OBJECTIVETo study the effect of endogeneous gangliosides (Gls) on integrin alpha2beta1-mediated adhesion of neuroblastoma cells to collagen (Col).

METHODSNeuroblastoma SK-N-SH cell line was cultured in the modified eagle's medium with the presence of 10 mum D-threo-1-phenyl-2-decanolamino-3-morphinolin-1-propanol (D-PDMP), an inhibitor of glucosylceramide synthase. Flow cytometry was used to detect the expression of integrin alpha2beta1 in the cell line. The effects of Mg2(+) and monoclonal antibodies to integrin alpha2beta1 on the adhesion of the cell line to immobilized Col were observed. The adhesion cell number was measured with the BCA method and presented with absorptance A570.

RESULTSThere was a high expression of integrin alpha2beta1 in the SK-N-SH cell line without D-PDMP treatment. Endogenous Gls in the cells were almost depleted after 6-day exposure to D-PDMP, but the integrin alpha2beta1 expression was not significantly changed. 1 mmoL/L Mg2(+) treatment increased significantly the number of adhesion cells in the SK-N-SH cell line. The adhesion to Col of the SK-N-SH cells exposed to D-PDMP which Gls was depleted was significantly reduced compared with the control SK-N-SH cells treated with 1 mmoL/L Mg2(+) (A570: 0.33 +/- 0.016 vs 0.57 +/- 0.033; P < 0.01). After endogeneous Gls was added into the Gls-depleted SK-N-SH cells, the adhesion of the cells was restored (A570: 0.52 +/- 0.035). The adhesion of SK-N-SH cells was significantly blocked by anti-alpha2 and anti-beta1 monoclonal antibodies, with A570 of 0.31 +/- 0.018 and 0.36 +/- 0.021 respectively.

CONCLUSIONSEndogenous tumor Gls increases neuroblastoma cell adhesion to Col by regulating the function of integrin alpha2beta1, but has no effects on the integrin expression. It is suggested that tumor Gls may play a role in migration, invasion and metastasis of tumor cells.

Antibodies, Monoclonal ; immunology ; Cell Adhesion ; Cell Line, Tumor ; Collagen ; physiology ; Gangliosides ; physiology ; Humans ; Integrin alpha2beta1 ; physiology ; Magnesium ; pharmacology ; Morpholines ; pharmacology ; Neuroblastoma ; pathology

Neuronal apoptosis induced by amyloid beta-peptide (A beta) plays an important role in the pathophysiology of Alzheimer's disease (AD). However, the molecular mechanism underlying A beta-induced apoptosis remains undetermined. The disialoganglioside GD3 involves ceramide-, Fas- and TNF-alpha-mediated apoptosis in lymphoid cells and hepatocytes. Although the implication of GD3 has been suggested, the precise role of GD3 in A beta-induced apoptosis is still unclear. Here, we investsigated the changes of GD3 metabolism and characterized the distribution and trafficking of GD3 during A beta-induced apoptosis using human brain-derived TE671 cells. Extracellular A beta induced apoptosis in a mitochondrial-dependent manner. GD3 level was negligible in the basal condition. However, in response to extracellular A beta, both the expression of GD3 synthase mRNA and the intracellular GD3 level were dramatically increased. Neosynthesized GD3 rapidly accumulated in cell surface lipid microdomains, and was then translocated to mitochondria to execute the apoptosis. Disruption of membrane lipid microdomains with methyl-beta-cyclodextrin significantly prevented both GD3 accumulation in cell surface and A beta-induced apoptosis. Our data suggest that rapidly accumulated GD3 in plasma membrane lipid microdomains prior to mitochondrial translocation is one of the key events in A beta-induced apoptosis.
Amyloid beta-Peptides/*pharmacology ; *Apoptosis ; Cell Line ; Gangliosides/*metabolism/physiology ; Humans ; Membrane Microdomains/*metabolism ; Mitochondria/*metabolism ; Sialyltransferases/genetics/metabolism ; beta-Cyclodextrins/pharmacology

In the injured brain, microglia is known to be activated and produce proinflammatory mediators such as interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS). We investigated the role of protein kinase A (PKA) in microglial activation by both plasminogen and gangliosides in rat primary microglia and in the BV2 immortalized murine microglial cell line. Both plasminogen and gangliosides induced IL-1beta, TNF-alpha and iNOS mRNA expression, and that this expression was inhibited by the addition of the PKA inhibitors, KT5720 and H89. Both plasminogen and gangliosides activated PKA and increased the DNA binding activity of the cAMP response element- binding protein (CREB). Furthermore, KT5720 and H89 reduced the DNA binding activities of CREB and NF-kappaB in plasminogen-treated cells. These results suggest that PKA plays an important role in plasminogen and gangliosides- induced microglial activation.
Animals ; Carbazoles/pharmacology ; Cell Line ; Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors/*physiology ; DNA-Binding Protein, Cyclic AMP-Responsive/metabolism ; DNA-Binding Proteins/metabolism ; Gangliosides/pharmacology/*physiology ; Gene Expression Regulation ; Indoles/pharmacology ; Interleukin-1/genetics ; Isoquinolines/pharmacology ; Mice ; Microglia/drug effects/*enzymology/*immunology ; NF-kappa B/metabolism ; Nitric-Oxide Synthase/genetics ; Plasminogen/pharmacology/*physiology ; Pyrroles/pharmacology ; RNA, Messenger/analysis/metabolism ; Rats ; Research Support, Non-U.S. Gov't ; Sulfonamides/pharmacology ; Tumor Necrosis Factor-alpha/genetics