Platelet derived growth factor recruits lactosylceramide to induce cell proliferation in UDP Gal:GlcCer: β1 → 4Galactosyltransferase (GalT-V) mutant Chinese hamster ovary cells

Recent molecular cloning studies have suggested the presence of at least two β4Gal transferase genes (β4GalT-V and β4GalT-VI) that may encode lactosylceramide synthase but whether they are functional in vivo and whether they mediate growth factor induced phenotypic change such as cell proliferation is not known. Our previous studies lead to the suggestion that various risk factors in atherosclerosis such as oxidized LDL, shear stress, nicotine, tumor necrosis factor-α converge upon LacCer synthase to induce critical phenotypic changes such as cell proliferation and cell adhesion [1]. However, whether platelet-derived growth factor also recruits LacCer synthase in mediating cell proliferation is not known. Here we have employed a Chinese hamster ovary mutant cell line Pro^-5Lec20 to determine whether this enzyme physiologically functions to mediate cell proliferation. We show that PDGF stimulates the activity of UDP galactose:glucosylceramide, β1,4galactosyltransferase. The activity of LacCer synthase increased about 2.5 fold within 2.5-5 min of incubation with PDGF in both wild type and Pro ^-5Lec20 cells. Concomitantly, there was an increase in the generation of superoxide radicals, p⁴⁴MAPK phosphorylation and cell proliferation in CHO cells. D-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), a potent inhibitor of GlcCer synthase/LacCer synthase impaired PDGF mediated induction of LacCer synthase activity, superoxide generation, p ⁴⁴ MAPK activation and cell proliferation in Pro^-5Lec20 cells. PDGF-induced superoxide generation was also mitigated by the use of diphenylene iodonium; an inhibitor of NADPH oxidase activity that is required for superoxide generation. This inhibition was bypassed by the addition of lactosylceramide. Thus, β4GalT-V gene produces a bona fide LacCer synthase that can function in vivo to generate LacCer. Moreover, this enzyme alone can mediate PDGF induced activation of a signal transduction cascade involving superoxide generation, p⁴⁴MAPK activation, phosphorylation of Akt and cell proliferation.