Intracellular Compartmentalization of Leukotriene Biosynthesis

MARC PETERS-GOLDEN and THOMAS G. BROCK

Pulmonary and Critical Care Medicine Division, University of Michigan, Ann Arbor, Michigan

	INTRODUCTION

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Leukotrienes (LTs) are potent bioactive lipids derived from the 5-lipoxygenase (5-LO) pathway of arachidonic acid (AA) metabolism. Although they have been conventionally viewed as paracrine mediators of inflammatory disease processes such as asthma (1), more recent information suggests that they are also important participants in disease processes characterized by cellular proliferation and fibrogenesis (2), and that they subserve a homeostatic role in antimicrobial host defense (5). In view of the actions and importance of LTs, substantial effort has been directed at increasing our understanding of the regulation of their synthesis.

An extensive body of research, reviewed elsewhere in this symposium, has identified three key factors that regulate LT synthesis: (1) the steady state levels of key proteins necessary for AA release and 5-LO metabolism; (2) posttranslational modifications (such as phosphorylation) that alter the catalytic activities of these proteins; and (3) the concentrations of small molecules (e.g., Ca²⁺, ATP, and glutathione) that serve as cofactors for certain of these proteins. One additional determinant of LT biosynthesis that has been recognized is the intracellular compartmentalization of these LT-forming proteins. This topic has become a focus of investigation in our own and other laboratories, and this article reviews the current state of knowledge in this area. Indeed, a number of unexpected findings that have emerged from these studies underscore the appropriateness of a perspective on LT biology that transcends conventional notions.

	THE TRANSLOCATION MODEL FOR 5-LIPOXYGENASE ACTIVATION

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The intracellular locale of the proteins necessary for LT synthesis went largely unstudied for many years. An important advance in our understanding of the mechanism of 5-LO activation was the demonstration that the enzyme undergoes a rapid Ca²⁺-dependent redistribution ("translocation") from its locale within a soluble intracellular compartment in resting cells to a membrane compartment after agonist activation (6). This process of translocation could reasonably be assumed to bring the enzyme in proximity to its membrane-derived substrate, AA. It was soon determined that cytosolic phospholipase A₂ (cPLA₂), a PLA₂ isoform considered an attractive candidate to mediate intracellular AA hydrolysis on the basis of its preference for arachidonoyl-containing phospholipids, likewise underwent a Ca²⁺-dependent redistribution from a soluble to a membrane compartment on stimulation (7). Since the helper protein, 5-LO activating protein (FLAP), was also present in the membrane fraction of cells both in the resting and stimulated states (8), a model was formulated in which agonist activation resulted in colocalization of the proteins necessary for arachidonate release and the initiation of LT synthesis. Since LTs were known to be efficiently secreted from cells, the site at which these proteins were colocalized was assumed to be the plasma membrane. In retrospect, however, it is apparent that these early subcellular fractionation studies were not capable of adequately resolving intracellular compartmentalization. Given the assumptions regarding the primacy of the plasma membrane, there was in fact no a priori reason to do so at the time.

	ROLE OF THE NUCLEAR ENVELOPE IN LEUKOTRIENE SYNTHESIS

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Surprisingly, when activated blood neutrophils were studied by immunoelectron microscopy, both 5-LO and FLAP were localized to the nuclear envelope (see Figure 1) (9). At about the same time, peritoneal macrophages were gently disrupted (so that the plasma membrane but not the nuclear membrane was ruptured) and separated into nuclear, cytosolic, and nonnuclear membrane fractions that were then subjected to immunoblot analysis. FLAP was found predominantly in the nuclear fraction of both resting and stimulated cells; furthermore, 5-LO was found to redistribute from the cytosolic to the nuclear fraction on activation (10). These results indicating the nuclear envelope to be the site of 5-LO and FLAP colocalization in activated leukocytes appear to reflect a universal phenomenon, now also verified not only for neutrophils (11) but also for alveolar macrophages (12, 13), blood monocytes (12), mast cells (14), and the rat basophilic leukemia (RBL) mast cell-like cell line (13). Localization of 5-LO at the nuclear envelope in activated cells has now also been confirmed in situ, since immunohistochemical analysis revealed an increased number of macrophages with this staining pattern in lung sections from patients with both idiopathic pulmonary fibrosis (3) and primary pulmonary hypertension (4), diseases characterized by constitutive overproduction of LTs.

View larger version (16K): [in this window] [in a new window]   Figure 1.   Role of the nucleus in LT synthesis. LT synthesis requires free AA, hydrolyzed from phospholipids by a PLA2. Here, arachidonoyl-selective cPLA2 is shown translocating from its resting site in the cytosol to the nuclear envelope on activation. Arachidonate released from nuclear envelope phospholipids is bound by FLAP, an integral nuclear envelope protein, to facilitate processing by 5-LO. On activation, 5-LO translocates from its resting locale(s) in the cytosol and/or nucleoplasm to the nuclear envelope, where it catalyzes the initial steps in LT synthesis. In some circumstances, 5-LO can shuttle through the nuclear pores between cytosol and nucleoplasm (dotted arrow) independent of the process of activation and subsequent translocation to the nuclear envelope. Not shown here is LTC4 synthase, also an integral nuclear envelope protein. LTs thus synthesized are then capable of either entering the nucleus or being exported out of the cell. Abbreviations are defined in text.

Early studies using crude fractionation techniques had suggested that the reversibility of membrane association of 5-LO depended on the intensity of the stimulus and the magnitude of its associated Ca²⁺ transient (15). The reversibility of nuclear envelope association was shown by immunofluorescence microscopy to depend on both the intensity and the duration of the stimulus (16). In RBL cells and alveolar macrophages, the addition of the Ca²⁺ chelator EGTA up to 5 min after the addition of the agonist Ca²⁺ ionophore resulted in the return of nuclear envelope-associated 5-LO to its resting locale, and a second stimulation was effective at causing another round of translocation to the nuclear envelope and LT generation. On the other hand, if the initial stimulation period lasted more than 5-10 min prior to addition of EGTA, nuclear envelope association was irreversible and restimulation was ineffective at inducing a second round of LT generation. It is likely that physiological agonists to which leukocytes are exposed in vivo result in the former, more transient type of enzyme translocation. This would imply that, in vivo, cellular secretion of LTs is distinguished from that of many other inflammatory mediators with respect to both its rapidity and its capacity for repeatability.

FLAP was originally thought to serve as a membrane "docking protein" with which 5-LO associated after agonist stimulation. This notion was based on the observation that the FLAP inhibitor MK-886 appeared to prevent redistribution of 5-LO to the membrane fraction of stimulated granulocytic cells while concomitantly inhibiting LT biosynthesis (17). However, these data were obtained by crude techniques for cellular disruption and subcellular fractionation, and subsequent immunofluorescence (16) and immunoelectron (12) microscopy analyses found no inhibitory effect of MK-886 on 5-LO translocation to the nuclear envelope of activated cells despite its inhibition of LT production. These latter results are consistent with the finding that membrane association of 5-LO occurred even in cells lacking FLAP (18). It was subsequently determined that FLAP is an AA-binding protein (19), and it is now thought that this helper protein somehow facilitates the presentation of substrate fatty acid to 5-LO.

Of course, the findings that 5-LO and FLAP were colocalized at the nuclear envelope in activated cells raised the question of whether cPLA₂ translocated to the same membrane. Studies using appropriate disruption and fractionation methods as well as immunofluorescence microscopy have indeed revealed that cPLA₂ is also localized primarily at the nuclear envelope in a variety of types of stimulated cells (10, 11, 20- 22). Importantly, translocation of cPLA₂ to the nuclear envelope has been shown to be associated with selective hydrolysis of nuclear membrane phospholipids (22). LTC₄ synthase has been shown to have a high degree of homology with FLAP; like FLAP, it too is an integral membrane protein located primarily at the nuclear envelope (23).

Taken together, there is now abundant evidence suggesting that the nuclear envelope is the site at which AA release (at least that mediated by cPLA₂), LTA₄ synthesis, and LTA₄ conversion to LTC₄ all occur. The mechanism by which the translocation of cPLA₂ and 5-LO, which originate in the cytosol is targeted to the nuclear envelope, as opposed to other intracellular membranes, remains to be elucidated. The suggestion that translocation of 5-LO might be regulated by protein tyrosine kinase-dependent interactions (24) and protein-protein interactions involving the Src homology 3 domain of the enzyme (25) are intriguing. However, these conclusions must be interpreted with caution since they are based exclusively on data obtained by crude disruption and fractionation methods that previously yielded misleading information about the effects of MK-886. In any case, these findings raise the important question of why mediators destined for extracellular secretion would be synthesized deep within the cell, and this is considered at the end of this article.

	CELL-SPECIFIC COMPARTMENTALIZATION OF 5-LIPOXYGENASE IN RESTING CELLS

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Initial studies in unstimulated blood neutrophils (26) and peritoneal macrophages (10) demonstrated that 5-LO was predominantly cytosolic, and this finding has been extended to blood monocytes (12) and blood eosinophils (27) (see Figure 1). Unexpectedly, subsequent fractionation as well as immunomicroscopy studies of isolated alveolar macrophages (12, 13), mast cell-like RBL cells (26), and primary mast cells (14) revealed abundant intranuclear 5-LO in addition to that found within the cytosol. Importantly, an intranuclear pool of 5-LO in alveolar macrophages has been confirmed in situ by immunohistochemical staining of normal human lung tissue (3). The same finding has been reported for epidermal Langerhans cells examined in situ (28). A portion of the intranuclear pool in RBL cells is insoluble and has biochemical characteristics suggesting that it is chromatin associated (26). Immunoelectron microscopy analysis of human alveolar macrophages demonstrated that intranuclear 5-LO was not randomly distributed, but was instead concentrated in the euchromatin region (12), that portion of the nucleus where actively transcribing genes are distributed.

That the intranuclear pool of 5-LO participates in cellular LT synthesis is suggested by the facts that it is catalytically active in cell-free assays and translocates to the nuclear envelope on agonist stimulation (13). In fact, although alveolar macrophages contain both intranuclear and cytosolic pools of 5-LO, only the former appears to translocate on stimulation; in contrast, both pools translocate with stimulation of RBL cells (13). The contribution of intranuclear 5-LO to LT synthesis is emphasized by the demonstration that, in adherent eosinophils, a minimal capacity for LTC₄ generation is associated with a minimal degree of translocation of this enzyme pool (27). These findings indicate that compartmentalization of 5-LO in unstimulated cells varies depending on the cell type, with some cells exhibiting exclusively cytosolic enzyme and others containing both cytosolic and intranuclear pools; in either case, activation is associated with translocation to the nuclear envelope. The mechanisms by which compartmentalization of 5-LO is differentially regulated in different cell types are not currently understood.

	DYNAMIC REGULATION OF 5-LIPOXYGENASE COMPARTMENTALIZATION

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Rather than a static model in which 5-LO compartmentalization is considered to be dictated solely by cell type, several lines of evidence indicate that it is, in fact, a dynamically regulated process even within a given cell type (see Figure 1). The first is based on data obtained with various mononuclear phagocyte populations. Blood monocytes, the precursors for all mature tissue macrophages, contain cytosolic 5-LO (12). While mature peritoneal macrophages retain this cytosolic distribution (10), alveolar macrophages do not (12, 13), indicating that nuclear import of this protein accompanies monocyte migration into the pulmonary alveolar space, but not the peritoneum. Interestingly, when alveolar macrophages were removed from the lung and cultured ex vivo for 3 d, there was a time-dependent loss of 5-LO from the nucleus and a corresponding accumulation in the cytoplasm (29), presumably reflecting the deprivation of those factors in the alveolar space that favor nuclear accumulation. Second, the recruitment of blood neutrophils into sites of inflammation (either pulmonary alveolus or peritoneum) is associated with rapid movement of 5-LO into the nucleus, which is not itself accompanied by LT synthesis (30). This can be mimicked by adherence of blood neutrophils to various surfaces for as little as 15 min (30). Third, nuclear import of 5-LO has also been observed when blood eosinophils were adhered to fibronectin for as little as 30 min (27).

The molecular mechanisms by which dynamic nuclear import and export of 5-LO are mediated are as yet undefined. However, movement of large proteins (> 50-60 kD) through the nuclear pores generally involves interactions with receptors or chaperones that are mediated by specific molecular motifs (31), and the same is likely to be true for 5-LO. Precedent also exists for the dynamic nature of intracellular protein compartmentalization to be explained by phosphorylation/ dephosphorylation reactions (31), and 5-LO has a number of potential phosphorylation sites.

Finally, it is interesting that nuclear import of both cPLA₂ (32) and cyclooxygenase 2 (33), an inducible form of the enzyme responsible for conversion of AA to prostaglandins, has been described. Thus, the phenomenon of dynamic modulation of 5-LO compartmentalization may serve as a model that also applies to other enzymes in the eicosanoid synthetic pathway.

	METABOLIC IMPLICATIONS OF 5-LIPOXYGENASE LOCALIZATION

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The fact that 5-LO and FLAP (along with other relevant proteins) are localized to the nuclear envelope of activated cells implies that LT synthesis is initiated at this site. As a result, the local concentrations of LTs within the nucleus are likely to be quite high. This would be especially true when 5-LO associates with the inner membrane of the nuclear envelope, as would occur when an intranuclear pool of enzyme is activated. Teleologically, these observations suggest that the autocrine actions of these bioactive lipid mediators, including those potentially mediated within the nucleus, may be more important than those paracrine actions that have been classically recognized. Indeed, a growing body of evidence implicates endogenously produced 5-LO metabolites as modulators of such fundamental processes as mitogenesis (34), apoptosis (2, 35), and the expression or activation of various transcription factors including NF-B (36) and Fos (34). Theoretically, these actions of LTs could involve their direct interaction with nucleic acids, transcription factors, or signaling pathways. The identification of a soluble nuclear receptor for LTB₄ exemplifies such a direct intranuclear interaction (39); interestingly, this receptor is a member of the steroid superfamily of transcription factors and its ligation induced gene transcription. Finally, it should also be recalled that reactive oxygen species are a by-product of arachidonate 5-lipoxygenation, and these reactive intermediates could themselves exert nuclear actions by activating transcription factors or otherwise modifying nuclear constituents. 5-LO-dependent activation of NF-B has been ascribed to reactive oxygen species in some (36, 38) but not all (37) experimental systems.

The propensity of particular LT-forming proteins to be localized at particular intracellular sites raises the possibility that metabolic coupling among proteins will be dictated by their topographic proximity. This notion posits that both the access of 5-LO/FLAP to free AA and their capacity to supply LTA₄ for LT synthesis will be enhanced by the proximity of PLA₂ and LT synthases, respectively, to the nuclear envelope. In this regard, it has long been recognized that different functional pools of AA exist within a given cell type, and it can be speculated that different metabolic fates for AA could reflect the topographic proximity of PLA₂s and downstream enzymes.

Dynamic nuclear import of 5-LO adds a further measure of complexity to the regulation of LT synthesis. It should be emphasized that nuclear import is not itself associated with LT synthesis. But how does this phenomenon in resting cells influence subsequent LT generation on activation? This may depend on the capacity of the imported enzyme to translocate on activation. Peripheral blood eosinophils in suspension, with predominantly cytosolic 5-LO, respond to stimulation with enzyme translocation to the nuclear envelope and abundant LT synthesis; in contrast, the intranuclear 5-LO that accumulates after adherence of these cells appears relatively unable to translocate and this is associated with a dramatic decline in LT synthetic capacity (27). On the other hand, alveolar macrophages (40) and recruited (or adherent) neutrophils (30) (all of which have an intranuclear pool of 5-LO that is capable of translocating) all display greater maximal capacities for LT production in response to stimuli than do the corresponding cells with cytosolic 5-LO. However, the cells in these two models with intranuclear 5-LO require a higher dose of ionophore to trigger LT synthesis than do the cells with cytosolic enzyme (30, 40). The lower sensitivity and greater capacity of these cells with an intranuclear distribution of 5-LO could reflect a lower hydroperoxide tone in the nuclear than cytosolic compartment. The lower sensitivity could alternatively reflect the likelihood that intranuclear Ca²⁺ concentrations are lower than cytosolic Ca²⁺ concentrations after the addition of an extracellular stimulus. Although much has yet to be learned about the underlying mechanisms, it is apparent that a growing body of evidence does suggest that compartmentalization of 5-LO is an important determinant of LT synthetic responses.

	NONMETABOLIC IMPLICATIONS OF 5-LIPOXYGENASE LOCALIZATION

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The compartmentalization of 5-LO could have biological implications that go beyond its catalytic products, LTs and reactive oxygen species. The facts that 5-LO can be found within the euchromatin region of nuclei (12) and can be demonstrated to be chromatin associated (26) suggest that the enzyme itself might interact directly with intranuclear proteins or, perhaps, genes. Likewise, possible interactions with signaling or cytoskeletal proteins (25) could also be dictated by intracellular compartmentalization.

	CONCLUSIONS

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Our understanding of the molecular mechanisms and regulation of LT synthesis has increased dramatically. In particular, investigations into the cell biology of this metabolic pathway have revealed an unexpected role for the nucleus. While many questions of a molecular and functional nature remain to be answered, this finding provides the impetus to explore novel intracellular actions of LTs beyond those traditionally appreciated.

	Footnotes

Correspondence and requests for reprints should be addressed to Marc Peters-Golden, M.D., Pulmonary and Critical Care Medicine Division, University of Michigan Medical School, 6301 MSRB III, Box 0642, Ann Arbor, MI 48109. E-mail: petersm{at}umich.edu

Acknowledgments: The authors thank Dr. Jilly Evans of Merck Frosst for providing antisera to 5-LO and FLAP. They also acknowledge the contributions of a number of individuals to the work presented from the authors' laboratories, including Michael Coffey, Peter Sporn, Marc Bailie, Randal Covin, John Woods, Robert Paine, and Robert McNish.

Supported by funds from the NIH (RO1 HL47391, P50 HL56402, R29 AI43574), the Parker B. Francis Fellowship Program, the American Lung Association, and the University of Michigan.

	References

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1. Lewis, R. A., K. F. Austen, and R. J. Soberman. 1990. Leukotrienes and other products of the 5-lipoxygenase pathway: biochemistry and relation to pathobiology in human disease. N. Engl. J. Med. 323: 645-655 [Medline].

2. Avis, I., M. Jett, T. Boyle, M. Vos, T. Moody, A. Treston, A. Martinez, and J. Mulshine. 1996. Growth control of lung cancer by interruption of 5-lipoxygenase-mediated growth factor signaling. J. Clin. Invest. 97: 806-813 [Medline].

3. Wilborn, J., M. Bailie, M. Coffey, M. Burdick, R. Strieter, and M. Peters-Golden. 1996. Constitutive activation of 5-lipoxygenase in the lungs of patients with idiopathic pulmonary fibrosis. J. Clin. Invest. 97: 1827-1836 [Medline].

4. Wright, L., R. Tuder, J. Wang, C. Cool, R. Lepley, and N. Voelkel. 1998. 5-Lipoxygenase and 5-lipoxygenase activating protein (FLAP) immunoreactivity in lungs from patients with primary pulmonary hypertension. Am. J. Respir. Crit. Care Med. 157: 219-229 [Abstract/Free Full Text].

5. Bailie, M., T. Standiford, L. Laichalk, M. Coffey, R. Strieter, and M. Peters-Golden. 1996. Leukotriene-deficient mice manifest enhanced lethality from Klebsiella pneumoniae in association with decreased alveolar macrophage phagocytic and bactericidal activities. J. Immunol. 157: 5221-5224 [Abstract].

6. Rouzer, C. A., and S. Kargman. 1988. Translocation of 5-lipoxygenase to the membrane in human leukocytes challenged with ionophore A23187. J. Biol. Chem. 263: 10980-10988 [Abstract/Free Full Text].

7. Channon, J., and C. Leslie. 1990. A calcium-dependent mechanism for associating a soluble arachidonoyl-hydrolyzing phospholipase A₂ with membrane in the macrophage cell line RAW 264.7.  J. Biol. Chem. 265: 5409-5413 [Abstract/Free Full Text].

8. Miller, D. K., J. W. Gillard, P. J. Vickers, S. Sadowski, C. Leveille, J. A. Mancini, P. Charleson, R. A. F. Dixon, A. W. Ford-Hutchinson, R. Fortin, J. Y. Gauthier, J. Rodkey, R. Rosen, C. Rouzer, I. S. Sigal, C. D. Strader, and J. F. Evans. 1990. Identification and isolation of a membrane protein necessary for leukotriene production. Nature (London) 343: 278-281 [Medline].

9. Woods, J., J. Evans, D. Ethier, S. Scott, P. Vickers, L. Hearn, S. Charleson, J. Heibein, and I. Singer. 1993. 5-Lipoxygenase and 5-lipoxygenase activating protein are localized in the nuclear envelope of activated human leukocytes. J. Exp. Med. 178: 1935-1946 [Abstract/Free Full Text].

10. Peters-Golden, M., and R. McNish. 1993. Redistribution of 5-lipoxygenase and cytosolic phospholipase A₂ to the nuclear fraction upon macrophage activation. Biochem. Biophys. Res. Commun. 196: 147-153 [Medline].

11. Pouliot, M., P. McDonald, E. Krump, J. Mancini, S. McColl, P. Weech, and P. Borgeat. 1996. Colocalization of cytosolic phospholipase A₂, 5-lipoxygenase, and 5-lipoxygenase-activating protein at the nuclear membrane of A23187-stimulated human neutrophils. Eur. J. Biochem. 238: 250-258 [Medline].

12. Woods, J., M. Coffey, T. Brock, I. Singer, and M. Peters-Golden. 1995. 5-Lipoxygenase is located in the euchromatin of the nucleus in resting human alveolar macrophages and translocates to the nuclear envelope upon cell activation. J. Clin. Invest. 95: 2035-2040 .

13. Brock, T. G., R. W. McNish, and M. Peters-Golden. 1995. Translocation and leukotriene synthetic capacity of nuclear 5-lipoxygenase in rat basophilic leukemia cells and alveolar macrophages. J. Biol. Chem. 270: 21652-21658 [Abstract/Free Full Text].

14. Chen, X.-S., T. Naumann, U. Kurre, N. Jenkins, N. Copeland, and C. Funk. 1995. cDNA cloning, expression, mutagenesis, intracellular localization, and gene chromosomal assignment of mouse 5-lipoxygenase. J. Biol. Chem. 270: 17993-17999 [Abstract/Free Full Text].

15. Malaviya, R., R. Malaviya, and B. Jakschik. 1993. Reversible translocation of 5-lipoxygenase in mast cells upon IgE/antigen stimulation. J. Biol. Chem. 268: 4939-4944 [Abstract/Free Full Text].

16. Brock, T., R. McNish, and M. Peters-Golden. 1998. Capacity for repeatable leukotriene generation after transient stimulation of mast cells and macrophages. Biochem. J. 329: 519-525 .

17. Rouzer, C. A., A. W. Ford-Hutchinson, H. E. Morton, and J. W. Gillard. 1990. MK886, a potent and specific leukotriene biosynthesis inhibitor blocks and reverses the membrane association of 5-lipoxygenase in ionophore-challenged leukocytes. J. Biol. Chem. 265: 1436-1442 [Abstract/Free Full Text].

18. Kargman, S., P. Vickers, and J. Evans. 1992. A23187-induced translocation of 5-lipoxygenase in osteosarcoma cells. J. Cell Biol. 119: 1701-1709 [Abstract/Free Full Text].

19. Abramovitz, M., E. Wong, M. Cox, C. Richardson, C. Li, and P. Vickers. 1993. 5-Lipoxygenase-activating protein stimulates the utilization of arachidonic acid by 5-lipoxygenase. Eur. J. Biochem. 215: 105-111 [Medline].

20. Glover, S., T. Bayburt, M. Jonas, E. Chi, and M. Gelb. 1995. Translocation of the 85-kDa phospholipase A₂ from cytosol to the nuclear envelope in rat basophilic leukemia cells stimulated with calcium ionophore or IgE/antigen. J. Biol. Chem. 270: 15359-15367 [Abstract/Free Full Text].

21. Schievella, A., M. Regier, W. Smith, and L. Lin. 1995. Calcium-mediated translocation of cytosolic phospholipase A₂ to the nuclear envelope and endoplasmic reticulum. J. Biol. Chem. 270: 30749-30754 [Abstract/Free Full Text].

22. Peters-Golden, M., K. Song, T. Marshall, and T. Brock. 1996. Translocation of cytosolic phospholipase A₂ to the nuclear envelope elicits topographically localized phospholipid hydrolysis. Biochem. J. 318: 797-803 .

23. Penrose, J., J. Spector, B. Lam, D. Friend, K. Xu, R. Jack, and K. Austen. 1995. Purification of human lung LTC₄ synthase and preparation of a polyclonal antibody. Am. J. Respir. Crit. Care Med. 152: 283-289 [Abstract].

24. Lepley, R., D. Muskardin, and F. Fitzpatrick. 1996. Tyrosine kinase activity modulates catalysis and translocation of cellular 5-lipoxygenase. J. Biol. Chem. 271: 6179-6184 [Abstract/Free Full Text].

25. Lepley, R. A., and F. Fitzpatrick. 1994. 5-lipoxygenase contains a functional Src homology 3-binding motif that interacts with the Src homology 3 domain of Grb2 and cytoskeletal proteins. J. Biol. Chem. 269: 24163-24168 [Abstract/Free Full Text].

26. Brock, T. G., R. Paine, and M. Peters-Golden. 1994. Localization of 5-lipoxygenase to the nucleus of unstimulated rat basophilic leukemia cells. J. Biol. Chem. 269: 22059-22066 [Abstract/Free Full Text].

27. Brock, T. G., J. A. Anderson, F. P. Fries, M. Peters-Golden, and P. H. S. Sporn. 1999. Decreased LTC₄ synthesis accompanies adherence- dependent nuclear import of 5-lipoxygenase in human blood eosinophils. J. Immunol. 162: 1169-1676 .

28. Spanbroek, R., H.-J. Stark, U. Janssen-Timmen, S. Kraft, M. Hildner, T. Andl, F.-X. Bosch, N. Fusenig, T. Bieber, O. Radmark, B. Samuelsson, and A. Habenicht. 1998. 5-Lipoxygenase expression in Langerhans cells of normal human epidermis. Proc. Natl. Acad. Sci. U.S.A. 95: 663-668 [Abstract/Free Full Text].

29. Covin, R., T. Brock, M. Bailie, and M. Peters-Golden. 1998. Altered expression and localization of 5-lipoxygenase accompany macrophage differentiation in the lung. Am. J. Physiol. (Lung Cell Mol. Physiol). 275: L303-L310 [Abstract/Free Full Text].

30. Brock, T., R. McNish, M. Bailie, and M. Peters-Golden. 1997. Rapid import of cytosolic 5-lipoxygenase into the nucleus of neutrophils after in vivo recruitment and in vitro adherence. J. Biol. Chem. 272: 8276-8280 [Abstract/Free Full Text].

31. Gorlich, D.. 1997. Nuclear protein import. Curr. Opin. Cell Biol. 9: 412-419 [Medline].

32. Sierra-Honigmann, M., J. Bradley, and J. Pober. 1996. "Cytosolic" phospholipase A₂ is in the nucleus of subconfluent endothelial cells but confined to the cytoplasm of confluent endothelial cells and redistributes to the nuclear envelope and cell junctions upon histamine stimulation. Lab. Invest. 74: 684-695 [Medline].

33. Coffey, R., C. Hawkey, L. Damstrup, R. Graves-Deal, V. Daniel, P. Dempsey, R. Chinery, S. Kirkland, R. DuBois, T. Jetton, and J. Morrow. 1997. Epidermal growth factor receptor activation induces nuclear targeting of cyclooxygenase-2, basolateral release of prostaglandins, and mitogenesis in polarizing colon cancer cells. Proc. Natl. Acad. Sci. U.S.A. 94: 657-662 [Abstract/Free Full Text].

34. Beno, D., J. Mullen, and B. Davis. 1995. Lipoxygenase inhibitors block PDGF-induced mitogenesis: a MAPK-independent mechanism that blocks Fos and Egr. Am. J. Physiol. 268: C604-C610 [Abstract/Free Full Text].

35. Hebert, M.-J., T. Takano, H. Holthofer, and H. Brady. 1996. Sequential morphologic events during apoptosis of human neutrophils: modulation by lipoxygenase-derived eicosanoids. J. Immunol. 157: 3105-3115 [Abstract].

36. Bonizzi, G., J. Piette, M.-P. Merville, and V. Bours. 1997. Distinct signal transduction pathways mediate nuclear factor-B induction by IL-1 in epithelial and lymphoid cells. J. Immunol. 159: 5264-5272 [Abstract].

37. Lee, S., K. Felts, G. Parry, L. Armacost, and R. Cobb. 1997. Inhibition of 5-lipoxygenase blocks IL-1-induced vascular adhesion molecule-1 gene expression in human endothelial cells. J. Immunol. 158: 3401-3407 [Abstract].

38. Los, M., H. Schenk, K. Hexel, P. Baeuerle, W. Droge, and K. Schulze-Osthoff. 1995. IL-2 gene expression and NF-B activation through CD28 requires reactive oxygen production by 5-lipoxygenase. EMBO J. 14: 3731-3740 [Medline].

39. Devchand, P., H. Keller, J. Peters, M. Vazquez, F. Gonzalez, and W. Wahli. 1996. The PPAR-leukotriene B₄ pathway to inflammation control. Nature (London) 384: 39-43 [Medline].

40. Peters-Golden, M., R. W. McNish, R. Hyzy, C. Shelly, and G. B. Toews. 1990. Alterations in the pattern of arachidonate metabolism accompany rat macrophage differentiation in the lung. J. Immunol. 144: 263-270 [Abstract].