RESUMO
Cellular repressor of E1A-stimulated genes (CREG) has been reported to be a secretory glycoprotein implicated in cellular growth and differentiation. We now show that CREG is predominantly localized within intracellular compartments. Intracellular CREG was found to lack an N-terminal peptide present in the secreted form of the protein. In contrast to normal cells, CREG is largely secreted by fibroblasts missing both mannose 6-phosphate receptors. This is not observed in cells lacking only one of them. Mass spectrometric analysis of recombinant CREG revealed that the protein contains phosphorylated oligosaccharides at either of its two N-glycosylation sites. Cellular CREG was found to cosediment with lysosomal markers upon subcellular fractionation by density-gradient centrifugation. In fibroblasts expressing a CREG-GFP fusion construct, the heterologous protein was detected in compartments containing lysosomal proteins. Immunolocalization of endogenous CREG confirmed that intracellular CREG is localized in lysosomes. Proteolytic processing of intracellular CREG involves the action of lysosomal cysteine proteinases. These results establish that CREG is a lysosomal protein that undergoes proteolytic maturation in the course of its biosynthesis, carries the mannose 6-phosphate recognition marker and depends on the interaction with mannose 6-phosphate receptors for efficient delivery to lysosomes.
Assuntos
Lisossomos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Animais , Células COS , Chlorocebus aethiops , Cisteína Endopeptidases/metabolismo , Glicosilação , Humanos , Insetos , Lisossomos/química , Manosefosfatos/química , Manosefosfatos/metabolismo , Espectrometria de Massas , Camundongos , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Inibidores de Proteases/metabolismo , Receptor IGF Tipo 2/genética , Receptor IGF Tipo 2/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Repressoras/química , Proteínas Repressoras/genéticaRESUMO
Erk/MAPK and TGFbeta signaling cause epithelial to mesenchymal transition (EMT) and metastasis in mouse mammary epithelial cells (EpH4) transformed with oncogenic Ras (EpRas). In trials to unravel underlying mechanisms, expression profiling for EMT-specific genes identified a secreted interleukin-related protein (ILEI), upregulated exclusively at the translational level. Stable overexpression of ILEI in EpH4 and EpRas cells caused EMT, tumor growth, and metastasis, independent of TGFbeta-R signaling and enhanced by Bcl2. RNAi-mediated knockdown of ILEI in EpRas cells before and after EMT (EpRasXT) prevented and reverted TGFbeta-dependent EMT, also abrogating metastasis formation. ILEI is overexpressed and/or altered in intracellular localization in multiple human tumors, an event strongly correlated to invasion/EMT, metastasis formation, and survival in human colon and breast cancer.
Assuntos
Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Citocinas/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células-Tronco Mesenquimais/citologia , Proteínas de Neoplasias/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Citocinas/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica/patologia , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Neoplasias/metabolismo , Neoplasias/patologia , Prognóstico , Biossíntese de Proteínas/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Taxa de Sobrevida , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Epithelial-to-mesenchymal transition (EMT), a switch of polarized epithelial cells to a migratory, fibroblastoid phenotype, is increasingly considered as an important event during malignant tumor progression and metastasis. To identify molecular players involved in EMT and metastasis, we performed expression profiling of a set of combined in vitro/in vivo cellular models, based on clonal, fully polarized mammary epithelial cells. Seven closely related cell pairs were used, which were modified by defined oncogenes and/or external factors and showed specific aspects of epithelial plasticity relevant to cell migration, local invasion and metastasis. Since mRNA levels do not necessarily reflect protein levels in cells, we used an improved expression profiling method based on polysome-bound RNA, suitable to analyse global gene expression on Affymetrix chips. A substantial fraction of all regulated genes was found to be exclusively controlled at the translational level. Furthermore, profiling of the above multiple cell pairs allowed one to identify small numbers of genes by cluster analysis, specifically correlating gene expression with EMT, metastasis, scattering and/or oncogene function. A small set of genes specifically regulated during EMT was identified, including key regulators and signaling pathways involved in cell proliferation, epithelial polarity, survival and trans-differentiation to mesenchymal-like cells with invasive behavior.