Signal transduction by tumor necrosis factor α is mediated through a guanine nucleotide-binding protein in osteoblast-like cell line, MC3T3-E1

Transmembrane signalling mechanisms of tumor necrosis factor α (TNFα) were examined with special reference to the involvement of G-protein, in intact and permeabilized murine osteoblast-like cells. TNFα stimulated the release of ³H radioactivity from intact cells labeled with [³H]arachidonic acid within 10 min in a dose dependent manner and the production of lyso forms of phospholipids, an event presumably mediated through the activation of phospholipase A₂. Production of cAMP and inositol 1,4,5-trisphosphate was not affected by TNFα. Pretreatment of the cells with pertussis toxin inhibited the liberation of [³H]arachidonate. GTPγS (guanosine 5'-3-O- (thio)triphosphate) reduced the binding affinity of [¹²⁵I]TNFα to β- escin-permeabilized cells. The addition of TNFα together with an unhydrolyzable analog of GTP, GTPγS, to the β-escin-permeabilized cells prelabeled with [³H]arachidonic acid led to a release of the ³H radioactivity. The production of prostaglandin E₂ (PGE₂) was markedly stimulated by TNFα in a dose over 100 ng/ml, with a latent time of about 3 h, and the stimulation was abolished by pretreatment with pertussis toxin. The time and dose requirements for this process differed from those for the possible activation of phospholipase A₂, thereby indicating that other process(es) in addition to the activation of phospholipase A₂ may be responsible for the enhanced production of PGE₂. The activity of cyclooxygenase (i.e. the combined activities of prostaglandin endoperoxide syntase and PGH₂-PGE₂ isomerase) was stimulated by TNFα with much the same time and dose requirements as for the production of PGE₂, and the activation was found to be due to the increased amount of the enzyme, as assessed by a Western blot analysis with anti-cyclooxygenase antibody. This process was also sensitive to pertussis toxin. Therefore, receptors for TNFα in MC3T3-E1 cells apparently couple to G-protein sensitive to pertussis toxin and the coupling regulates the activations of phospholipase A₂ and the de novo synthesis of cyclooxygenase.