Daidzein phosphorylates and activates 5-lipoxygenase via the MEK/ERK pathway: a mechanism for inducing the production of 5-lipoxygenase metabolite that inhibit influenza virus intracellular replication

We previously reported that the soy isoflavone daidzein (Dz) suppresses influenza virus replication in MDCK cells [1]. 5-Hydroxyeicosatetraenoic acid (5-HETE), a 5-lipoxygenase (5-LOX) product derived from arachidonic acid (AA), was significantly increased in Dz-treated cells. Influenza virus replication was suppressed by the administration of 5-hydroperoxyeicosatetraenoic acid (5-HpETE), a primary product of 5-LOX and a precursor of 5-HETE. Furthermore, the inhibitory effect of Dz on viral replication was reduced when 5-LOX activity was suppressed by siRNA. These results suggested that 5-LOX and its metabolite derived from AA, are mechanistically involved in Dz-mediated suppression of influenza virus replication. Experiments carried out to examine the process of viral replication inhibited by Dz by differentiating the timing of Dz addition to MDCK cells into early (0–4 h) and late (4–8 h) post-infection showed an inhibitory effect in the late stage (assembly processes or subsequent process) of viral replication [2]. The mechanism by which Dz (or 5-HpETE or 5-HETE) inhibits viral replication or by which Dz increases 5-HETE in cells remains unclear. In this study, we focused on exploring the mechanisms by which Dz activates 5-LOX.

Some of the lipid oxidation products synthesized via LOXs suppress the replication of influenza virus in cells [3]. Protectin D1 derived from docosahexaenoic acid (DHA) via 15-LOX was shown to inhibit influenza virus replication by suppressing the extranuclear migration of viral RNA synthesized in the nucleus [3]. Although and 5-HETE, which demonstrated an inhibitory effect against virus replication in our previous study [1], and protectin D1 involve different substrates and LOXs, some of the LOX-related lipid oxidation products represent a new strategy for the treatment and the prevention against influenza infection.

LOX is an oxygenase that recognizes and adds an oxygen molecule to the pentadiene structure of polyunsaturated fatty acids [4]. Depending on the oxygenation position when AA is used as a substrate of LOX, humans possess the following six types of LOXs (5-LOX, platelet-type 12-LOX, epidermal-type 12R-LOX, 15-LOX-1, 15-LOX-2, and eLOX3) [5]. 5-LOX is present in various leukocytes, including monocytes/macrophages, neutrophils, and eosinophils [6]. 5-LOX metabolizes AA to 5-HpETE (Fig. 1A). The platelet-type 12-LOX produces 12(S)-HpETE enantiomer, whereas the epidermal-type 12R-LOX generates 12(R)-HpETE enantiomer [7]. 15-LOX-1 not only metabolizes AA mainly to 15-HpETE but also converts LA to 13-(Z,E)-HpODE [8]. 15-LOX-2 has a strong ability to metabolize AA mainly to 15-HpETE and a weak activity in producing 13-(Z,E)-HpODE from LA [9]. eLOX3 is present in the epidermis of the skin and exhibits potential dioxygenase activity [10].

Through a free radical-mediated reaction, AA is oxidized to 8-HpETE, 9-HpETE, 11-HpETE and 8-isoprostagrandin F2α (8-iso-PGF2α) in addition to 5-, 12-, and 15-HpETE. Free radical-mediated reactions with LA produced equal amounts of 9-(E,Z)- and 13-(Z,E)-HpODE and equal amounts of 9-(E,E)- and 13-(E,E)-HpODE (Fig. 1B). Through nonradical reactions mediated by singlet oxygen, 9-(E,Z)-, 13-(Z,E)-, 10-(E,Z)-, and 12-(Z,E)-HpODE were produced from LA (Fig. 1B). HpETEs and HpODEs, which are peroxides of AA and LA, respectively, were reduced and converted into HETEs and HODEs, respectively.

Among the six types of LOXs, 5-LOX has been well investigated for the regulation of its enzymatic activity [11]. This is because 5-LOX produces leukotriene (LT) A4, which is the starting point for the production of LTs that have physiological effects, such as bronchial asthma, allergic reactions, and maintenance of inflammatory reactions [11].

Leukocytes express 5-LOX, 5-lipoxygenase-activating protein (FLAP), and cytosolic phospholipase A2 (cPLA2) for the biosynthesis of 5-LOX products [6]. 5-LOX is widely distributed in the cytoplasm or the nucleus in resting cells. FLAP is bound to the nuclear membrane and function as a scaffold protein for 5-LOX [12]. In stimulated leukocytes, both 5-LOX and cPLA2 translocate to the nuclear envelope and congregate near FLAP. After binding to the membrane, cPLA2 cleaves AA from arachidonyl-phosphatidylcholine (PC) in the nuclear envelope, which is then passed to membrane-attached 5-LOX via FLAP as a substrate for enzyme activity.

Drugs such as zileuton and MK-886, 5-LOX and FLAP inhibitor, respectively, have been developed as candidates for the therapeutic agents against bronchial asthma and allergy-related diseases controlled by LTs. However, to the best of our knowledge, no compound that can induce 5-LOX activation has yet been identified.

Several intracellular factors regulate 5-LOX [6]. The mammalian 5-LOX protein has a C2-like regulatory domain, a phospholipid-binding domain [13]. This domain binds Ca2+, and Ca2+ binding increases the hydrophobicity resulting in 5-LOX binding to the nuclear membrane [14]. Although 5-LOX has no known ATP-binding motif, it has been reported that binding to ATP increases enzyme activity [15]. ATP hydrolysis is not involved in the 5-LOX activity [16], and the mechanism by which ATP accelerates its enzymatic activity remains unclear. Phosphatidylcholine and diacylglycerol contained in the phospholipids of the nuclear envelope are involved in enzyme activity and are associated with 5-LOX cohesion to the nuclear membrane [17]. 5-LOX proteins are phosphorylated at multiple sites, such as serine 271, 523, and 663 and tyrosine 42, 53, 94, and 445 by various kinases, and phosphorylation at different sites contribute to different functions of 5-LOX [18]. The enzymatic activity of 5-LOX is regulated by these intracellular factors.

In this study, we elucidated the mechanism of activation of 5-LOX by Dz.

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