INHIBITION OF IMMEDIATE-EARLY-GENE INDUCTION IN RENAL MESANGIAL CELLSBY DEPLETION OF INTRACELLULAR POLYAMINES

Mitogens have been shown to stimulate the activity of the rate-limitin g enzyme for polyamine synthesis, ornithine decarboxylase (ODC), and O DC mRNA expression in cultured rat mesangial cells (MCs). In addition, inhibition of ODC by alpha-difluoromethylornithine (DFMO) results in growth arrest of MCs. To elucidate the mechanisms involved in the inhi bition of MC proliferation due to polyamine depletion, we studied the effects of DFMO on the activation of phospholipase C and induction of the immediate early genes (IEGs), c-fos, c-jun and Egr-1, which are th ought to regulate cell growth. Mitogenic 10% fetal-calf serum (FCS) an d 1 unit/ml thrombin activated phospholipase C in MCs within 30 s, as assessed by generation of [H-3]inositol phosphates. This activation wa s not affected by DFMO. mRNAs of the IEGs c-fos, c-jun and Egr-1 were induced by FCS within 15 min. Expression of these genes reached a peak at 60 min and disappeared at 3 h. Treatment of MCs with a growth-supp ressing dose of DFMO (5 mM) inhibited mRNAs of all three IEGs by 52-87 % at 1 h. Total expression of Egr-1 over 20-120 min was diminished by 41%, and the time point of maximal expression was delayed by 40 min. T his inhibitory effect was abolished in a time-dependent manner (1-3 da ys) by prior addition of 200 muM putrescine, the reaction product of O DC. Egr-1 mRNA expression was superinduced by the inhibitor of protein synthesis, cycloheximide. This effect was also blocked by DFMO. The r esults indicate that the DFMO-induced process of MC growth inhibition involves steps necessary for IEG activation. The signal-transduction s tep sensitive to polyamines occurs distal to the activation of phospho lipase C. Since reconstitution of normal induction of IEGs requires 3 days, it seems likely that polyamine depletion affects the regulation of IEG expression in an indirect fashion. We conclude that activation of IEGs requires the presence of polyamines and plays a significant ro le in the induction of MC replication.