(Received for publication, May 8, 1995; and in revised form, July 5, 1995)
From the
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) co-localizes 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.
Leukotrienes (LTs) ()are lipid mediators with
important roles in both host defense and inflammatory disease
states(1, 2) . The synthesis of LTs from arachidonic
acid is catalyzed by the enzyme 5-lipoxygenase (5-LO). Activation of
5-LO is thought to be initiated by the translocation of the enzyme from
a soluble pool to a bound site(3, 4) . This process of
translocation places 5-LO in close proximity to its substrate,
arachidonic acid, and to the 5-lipoxygenase-activating protein
(FLAP)(5, 6) , an integral membrane protein which
increases the leukotriene synthetic capacity of 5-LO in intact
cells(7, 8, 9) .
Although translocation of 5-LO has generally been assumed to involve movement from the cytosol to a membrane(3, 10) , actual identification of the soluble site in resting cells and the bound site in activated cells has only recently been attempted. Using immunoelectron microscopy, Woods et al.(11) have demonstrated that 5-LO co-localized at the nuclear envelope (NE) with FLAP in activated leukocytes. However, 5-LO could not be detected in resting cells by this technique, presumably because of loss of soluble protein during cell processing. Using indirect immunofluorescent microscopy and cell fractionation combined with immunoblotting, we recently demonstrated that 5-LO is indeed in the cytosol of resting neutrophils(12) . Together, these data indicate that 5-LO moves from the cytosol to the NE in PMNs. This pattern, which we have also recognized in rat peritoneal macrophages(13) , forms the basis for the current model for 5-LO translocation during cell activation.
Recently, we have examined the localization of 5-LO in two other cell types that produce abundant LTs in response to activation, RBL cells and AMs. Using a variety of techniques, we have demonstrated the presence of abundant 5-LO within the nucleus, as well as in the cytosol, of both resting RBL cells (12) and AMs(14) . It is unknown whether the nuclear pool of 5-LO functions in LT synthesis. Since translocation is an important early event in 5-LO action, translocation of nuclear 5-LO would support a role in LT synthesis. In previous studies, 5-LO translocation has been observed in RBL cells(15, 16) , but not in AMs(17) . However, in none of these studies was nuclear material examined. In fact, cells typically were disrupted using harsh techniques that disrupted nuclei, thus mixing nuclear soluble proteins with cytosol; moreover, much nuclear material was discarded in early processing steps. Thus, the fate of nuclear 5-LO has never been considered.
Efficient arachidonate processing by 5-LO requires three features: 1) translocation of 5-LO, 2) co-localization with FLAP, and 3) the ability to enzymatically convert arachidonate to 5-LO products. In the present study, we have used a variety of techniques to assess the ability of nuclear 5-LO, as well as cytosolic 5-LO, to contribute to LT synthesis in RBL cells and AMs, using these criteria. For comparison, we have performed parallel experiments with PMNs.
Figure 1: Presence of 5-LO and FLAP in subcellular fractions from resting and activated RBL cells. A, representative immunoblot of 5-LO content in subcellular fractions. Resting or activated (1 µM A23187, 15 min, 37 °C) RBL cells were disrupted by cavitation and separated into cytosolic (C), membrane (M), or nuclear (N) fractions, as described under ``Experimental Procedures.'' Nuclei were further disrupted and separated into soluble (s) and pelletable (p) fractions. Fractions were then probed for 5-LO and FLAP by immunoblot analysis. B, densitometric analysis of 5-LO content in subcellular fractions from three independent experiments. Values represent means ± S.E.; **statistically different from values in same fractions from resting cells (p < 0.05). C, immunoblot analysis of Triton X-100 extractable 5-LO in the pelletable nuclear fractions from resting and activated RBL cells. Pellets (Np) from sonicated nuclei were solubilized in 5% Triton X-100, separated into soluble (sol) and pelletable (pel) fractions by ultracentrifugation, and probed for FLAP or 5-LO by immunoblotting. D, representative immunoblot of FLAP protein in subcellular fractions. Conditions and fractions as in A. For all blots, equal amounts of protein were loaded in each lane. Migration of molecular weight markers indicated at left. All blots are representative of at least three independent experiments.
FLAP protein was localized primarily in the nuclear pelletable fraction of resting RBL cells, with some detectable protein also in the membrane fraction (Fig. 1D). As mentioned above, the membrane fraction should include endoplasmic reticulum, which has previously been identified as being a minor site for FLAP protein(11) . Cell activation with ionophore did not alter the distribution of FLAP (Fig. 1D). Detergent treatment extracted FLAP from the nuclear pelletable fractions of either resting or activated cells (data not shown). Similar results, for 5-LO and FLAP, were obtained when cells were initially disrupted using Dounce homogenization, rather than nitrogen cavitation (data not shown).
These results using immunoblot analysis indicated that, upon activation, 5-LO, originating from two distinct soluble pools, translocated to the NE, where it co-localized with FLAP. To confirm this interpretation, the localization of 5-LO in intact, individual cells was studied using indirect immunofluorescent microscopy. RBL cells mounted on slides were immersed in medium with or without ionophore. At given times, cells were immersed in fixative to stop the process of translocation. As reported previously(12) , resting cells consistently displayed a characteristic ``granular'' distribution of 5-LO within the nucleus, with a less pronounced, diffuse staining in the cytosol (Fig. 2A). Within 10 s after ionophore addition, two changes were evident, cytosolic staining for 5-LO had diminished, and within the nucleus, the pattern of 5-LO staining changed from granular to diffuse (Fig. 2B). Residual cytosolic staining, particularly around the nucleus, as well as some persistent granular staining within the nucleus, was apparent in a minority of cells (<20%) at this early time point. By 5 min, essentially all cytosolic 5-LO was depleted, and some 5-LO appeared to reside at the NE, as evidenced by the appearance of membrane folds in some cells (Fig. 2C). This pattern was consistently seen in most cells examined at 7.5 and 10 min after ionophore addition (not shown).
Figure 2: Localization of 5-LO and FLAP in resting and activated RBL cells by indirect immunofluorescent microscopy. Slide-mounted RBL cells were fixed before (A) or after 10 s (B), 5 min (C), or 15 min (D-F) of activation with 1 µM A23187. Preparations were then stained with anti-5-LO (A-D), anti-FLAP (E), or nonimmune serum (F). Bar corresponds to 50 µm for all micrographs.
With longer activation times, a second change in 5-LO distribution became apparent. By 15 min after ionophore addition, 5-LO was no longer diffusely dispersed throughout the nucleus. Instead, larger amounts of 5-LO were concentrated at the NE, as indicated by a pronounced nuclear ``rimming'' pattern of fluorescence (Fig. 2D). This pattern of 5-LO distribution was quite similar to that of FLAP in RBL cells (Fig. 2E). In contrast to the staining for 5-LO or FLAP, control staining with nonimmune serum was minimal (Fig. 2F).
The change in 5-LO pattern between 5 and 15 min of activation could result from the translocation of a dispersed intranuclear pool to the nuclear envelope during that interval. To address this possibility, we used confocal microscopy to obtain thin optical sections through the nucleus 5 and 15 min after activation. As compared with resting cells (Fig. 3A), thin sections of nuclei from RBL cells activated for 5 min (Fig. 3B) were characterized by diffuse fluorescence across the nucleus, with no pronounced signal at the nuclear envelope. Scans of nuclei from cells activated for 15 min showed much less 5-LO inside the nucleus and much more at the NE (Fig. 3C). Thus, translocation of nuclear 5-LO involved two steps, the first being the rapid dispersion of 5-LO throughout the nucleus, followed by a slower movement to the NE.
Figure 3: Visualization of nuclear pools of 5-LO in activated RBL cells by optical sectioning using confocal microscopy. Resting cells (A) or cells activated for 5 min (B) or 15 min (C) with 1 µM A23187 were stained with anti-5-LO and visualized by confocal microscopy. Aperture for laser excitation was adjusted to provide the minimum thickness of optical slice (approximately 0.3 µm). Arrowhead in A indicates a cell undergoing mitosis.
Figure 4: Presence of 5-LO and FLAP in subcellular fractions from resting and activated AMs (A) and PMNs (B). Representative immunoblots of cytosolic (C), membrane (M), nuclear soluble (Ns), and nuclear pelletable (Np) fractions, probed for 5-LO (upper) or FLAP (lower). Cells were activated with 1 µM A23187 (15 min, 37 °C), and fractions prepared as in Fig. 1.
Immunofluorescent microscopy confirmed that 5-LO translocated to the NE upon cell activation in both AMs and PMNs. Resting AMs showed the same pattern of 5-LO distribution as RBL cells: diffuse cytosolic staining combined with granular nuclear staining (Fig. 5A). Following activation, the nuclear 5-LO rapidly dispersed in over 90% of all cells, as had been observed in RBL cells; however, unlike RBL cells, cytosolic 5-LO did not change (Fig. 5B). The image presented in Fig. 5B was selected because it contains two cells which stain exactly like resting AMs and thus can be compared with the single activated cell to demonstrate that there is no change in the cytosolic 5-LO pool. With longer incubations, the dispersed 5-LO within the nucleus moved to the NE (Fig. 5C), resulting in a pattern of distribution similar to that seen for FLAP (Fig. 5D). In PMNs, 5-LO moved from the cytosol in resting cells (Fig. 5E) to the NE of the multilobed nucleus following cell activation (Fig. 5F), again co-localizing with FLAP (Fig. 5G).
Figure 5: Localization of 5-LO and FLAP in resting and activated AMs and PMNs by indirect immunofluorescent microscopy. Slide-mounted AMs (A-D) and PMNs (E-G) were incubated in medium alone (A, D, E, and G) or activated (1 µM A23187) for 10 s (B) or 10 min (C and F), then fixed and stained for 5-LO (A-C, E, and F) or FLAP (D and G). All images are from representative fields, except in B, where two atypical cells, demonstrating a staining pattern for 5-LO that matched that seen in resting cells, are close to the typical activated cell, so that the lack of change in cytosolic 5-LO following activation can be appreciated. Bar corresponds to 50 µm for all micrographs.
Figure 6:
Cell-free 5-LO activity of cytosolic and
nuclear fractions from RBL cells and inhibition by zileuton. Presented
are representative HPLC elution profiles of radiolabeled products
generated from [H]arachidonic acid by cytosol (upper panel) or nuclear soluble proteins (lower
panel), without (filled circles) or with 3 µM zileuton (open circles). Samples were prepared from
resting RBL cells. Peak A co-elutes with standards for
LTB
and LTA
metabolites. Peak B co-elutes with 5-HETE. Peak C co-elutes with free
arachidonic acid.
The current paradigm for 5-LO action is that cell activation
causes the translocation of 5-LO from a soluble pool to a bound site,
where it can interact with FLAP to efficiently produce LTA from free arachidonate. Recent work with neutrophils has
indicated that the target of translocation is the NE(11) . In
this study, we have extended our understanding of this process. First
and foremost, our data establish that there is no single model for 5-LO
translocation. Instead, we have described three patterns of
translocation, in three different cells types. Second, we have shown
that some pools are able to translocate, and that others do not under
the conditions tested. Third, we have demonstrated that, despite this
heterogeneity in patterns of translocation, the NE appears to be the
target of translocation in all cases. Fourth, we have confirmed the
localization of FLAP to the NE and contiguous endoplasmic reticulum.
Fifth, we have shown that nuclear 5-LO is enzymatically active and can
be inhibited by the specific 5-LO inhibitor zileuton. Finally, we have
demonstrated that, although all translocating pools of 5-LO may possess
the capacity for activity, enzymatically active 5-LO may not always
translocate.
Of the three patterns of translocation, neutrophils fit the simplest model, in that the single cytosolic pool of 5-LO moves to the NE following activation. In AMs, which have both cytosolic and intranuclear 5-LO pools, only the nuclear pool moves following cell activation. RBL cells, like AMs, have both cytosolic and nuclear pools; unlike AMs, both pools translocate. In all cases, the primary target of translocation is the NE. Interestingly, the response in RBL cells appears to involve two steps. Both the translocation of cytosolic 5-LO and the change in nuclear 5-LO, from a granular to dispersed distribution, are immediate and therefore may be a direct response to elevated calcium levels. These changes may represent a phase of translocation which is reversible upon removal of agonist, described previously(21, 22) . On the other hand, movement of the dispersed intranuclear 5-LO to the NE is a slower event, requiring several minutes. This delay suggests that steps subsequent to the calcium flux are necessary to drive translocation of the nuclear pool. These steps remain to be elucidated, but may include binding of 5-LO to a chaperone or docking protein, or cytoskeletal elements, via its Src homology 3 (SH3) binding motif(23) .
The failure of the cytosolic 5-LO pool to translocate upon activation in AMs ( Fig. 4and Fig. 5) has been suggested previously from cruder studies(17) . The finding that this pool is nonetheless enzymatically active suggests that its function may be different from the translocating nuclear 5-LO pool that co-localizes with FLAP. 5-LO can metabolize arachidonate derived from extracellular sources even when FLAP is effectively inhibited(9, 24, 25) ; thus, FLAP activity and, conceivably, co-localization with FLAP may not be necessary for 5-LO to process exogenously derived arachidonate. We suggest that the nontranslocating cytosolic pool of 5-LO in AMs could therefore play a role in processing arachidonate from such exogenous sources. This hypothesis would be consistent with the observation that AMs can efficiently use free arachidonate released by adjacent lung epithelial cells to generate 5-LO products(26) . On the other hand, utilization of arachidonate from intracellular (endogenous) sources may depend on FLAP-mediated delivery of the fatty acid to translocated 5-LO for efficient LT synthesis, and the intranuclear 5-LO pool may be preferentially involved in this process.
Translocation of both
cytosolic and intranuclear 5-LO to the NE underscores the role of this
membrane system in arachidonate metabolism. Previous studies have
demonstrated that arachidonate released following agonist addition
originates from NE phospholipids (27) and that an
arachidonate-selective phospholipase A binds to the nuclear
fraction following cell activation(13) . It is thus possible
that the substrate for 5-LO comes from NE phospholipids. Furthermore,
FLAP is at the NE. Using immunoelectron microscopy, Woods et al.(11) found FLAP on both the inner and outer NE membranes
in leukocytes, as well as the endoplasmic reticulum. Thus FLAP should
be able to utilize its arachidonate binding capacity (28) to
deliver arachidonate to, and thus ``activate,'' 5-LO on
either membrane of the NE.
The results presented here support roles for both nuclear and cytosolic pools of 5-LO in LT synthesis. Although the advantages of 5-LO compartmentation are unclear, there are several possibilities relating to both known catalytic functions as well as possible noncatalytic functions. In terms of catalytic functions, distinct 5-LO pools could generate different amounts or profiles of products. Likewise, pools may be differentially regulated, having unique rates or magnitudes of response to different agonists. Or, 5-LO pools may have unique catalytic functions within their specific compartments. Since free arachidonic acid is a potent intracellular effector(29, 30, 31) , pools of 5-LO may scavenge arachidonate within a given compartment. Similarly, 5-LO-generated products may act directly at the point of synthesis, as has recently been suggested(32, 33) .
Novel noncatalytic functions of particular intracellular pools of 5-LO could be mediated by direct association of the protein with specific molecular targets, perhaps via its SH3-binding domain. 5-LO interacts specifically with SH3 sites on select cell signaling proteins and cytoskeletal elements in vitro(23) , suggesting a role for cytosolic 5-LO protein in related processes. Similarly, within the nucleus, 5-LO may interact with signaling or structural proteins, or nucleic acids within the euchromatin region, where 5-LO protein has been observed(14) . These possibilities provide the basis for considering the specific functions subserved by different 5-LO pools in future studies.