Interferon-γ (IFN-γ) is a potent growth-inhibitory cytokine also endowed with differentiating activity on neural cells. Binding of IFN-γ to its high-affinity receptor induces a rapid and transient activation of phospholipase A2 (PLA2). The mechanism coupling the IFN-γ receptor (IFN-γ-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-γ-R to PLA2 in the human neuroblastoma (NB) cell line LAN-5. Incubation of NB cells with IFN-γ resulted in a rapid increase in [3H]arachidonic acid (AA) release, and this effect was blocked by pretreatment with anti-IFN-γ antibodies. IFN-γ-stimulated AA release was still observed in permeabilized cells that were blocked by pretreatment with anti-IFN-γ-R antibodies. Exposure of permeabilized LAN-5 cells to guanosine 5'-[γ-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-γ-stimulated AA release was completely blocked by the guanine nucleotide analogue that inhibits G-protein function, guanosine 5' [B-thio]diphosphate (GDP[S]). A role for G-proteins in IFN-γ-R coupling to PLA2 was further supported by the inhibition of IFN-γ-induced [3H]AA release by treatment of permeabilized cells with pertussis toxin and with the antiserum against the common α-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-γ-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-γ-stimulated PLA2 activity, since incubating permeabilized cells in different concentrations of Ca2+ induced AA release without affecting the IFN-γ response. Altogether, these findings suggest the existence of IFN-γ-R, which couples a Ca2+-independent PLA2 activation via pertussis-toxin-sensitive G-proteins.
|Number of pages||6|
|Publication status||Published - 1993|
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