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.
|ジャーナル||Journal of Biological Chemistry|
|出版ステータス||出版済み - 1992|
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