Translocation and leukotriene synthetic capacity of nuclear 5-lipoxygenase in rat basophilic leukemia cells and alveolar macrophages.
Leukotriene (LT) synthesis involves the translocation of enzymatically active 5-lipoxygenase (5-LO) from a soluble site to a bound site, where it interacts with 5-lipoxygenase-activating protein (FLAP). In human polymorphonuclear leukocytes (PMNs), 5-LO moves from the cytosol to the nuclear envelope (NE) to interact with FLAP. However, 5-LO has recently been found within the nucleus, as well as the cytosol, of rat basophilic leukemia (RBL) cells and alveolar macrophages (AMs). To assess whether nuclear 5-LO can contribute to LT synthesis in these cells, we investigated whether this enzyme pool 1) translocates upon cell activation, 2) colocalizes with FLAP, and 3) is enzymatically active. By cell fractionation followed by immunoblotting, both cytosolic and nuclear soluble 5-LO decreased dramatically in RBL cells following activation with the calcium ionophore A23187. Concurrently, 5-LO increased in the pelletable nuclear pool, where FLAP was also detected. The loss of both cytosolic and nuclear soluble 5-LO, with concomitant increase exclusively at the NE, as well as co-localization with FLAP, were confirmed by indirect immunofluorescent and confocal microscopy. In AMs, the nuclear soluble pool of 5-LO moved to the NE, where FLAP was also found; however, the cytosolic 5-LO pool did not translocate. Application of these methods to PMNs confirmed that cytosolic 5-LO moved to the nuclear envelope and co-localized with FLAP. By cell-free assay, nuclear soluble proteins from both RBL cells and AMs, but not PMNs, were able to generate 5-LO products from arachidonate, and this was inhibited by the direct 5-LO inhibitor zileuton. Cytosolic proteins from all cell types also showed cell-free 5-LO activity. These results demonstrate three distinct patterns of 5-LO translocation that are specific for each cell type: translocation of only a cytosolic pool in PMNs, of only a nuclear pool in AMs, and of both cytosolic and nuclear pools in RBL cells. By virtue of its enzymatic activity and ability to translocate, nuclear 5-LO has the potential to contribute to LT synthesis in RBL cells and AMs. Finally, these results provide a foundation for considering the individual functions of discrete pools of 5-LO in future studies.
Brock
T G
TG
Department of Internal Medicine, University of MIchigan Medical Center, Ann Arbor 48109-0652, USA.
McNish
R W
RW
Peters-Golden
M
M
D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002458:Cell Fractionation; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology*; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D006801:Humans; D007700:Kinetics; D015471:Leukemia, Basophilic, Acute / Q000201:enzymology*; D015289:Leukotrienes / Q000096:biosynthesis*; D016676:Macrophages, Alveolar / Q000201:enzymology*; D018613:Microscopy, Confocal; D009504:Neutrophils / Q000201:enzymology; D051381:Rats; D014407:Tumor Cells, Cultured
D015289:Leukotrienes; D000001:Calcimycin; D001094:Arachidonate 5-Lipoxygenase