SEQUENTIAL INDUCTION OF 5-LIPOXYGENASE GENE-EXPRESSION AND ACTIVITY IN MONO-MAC-6 CELLS BY TRANSFORMING GROWTH-FACTOR-BETA AND 1,25-DIHYDROXYVITAMIN-D3

5-Lipoxygenase (5-LO; EC 1.13.11.34) activity in the human monocytic c ell line Mono Mac 6 was upregulated by combined treatment with transfo rming growth factor beta 1 (TGF-beta) and 1,25-dihydroxyvitamin D-3 (V D3). In undifferentiated cells, 5-LO enzyme activity was undetectable. After the addition of TGF-beta plus VD3, the activity of intact cells was 800 ng per 10(6) cells-500 times more than the assay detection li mit. Also 5-LO protein and mRNA expression were induced >128-fold and 64-fold, respectively, as compared to undifferentiated cells. Both TGF -beta and VD3 were required for these prominent responses. Either agen t alone gave small amounts of 5-LO protein and mRNA but very low 5-LO activities. After the addition of TGF-beta and VD3, the induction of 5 -LO protein was obvious after 1 day, but the increase in activity was delayed and did not appear until the second day. Pretreatment of cells with TGF-beta or VD3 alone for 2 days led to 5-LO protein expression but very low enzyme activity. Addition of the lacking second inducer w as required for full induction of 5-LO protein expression and for upre gulation of enzyme activity, partial purification of 5-LO from Mono Ma c 6 cells and recombination with soluble cellular proteins from differ ent sources indicated the presence of cytosolic factors that affect th e activity of 5-LO.