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Inhibition of long-chain acyl-CoA synthetase 4 facilitates production of 5, 11-dihydroxyeicosatetraenoic acid via the cyclooxygenase-2 pathway
Biochemical and Biophysical Research Communications xxx (2015) 1e6
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Inhibition of long-chain acyl-CoA synthetase 4 facilitates production of 5, 11-dihydroxyeicosatetraenoic acid via the cyclooxygenase-2 pathway Hiroshi Kuwata*, Shuntaro Hara Division of Health Chemistry, Department of Healthcare and Regulatory Sciences, School of Pharmacy, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 August 2015 Accepted 12 August 2015 Available online xxx
Long chain acyl-CoA synthetases (ACSLs) are a family of enzymes that convert free long chain fatty acids into their acyl-CoA forms. Among ACSL enzymes, ACSL4 prefers arachidonic acid (AA) as a substrate and plays an important role in re-esterification of free AA. We previously reported that the suppression of ACSL4 activity by treatment with an ACSL inhibitor or a small interfering RNA markedly enhanced interleukin-1b (IL-1b)-dependent prostaglandin (PG) biosynthesis in rat fibroblastic 3Y1 cells. We show here that in addition to these prostanoids, cytokine-dependent production of 5,11dihydroxyeicosatetraenoic acid (5,11-diHETE), a cyclooxygenase product of 5-hydroxyeicosatetraenoic acid (5-HETE), was enhanced by the inhibition of ACSL4 activity. Treatment of several types of cells with an ACSL inhibitor, triacsin C, markedly enhanced IL-1b-dependent production of 5,11-diHETE. siRNA-mediated knockdown of ACSL4 also enhanced IL-1b-dependent production of 5,11-diHETE from 3Y1 cells. The production of 5,11-diHETE was significantly decreased by a cyclooxygenase (COX)-2 selective inhibitor, NS-398, but not by a 5-lipoxygenase activating protein (FLAP) inhibitor, MK-886. The inhibition of ACSL enzymes significantly facilitated release of not only 5-HETE but also 8-HETE, 9-HETE, 11-HETE, 12-HETE, and 15-HETE, independently of IL-1b stimulation. In vitro analysis showed that a recombinant COX-2 enzyme more effectively metabolized 5(S)-HETE to 5-11-diHETE compared to COX-1 enzyme. From these results, we proposed the following mechanism of 5,11-diHETE biosynthesis in these cells: 1) inhibition of ACSL4 causes accumulation of free AA; 2) the accumulated AA is nonspecifically converted into various HETEs; and 3) among these HETEs, 5-HETE is metabolized into 5,11-diHETE by cytokine-induced COX-2. © 2015 Elsevier Inc. All rights reserved.
Keywords: Long-chain acyl-CoA synthetase 4 Arachidonic acid Cyclooxygenase 5,11-Dihydroxyeicosatetraenoic acid 5-Hydroxyeicosatetraenoic acid Triacsin C
1. Introduction Arachidonic acid (AA) released from membrane phospholipids is converted into various bioactive lipid mediators, such as prostaglandins (PGs) and leukotrienes, by either the cyclooxygenase (COX) or lipoxygenase (LOX) pathway [1]. To control the levels of these bioactive lipid mediators, the free AA levels are strictly regulated by the deacylation/reacylation pathways, and several enzymes have been shown to be involved in these pathways, including phospholipase A2, acyl-CoA synthetases, and acyl-CoA acyltransferases [2]. The unmetabolized free AA is incorporated
* Corresponding author. E-mail address: [email protected] (H. Kuwata).
into cellular lipids at least in part via the actions of the phospholipid remodeling pathway. Among the enzymes involved in this pathway, acyl-CoA synthetase long-chain family members (ACSL) catalyze the conversion of free long-chain fatty acids into their acylCoA forms. Five ACSL isozymes have been identified in humans and rodents [3], and each isoform is thought to have its own functions. In particular, ACSL4, an isoform that prefers AA compared to other long-chain fatty acids [4], has been shown to participate in the incorporation of AA into cellular lipids and in steroidogenesis [5e7]. We have previously reported that pharmacological inhibition of ACSL isoforms or small interfering RNA (siRNA)-mediated knockdown of ACSL4 markedly enhanced cytokine-mediated biosynthesis of PGs, including PGE2, PGD2, and PGF2a, from IL-1bstimulated rat fibroblastic 3Y1 cells [8]. These results suggest that
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Please cite this article in press as: H. Kuwata, S. Hara, Inhibition of long-chain acyl-CoA synthetase 4 facilitates production of 5, 11dihydroxyeicosatetraenoic acid via the cyclooxygenase-2 pathway, Biochemical and Biophysical Research Communications (2015), http:// dx.doi.org/10.1016/j.bbrc.2015.08.054
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Fig. 1. Effects of ACSL inhibitor on biosynthesis of HETEs and 5,11-diHETE. A, 3Y1 cells grown in 24-well plate were incubated with (gray bar) or without 5 mM triacsin C (open bar) for 24 h, and the levels of HETEs released into culture medium were assessed. B-E, 3Y1 cells (B), WI-38 cells (C), Colon-26 cells (D), or LLC cells (E) were stimulated with or without IL-1b in the presence or absence of 5 mM triacsin C (T.C) for 24 h, and 5,11-diHETE release was examined (left panels) (mean ± S.D. (n ¼ 3); *, p < 0.05; **, p < 0.01). Expression of COX-1, COX-2, and b-actin was assessed by immunoblotting (right panels).
ACSL4 is participated in the elimination of free AA after stimulation, and that inhibition of the ACSL4 pathway altered the production profile of bioactive lipid mediators. In addition to these bioactive lipid mediators, in the present study we found that the formation of 5,11-dihydroxyeicosatetraenoic acid (5,11-diHETE), a COX product of 5-HETE [9], was markedly enhanced by inhibition of the ACSL4 pathway. We also showed that the formation of 5,11-diHETE in rat fibroblasts was independent of the 5-lipoxygenase (5-LOX) pathway.
2. Materials and methods 2.1. Materials Mouse IL-1b was purchased from R & D Systems (Abingdon, UK). Mouse monoclonal antibody against b-actin was purchased form Sigma (St. Louis, MO, USA). Goat polyclonal antibodies against COX1, COX-2, and 5-LOX, rabbit polyclonal antibody against ACSL4, and triacsin C were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Lipofectamine RNAiMAX reagent and Opti-MEM
medium were obtained from Invitrogen (Carlsbad, CA, USA). NS398, MK-886, various HETE standards, deuterium-labeled leukotriene B4 (LTB4-d4), COX-1 (ovine), and COX-2 (human) were purchased from Cayman Chemicals (Ann Arbor, MI, USA).
2.2. Cell culture, treatment, and transfection Conditions for the maintenance of 3Y1 cells (rat embryonic fibroblasts) and WI-38 cells (human lung fibroblasts) were described previously [8]. LLC (Lewis lung carcinoma) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal calf serum (FCS), penicillin/streptomycin (100 units/ml and 100 mg/ml, respectively), and 2 mM glutamine (Invitrogen) under a humidified atmosphere containing 5% CO2. Colon-26 cells (a colon cancer cell line) were cultured in RPMI 1640 with the same supplements. The media of the cells that had attained 80% confluence in 24-well plates were replaced with 0.5 ml of DMEM supplemented with 2% (v/v) FCS. After 24 h culture, 1 ng/ml mouse IL-1b (3Y1, LLC, and colon-26 cells) or 1 ng/ml human IL-1b (WI-38 cells) was added to the cultures to assess the eicosanoid biosynthetic
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COX-2, 5-LOX, and b-actin). The separated proteins were electroblotted onto nitrocellulose membranes (Schleicher & Schuell) with a bath-type blotter. After blocking for 1 h with 5% (w/v) skimmed milk in phosphate-buffered saline (PBS) containing 0.05% Tween 20 (TPBS), the membranes were probed for 2 h with the respective antibodies (1:2000 for ACSL4 and 5-LOX, 1:5000 for COX-1 and COX-2, 1:10,000 for b-actin), followed by incubation with horseradish peroxidase-conjugated anti-mouse (1:2000 for b-actin), anti-rabbit (1:5000 for ACSL4), or anti-goat (1:5000 for COX-1 and COX-2) IgG. After washing, the membranes were visualized with Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Wellesley, MA, USA). 2.4. Sample preparation Extraction of eicosanoids from culture medium was performed as described previously with minor modification [8]. Briefly, an internal standard (50 ng of LTB4-d4) was added to the medium (500 ml). The medium was acidified by addition of 100 ml of 0.2% (v/ v) formic acid followed by 500 ml of ethyl acetate. Samples were mixed and centrifuged at 15,000 rpm for 10 min. The organic layer was retrieved and evaporated to dryness with a vacuum evaporator. Samples were resuspended in 50 ml of mobile phase A (water/ acetonitrile/formic acid (63:37:0.02, v/v/v)) and injected into an LCESI-MS/MS system. 2.5. Electrospray ionization (ESI) mass spectrometry analysis Quantification of eicosanoids, such as 5,11-diHETE, were performed using LC-ESI-MS/MS via multiple-reaction monitoring (MRM) in negative-ion mode as described previously with minor modification [8]. MRM transitions for quantification of 5,11-diHETE, 5-HETE, 8-HETE, 9-HETE, 11-HETE, 12-HETE, 15-HETE, and LTB4-d4 (internal standard) were 335/183, 319/115, 319/155, 319/151, 319/ 167, 319/179, 319/175, and 339/197, respectively. Because a 5,11diHETE standard is not available currently, the levels of this eicosanoid were expressed as fold-change compared to control levels. 2.6. Synthesis of 5,11-diHETE
Fig. 2. Effect of knockdown of ACSL4 expression on biosynthesis of 5,11-diHETE in 3Y1 cells. 3Y1 cells grown in 24-well plate were transfected wit ACSL4 or control siRNA. Two days after transfection, the expression level of ACSL4 protein was assessed by immunoblotting (A). An equal loading of samples in each lane was immunoblotted with anti-b-actin antibody. B and C, effects of siRNA against ACSL4 expression on IL-1binduced 5,11-diHETE (B) or 5-HETE (C) (mean ± S.D. (n ¼ 3); **, p < 0.01; N.S., not significant).
response. RBL-2H3 cells (a rat basophilic leukemia cell line) were maintained with DMEM with the same supplements. The media of RBL-2H3 cells that had attained 60e80% confluence in 24-well plates were stimulated with 1 mM A23187 for 30 min in serumfree DMEM. In studies using inhibitors, these cells were pretreated with various inhibitors for 1 h and then treated with IL-1b for 24 h or A23187 for 30 min. Small interfering RNA (siRNA) against ACSL4 (siRNA ID numbers SASI_Rn01_00041844; Sigma) or a negative control siRNA (Sigma) was transfected into 3Y1 cells using Lipofectamine RNAiMAX according to our protocol [8]. 2.3. Immunoblotting Cell lysates (105 cells equivalent) were subjected to SDS-PAGE using 10% (w/v) gels under reducing conditions (for ACSL4, COX-1,
Incubation of AA (1 mM) or 5(S)-HETE (1 mM) was conducted in 300 ml of 50 mM TriseHCl pH7.4 containing 5 mM tryptophan, 2 mM hematin, and ovine COX-1 (1 unit) or human COX-2 (1 unit) for 30 min at 37 C. After reactions were terminated by the addition of 0.2% (v/v) formic acid, the eicosanoids were extracted as described above. 2.7. Statistical analysis Results are expressed as the means ± standard deviation (SD). Comparisons between two groups were made with an unpaired Student's t-test, and comparisons among three or more groups were made with two-way analysis of variance and TukeyeKramer post-test. P values of <0.05 were considered statistically significant. 3. Results and discussion Each ACSL enzyme is thought to have distinct biological functions, such as free fatty acid incorporation into cellular lipids [10], control of the composition of esterified fatty acids in neutral lipids and phospholipids [11], and b-oxidation [12]. We have previously reported that inhibition of ACSL4, an AA preferred ACSL isozyme, markedly enhanced IL-1b-dependent production of several COX-2 metabolites, such as PGE2, PGD2, and PGF2a [8]. These results prompted us to ascertain whether the other AA metabolites,
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Fig. 3. Effects of COX-2 or FLAP inhibitor on biosynthesis of 5,11-diHETE. A and B, 3Y1 cells were pretreated with or without 10 ng/ml NS-398 or 5 mM MK-886 for 1 h in the presence or absence of 5 mM triacsin C and then stimulated for 24 h with or without IL-1b. After collecting culture supernatants, the release of 5,11-diHETE was assessed (mean ± S.D. (n ¼ 3); **, p < 0.01). C, RBL-2H3 cells grown in 24-well plate were stimulated with or without A23187 for 30 min in the presence or absence of 5 mM MK-886. The amounts of 5-HETE released into culture medium were assessed (mean ± S.D. (n ¼ 3); **, p < 0.01). D, Expressions of 5-LOX and b-actin in 3Y1, RBL-2H3, colon-26, LLC, or WI38 cells were assessed by immunoblotting.
including even minor lipids, are also enhanced by the inhibition of ACSL enzymes. As shown in Fig. 1A, when rat fibroblastic 3Y1 cells were treated with the ACSL inhibitor triacsin C, the release of HETEs into the culture medium was significantly enhanced compared to that from control cells. The enhanced formation of HETEs by triacsin C treatment was also observed in several other cell lines, including human fibroblastic WI-38 cells and colon-26 mouse colon cancer cells (data not shown). Among these HETEs, 11-HETE and 15-HETE are known as by-products of the reaction between COX and AA [13], and their release is attenuated by treatment with the COX-2 selective inhibitor NS-398 in these cells [8]. On the other hand, it appears that other HETEs are produced via either the AA LOX pathway or a non-enzymatic pathway. Because 5-HETE is metabolized into 5(S),11(R)-diHETE, 5(S),15(S)-diHETE, and 5(S),15(R)-diHETE by COX-2 [9,14], we next examined the effects of triacsin C on the production of these diHETEs. As shown in Fig. 1B, when 3Y1 cells were treated with triacsin C, the level of 5,11-diHETE increased 2-fold compared to that in control cells, and was further enhanced to 8-fold after treatments with triacsin C and IL-1b. As we previously reported [15], the expression of COX-2, but not COX-1, protein was increased after IL-1b treatment in these cells (Fig. 1B, right panel). The enhanced formation of 5,11-diHETE observed in cells treated with triacsin C and IL-1b seemed to be mediated by IL-1b-induced COX-2 protein. Furthermore, the biosynthesis of 5,11-diHETE was also analyzed in WI-38 human fibroblasts (Fig. 1C), Colon-26 mouse colon cancer cells (Fig. 1D), and LLC mouse Lewis lung carcinoma cells (Fig. 1E). As shown in Fig. 1CeE, both the IL-1b-dependent and
the IL-1b-independent biosyntheses of 5,11-diHETE were significantly enhanced by treatment with triacsin C in these cells. These results suggested that the enhanced production of 5,11-diHETE via inhibition of the ACSL pathway seems to occur in a wide variety of cells. In contrast to 5,11-diHETE, 5,15-diHETEs were hardly detected in the culture media of these cells (data not shown). We next examined whether knockdown of the ACSL4 protein would affect formation of 5,11-diHETE from 3Y1 cells. Treatment of 3Y1 cells with siRNAs against ACSL4 attenuated the expression of ACSL4 proteins compared to control siRNA-transfected cells (Fig. 2A). To assess the effect of ACSL4 knockdown on 5,11-diHETE formation, we analyzed the 5,11-diHETE levels after IL-1b stimulation. As shown in Fig. 2B, treatment of ACSL4-knockdown cells with IL-1b for 24 h markedly enhanced the production of 5,11diHETE, whereas the level of 5,11-diHETE in control siRNAtransfected cells was barely increased. In addition, the formation of 5-HETE, a precursor of 5,11-diHETE, was significantly enhanced by the knockdown of ACSL4, although it was not increased after IL1b stimulation (Fig. 2C). From these results we conjectured that inhibition of ACSL4 enhanced the formation of 5-HETE, and, in turn, 5-HETE may be metabolized to 5,11-diHETE by the cytokineinduced COX-2 protein. Because the above results raised the possibility that both the 5LOX and COX-2 pathway participated in the formation of 5,11diHETE in IL-1b-stimulated ACSL4-knockdown cells, we next examined the effects of COX-2 and FLAP inhibitors. As shown in Fig. 3A, when triacsin C-pretreated 3Y1 cells were stimulated with IL-1b in the presence of the COX-2 selective inhibitor NS-398, the
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HETE, a by-product of COX reaction, were nearly equivalent (Fig. 4B). Thus, the selectivity of COX enzymes against 5-HETE explains why the release of 5,11-diHETE is enhanced under the IL-1bstimulated condition (Fig. 1), even though the level of 5-HETE is comparable between these conditions (Fig. 2). From these results, we propose that the induction of COX-2 protein rather than 5-HETE synthesis is the rate-limiting step for the production of 5,11diHETE. In conclusion, our findings suggest that inhibition of ACSL4 activity perturbs the production of various eicosanoids [8], including 5,11-diHETE (this study). These events appear to be involved in several pathophysiological conditions that are observed in ACSL4 dysfunction, including X-linked mental retardation [16]. Further studies are needed to identify the actual role of the ACSL4 enzyme. Conflict of interest The authors declare no conflict of interest. Acknowledgments We thank the members of our lab for their helpful discussions and comments. This work was supported in part by Grantsein Aid for Scientific Research (B) (25293033) from the Japan Society for the Promotion of Science, and by a Grant-in-Aid for Scientific Research on Innovative Areas (25116720), and a grant for a Private University High Technology Research Center Project from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. References Fig. 4. In vitro synthesis of 5,11-diHETE by COX isozymes. Ovine COX-1 or human COX-2 were incubated with 1 mM AA or 1 mM 5(S)-HETE for 30 min and then the products were extracted as described “material and methods”. Data represent as means ± S.D.
production of 5,11-diHETE was significantly attenuated compared to that in control cells. By contrast, treatment of these cells with the FLAP inhibitor MK-886 (5 mM) failed to inhibit the formation of 5,11-diHETE (Fig. 3B). The release of 5-HETE from triacsin C-treated 3Y1 cells was unaffected by 5 mM MK-886 (data not shown), whereas its release from Ca2þ ionophore A23187-stimulated RBL2H3 cells was abolished by 5 mM MK-886 treatment (Fig. 3C). These results suggested that the COX-2 pathway does indeed participate in the biosynthesis of 5,11-diHETE in 3Y1 cells, but the 5-LOX pathway does not. Furthermore, the immunoblot analysis revealed that 5-LOX was expressed in RBL-2H3 cells (the positive control), whereas the 5-LOX signal was undetectable in 3Y1 cells (Fig. 3D). In addition, we failed to detect expression of the 5-LOX protein in WI-38 cells, colon-26 cells, or LLC cells (Fig. 3D), all of which can produce 5,11-diHETE (Fig. 1CeE). Thus, we concluded that the 5-LOX pathway is not involved in the formation of 5,11diHETE in 3Y1 cells. As mentioned above, the release of 5,11-diHETE from triacsin Ctreated 3Y1 cells was enhanced by IL-1b treatment, whereas that of 5-HETE was unaltered by cytokine stimulation. Thus, we speculated that the region downstream of 5-HETE generation, perhaps including the COX-2 pathway, is the rate-limiting step of 5,11diHETE biosynthesis. As shown in Fig. 4A, when recombinant human COX-2 or purified ovine COX-1 enzyme was incubated with 5(S)-HETE, 5,11-diHETE was formed. The level of 5,11-diHETE produced by COX-2 was about 4-fold greater than that of COX-1. The efficacies of COX-1 and COX-2 on the conversion of AA into 11-
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Inhibition of long-chain acyl-CoA synthetase 4 facilitates production of 5, 11-dihydroxyeicosatetraenoic acid via the cyclooxygenase-2 pathway Hiroshi Kuwata*, Shuntaro Hara Division of Health Chemistry, Department of Healthcare and Regulatory Sciences, School of Pharmacy, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 August 2015 Accepted 12 August 2015 Available online xxx
Long chain acyl-CoA synthetases (ACSLs) are a family of enzymes that convert free long chain fatty acids into their acyl-CoA forms. Among ACSL enzymes, ACSL4 prefers arachidonic acid (AA) as a substrate and plays an important role in re-esterification of free AA. We previously reported that the suppression of ACSL4 activity by treatment with an ACSL inhibitor or a small interfering RNA markedly enhanced interleukin-1b (IL-1b)-dependent prostaglandin (PG) biosynthesis in rat fibroblastic 3Y1 cells. We show here that in addition to these prostanoids, cytokine-dependent production of 5,11dihydroxyeicosatetraenoic acid (5,11-diHETE), a cyclooxygenase product of 5-hydroxyeicosatetraenoic acid (5-HETE), was enhanced by the inhibition of ACSL4 activity. Treatment of several types of cells with an ACSL inhibitor, triacsin C, markedly enhanced IL-1b-dependent production of 5,11-diHETE. siRNA-mediated knockdown of ACSL4 also enhanced IL-1b-dependent production of 5,11-diHETE from 3Y1 cells. The production of 5,11-diHETE was significantly decreased by a cyclooxygenase (COX)-2 selective inhibitor, NS-398, but not by a 5-lipoxygenase activating protein (FLAP) inhibitor, MK-886. The inhibition of ACSL enzymes significantly facilitated release of not only 5-HETE but also 8-HETE, 9-HETE, 11-HETE, 12-HETE, and 15-HETE, independently of IL-1b stimulation. In vitro analysis showed that a recombinant COX-2 enzyme more effectively metabolized 5(S)-HETE to 5-11-diHETE compared to COX-1 enzyme. From these results, we proposed the following mechanism of 5,11-diHETE biosynthesis in these cells: 1) inhibition of ACSL4 causes accumulation of free AA; 2) the accumulated AA is nonspecifically converted into various HETEs; and 3) among these HETEs, 5-HETE is metabolized into 5,11-diHETE by cytokine-induced COX-2. © 2015 Elsevier Inc. All rights reserved.
Keywords: Long-chain acyl-CoA synthetase 4 Arachidonic acid Cyclooxygenase 5,11-Dihydroxyeicosatetraenoic acid 5-Hydroxyeicosatetraenoic acid Triacsin C
1. Introduction Arachidonic acid (AA) released from membrane phospholipids is converted into various bioactive lipid mediators, such as prostaglandins (PGs) and leukotrienes, by either the cyclooxygenase (COX) or lipoxygenase (LOX) pathway [1]. To control the levels of these bioactive lipid mediators, the free AA levels are strictly regulated by the deacylation/reacylation pathways, and several enzymes have been shown to be involved in these pathways, including phospholipase A2, acyl-CoA synthetases, and acyl-CoA acyltransferases [2]. The unmetabolized free AA is incorporated
* Corresponding author. E-mail address: [email protected] (H. Kuwata).
into cellular lipids at least in part via the actions of the phospholipid remodeling pathway. Among the enzymes involved in this pathway, acyl-CoA synthetase long-chain family members (ACSL) catalyze the conversion of free long-chain fatty acids into their acylCoA forms. Five ACSL isozymes have been identified in humans and rodents [3], and each isoform is thought to have its own functions. In particular, ACSL4, an isoform that prefers AA compared to other long-chain fatty acids [4], has been shown to participate in the incorporation of AA into cellular lipids and in steroidogenesis [5e7]. We have previously reported that pharmacological inhibition of ACSL isoforms or small interfering RNA (siRNA)-mediated knockdown of ACSL4 markedly enhanced cytokine-mediated biosynthesis of PGs, including PGE2, PGD2, and PGF2a, from IL-1bstimulated rat fibroblastic 3Y1 cells [8]. These results suggest that
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Fig. 1. Effects of ACSL inhibitor on biosynthesis of HETEs and 5,11-diHETE. A, 3Y1 cells grown in 24-well plate were incubated with (gray bar) or without 5 mM triacsin C (open bar) for 24 h, and the levels of HETEs released into culture medium were assessed. B-E, 3Y1 cells (B), WI-38 cells (C), Colon-26 cells (D), or LLC cells (E) were stimulated with or without IL-1b in the presence or absence of 5 mM triacsin C (T.C) for 24 h, and 5,11-diHETE release was examined (left panels) (mean ± S.D. (n ¼ 3); *, p < 0.05; **, p < 0.01). Expression of COX-1, COX-2, and b-actin was assessed by immunoblotting (right panels).
ACSL4 is participated in the elimination of free AA after stimulation, and that inhibition of the ACSL4 pathway altered the production profile of bioactive lipid mediators. In addition to these bioactive lipid mediators, in the present study we found that the formation of 5,11-dihydroxyeicosatetraenoic acid (5,11-diHETE), a COX product of 5-HETE [9], was markedly enhanced by inhibition of the ACSL4 pathway. We also showed that the formation of 5,11-diHETE in rat fibroblasts was independent of the 5-lipoxygenase (5-LOX) pathway.
2. Materials and methods 2.1. Materials Mouse IL-1b was purchased from R & D Systems (Abingdon, UK). Mouse monoclonal antibody against b-actin was purchased form Sigma (St. Louis, MO, USA). Goat polyclonal antibodies against COX1, COX-2, and 5-LOX, rabbit polyclonal antibody against ACSL4, and triacsin C were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Lipofectamine RNAiMAX reagent and Opti-MEM
medium were obtained from Invitrogen (Carlsbad, CA, USA). NS398, MK-886, various HETE standards, deuterium-labeled leukotriene B4 (LTB4-d4), COX-1 (ovine), and COX-2 (human) were purchased from Cayman Chemicals (Ann Arbor, MI, USA).
2.2. Cell culture, treatment, and transfection Conditions for the maintenance of 3Y1 cells (rat embryonic fibroblasts) and WI-38 cells (human lung fibroblasts) were described previously [8]. LLC (Lewis lung carcinoma) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal calf serum (FCS), penicillin/streptomycin (100 units/ml and 100 mg/ml, respectively), and 2 mM glutamine (Invitrogen) under a humidified atmosphere containing 5% CO2. Colon-26 cells (a colon cancer cell line) were cultured in RPMI 1640 with the same supplements. The media of the cells that had attained 80% confluence in 24-well plates were replaced with 0.5 ml of DMEM supplemented with 2% (v/v) FCS. After 24 h culture, 1 ng/ml mouse IL-1b (3Y1, LLC, and colon-26 cells) or 1 ng/ml human IL-1b (WI-38 cells) was added to the cultures to assess the eicosanoid biosynthetic
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COX-2, 5-LOX, and b-actin). The separated proteins were electroblotted onto nitrocellulose membranes (Schleicher & Schuell) with a bath-type blotter. After blocking for 1 h with 5% (w/v) skimmed milk in phosphate-buffered saline (PBS) containing 0.05% Tween 20 (TPBS), the membranes were probed for 2 h with the respective antibodies (1:2000 for ACSL4 and 5-LOX, 1:5000 for COX-1 and COX-2, 1:10,000 for b-actin), followed by incubation with horseradish peroxidase-conjugated anti-mouse (1:2000 for b-actin), anti-rabbit (1:5000 for ACSL4), or anti-goat (1:5000 for COX-1 and COX-2) IgG. After washing, the membranes were visualized with Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Wellesley, MA, USA). 2.4. Sample preparation Extraction of eicosanoids from culture medium was performed as described previously with minor modification [8]. Briefly, an internal standard (50 ng of LTB4-d4) was added to the medium (500 ml). The medium was acidified by addition of 100 ml of 0.2% (v/ v) formic acid followed by 500 ml of ethyl acetate. Samples were mixed and centrifuged at 15,000 rpm for 10 min. The organic layer was retrieved and evaporated to dryness with a vacuum evaporator. Samples were resuspended in 50 ml of mobile phase A (water/ acetonitrile/formic acid (63:37:0.02, v/v/v)) and injected into an LCESI-MS/MS system. 2.5. Electrospray ionization (ESI) mass spectrometry analysis Quantification of eicosanoids, such as 5,11-diHETE, were performed using LC-ESI-MS/MS via multiple-reaction monitoring (MRM) in negative-ion mode as described previously with minor modification [8]. MRM transitions for quantification of 5,11-diHETE, 5-HETE, 8-HETE, 9-HETE, 11-HETE, 12-HETE, 15-HETE, and LTB4-d4 (internal standard) were 335/183, 319/115, 319/155, 319/151, 319/ 167, 319/179, 319/175, and 339/197, respectively. Because a 5,11diHETE standard is not available currently, the levels of this eicosanoid were expressed as fold-change compared to control levels. 2.6. Synthesis of 5,11-diHETE
Fig. 2. Effect of knockdown of ACSL4 expression on biosynthesis of 5,11-diHETE in 3Y1 cells. 3Y1 cells grown in 24-well plate were transfected wit ACSL4 or control siRNA. Two days after transfection, the expression level of ACSL4 protein was assessed by immunoblotting (A). An equal loading of samples in each lane was immunoblotted with anti-b-actin antibody. B and C, effects of siRNA against ACSL4 expression on IL-1binduced 5,11-diHETE (B) or 5-HETE (C) (mean ± S.D. (n ¼ 3); **, p < 0.01; N.S., not significant).
response. RBL-2H3 cells (a rat basophilic leukemia cell line) were maintained with DMEM with the same supplements. The media of RBL-2H3 cells that had attained 60e80% confluence in 24-well plates were stimulated with 1 mM A23187 for 30 min in serumfree DMEM. In studies using inhibitors, these cells were pretreated with various inhibitors for 1 h and then treated with IL-1b for 24 h or A23187 for 30 min. Small interfering RNA (siRNA) against ACSL4 (siRNA ID numbers SASI_Rn01_00041844; Sigma) or a negative control siRNA (Sigma) was transfected into 3Y1 cells using Lipofectamine RNAiMAX according to our protocol [8]. 2.3. Immunoblotting Cell lysates (105 cells equivalent) were subjected to SDS-PAGE using 10% (w/v) gels under reducing conditions (for ACSL4, COX-1,
Incubation of AA (1 mM) or 5(S)-HETE (1 mM) was conducted in 300 ml of 50 mM TriseHCl pH7.4 containing 5 mM tryptophan, 2 mM hematin, and ovine COX-1 (1 unit) or human COX-2 (1 unit) for 30 min at 37 C. After reactions were terminated by the addition of 0.2% (v/v) formic acid, the eicosanoids were extracted as described above. 2.7. Statistical analysis Results are expressed as the means ± standard deviation (SD). Comparisons between two groups were made with an unpaired Student's t-test, and comparisons among three or more groups were made with two-way analysis of variance and TukeyeKramer post-test. P values of <0.05 were considered statistically significant. 3. Results and discussion Each ACSL enzyme is thought to have distinct biological functions, such as free fatty acid incorporation into cellular lipids [10], control of the composition of esterified fatty acids in neutral lipids and phospholipids [11], and b-oxidation [12]. We have previously reported that inhibition of ACSL4, an AA preferred ACSL isozyme, markedly enhanced IL-1b-dependent production of several COX-2 metabolites, such as PGE2, PGD2, and PGF2a [8]. These results prompted us to ascertain whether the other AA metabolites,
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Fig. 3. Effects of COX-2 or FLAP inhibitor on biosynthesis of 5,11-diHETE. A and B, 3Y1 cells were pretreated with or without 10 ng/ml NS-398 or 5 mM MK-886 for 1 h in the presence or absence of 5 mM triacsin C and then stimulated for 24 h with or without IL-1b. After collecting culture supernatants, the release of 5,11-diHETE was assessed (mean ± S.D. (n ¼ 3); **, p < 0.01). C, RBL-2H3 cells grown in 24-well plate were stimulated with or without A23187 for 30 min in the presence or absence of 5 mM MK-886. The amounts of 5-HETE released into culture medium were assessed (mean ± S.D. (n ¼ 3); **, p < 0.01). D, Expressions of 5-LOX and b-actin in 3Y1, RBL-2H3, colon-26, LLC, or WI38 cells were assessed by immunoblotting.
including even minor lipids, are also enhanced by the inhibition of ACSL enzymes. As shown in Fig. 1A, when rat fibroblastic 3Y1 cells were treated with the ACSL inhibitor triacsin C, the release of HETEs into the culture medium was significantly enhanced compared to that from control cells. The enhanced formation of HETEs by triacsin C treatment was also observed in several other cell lines, including human fibroblastic WI-38 cells and colon-26 mouse colon cancer cells (data not shown). Among these HETEs, 11-HETE and 15-HETE are known as by-products of the reaction between COX and AA [13], and their release is attenuated by treatment with the COX-2 selective inhibitor NS-398 in these cells [8]. On the other hand, it appears that other HETEs are produced via either the AA LOX pathway or a non-enzymatic pathway. Because 5-HETE is metabolized into 5(S),11(R)-diHETE, 5(S),15(S)-diHETE, and 5(S),15(R)-diHETE by COX-2 [9,14], we next examined the effects of triacsin C on the production of these diHETEs. As shown in Fig. 1B, when 3Y1 cells were treated with triacsin C, the level of 5,11-diHETE increased 2-fold compared to that in control cells, and was further enhanced to 8-fold after treatments with triacsin C and IL-1b. As we previously reported [15], the expression of COX-2, but not COX-1, protein was increased after IL-1b treatment in these cells (Fig. 1B, right panel). The enhanced formation of 5,11-diHETE observed in cells treated with triacsin C and IL-1b seemed to be mediated by IL-1b-induced COX-2 protein. Furthermore, the biosynthesis of 5,11-diHETE was also analyzed in WI-38 human fibroblasts (Fig. 1C), Colon-26 mouse colon cancer cells (Fig. 1D), and LLC mouse Lewis lung carcinoma cells (Fig. 1E). As shown in Fig. 1CeE, both the IL-1b-dependent and
the IL-1b-independent biosyntheses of 5,11-diHETE were significantly enhanced by treatment with triacsin C in these cells. These results suggested that the enhanced production of 5,11-diHETE via inhibition of the ACSL pathway seems to occur in a wide variety of cells. In contrast to 5,11-diHETE, 5,15-diHETEs were hardly detected in the culture media of these cells (data not shown). We next examined whether knockdown of the ACSL4 protein would affect formation of 5,11-diHETE from 3Y1 cells. Treatment of 3Y1 cells with siRNAs against ACSL4 attenuated the expression of ACSL4 proteins compared to control siRNA-transfected cells (Fig. 2A). To assess the effect of ACSL4 knockdown on 5,11-diHETE formation, we analyzed the 5,11-diHETE levels after IL-1b stimulation. As shown in Fig. 2B, treatment of ACSL4-knockdown cells with IL-1b for 24 h markedly enhanced the production of 5,11diHETE, whereas the level of 5,11-diHETE in control siRNAtransfected cells was barely increased. In addition, the formation of 5-HETE, a precursor of 5,11-diHETE, was significantly enhanced by the knockdown of ACSL4, although it was not increased after IL1b stimulation (Fig. 2C). From these results we conjectured that inhibition of ACSL4 enhanced the formation of 5-HETE, and, in turn, 5-HETE may be metabolized to 5,11-diHETE by the cytokineinduced COX-2 protein. Because the above results raised the possibility that both the 5LOX and COX-2 pathway participated in the formation of 5,11diHETE in IL-1b-stimulated ACSL4-knockdown cells, we next examined the effects of COX-2 and FLAP inhibitors. As shown in Fig. 3A, when triacsin C-pretreated 3Y1 cells were stimulated with IL-1b in the presence of the COX-2 selective inhibitor NS-398, the
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HETE, a by-product of COX reaction, were nearly equivalent (Fig. 4B). Thus, the selectivity of COX enzymes against 5-HETE explains why the release of 5,11-diHETE is enhanced under the IL-1bstimulated condition (Fig. 1), even though the level of 5-HETE is comparable between these conditions (Fig. 2). From these results, we propose that the induction of COX-2 protein rather than 5-HETE synthesis is the rate-limiting step for the production of 5,11diHETE. In conclusion, our findings suggest that inhibition of ACSL4 activity perturbs the production of various eicosanoids [8], including 5,11-diHETE (this study). These events appear to be involved in several pathophysiological conditions that are observed in ACSL4 dysfunction, including X-linked mental retardation [16]. Further studies are needed to identify the actual role of the ACSL4 enzyme. Conflict of interest The authors declare no conflict of interest. Acknowledgments We thank the members of our lab for their helpful discussions and comments. This work was supported in part by Grantsein Aid for Scientific Research (B) (25293033) from the Japan Society for the Promotion of Science, and by a Grant-in-Aid for Scientific Research on Innovative Areas (25116720), and a grant for a Private University High Technology Research Center Project from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. References Fig. 4. In vitro synthesis of 5,11-diHETE by COX isozymes. Ovine COX-1 or human COX-2 were incubated with 1 mM AA or 1 mM 5(S)-HETE for 30 min and then the products were extracted as described “material and methods”. Data represent as means ± S.D.
production of 5,11-diHETE was significantly attenuated compared to that in control cells. By contrast, treatment of these cells with the FLAP inhibitor MK-886 (5 mM) failed to inhibit the formation of 5,11-diHETE (Fig. 3B). The release of 5-HETE from triacsin C-treated 3Y1 cells was unaffected by 5 mM MK-886 (data not shown), whereas its release from Ca2þ ionophore A23187-stimulated RBL2H3 cells was abolished by 5 mM MK-886 treatment (Fig. 3C). These results suggested that the COX-2 pathway does indeed participate in the biosynthesis of 5,11-diHETE in 3Y1 cells, but the 5-LOX pathway does not. Furthermore, the immunoblot analysis revealed that 5-LOX was expressed in RBL-2H3 cells (the positive control), whereas the 5-LOX signal was undetectable in 3Y1 cells (Fig. 3D). In addition, we failed to detect expression of the 5-LOX protein in WI-38 cells, colon-26 cells, or LLC cells (Fig. 3D), all of which can produce 5,11-diHETE (Fig. 1CeE). Thus, we concluded that the 5-LOX pathway is not involved in the formation of 5,11diHETE in 3Y1 cells. As mentioned above, the release of 5,11-diHETE from triacsin Ctreated 3Y1 cells was enhanced by IL-1b treatment, whereas that of 5-HETE was unaltered by cytokine stimulation. Thus, we speculated that the region downstream of 5-HETE generation, perhaps including the COX-2 pathway, is the rate-limiting step of 5,11diHETE biosynthesis. As shown in Fig. 4A, when recombinant human COX-2 or purified ovine COX-1 enzyme was incubated with 5(S)-HETE, 5,11-diHETE was formed. The level of 5,11-diHETE produced by COX-2 was about 4-fold greater than that of COX-1. The efficacies of COX-1 and COX-2 on the conversion of AA into 11-
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