Interleukin 2 mRNA induction in human lymphocytes: analysis of the synergistic effect of a calcium ionophore A23187 and a phorbol ester.
Induction of interleukin 2 (IL2) mRNA in human tonsillar lymphocytes under various conditions was examined by cytoplasmic dot hybridization using a 32P-labeled IL2 cDNA probe to study the signal transduction mechanisms which lead to IL2 gene expression. A tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate (TPA), acted synergistically with a Ca2+ ionophore A23187 or phytohemagglutinin (PHA) to induce a high level of IL2 mRNA in lymphocytes, whereas each of them by itself could not induce the mRNA production. In two-step culture experiments the lymphocytes pulse-incubated with TPA for 1 h (the first culture) could efficiently initiate IL2 mRNA production by subsequent culture with A23187 or PHA (the second culture). Results obtained by removal of extracellular Ca2+ from either the first or second culture revealed that Ca2+ was not necessarily required during the first culture with TPA, but it is essential in the second culture with A23187 or PHA, regardless of the presence or absence of Ca2+ in the first culture. A reagent known to be a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), almost completely inhibited the IL2 mRNA induction in A23187-TPA-stimulated lymphocytes at a concentration of 25 microM, whereas N-(6-aminohexyl)-1-naphthalenesulfonamide that has much lower affinity for calmodulin than W-7 did not inhibit at this concentration. The IL2 mRNA induction was also blocked by the addition of 50 microM of 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride which is known to block the release of Ca2+ from intracellular storage sites. These results show that mobilization of Ca2+ and the calmodulin-dependent regulatory system appear to work synergistically with TPA which probably activates protein kinase C in the pathway to IL2 gene expression.
D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000502:physiology; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002478:Cells, Cultured; D004357:Drug Synergism; D005707:Gallic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D007376:Interleukin-2 / Q000235:genetics*; D014066:Palatine Tonsil / Q000166:cytology; D010704:Phorbols / Q000494:pharmacology*; D011493:Protein Kinase C / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D002147:Calmodulin; D007376:Interleukin-2; D010704:Phorbols; D012333:RNA, Messenger; D000001:Calcimycin; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005707:Gallic Acid; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium