Abstract
Arachidonate metabolism by 5-lipoxygenase (5-LO) coincides with the translocation of the enzyme from a soluble to a pelletable fraction in thoroughly disrupted granulocytic cells. While immunoelectron microscopy has identified the nuclear membrane as the site at which 5-LO, as well as 5-LO activating protein (FLAP), are localized in activated cells, the locale of soluble 5-LO in unstimulated cells could not be established by this technique. We asked whether the nucleus might also be the site for soluble 5-LO in unstimulated cells, and utilized rat basophilic leukemia (RBL) cells as model granulocytic cells to address this question. Using three different techniques to disrupt cells while leaving nuclei intact (mild nitrogen cavitation, Dounce homogenization, and detergent lysis), immunoblot analysis indicated abundant 5-LO in isolated nuclei. Within purified nuclei, 5-LO existed in two pools: a soluble pool that was readily released upon nuclear disruption and a bound pool that was not removed by 300 mM NaCl treatment. In all cases, 5-LO was also found in cytosolic and non-nuclear membrane fractions. Indirect immunofluorescent microscopy confirmed the presence of abundant 5-LO within the nucleus with minimal extranuclear signal in most cells. However, a minority of cells, characterized by condensed chromatin, showed no nuclear-associated staining with increased cytoplasmic staining for 5-LO. This suggested that some of the cytosolic 5-LO found by cell fractionation resulted from these dividing cells. When the contribution from dividing cells was minimized, either by overnight serum deprivation or by isolating cytoplasts of nucleus-containing cells, 5-LO was prominent in the nuclear fraction but negligible in the cytosolic fraction. In contrast to this distribution in RBL cells, 5-LO in unstimulated human neutrophils was predominantly cytosolic, by both immunoblot and immunofluorescence analyses. In both RBL cells and human neutrophils, FLAP was localized at the nuclear membrane and the endoplasmic reticulum. These data provide the first evidence for the localization of 5-LO in unstimulated granulocytic cells. The finding that a substantial proportion of enzyme is localized within the nucleus of unstimulated RBL cells suggests potentially novel roles for 5-LO or its products within the nucleus.
Highlights
From the Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan and Department of Veterans Affairs Medical Centers, Ann Arbor, Michigan 48109-0360
We asked whether the nucleus might bethe site for soluble 5-LO in unstimulated cells, and utilized rat basophilic leukemia (RBL) cells as model granulocytic cellsto address this question
When the contribution fromdividingcellswasminimized, either by overnight serum deprivation or by isolating cytoplasts of nucleus-containing cells,5-LO was prominent in the nuclear fraction but negligible in the cytosolic fraction. In contrast to this distribution in RBL cells, 5-LO in unstimulated human neutrophils was predominantly cytosolic, by both immunoblot and immunofluorescence analyses. In both RBL cells and human neutrophils, FLAP was localized at the nuclear membrane and the endoplasmic reticulum
Summary
RBL-1 cells (American Type Culture CollectionNo. 353011, Batch F-9128,ATCC(R) 1378-CRL) were seedeadt 1 x lo5cells ml-'in a-MEM (Life Technologies,Inc.) supplemented with 10%heat-inactivated fetal calf serum (Life Technologies, Inc.) and penicillidstreptomycin, fed 2 days later and harvested the third day.At time of harvest, the cell population was in log-phase growth. In some experiments cells were pelleted, resuspended in a half-volume of serum-free a-MEM on the third day to render them quiescent and harvested the day. Human neutrophils (PMN)were isolated from venousblood drawn from normal, healthy volunteers as described previously[8].Heparinized whole blood was centrifugedthrough a Ficoll-Paque(Pharmacia LKB Biotechnology, Inc.) cushion, the plasma and mononuclear layers removed, and the lower layer was subjected to dextran sedimentation. The remaining PMN were pelleted, washed, and resuspended in phosphate-buffered saline (PBS). Cell viability was tested by exclusion of trypan blue (0.2%in PBS)
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