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Depletion of phospholipid hydroperoxide glutathione peroxidase up-regulates arachidonate metabolism by 12(S)-lipoxygenase and cyclooxygenase 1 in human epidermoid carcinoma A431 cells¹

Departments of Pharmacology and
^* Medical Technology, College of Medicine, National Cheng Kung University, Tainan 704, Taiwan

²Correspondence: Department of Pharmacology, College of Medicine, National Cheng Kung University, 138 Sheng-Li Road, Tainan 704, Taiwan. E-mail: wcchang{at}mail.ncku.edu.tw

SPECIFIC AIMS

In this study, we established a stable cell line that overexpressed anti-phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA to specifically block the formation of endogenous PHGPx and studied the functional role of endogenous PHGPx in the regulation of 12(S)-lipoxygenase and cyclooxygenase 1 activities in human epidermoid carcinoma A431 cells.

PRINCIPAL FINDINGS

1. Arachidonate metabolism in anti-PHGPx mRNA transfectants
A retroviral expression vector designated as L1-3, wherein cDNA of PHGPx was reversely inserted into pFB-ERV in antisense orientation, was constructed. Expression vectors of L1-3 and pFB-ERV were used to transfect Amphopack-293 cells to produce anti-PHGPx mRNA and vector control virions, respectively. Stable cell line ANR-10 of Amphopack-293 cells, which could produce high titer of anti-PHGPx mRNA, was established. A control cell line AER-18, which produced vector control virions, was also produced. By using antisense virions produced by ANR-10 cell line for infection of A431 cells, we generated several stable clones including NR8 and NR101. A vector control transfectant NER4 was generated by using vector control virions produced by AER-18 cell line for infection of A431 cells. Expression of PHGPx antisense mRNA in these transfectants was detected by RT-PCR analysis using specific primers designed for His-tag antisense-PHGPx mRNA. His-tag antisense PHGPx mRNA was expressed in anti-PHGPx mRNA transfectants but not in vector control transfectant. The immunoblot analysis confirmed that PHGPx was almost depleted in anti-PHGPx mRNA transfectants but not in vector control transfectant (Fig. 1 a). The functional role of PHGPx in arachidonate metabolism in cells was then studied. As shown in Fig. 1c , in an intact cell assay, the metabolism of arachidonic acid to 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) and prostaglandin (PG) E₂ significantly increased in anti-PHGPx mRNA transfectants compared with that of vector control transfectant. The expression of 12(S)-lipoxygenase and cyclooxygenase 1 in these transfectants did not change (Fig. 1a ). No significant change of GPx1 was observed (Fig. 1b ). Therefore, the low expression of PHGPx resulted in an elevated level of arachidonate metabolites in anti-PHGPx mRNA transfectants.

View larger version (30K): [in this window] [in a new window]   Figure 1. Effect of overexpressed anti-PHGPx mRNA on metabolism of arachidonic acid to PG E2 and 12(S)-HETE in transfectants. a) Total cell lysates of these transfectants were prepared. 50 µg of protein was loaded and analyzed by Western blot using anti-PHGPx, 12(S)-lipoxygenase (12(S)-LOX), cyclooxygenase 1 (COX 1), and ß-actin polyclonal antibody to detect PHGPx, 12(S)-lipoxygenase, cyclooxygenase 1, and ß-actin, respectively. b) 50 µg of protein of cell lysates was loaded and analyzed by Western blot using anti-GPx1 monoclonal and ß-actin polyclonal antibodies to detect GPx1 and ß-actin, respectively. c) Confluent cells in 12-multiwell plastic dishes were incubated with 2.5 µg of [1-14C] arachidonic acid (0.125 µCi) and 10 µM calcium ionophore A23187 in 0.5 mL of HEPES buffer for 30 min. Formations of PG E2 and 12(S)-HETE in buffer were analyzed by thin-layer chromatographic analysis. Each value represents the mean ± SE from triplicate assays. **P < 0.01; *P < 0.05.

2. Formation of intracellular hydroperoxides in stable transfectants
Since many reports indicate that lipoxygenases and cyclooxygenases need a certain "peroxide tone" for the initial reaction of arachidonate catabolism, the intracellular level of hydroperoxides in these transfectants was determined by a flow cytometric analysis using 2',7'-dichlorofluorescin diacetate (DCFH-DA) as a fluorescent probe. The basal fluorescence intensity of dichlorofluorescein (DCF) in anti-PHGPx mRNA transfectants was significantly higher than that observed in the vector control transfectant. We recently reported that treatment of A431 cells with arsenite fostered the intracellular formation of hydroperoxides. The effect of the depletion of endogenous PHGPx on arsenite-induced formation of intracellular hydroperoxides was then studied. As shown in Fig. 2 , the increase in fluorescence intensity in anti-PHGPx mRNA transfectants was significantly higher than that in vector control transfectant upon arsenite treatment. These results support the notion that the endogenous PHGPx plays a functional role in regulating hydroperoxides.

View larger version (19K): [in this window] [in a new window]   Figure 2. Effect of arsenite on hydroperoxides production in anti-PHGPx mRNA and vector control transfectants. To assess levels of intracellular peroxides, flow cytometric analysis was performed with the fluorescent probe DCFH-DA. Vector control transfectant (NER4) and anti-PHGPx mRNA transfectants (NR8 and NR101) were preincubated with 50 µM arsenite for 1 h at 37°C, then incubated with 100 µM DCFH-DA for 1 h. The intensity of fluorescence of DCF in cells was quantified by flow cytometry.

CONCLUSIONS AND SIGNIFICANCE

In a series of studies of the regulation of arachidonate metabolism by PHGPx in cells, direct evidence to ascertain the functional role of endogenous PHGPx in the regulation of arachidonate metabolism has not been provided. So far, several reports have used PHGPx overexpression to study this notion. Overexpression of PHGPx in RBL-2H3 cells suppressed the leukotriene formation through 5-lipoxygenase and attenuated the activation of cyclooxygenase 2. We recently reported that overexpression of PHGPx in A431 cells attenuated the 12(S)-lipoxygenase and cyclooxygenase 1 activities. Although results of these three reports agree that PHGPx overexpression in cells could attenuate the arachidonate metabolism, the results are not enough to directly indicate the functional role of endogenous PHGPx in arachidonate metabolism in cells. Therefore, in this study we established a stable cell line with deletion of PHGPx by an antisense approach. This is the first report to use a stable cell line with PHGPx depletion to answer the notion about the functional role of endogenous PHGPx in cells. Our results strongly suggest that depletion of endogenous PHGPx in A431 cells enhances not only 12(S)-lipoxygenase activity, but also cyclooxygenase 1 activity. These results provide the direct evidence for the participation of the endogenous PHGPx in the regulation of arachidonate metabolism through 12(S)-lipoxygenase and cyclooxygenase 1. Taken together, the results of PHGPx overexpression and the present antisense study lead to the conclusion that the level of the endogenous PHGPx indeed plays a pivotal role in regulating the arachidonate metabolism in cells.

Prior to structural identification of the cytosolic inhibitor of arachidonate metabolism as PHGPx, the putative inhibitor is observed in the primary culture of ovine tracheal epithelial cells in addition to A431 cells. The activity of putative inhibitor in inhibiting arachidonate metabolism in ovine tracheal epithelial is attenuated by the supplementation of lipid hydroperoxides in cell culture medium, indicating that inhibitor activity depends on the changes in cellular oxidation reduction conditions. In studying the arachidonate metabolism, many reports indicate that lipoxygenases and cyclooxygenases need a certain peroxide tone for their enzyme activities. PHGPx is a glutathione peroxidase and is able to reduce peroxides, including phospholipid hydroperoxides and other lipid hydroperoxides. Therefore, it is reasonable to deduce that PHGPx is an inhibitor of arachidonate metabolism and attenuate the peroxides tone, a step required for the activities of 12(S)-lipoxygenase and cyclooxygenases 1 that result in the attenuation of arachidonate metabolism in cells (Fig. 3 ). Our present study has provided another piece of evidence to support this reasoning. Depletion of the endogenous PHGPx enhanced the basal level of cellular hydroperoxides; accumulation of cellular hydroperoxides upon arsenite treatment was more significant in PHGPx-depleted cells than that in vector control cells (Fig. 2) . These results clearly indicate that the endogenous PHGPx could play a functional role in reducing peroxides tone.

View larger version (17K): [in this window] [in a new window]   Figure 3. Schematic diagram of possible mechanisms of PHGPx-inhibited lipid oxygenation in A431 cells.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0847fje; doi: 10.1096/fj.02-0847fje

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This Article Full Text (PDF) All Versions of this Article: 17/12/1694 02-0847fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by CHEN, C.-J. Articles by CHANG, W.-C. Search for Related Content PubMed PubMed Citation Articles by CHEN, C.-J. Articles by CHANG, W.-C.