RESUMO
EpCAM was found to be overexpressed on epithelial progenitors, carcinomas and cancer-initiating cells. The role of EpCAM in proliferation, and its association with cancer is poorly explained by proposed cell adhesion functions. Here we show that regulated intramembrane proteolysis activates EpCAM as a mitogenic signal transducer in vitro and in vivo. This involves shedding of its ectodomain EpEX and nuclear translocation of its intracellular domain EpICD. Cleavage of EpCAM is sequentially catalysed by TACE and presenilin-2. Pharmacological inhibition or genetic silencing of either protease impairs growth-promoting signalling by EpCAM, which is compensated for by EpICD. Released EpICD associates with FHL2, beta-catenin and Lef-1 to form a nuclear complex that contacts DNA at Lef-1 consensus sites, induces gene transcription and is oncogenic in immunodeficient mice. In patients, EpICD was found in nuclei of colon carcinoma but not of normal tissue. Nuclear signalling of EpCAM explains how EpCAM functions in cell proliferation.
Assuntos
Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Mitose/fisiologia , Transdução de Sinais/fisiologia , Proteínas ADAM/metabolismo , Proteína ADAM17 , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Antígenos de Neoplasias/genética , Carcinoma/genética , Carcinoma/metabolismo , Moléculas de Adesão Celular/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Núcleo Celular/genética , Transformação Celular Neoplásica/genética , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Molécula de Adesão da Célula Epitelial , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas com Homeodomínio LIM , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Masculino , Camundongos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Células NIH 3T3 , Presenilina-2/metabolismo , Estrutura Terciária de Proteína , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
We have identified in organic solvent extracts of whole cells of the gram-positive pathogen Rhodococcus equi two channel-forming proteins with different and complementary properties. The isolated proteins were able to increase the specific conductance of artificial lipid bilayer membranes made from phosphatidylcholine-phosphatidylserine mixtures by the formation of channels able to be permeated by ions. The channel-forming protein PorA(Req) (R. equi pore A) is characterized by the formation of cation-selective channels, which are voltage gated. PorA(Req) has a single-channel conductance of 4 nS in 1 M KCl and shows high permeability for positively charged solutes because of the presence of negative point charges. According to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein has an apparent molecular mass of about 67 kDa. The analysis (using the effect of negative charges on channel conductance) of the concentration dependence of the single-channel conductance suggested that the diameter of the cell wall channel is about 2.0 nm. The second channel (formed by PorB(Req) [R. equi pore B]) shows a preferred movement of anions through the channel and is not voltage gated. This channel shows a single-channel conductance of 300 pS in 1 M KCl and is characterized by the presence of positive point charges in or near the channel mouth. Based on SDS-PAGE, the apparent molecular mass of the channel-forming protein is about 11 kDa. Channel-forming properties of the investigated cell wall porins were compared with those of others isolated from mycolic acid-containing actinomycetes. We present here the first report of a fully characterized anion-selective cell wall channel from a member of the order Actinomycetales.