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 cell line Mono Mac 6 was upregulated by combined treatment with transforming growth factor beta 1 (TGF-beta) and 1,25-dihydroxyvitamin D3 (VD3). 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 limit. 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 agent 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 was required for full induction of 5-LO protein expression and for upregulation of enzyme activity. Partial purification of 5-LO from Mono Mac 6 cells and recombination with soluble cellular proteins from different sources indicated the presence of cytosolic factors that affect the activity of 5-LO.
Brungs
M
M
Department of Pharmaceutical Chemistry, University of Tübingen, Germany.
Rådmark
O
O
Samuelsson
B
B
Steinhilber
D
D
D001094:Arachidonate 5-Lipoxygenase / Q000096:biosynthesis* / Q000302:isolation & purification; D001483:Base Sequence; D002454:Cell Differentiation; D002460:Cell Line; D002762:Cholecalciferol / Q000494:pharmacology*; D002846:Chromatography, Affinity; D017931:DNA Primers; D004347:Drug Interactions; D004790:Enzyme Induction / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D017931:DNA Primers; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D002762:Cholecalciferol; D001094:Arachidonate 5-Lipoxygenase