RESUMEN
The L1 adhesion molecule plays an important role in axon guidance and cell migration in the nervous system. L1 is also expressed by many human carcinomas. In addition to cell surface expression, the L1 ectodomain can be released by a metalloproteinase, but the biological function of this process is unknown. Here we demonstrate that membrane-proximal cleavage of L1 can be detected in tumors and in the developing mouse brain. The shedding of L1 involved a disintegrin and metalloproteinase (ADAM)10, as transfection with dominant-negative ADAM10 completely abolishes L1 release. L1-transfected CHO cells (L1-CHO) showed enhanced haptotactic migration on fibronectin and laminin, which was blocked by antibodies to alpha v beta 5 and L1. Migration of L1-CHO cells, but not the basal migration of CHO cells, was blocked by a metalloproteinase inhibitor, indicating a role for L1 shedding in the migration process. CHO and metalloproteinase-inhibited L1-CHO cells were stimulated to migrate by soluble L1-Fc protein. The induction of migration was blocked by alpha v beta 5-specific antibodies and required Arg-Gly-Asp sites in L1. A 150-kD L1 fragment released by plasmin could also stimulate CHO cell migration. We propose that ectodomain-released L1 promotes migration by autocrine/paracrine stimulation via alpha v beta 5. This regulatory loop could be relevant for migratory processes under physiological and pathophysiological conditions.
Asunto(s)
Movimiento Celular/fisiología , Integrinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Receptores de Vitronectina , Secretasas de la Proteína Precursora del Amiloide , Animales , Anticuerpos Monoclonales/inmunología , Ácido Aspártico Endopeptidasas , Comunicación Autocrina , Sitios de Unión , Transporte Biológico , Encéfalo/embriología , Encéfalo/metabolismo , Células CHO , Membrana Celular/metabolismo , Cricetinae , Citoplasma/metabolismo , Endopeptidasas/metabolismo , Fibrinolisina/metabolismo , Expresión Génica , Humanos , Integrinas/inmunología , Complejo de Antígeno L1 de Leucocito , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Ratones , Moléculas de Adhesión de Célula Nerviosa/antagonistas & inhibidores , Moléculas de Adhesión de Célula Nerviosa/genética , Oligopéptidos/metabolismo , Fragmentos de Péptidos/metabolismo , Solubilidad , Células Tumorales CultivadasRESUMEN
Ovarian carcinoma is the leading cause of death from gynecological cancers in many countries. Fucosylated glycoconjugates have been associated with various carcinomas. In the present study, we have characterized the expression of alpha3/4 fucosyltransferases transcripts and their products, the Lewis carbohydrate determinants, and their in vitro specificity towards synthetic acceptors using ovarian carcinoma cell lines OVM, m130, GG and SKOV3. We found different expression patterns: GG cells expressed mostly Lewisx (Lex), Lewisy (Ley), sLea and Leb, and m130 cells expressed mostly Lex and Ley. The detection was on the plasma membrane and in intracellular vesicles. OVM and SKOV3 cells had very low amounts of staining. From RT-PCR studies, enzyme specificity of cellular extracts towards a panel of synthetic carbohydrate acceptors and Western blot analysis we concluded that Lea, sLea and Leb were synthesised by FUT3, whereas Lex and Ley were synthesized by FUT4 and FUT9 in both cell lines. The GG and m130 cell lines are adequate models to investigate the role of Lex, Ley, sLea and Leb in ovarian carcinoma development.
Asunto(s)
Fucosiltransferasas/genética , Regulación Neoplásica de la Expresión Génica , Antígenos del Grupo Sanguíneo de Lewis/genética , Neoplasias Ováricas/genética , Adenocarcinoma Mucinoso/genética , Adenocarcinoma Mucinoso/metabolismo , Adenocarcinoma Mucinoso/patología , Western Blotting , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patología , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patología , Femenino , Fucosa/metabolismo , Fucosiltransferasas/metabolismo , Humanos , Lectinas/metabolismo , Antígenos del Grupo Sanguíneo de Lewis/metabolismo , Microscopía Fluorescente , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Exosomes are small membrane vesicles that are secreted from a variety of cell types into various body fluids including the blood and urine. These vesicles are thought to play a role in cell-cell interactions. CD24 is a small but extensively glycosylated protein linked to the cell surface by means of a glycosyl-phosphatidylinositol anchor. In this study we found that CD24 is present in membrane vesicles characterized as exosomes that were isolated from the urine of normal individuals. CD24 was expressed by both tubule cells and podocytes and treatment of the latter with a cholesterol-extracting agent, but not with a calcium ionophore, caused the release of CD24-containing exosomes. Using CD24 as a marker, we found exosomes in the urine of newborn infants and in the amniotic fluid of pregnant women with similar findings made in mice. Interestingly, studies with CD24 knockout mice showed that the exosomes are released from the fetus but not from the mother; however, exosome release was similar from both the knockout and the wild-type mice. This indicates that CD24 is not essential for exosome formation or release but may be a convenient exosome marker. Our studies suggest that exosomal secretion from the embryonic kidney could play a biological role at the fetal-maternal interphase.