic crypts independently of stimulating increased cell proliferation. The results for the skin are consistent with this. An increase of IGF2 dose increases the proportion of dividing cells in the basal layer, the thickness of the epidermis and the total area of the epidermis. By comparison with Drosophila, these results show no effects on cell size, but do show the possibility of inducing disproportionate growth. These differences may represent properties of the SPU organisation that is characteristic of vertebrate tissues.