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Phosphorylation- and stimulus-dependent inhibition of cellular 5-lipoxygenase activity by nonredox-type inhibitors¹

LUTZ FISCHER, DAGMAR SZELLAS, OLOF RÅDMARK, DIETER STEINHILBER and OLIVER WERZ^,2

Institute of Pharmaceutical Chemistry, University of Frankfurt, D-60439 Frankfurt, Germany; and
Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, S-171 77 Stockholm, Sweden

²Correspondence: Institute of Pharmaceutical Chemistry, University of Frankfurt, Marie-Curie Strasse 9, D-60439 Frankfurt, Germany. E-mail: o.werz{at}pharmchem.uni-frankfurt.de

SPECIFIC AIMS

5-Lipoxygenase (LO), the key enzyme in the biosynthesis of proinflammatory leukotrienes (LTs), is activated in the cell by Ca²⁺ but also via phosphorylation by mitogen-activated protein kinase-activated protein kinase (MK)-2/3 and extracellular signal-regulated kinases (ERKs). In this study we attempted to determine whether the efficacy of pharmacological 5-LO inhibitors depends on the activation pathway of the 5-LO enzyme.

PRINCIPAL FINDINGS

1. 5-LO inhibition by nonredox-type inhibitors is stimulus dependent
The efficacy of various types of 5-LO inhibitors was evaluated in isolated human polymorphonuclear leukocytes (PMNL). For the nonredox-type inhibitors ZM230487 and L-739.010, 10- to 100-fold higher concentrations were required for efficient inhibition of 5-LO product formation induced by phosphorylation-dependent stimuli [sodium arsenite (SA), hyperosmotic NaCl, or 60 µM AA] than when Ca²⁺ ionophore A23187 (leading to prominent cellular Ca²⁺ influx) was used as stimulus (Fig. 1 A, B). In contrast, BWA4C (Fig. 1C ), an iron ligand-type 5-LO inhibitor, as well as the novel-type 5-LO inhibitors hyperforin and AKBA (not shown) revealed no such differences.

View larger version (17K): [in this window] [in a new window]   Figure 1. Efficacy of nonredox-type 5-LO inhibitors in PMNL is stimulus dependent. Isolated PMNL from leukocyte concentrates were preincubated with the indicated concentrations of compounds for 15 min at 37°C. SA (10 µM) and NaCl (300 mM) were added 3 min before addition of 40 µM AA, ionophore (2.5 µM) was added together with AA. Alternatively, PMNL resuspended in buffer containing 1 mM EDTA were stimulated with 60 µM AA alone. After 10 min at 37°C, 5-LO products (LTB4 and its all-trans isomers as well as 5-H(p)ETE) were determined by HPLC. Results are given as mean ± SE, n = 3–4.

2. Ca²⁺ and elevated peroxide levels are no prerequisite for potent 5-LO inhibition
Elevated concentrations of Ca²⁺ might be required for efficient inhibition by nonredox-type inhibitors—for example, to provide 5-LO a susceptible conformation. Removal of Ca²⁺ by EDTA and/or BAPTA/AM further impaired the efficacy of ZM230487 and L-739.010 (but not of BWA4C) in SA- or NaCl-stimulated PMNL vs. cells receiving Ca²⁺. Moreover, the IC₅₀ value of ZM230487 for PMNL prestimulated with SA and subsequently challenged with ionophore is consistent with values obtained from cells that received only SA. Furthermore, ZM230487 was equipotent in the presence and absence of Ca²⁺ when partially purified 5-LO was investigated in vitro at low peroxide levels (addition of GPx/GSH). Thus, elevated Ca²⁺ is no prerequisite for high efficacy of ZM230487.

Since elevated peroxide levels impair the efficacy of ZM230487 in PMNL, cell stress could lower the efficacy of ZM230487 by induction of peroxide formation. However, stimulation of PMNL with SA or hyperosmotic NaCl did not enhance the release of peroxides compared with unstimulated PMNL (determined by an assay using the peroxide-sensitive fluorescence dye 2',7'-dichlorofluorescein diacetate). The peroxide scavenger n-acetylcysteine (1 or 5 mM) could not improve the efficacy of ZM230487 in cell stress-activated PMNL. Accordingly, an elevated peroxide tone is not the reason for the impaired efficacy of nonredox 5-LO inhibitors in cell stress-treated PMNL.

3. Kinetic analysis of 5-LO product formation and 5-LO kinase activation in PMNL
In contrast to cell stress, the kinetics of ionophore-induced 5-LO kinase activation did not correlate to 5-LO product synthesis. Thus, 5-LO product formation in ionophore-activated PMNL was half-maximal after 30 s, whereas 5-LO kinases (as determined by in-gel kinase assay) first became active after 1 min, indicating that 5-LO phosphorylation should not primarily contribute to 5-LO activation. Consequently, 5-LO phosphorylation events should not affect the efficacy of nonredox-type 5-LO inhibitors in ionophore-challenged PMNL.

4. Inhibition of wt-5-LO and S271A/S663A-5-LO in transfected HeLa cells
Ser-271 and Ser-663 are phosphorylation sites in 5-LO for MKs and ERKs, respectively. As shown in Fig. 2 , 6- to 10-fold lower concentrations of ZM230487 and L-739.010 were required to inhibit the mutated nonphosphorylatable S271A/S663A-5-LO than with the phosphorylatable wt-5-LO. For BWA4C, the IC₅₀ values were practically the same for wt-5-LO and S271A/S663A-5-LO.

View larger version (16K): [in this window] [in a new window]   Figure 2. Inhibition of wt-5-LO and S271A/S663A-5-LO transfected in HeLa cells. HeLa cells were transiently transformed with the plasmids pcDNA3.1–5LO or pcDNA3.1–5LO-S271A/S663A. Cells were preincubated with the indicated concentrations of the compounds for 15 min at 37°C, then stimulated with 40 µM AA for another 10 min. 5-LO products were determined by HPLC. Results are given as mean ± SE, n = 4.

5. 5-LO phosphorylation alone does not affect 5-LO inhibition by ZM230487 in vitro
In vitro, enzyme phosphorylation by MK2 or ERK2 (or both) does not significantly alter the enzymatic activity of 5-LO. There was no significant shift in the IC₅₀ value of ZM230487 for phosphorylated (MK2 and ERK) 5-LO compared with unphosphorylated enzyme. Therefore, phosphate incorporation itself does not protect 5-LO catalytic activity against ZM230487 in vitro, suggesting that the cellular environment or cellular constituents might be operative.

CONCLUSIONS

5-LO metabolites are bioactive lipids that possess pivotal functions in the pathophysiology of inflammation and allergy, but also regulate carcinogenesis and survival of various cell types and tissues, the metabolism of bone, and vascular homeostasis. Thus, pharmacological intervention with 5-LO activity was shown to be beneficial in inflammatory and allergic diseases but may also possess potential for prevention and therapy of cancer, osteoporosis, and vascular diseases. Nonredox-type 5-LO inhibitors such as ZM230487 and L-739.010 are highly potent inhibitors of 5-LO metabolite formation in several ex vivo and in vitro models and are able to significantly reduce acute inflammatory responses. Nevertheless, these compounds failed to inhibit more chronic inflammatory processes.

Our present findings suggest that phosphorylation of 5-LO impairs the efficacy of nonredox-type inhibitors. Thus, compared with 5-LO product synthesis induced by the Ca²⁺-mobilizing agent ionophore A23187, cell stress-induced 5-LO product formation involving 5-LO kinase pathways required 10- to 100-fold higher concentrations of ZM230487 or L-739.010 for comparable 5-LO inhibition (Fig. 1) . Activation of cellular 5-LO may occur by at least two different pathways (which may act in conjunction): either by elevation of intracellular Ca²⁺ or by Ca²⁺-independent pathways involving 5-LO phosphorylation by ERKs and/or p38 MAPK-regulated MKs. Many priming agents (PMA, GM-CSF, TNF-) and natural ligands (fMLP, PAF, C5a) activate p38 MAPK as well as ERKs in PMNL and, in turn, enhance or induce LT synthesis. Thus, activation of 5-LO by these kinases might be of relevance for LT synthesis in vivo under inflammatory conditions associated with the release of inflammatory cytokines and chemotactic factors as well as with cell stress. Consequently, such phosphorylation-dependent 5-LO catalysis may be less susceptible to nonredox inhibitors, which also might explain the reported loss of efficacy of ZM230487 in chronic inflammatory diseases.

Although our experiments with wt-5-LO and the mutated S271A/S663A-5-LO support the concept that phosphorylation of 5-LO impairs the efficacy of nonredox-type inhibitors in intact cells, in vitro studies using purified enzyme show that phosphorylation alone is not sufficient to alter the sensitivity of 5-LO toward ZM230487. Apparently cellular integrity or cellular components are operative. Indeed, 5-LO interacts with several cellular proteins, and such interactions might be affected by the 5-LO phosphorylation status.

The precise mode of action of ZM230487 and L-739.010 is unclear. In cell-free systems under reducing conditions or in intact cells, the compounds act as noncompetitive inhibitors by binding to a putative regulatory domain, probably at a fatty acid (hydroperoxide) binding site, which is responsible for the high efficacy of ZM230487. Phosphate incorporation in close proximity to such a regulatory fatty acid (hydroperoxide) binding site could allow the enzyme to interfere with a cellular protein/factor as outlined above. It appears reasonable that such (protein/protein) interactions may hamper the interference of ZM230487 and L-739.010 (but not of BWA4C) with 5-LO by facilitating the binding or formation of activating fatty acid hydroperoxides, which in turn could compete with nonredox-type inhibitors at this regulatory site. Further studies are necessary to explore the underlying molecular mechanisms.

Together, we show that the efficacy of nonredox-type inhibitors depends on the 5-LO activation pathway. The findings could be relevant for the development of 5-LO inhibitors, in particular for establishing of alternative assays suitable for inhibitor evaluation. An increased 5-LO phosphorylation state, often associated with inflammation, malignant cell proliferation, increased bone resorption, and macrophage activation at atherosclerotic sites, should be taken into account for pharmacological targeting of the 5-LO pathway in order to control inflammatory and allergic reactions, cancer, osteoporosis, and vascular diseases.

View larger version (28K): [in this window] [in a new window]   Figure 3. Mechanisms of 5-LO activation and effects on the pharmacological inhibition. Cellular 5-LO can be activated by elevation of Ca2+ or via phosphorylation by members of the MAPK family, depending on the nature of the stimulus. ZM230487 potently suppresses Ca2+-activated 5-LO (IC5020 nM), whereas 5-LO activated by phosphorylation requires much higher concentrations (IC50800 nM). Apparently, an unknown cellular component is operative to impair the susceptibility of phosphorylated 5-LO against ZM230487.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0815fje; to cite this article, use FASEB J. (March 28, 2003) 10.1096/fj.02-0815fje

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