Regulation of 5-lipoxygenase and 5-lipoxygenase-activating protein expression in HL-60 cells.
5-Lipoxygenase performs the first two enzymic reactions in the biosynthetic pathway for the leukotrienes. We have utilized HL-60 cells to study the mechanisms regulating expression of 5-lipoxygenase and the recently described 18 kDa membrane protein, 5-lipoxygenase-activating protein (FLAP). Differentiation of HL-60 cells into granulocyte-like cells with dimethyl sulphoxide (Me2SO), retinoic acid or dibutyryl cyclic AMP (Bt2-cAMP) resulted in a 2-3-fold increase in 5-lipoxygenase enzyme activity and a 4-fold increase in leukotriene B4 synthesis. Differentiation of HL-60 cells into monocyte-like cells with 1,25-dihydroxyvitamin D3 induced 5-lipoxygenase activity 5-fold, but leukotriene B4 production was only increased 2-fold. Differentiation of HL-60 cells with phorbol 12-myristate 13-acetate (PMA) into macrophage-like cells failed to induce 5-lipoxygenase or leukotriene B4 production. Examination of the levels of the transcript encoding 5-lipoxygenase and FLAP demonstrated that differentiation of HL-60 cells into granulocytes resulted in a co-ordinate induction of 5-lipoxygenase and FLAP mRNA. In contrast, differentiation of HL-60 cells into monocytes resulted in discordant regulation of 5-lipoxygenase and FLAP mRNA. Treatment with 1,25-dihydroxyvitamin D3 resulted in a 6-fold increase in 5-lipoxygenase mRNA and a 1.3-fold increase in FLAP mRNA, while treatment with phorbol ester did not induce 5-lipoxygenase mRNA but did increase FLAP mRNA 2-fold. The transcriptional rate of the 5-lipoxygenase and FLAP genes did not change upon Me2SO or 1,25-dihydroxyvitamin D3 treatment, suggesting that the increase of the mRNA coding for these proteins was not due to transcriptional activation of their respective genes. The mRNA half-life for 5-lipoxygenase did not change significantly upon treatment with Me2SO or 1,25-dihydroxyvitamin D3. The FLAP mRNA half-life increased from approx. 3.5 h to 4.5 h in cells treated with either Me2SO or 1,25-dihydroxyvitamin D3. These data suggest that expression of 5-lipoxygenase and FLAP is controlled by a post-transcriptional event other than stabilization of the mRNA.
Department of Molecular and Cellular Biology, ISIS Pharmaceuticals, Carlsbad, CA 92008.
C F
CF
Bennett
M Y
MY
Chiang
B P
BP
Monia
S T
ST
Crooke
D058927:5-Lipoxygenase-Activating Proteins; D001094:Arachidonate 5-Lipoxygenase / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D002454:Cell Differentiation / Q000187:drug effects; D004277:DNA, Single-Stranded; D004790:Enzyme Induction; D015971:Gene Expression Regulation, Enzymologic; D006098:Granulocytes / Q000166:cytology / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D007700:Kinetics; D007975:Leukotriene B4 / Q000096:biosynthesis; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D058927:5-Lipoxygenase-Activating Proteins; C548592:ALOX5AP protein, human; D002352:Carrier Proteins; D004277:DNA, Single-Stranded; D008565:Membrane Proteins; D012333:RNA, Messenger; D007975:Leukotriene B4; D001094:Arachidonate 5-Lipoxygenase