Identification of a lipoxygenase inhibitor in A431 cells as a phospholipid hydroperoxide glutathione peroxidase

Huang, Hei Sheng; Chen, Ching Jiunn; Lu, Hsieng S.; Chang, Wen Chang

doi:10.1016/s0014-5793(98)00130-6

Cited by 35 publications

(22 citation statements)

References 29 publications

(25 reference statements)

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“…In accordance, 13(S)-HPODE but not H 2 O 2 counteracted M-DSP-or GPx-1-induced 5-LO suppression. Although GPx-4 has been reported to inhibit 5-LO activity in differentiated HL-60 and BL41-E95-A cells (11), RBL-2H3 cells (14) and A431 cells (13), it is unlikely that this peroxidase is the M-DSP, since GPx-4 is a monomeric 19 kDa protein (48) that is not sensitive to mercaptosuccinate (5) and requires millimolar concentrations of thiols for efficient inhibition of 5-LO activity (11,12). Interestingly, Coffey et al (49) reported about a cytosolic protein of mononuclear phagocytes that reduced 5-LO activity in broken cell preparations and prolonged the lag phase of soybean LO, which was reversed by addition of 13(S)-HPODE.…”

Section: Activation Of 5-lipoxygenase By Ca 2ϩmentioning

confidence: 99%

The C2-like β-Barrel Domain Mediates the Ca2+-dependent Resistance of 5-Lipoxygenase Activity Against Inhibition by Glutathione Peroxidase-1

Bürkert

Arnold

Hammarberg

et al. 2003

Journal of Biological Chemistry

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Recently, we reported that in crude enzyme preparations, a monocyte-derived soluble protein (M-DSP) renders 5-lipoxygenase (5-LO) activity Ca 2؉ -dependent. Here we provide evidence that this M-DSP is glutathione peroxidase (GPx)-1. Thus, the inhibitory effect of the M-DSP on 5-LO could be overcome by the GPx-1 inhibitor mercaptosuccinate and by the broad spectrum GPx inhibitor iodoacetate, as well as by addition of 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-HPODE). Also, the chromatographic characteristics and the estimated molecular mass (80 -100 kDa) of the M-DSP fit to GPx-1 (87 kDa), and GPx-1, isolated from bovine erythrocytes, mimicked the effects of the M-DSP. Intriguingly, only a trace amount of thiol (10 M GSH) was required for reduction of 5-LO activity by GPx-1 or the M-DSP. Moreover, the requirement of Ca 2؉ allowing 5-LO product synthesis in various leukocytes correlated with the respective GPx-1 activities. Mutation of the Ca 2؉ binding sites within the C2-like domain of 5-LO resulted in strong reduction of 5-LO activity by M-DSP and GPx-1, also in the presence of Ca 2؉ . In summary, our data suggest that interaction of Ca 2؉ at the C2-like domain of 5-LO protects the enzyme against the effect of GPx-1. Apparently, in the presence of Ca 2؉ , a low lipid hydroperoxide level is sufficient for 5-LO activation. 5-Lipoxygenase (5-LO)1 catalyzes the initial steps in the biosynthesis of leukotrienes (LTs) and 5(S)-hydro(pero)xyeicosatetraenoic acid (5(S)-H(P)ETE) from arachidonic acid (AA) (for review, see Ref. 1). Due to the pivotal biological functions of 5-LO metabolites (2, 3), the activity of 5-LO is tightly regulated. In intact cells, Ca 2ϩ and phosphorylation seem to be primary signals that activate 5-LO. Moreover, the membranebound 5-LO-activating protein (FLAP) (4) and the redox state (5, 6) have a strong impact on cellular 5-LO product formation.In cell-free systems Ca 2ϩ , ATP, phospholipids (membranes), lipid hydroperoxides (LOOH), and leukocyte-derived proteins have been shown to enhance 5-LO catalysis (reviewed in Ref. 1). However, the degree of stimulation by each of these components depends on the assay conditions, i.e. the source of 5-LO (isolated, in crude homogenates or cellular fractions), presence of other cofactors, the concentration of AA, etc. LOOH are of importance for the initial conversion of the active site iron from the ferrous (resting) to the ferric (active) state (7, 8). Accordingly, glutathione peroxidases (GPx) that reduce LOOH inhibit 5-LO product synthesis in vitro and in intact cells (5, 9 -15), and conditions that are associated with an increased peroxidetone promote 5-LO product formation (6,16,17). Two Ca 2ϩ ions bind to the N-terminal C2-like domain of 5-LO with a K d of 6 M (18, 19). Half-maximal activation of purified 5-LO was determined at 1-2 M Ca 2ϩ , whereas 4 -10 M Ca 2ϩ causes maximal activation of the enzyme (20,21). In intact cells lower concentrations of Ca 2ϩ (200 -300 nM) seem to be sufficient for 5-LO activation (22,23). It was shown that C...

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Section: Activation Of 5-lipoxygenase By Ca 2ϩmentioning

confidence: 99%

The C2-like β-Barrel Domain Mediates the Ca2+-dependent Resistance of 5-Lipoxygenase Activity Against Inhibition by Glutathione Peroxidase-1

Bürkert

Arnold

Hammarberg

et al. 2003

Journal of Biological Chemistry

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“…Moreover, purified inhibitor exhibits peroxidase activity using phosphatidylcholine hydroperoxides as the substrate. We therefore conclude that the endogenous inhibitor of arachidonate metabolism present in A431 cells is indeed a PHGPx [23]. PHGPx was first purified from pig liver [53] and then from rat testis [43].…”

Section: Structural Determination and Oxygenase-inhibitory Specificitmentioning

confidence: 72%

“…Since the discovery of the endogenous inhibitor from the cytosolic fraction of A431 cells, which regulates the cellular arachidonate metabolism [5], two major breakthroughs have been achieved in our series of studies. The first one is the purification of the endogenous inhibitor from the cytosolic fraction of cells which regulates the arachidonate metabolism [9], and the subsequent identification of the entity of this inhibitor as a PHGPx [23]. The second one is the creation of a stable PHGPx-depleted A431 cell line, which provides direct evidence showing that the endogenous PHGPx indeed plays a functional role in down-regulating the 12(S)-lipoxygenase and cyclooxygenase 1 activities in A431 cells [7].…”

Section: Discussionmentioning

confidence: 99%

Identification of an Endogenous Inhibitor of Arachidonate Metabolism in Human Epidermoid Carcinoma A431 Cells

Chang

2003

J Biomed Sci

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Eicosanoids, which include prostaglandins, thromboxanes, and leukotrienes, are produced from arachidonic acid by three main pathways in cells, including cyclooxygenases and lipoxygenases, and cytochrome P450 enzymes. Accumulated evidence indicates that a certain peroxide tone is required for the initiation of reaction by lipoxygenases and cyclooxygenases. An endogenous inhibitor of arachidonate oxygenation was suspected in the cytosolic fraction of human epidermoid carcinoma A431 cells. After a series of studies, the existence of this inhibitor was confirmed, while it was purified and characterized. By amino acid sequence analysis, the inhibitor in A431 cells was subsequently identified as a phospholipid hydroperoxide glutathione peroxidase (PHGPx). Depletion of cellular glutathione in cells by diethyl maleate or by dibuthionine-sulfoximine results in an increase in enzyme activities of 12(S)-lipoxygenase and cyclooxygenase, suggesting that glutathione-depleting agents abolish the enzyme activity of PHGPx in cells. Stable transfectants of A431 cells with overexpression and depletion of PHGPx have been constructed, respectively. Reduction of arachidonate metabolism through 12(S)-lipoxygenase and cyclooxygenase 1 and that of the arsenite-induced generation of reactive oxygen species are observed in cells overexpressing PHGPx. On the other hand, enhancement of arachidonate metabolism and the arsenite-induced generation of reactive oxygen species is detected in PHGPx-depleted cells. In conclusion, the endogenous inhibitor of arachidonate metabolism present in A431 cells is a PHGPx, which plays a functional role in the down-regulation of arachidonate oxygenation catalyzed by 12(S)-lipoxygenase and cyclooxygenase 1 through the reduction of the level of intracellular lipid hydroperoxides. The latter acts as the peroxide tone for arachidonate metabolism in A431 cells.

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“…The sequence of the purified inhibitor protein was analyzed by a high-sensitivity microsequencer and mass spectrometer, and the sequence data matched those of human PHGPx [8]. Moreover, the purified inhibitor exhibited peroxidase activity using phosphatidylcholine hydroperoxides as a substrate [8]. We therefore concluded that the 12-lipoxygenase inhibitor present in A431 cells was PHGPx.…”

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confidence: 82%

Inhibition of Arachidonate Metabolism in Human Epidermoid Carcinoma A431 Cells Overexpressing Phospholipid Hydroperoxide Glutathione Peroxidase

Chen¹,

Huang²,

Chang³

2002

J Biomed Sci

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Phospholipid hydroperoxide glutathione peroxidase (PHGPx), a selenium-dependent glutathione peroxidase, can interact with lipophilic substrates, including phospholipid hydroperoxides, fatty acid hydroperoxides and cholesterol hydroperoxides, and can reduce them to hydroxide compounds. It also seems to be a major regulator of lipid oxygenation in human epidermoid carcinoma A431 cells. In order to study the functional role of PHGPx in the regulation of 12-lipoxygenase and cyclooxygenase, cDNA of PHGPx was inserted into pcDNA3.1/His, and a plasmid designated as S4 with the His-tag sequence inserted between PHGPx and its 3′-untranslated region was constructed. A number of stable transfectants of A431 cells that could express the tag-PHGPx were generated using plasmid S4. Using an intact cell assay system, the metabolism of arachidonic acid to prostaglandin E₂ significantly decreased in stable transfectants of overexpressing PHGPx compared to that in a vector control cell line. If the intact cell assay was carried out in the presence of 13-hydroperoxyoctadecadienoic acid as a stimulator of lipid peroxidation, formation of 12-hydroxyeicosatetraenoic acid from arachidonic acid also significantly decreased in stable transfectants of overexpressing PHGPx compared to that in a vector control cell line, indicating that PHGPx could downregulate the 12-lipoxygenase activity in cells. These results support the hypothesis that PHGPx plays a pivotal role in the regulation of arachidonate metabolism in A431 cells.

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Identification of a lipoxygenase inhibitor in A431 cells as a phospholipid hydroperoxide glutathione peroxidase

Cited by 35 publications

References 29 publications

The C2-like β-Barrel Domain Mediates the Ca2+-dependent Resistance of 5-Lipoxygenase Activity Against Inhibition by Glutathione Peroxidase-1

The C2-like β-Barrel Domain Mediates the Ca2+-dependent Resistance of 5-Lipoxygenase Activity Against Inhibition by Glutathione Peroxidase-1

Identification of an Endogenous Inhibitor of Arachidonate Metabolism in Human Epidermoid Carcinoma A431 Cells

Inhibition of Arachidonate Metabolism in Human Epidermoid Carcinoma A431 Cells Overexpressing Phospholipid Hydroperoxide Glutathione Peroxidase

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