Gangliosides inhibit platelet-derived growth factor-stimulated growth, receptor phosphorylation, and dimerization in neuroblastoma SH-SY5Y cells.
SH-SY5Y is a thrice cloned cell line originally derived from the human neuroblastoma cell line SK-N-SH. It grows well in serum-containing medium and undergoes neuritogenesis in response to several trophic factors. Because it has been reported that this clonal line does not have receptors for platelet-derived growth factor (PDGF), it has been unclear what the major mitogenic factor in serum is for these cells. In competitive binding studies using radiolabeled PDGF-BB, we found that SH-SY5Y cells specifically bind PDGF with a KD = 0.14 +/- 0.06 nM and Bmax = 7.3 +/- 2.3 pM. Functionality of these receptors was demonstrated by an increased [3H]-thymidine incorporation in response to PDGF (stimulation index = 2.5). At concentrations of PDGF-BB between 5 and 100 ng/ml, maximum stimulation occurred with 20 ng/ml. Maximum DNA synthesis occurred after 12-24-h exposure to PDGF. Gangliosides GM3 and GT1b greatly inhibited [3H]thymidine incorporation, which was also inhibited to a lesser extent by GM1. Phosphorylation on tyrosine of a 170-kDa protein in response to PDGF stimulation of intact cells was demonstrated by western blot analysis probing with anti-phosphotyrosine antibody. Immunoprecipitation with anti-PDGF beta-receptor antibody and visualization on a western blot with an anti-phosphotyrosine antibody also revealed a 170-kDa protein. Maximum phosphorylation of the 170-kDa protein occurred after 5-min exposure to 20 ng/ml PDGF. This phosphorylation was inhibited by gangliosides GM1, GM2, GD1a, and GT1b but not by GM3. Receptor dimerization was also inhibited by GM1. These results show that SH-SY5Y cells have specific receptors for PDGF-BB that are functional, and can be modulated by gangliosides.
Hynds
D L
DL
Department of Pathology (Neuropathology), Ohio State University, Columbus, USA.
Summers
M
M
Van Brocklyn
J
J
O'Dorisio
M S
MS
Yates
A J
AJ
D001667:Binding, Competitive; D002455:Cell Division / Q000187:drug effects; D004247:DNA / Q000096:biosynthesis; D005732:Gangliosides / Q000494:pharmacology*; D006801:Humans; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation / Q000187:drug effects; D010982:Platelet-Derived Growth Factor / Q000037:antagonists & inhibitors* / Q000494:pharmacology*; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D005732:Gangliosides; D010982:Platelet-Derived Growth Factor; D004247:DNA; D011505:Protein-Tyrosine Kinases; D017479:Receptors, Platelet-Derived Growth Factor