Induction of gene expression by IL-1 in NIH 3T3 cells. Possible requirement of protein kinase C activity and independence from arachidonic acid metabolism.

NIH 3T3 fibroblasts were transfected with the chloramphenicol-acetyltransferase (CAT) gene under the control of the SV40 early promoter, which can be stimulated by IL-1. CAT activity in cell lysates and PGE2 release in the supernatants were measured in control and stimulated cell cultures in parallel. Human IL-1 beta (180 pM) and human rTNF-alpha (3 nM) significantly stimulated both CAT activity and PGE2 release. The combined incubation of the two cytokines resulted in a synergistic effect on PGE2 release. The addition of AA (30 microM) greatly stimulated PGE2 release without affecting CAT activity. Similarly, drugs interfering with AA metabolism were without effect on CAT activity although profoundly reducing PGE2 release. Forskolin (0.1 microM) did not modify either parameter. The glucocorticoid fluocinolone (20 nM) was able to decrease both parameters. Protein kinase inhibitors H7 (5-50 microM) and sphingosine (50 microM) inhibited only IL-1-induced CAT activity, whereas H8 (5-50 microM) and HA1004 (50 microM) were ineffective on both parameters. PMA (0.5 microM) and R59 022, a diacylglycerol kinase inhibitor (10 microM), did not modify either control or IL-1-induced CAT activity. IL-1-stimulated PGE2 release was potentiated by PMA, although this effect was not inhibited by H7. The data suggest that: 1) in NIH 3T3 cells the activation of AA metabolism by IL-1 is not involved in IL-1-induced gene expression; 2) protein kinase C activity is required but not sufficient for IL-1-induced gene expression; and 3) PMA may stimulate AA metabolism by a mechanism in part independent of protein kinase activity.