Kazuhisa Iwabuchi

Glycosphingolipid-enriched signaling domain in mouse neuroblastoma Neuro2a cells. Mechanism of ganglioside-dependent neuritogenesis.

Differentiation and neuritogenesis of mouse neuroblastoma Neuro2a cells are induced by exogenous ganglioside but are not induced by nerve growth factor because its receptor is absent in these cells. In view of the emerging concept of the “glycosphingolipid-enriched domain” (GEM), we studied the mechanism of the ganglioside effect, focusing on the structure and function of such a domain. GEM in Neuro2a cells, separated as a low density membrane fraction, contains essentially all glycosphingolipids and sphingomyelin, together with five signal transducer molecules (c-Src, Lyn, Csk, Rho A, Ha-Ras). 3H-Labeled Il3NeuAc-LacCer (GM3), Gb4Cer (globoside), and Il3NeuAc-Gg4Cer (GM1) added exogenously to cells were incorporated and concentrated in the low density GEM fraction. In contrast, more than 50% of glycerophospholipids and 30% of cholesterol were found in the high density fraction.3H-Labeled phosphatidylcholine added exogenously to cells was incorporated exclusively in the high density fraction. c-Src, the predominant signal transducer in the microdomain, was coimmunoprecipitated with anti-GM3 antibody DH2 or with anti-Csk; reciprocally, Csk was coimmunoprecipitated with anti-c-Src, indicating a close association of GM3, c-Src, and Csk. Brief stimulation of an isolated GEM fraction by the exogenous addition of GM3, but not lactosylceramide, caused enhanced c-Src phosphorylation with a concomitant decrease of Csk level in GEM. A decreased Csk/c-Src ratio in GEM may cause activation of c-Src because Csk is a negative regulator of c-Src. The effect of exogenous GM3 on c-Src activity was also observed in intact Neuro2a cells. Activation of c-Src was followed by rapid and prolonged (60 min) enhancement of mitogen-activated protein kinase activity leading to neuritogenesis. Thus, the ganglioside induction of neuritogenesis in Neuro2a cells is mediated by GEM structure and function.

VEGF regulates the proliferation of acid-exposed alveolar lining epithelial cells

Background: Acid induced pneumonitis resulting in acute respiratory distress syndrome (ARDS) is characterised by increased alveolar permeability and accumulation of neutrophils. It is hypothesised that vascular endothelial growth factor (VEGF) is involved in the development of lung oedema. Furthermore, lower levels of VEGF are detected in bronchoalveolar lavage fluid from patients with ARDS than from non-ARDS patients. We hypothesised that VEGF acts cytoprotectively and have investigated this possibility in vitro with A549 cells. Methods: A549 cells were incubated in 24 well culture dishes 24 hours before exposure to acid, then incubated with serum free medium containing various concentrations of HCl for 30 minutes at 37°C in 5% CO2. The acidified medium was changed to normal complete medium; at specified incubation periods the supernatants were collected and the VEGF concentration measured and the number of adherent cells counted. Results: Proliferation of A549 cells and VEGF production were suppressed for at least 48 hours in HCl at a concentration of 50 mM. Restoration of cellular proliferation occurred following exogenous administration of VEGF (concentration of 1–250 ng/ml) and was inhibited by co-incubation with neutralising anti-VEGF antibody, indicating an interaction between VEGF molecules and A549 cells. Control cells were not influenced by administration of exogenous VEGF or anti-VEGF antibody. Treatment with neutralising anti-VEGF receptor (VEGFR) antibodies against VEGFR-1 and VEGFR-2 suppressed proliferation of acid exposed A549 cells but had no effect on control cells. Conclusions: Exogenous VEGF interacts with VEGFR-1 and VEGFR-2 on the surface and regulates the proliferation of injured alveolar lining epithelial cells in an autocrine or paracrine fashion.