NORDIHYDROGUAIARETIC ACID INHIBITS UROKINASE SYNTHESIS BY PHORBOL-MYRISTATE ACETATE-STIMULATED LLC-PK1 CELLS

Protein kinase C (PKC) activation is regulated by Ca2+, phospholipids, diacylglycerol (DAG) and fatty acids. Phorbol myristate acetate (PMA) which mimics the effect of DAG on PKC induces transcriptional activation of the urokinase-type plasminogen activator (u-PA) gene in LLC-PK1 cells. We examined in the present work the relationships between PKC activity, fatty acids, and u-PA synthesis in this cell line. We showed that H7, an inhibitor of PKC, inhibited the PMA-induced u-PA synthesis by LLC-PK1 cells. PMA-induced u-PA synthesis was enhanced by eicosatetraynoic acid (ETYA), a competitive inhibitor of both the lipoxygenase and cyclooxygenase pathways and inhibited by nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase pathway. Three other unrelated lipoxygenase inhibitors (phenidone 100 μM, BW755 50 μM and diethylcarbamazine 50 μM) had no effect on u-PA biosynthesis. Two polyunsaturated fatty acids other than ETYA, arachidonic acid and linoleic acid, also potentiated the PMA effect and a lipoxygenase derivative, 12 hydroxyeicosatetraenoic acid (12 HETE), did not modify the basal and PMA-stimulated u-PA syntheses. PKC activity purified from cytosol of LLC-PK1 cells was stimulated by addition of 16 nM PMA in vitro and this effect was blunted by simultaneous addition of 5 μM NDGA. By Northern blot analysis using a pig u-PA cDNA probe we found that PMA increased the steady state level of u-PA mRNA after 2 h of incubation and that NDGA inhibited this effect. These data suggest that NDGA inhibits PMA-stimulated PKC activity in intact cells leading to a decrease of u-PA mRNA level and u-PA biosynthesis in PMA-stimulated LLC-PK1 cells. Polyunsaturated fatty acids have opposite effects. © 1990.