Interferon-gamma-stimulated and GTP-binding-proteins-mediated phospholipase A2 activation in human neuroblasts.
Interferon-gamma (IFN-gamma) is a potent growth-inhibitory cytokine also endowed with differentiating activity on neural cells. Binding of IFN-gamma to its high-affinity receptor induces a rapid and transient activation of phospholipase A2 (PLA2). The mechanism coupling the IFN-gamma receptor (IFN-gamma-R) to PLA2 activation is not clearly defined, and no information is available on this mechanism in neuroblast cells. We have tested the hypothesis that GTP-binding proteins (G-proteins) may couple the IFN-gamma-R to PLA2 in the human neuroblastoma (NB) cell line LAN-5. Incubation of NB cells with IFN-gamma resulted in a rapid increase in [3H]arachidonic acid (AA) release, and this effect was blocked by pretreatment with anti-IFN-gamma antibodies. IFN-gamma-stimulated AA release was still observed in permeabilized cells that were blocked by pretreatment with anti-IFN-gamma-R antibodies. Exposure of permeabilized LAN-5 cells to guanosine 5'-[gamma-thio]triphosphate (GTP[S]), a non-hydrolysable GTP analogue, induced a dose-dependent release of [3H]AA. A non-specific nucleotide effect was excluded, since similar stimulatory effects on AA mobilization were not observed by GTP, ATP, CTP, ADP and GDP. IFN-gamma-stimulated AA release was completely blocked by the guanine nucleotide analogue that inhibits G-protein function, guanosine 5'-[beta-thio]diphosphate (GDP[S]). A role for G-proteins in IFN-gamma-R coupling to PLA2 was further supported by the inhibition of IFN-gamma-induced [3H]AA release by treatment of permeabilized cells with pertussis toxin and with the antiserum against the common alpha-subunits of G-proteins. To determine a possible contribution to AA mobilization by the phospholipase C and diacyglycerol lipase pathway or by protein kinase C activation, the effects of neomycin, a phospholipase C inhibitor, and PMA (phorbol 12-myristate 13-acetate), a direct activator of protein kinase C, were investigated. Neither neomycin nor PMA affected either basal or IFN-gamma-stimulated AA release. Ca2+ concentration, which has been shown to regulate the activity of some PLA2s, does not appear to play an important role in the regulation of the IFN-gamma-stimulated PLA2 activity, since incubating permeabilized cells in different concentrations of Ca2+ induced AA release without affecting the IFN-gamma response. Altogether, these findings suggest the existence of IFN-gamma-R, which couples a Ca(2+)-independent PLA2 activation via pertussis-toxin-sensitive G-proteins.
Oncology Research Laboratory, G. Gaslini Children's Hospital, Genoa, Italy.
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Ponzoni
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Cornaglia-Ferraris
D016718:Arachidonic Acid / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D009355:Neomycin / Q000494:pharmacology; D009474:Neurons / Q000201:enzymology*; D009711:Nucleotides / Q000494:pharmacology; D037342:Pertussis Toxin; D010716:Phosphatidylinositols / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011493:Protein Kinase C / Q000502:physiology; D017471:Receptors, Interferon / Q000502:physiology; D011994:Recombinant Proteins; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010738:Type C Phospholipases / Q000502:physiology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D000096743:Interferon gamma Receptor
D016718:Arachidonic Acid; D002118:Calcium; D019204:GTP-Binding Proteins; D007371:Interferon-gamma; D009355:Neomycin; D009711:Nucleotides; D037342:Pertussis Toxin; D010716:Phosphatidylinositols; D010741:Phospholipases A; D054467:Phospholipases A2; D011493:Protein Kinase C; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D013755:Tetradecanoylphorbol Acetate; D010738:Type C Phospholipases; D010566:Virulence Factors, Bordetella; D000096743:Interferon gamma Receptor