RESUMO
Phosphoinositide 3-kinases produce 3'-phosphorylated phosphoinositides that act as second messengers to recruit other signalling proteins to the membrane. Pi3ks are activated by many extracellular stimuli and have been implicated in a variety of cellular responses. The Pi3k gene family is complex and the physiological roles of different classes and isoforms are not clear. The gene Pik3r1 encodes three proteins (p85 alpha, p55 alpha and p50 alpha) that serve as regulatory subunits of class IA Pi3ks (ref. 2). Mice lacking only the p85 alpha isoform are viable but display hypoglycaemia and increased insulin sensitivity correlating with upregulation of the p55 alpha and p50 alpha variants. Here we report that loss of all protein products of Pik3r1 results in perinatal lethality. We observed, among other abnormalities, extensive hepatocyte necrosis and chylous ascites. We also noted enlarged skeletal muscle fibres, brown fat necrosis and calcification of cardiac tissue. In liver and muscle, loss of the major regulatory isoform caused a great decrease in expression and activity of class IA Pi3k catalytic subunits; nevertheless, homozygous mice still displayed hypoglycaemia, lower insulin levels and increased glucose tolerance. Our findings reveal that p55 alpha and/or p50 alpha are required for survival, but not for development of hypoglycaemia, in mice lacking p85 alpha.
Assuntos
Anormalidades Múltiplas/genética , Ascite Quilosa/genética , Genes Letais , Hipoglicemia/genética , Fígado/patologia , Fosfatidilinositol 3-Quinases/deficiência , Isoformas de Proteínas/deficiência , Tecido Adiposo Marrom/patologia , Animais , Animais não Endogâmicos , Calcinose/genética , Cardiomiopatias/genética , Catálise , Cruzamentos Genéticos , Dimerização , Indução Enzimática , Feminino , Genes , Genótipo , Vida Livre de Germes , Glucose/metabolismo , Glucose/farmacologia , Hipertrofia , Insulina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Fibras Musculares Esqueléticas/patologia , Necrose , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/fisiologia , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Processamento de Proteína Pós-Traducional/genética , Subunidades Proteicas , Sistemas do Segundo Mensageiro/genéticaRESUMO
Phosphoinositide 3-kinase (PI3K) activation has been implicated in many cellular responses, including fibroblast growth, transformation, survival, and chemotaxis. Although PI3K is activated by several agents that stimulate T and B cells, the role of PI3K in lymphocyte function is not clear. The mouse gene encoding the PI3K adapter subunit p85alpha and its splice variants p55alpha and p50alpha was disrupted. Most p85alpha-p55alpha-p50alpha-/- mice die within days after birth. Lymphocyte development and function was studied with the use of the RAG2-deficient blastocyst complementation system. Chimeric mice had reduced numbers of peripheral mature B cells and decreased serum immunoglobulin. The B cells that developed had diminished proliferative responses to antibody to immunoglobulin M, antibody to CD40, and lipopolysaccharide stimulation and decreased survival after incubation with interleukin-4. In contrast, T cell development and proliferation was normal. This phenotype is similar to defects observed in mice lacking the tyrosine kinase Btk.
Assuntos
Linfócitos B/imunologia , Imunoglobulinas/sangue , Ativação Linfocitária , Fosfatidilinositol 3-Quinases/metabolismo , Tirosina Quinase da Agamaglobulinemia , Animais , Apoptose , Linfócitos B/citologia , Linfócitos B/enzimologia , Domínio Catalítico , Ciclo Celular , Quimera , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Marcação de Genes , Antígenos Comuns de Leucócito/análise , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Baço/imunologia , Linfócitos T/citologia , Linfócitos T/enzimologia , Linfócitos T/imunologiaRESUMO
Insulin-like growth factor (IGF)-II is a mitogenic peptide that has been reported to play an important role in the formation and growth of a variety of tumors. In most tissues, the IGF-II gene (IGF2) is parentally imprinted, with only the paternal allele being expressed. IGF2 messenger RNA (mRNA) and protein are overexpressed in some benign and malignant tumors. H19, a tumor suppressor gene located directly downstream from IGF2, is also genomically imprinted, but the paternal allele is silenced. It has been suggested that alterations in the imprinting of these two genes, which are located at chromosome 11p15.5, may lead to a malignant diathesis. We examined 18 fresh-frozen (FF) breast tumors with their adjacent normal breast tissue and 14 sets of paraffin-embedded formalin-fixed tissues for IGF2 and H19 gene expression and imprinting. IGF2 mRNA and H19 RNA could be quantitated in 15 of the 18 FF tumors. Although three of these tumors showed a > or = 2-fold increase in IGF2 expression when compared with the normal control tissues, the average abundance of IGF2 mRNA in 8 of 15 FF samples was < 50% that observed in the normal tissue. The expression of H19 RNA in these tumors was increased by > or = 2-fold in 5 tumors, but decreased by < or = 50% in 6 tumors when compared with normal adjacent tissue. By examining the ApaI and CA-repeat polymorphisms in the IGF2 gene, we found that the imprinting of IGF2 was maintained in all but 2 of the 17 informative subjects. H19 imprinting was maintained in all 18 informative fresh-frozen and paraffin-embedded formalin-fixed samples. Our data suggest that alterations in IGF2 and H19 gene expression and loss of imprinting do not occur reliably in breast cancer.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Mama/metabolismo , Expressão Gênica , Genes Supressores de Tumor , Impressão Genômica , Fator de Crescimento Insulin-Like II/biossíntese , Proteínas Musculares/biossíntese , Polimorfismo Genético , RNA não Traduzido , Alelos , Sequência de Bases , Primers do DNA , DNA Complementar , Desoxirribonucleases de Sítio Específico do Tipo II , Éxons , Feminino , Humanos , Fator de Crescimento Insulin-Like II/genética , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , RNA Longo não Codificante , Sequências Repetitivas de Ácido NucleicoRESUMO
Phosphoinositide 3-kinase (PI3K) has been shown to regulate cell and organ size in Drosophila, but the role of PI3K in vertebrates in vivo is not well understood. To examine the role of PI3K in intact mammalian tissue, we have created and characterized transgenic mice expressing constitutively active or dominant-negative mutants of PI3K in the heart. Cardiac- specific expression of constitutively active PI3K resulted in mice with larger hearts, while dominant-negative PI3K resulted in mice with smaller hearts. The increase or decrease in heart size was associated with comparable increase or decrease in myocyte size. Cardiomyopathic changes, such as myocyte necrosis, apoptosis, interstitial fibrosis or contractile dysfunction, were not observed in either of the transgenic mice. Thus, the PI3K pathway is necessary and sufficient to promote organ growth in mammals.