Leukotriene B(4) stimulates Rac-ERK cascade to generate reactive oxygen species that mediates chemotaxis.

Leukotriene B(4) is a potent chemoattractant known to be involved mainly in inflammation, immune responses, and host defense against infection, although the exact signaling mechanisms by which it exerts its effects are not well understood. Here we show that exogenous leukotriene B(4) induces reactive oxygen species (ROS) generation via a Rac-dependent pathway, and that stable expression of Rac(N17), a dominant negative Rac1 mutant, completely blocks leukotriene B(4)-induced ROS generation. In addition, leukotriene B(4)-induced ROS generation is selectively blocked by inhibition of ERK or cytosolic phospholipase A(2), but not p38 kinase, which is indicative of its dependence on ERK activation and synthesis of arachidonic acid. Consistent with those findings, leukotriene B(4) Rac-dependently stimulates ERK and cytosolic phospholipase A(2) activity, and transient transfection with plasmid expressing Rac(V12), a constitutively activated Rac1 mutant, also dose-dependently stimulates ERK activity. Our findings suggest that ERK and cytosolic phospholipase A(2) are situated downstream of Rac, and we conclude that Rac, ERK, and cytosolic phospholipase A(2) all play pivotal roles in mediating the ROS generation that appears to be a prerequisite for leukotriene B(4)-induced chemotaxis and cell proliferation.

LTs 1 are potent biological mediators of inflammation generated from arachidonic acid via the 5-LO pathway (1,2). Among them, LTB 4 is one of the most potent chemoattractants known, acting mainly on neutrophils and eosinophils, but also on mast cells and endothelial cells (3)(4)(5). LTB 4 stimulates a number of cellular functions in addition to chemotaxis, including release of lysosomal enzymes and production of ROS (6 -8); it also promotes cell adhesion to vascular endothelial cells and transmigration, which amplifies inflammatory responses. Although LTB 4 -induced leukocyte recruitment is thought to play a protective role in the host defense against various pathogens, it is also involved in the pathogenesis of such inflammatory diseases as bronchial asthma (9,10), inflammatory bowel diseases (11,12), and psoriasis (13,14).
Despite many reports on the cellular functions of LTB 4 , the exact signaling pathway along which its biological activities are transduced remains largely unknown. It is known, however, that LTB 4 acts via two G protein-coupled receptors, BLT1 and BLT2 (15)(16)(17)(18)(19)(20). The former is a high affinity LTB 4 receptor expressed mainly in polymorphonuclear leukocytes, whereas the latter is a ubiquitous, low affinity receptor whose expression is highest in spleen (17,18). The details of the cellular functions of BLT1 and BLT2 are still largely unknown. Recently, however, LTB 4 -induced chemotaxis was shown to be completely inhibited in cells pretreated with PTX (100 ng/ml), indicating the participation of a PTX-sensitive G protein in LTB 4 signaling to chemotaxis (16). LTB 4 also elicits increases in intracellular free Ca 2ϩ and inositol 1,4,5-triphosphate, but these are apparently not involved in the chemotactic response by LTB 4 (15). In addition to the BLTs, LTB 4 can also bind to and activate the intranuclear transcription factor peroxisome proliferator-activated receptor-␣, resulting in the activation of genes that terminate inflammatory processes (21,22).
We previously observed that LTB 4 plays a role in mediating TNF-␣-induced ROS generation in Rat-2 fibroblasts (23). LTB 4 likewise induces ROS generation in neutrophils, eosinophils, and other fibroblasts (6 -8, 23, 24), but the signaling pathway via which this effect is exerted as well as the precise cellular function of the increased ROS levels remains largely unknown. Although NADPH oxidase was proposed to play a role in the generation of ROS in response to LTB 4 in eosinophils, the detailed signaling mechanism is still unclear (7). Previously, we and others have shown that Rac, a member of the Rho family GTPases, plays a crucial role in ROS generation in fibroblasts (23)(24)(25). Additionally, the generation of ROS by Rac was shown to be mediated mainly by cPLA 2 -linked cascade (24), suggesting a possible role of cPLA 2 as a downstream mediator of Rac in the signaling to ROS generation. Therefore, in an effort to broaden our understanding of LTB 4 -induced signaling, we studied the pathway via which exogenous LTB 4 induces the generation of ROS in Rat-2 fibroblasts. Our results suggest that Rac, ERK, and cPLA 2 all play pivotal roles in the LTB 4 -induced generation of ROS required for the chemotactic activity and proliferation elicited by exogenous LTB 4 .

EXPERIMENTAL PROCEDURES
Chemicals and Plasmids-2Ј,7Ј-Dichlorofluorescein diacetate was purchased from Molecular Probes. MK-886, genistein, herbimycin, and AACOCF 3 were from BIOMOL. LTB 4 and cysLTs were from Cayman Chemical Co. LPA, LY294002, wortmannin, DPI, and NAC were from Sigma. FBS, DMEM, phenol red-free DMEM, gentamicin, and nonessential amino acids were from Invitrogen. ZK 158252, a specific BLT antagonist, was kindly provided by Dr. Claudia Giesen (Experimental Dermatology, Schering AG, Berlin, Germany). All other chemicals were from standard sources and were molecular biology grade or higher. The pEXV and pEXV-Rac V12 plasmids were gifts from Dr. A. Hall (University College, London, United Kingdom).
Cell Culture and DNA Transfection-Rat-2 fibroblasts were obtained from the American Type Culture Collection (ATCC, CRL 1764), and the cells were grown in DMEM supplemented with 0.1 mM nonessential amino acids, 10% FBS, penicillin (50 units/ml), and streptomycin (50 g/ml) at 37°C under a humidified 95%, 5% (v/v) mixture of air and CO 2 . Stable Rat2-Rac N17 clones expressing Rac N17 , a dominant negative Rac1 mutant, were described previously (23,26). Transient transfection was carried out by plating ϳ5 ϫ 10 5 cells in 100-mm dishes for 24 h and then adding calcium phosphate:DNA precipitates prepared with 20 g of DNA/dish. To control for variations in cell number and transfection efficiency, all clones were cotransfected with 1 g of pCMV-␤GAL, a eukaryotic expression vector containing the Escherichia coli ␤-galactosidase (lacZ) structural gene under the transcriptional control of the cytomegalovirus promoter. The quantity of DNA used in each transfection was held constant (20 g) by adding sonicated calf thymus DNA (Sigma). To measure ERK kinase activity with PathDetect trans-reporting system (Stratagene catalog no. 219005), Elk1 fused to trans-activator plasmid was co-transfected with pFR-Luc reporter plasmid according to the manufacturer's protocol. After incubating with the calcium phosphate:DNA precipitates for 6 h, the cells were rinsed twice with PBS before incubating them in DMEM supplemented with 0.5% FBS for additional 24 h. Each dish of cells was then rinsed twice with PBS and lysed in 0.1 ml of lysis solution (0.2 M Tris, pH 7.6, plus 0.1% Triton X-100), after which the supernatants were assayed for luciferase activity as well as protein concentration and ␤-galactosidase activity.
Measurement of Intracellular H 2 O 2 -Intracellular H 2 O 2 was measured as a function of DCF fluorescence using the procedures of Ohba et al. (27). Briefly, cells were grown on coverslips for 2 days and then serum-starved in DMEM supplemented with 0.5% (v/v) FBS for an additional 2 days. They were then stabilized in serum-free DMEM without phenol red for at least 30 min before exposure to agonists (LTB 4 or cysLTs) for the indicated times. When assessing the effects of inhibitors, cells were pretreated with the respective inhibitor for 30 min. To measure intracellular H 2 O 2 , cells were then incubated for 10 min with the H 2 O 2 -sensitive fluorophore 2Ј,7Ј-dichlorofluorescein diacetate (5 g/ ml), which when taken up fluorescently labels intracellular H 2 O 2 with DCF. The cells were then immediately observed under a laser-scanning confocal microscope (Carl Zeiss LSM 410); DCF fluorescence was excited at 488 nm using an argon laser, and the evoked emission was filtered with a 515-nm long pass filter. DCF fluorescence was measured in 30 randomly selected cells.
Rac1 Activity Assays-Rac1 activation was measured using a GST-(PAK)-PBD fusion protein that binds GTP-bound, activated Rac1 as described previously (28). Briefly, the fusion protein was expressed in E. coli BL21 transformed with pGEX-4T3 plasmid by isopropyl-1-thio-␤-D-galactopyranoside induction and then purified by column chromatography using glutathione-Sepharose-4B. Rat-2 cells were serum-starved for 36 h prior to stimulation with LTB 4 for the indicated time periods, after which cell lysates were prepared in lysis buffer (50 mM HEPES, pH 7.4, 10 mM NaF, 75 mM NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na 2 VO 3 ), centrifuged for 20 s at 12,000 ϫ g, and the supernatant was incubated on ice for 3 min with the GST-(PAK)-PBD fusion protein, which had been freshly coupled to glutathione-agarose beads. Proteins complexed to the beads were recovered by centrifugation, washed twice with the lysis buffer, and resuspended in sample buffer. The proteins were resolved by 15% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were then probed with anti-Rac1 antibody (1:2000 dilution) and detected using horseradish peroxidase-conjugated donkey anti-rabbit antibody and an enhanced chemiluminescence detection kit (ECL, Amersham Biosciences, Inc.).
SDS-PAGE and Immunoblot Analysis-Protein samples were heated at 95°C for 5 min and then analyzed by SDS-PAGE performed on 8% (for cPLA 2 ) or 10% (for ERKs or p38) acrylamide gels, followed by transfer to polyvinylidene difluoride membranes using a Novex wet transfer unit (for 2 h at 100 V). The membranes were blocked for 1 h with Tris-buffered saline containing 0.05% (v/v) Tween 20 plus 5% (w/v) nonfat dry milk, incubated first for 2 h with the primary antibody (1:1000 dilutions for cPLA 2 ; 1:2000 dilutions for phospho-ERKs) in Tris-buffered saline containing 0.05% (v/v) Tween 20 plus 3% (w/v) BSA, and then for 1 h with horseradish peroxidase-conjugated secondary antibody prior to development using an ECL kit. Bands corresponding to cPLA 2 , p38, and ERKs on XAR-5 film (Eastman Kodak Co.) were measured by densitometry.
Translocation of cPLA 2 -To visualize the localization of endogenous cPLA 2 , cells were plated on coverslips and grown for 24 h in DMEM containing 10% FBS. They were then starved in serum-free DMEM for 16 h before exposure to an agonist. Thereafter the cells were washed with cold PBS, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked in 1% BSA (solubilized in PBS), and labeled with mouse anti-cPLA 2 antibody (1:150). The immunolabeled cells were then washed with PBS and labeled with a tetramethylrhodamine B isothiocyanate-conjugated, anti-mouse secondary antibody (1:200). After washing again with cold PBS, the cells were mounted on a slide glass for observation under a fluorescence microscope.
Subcellular Fractionation of Cell Lysates-Rat-2 cells were serumstarved in DMEM containing 0.5% FBS for 24 h and then exposed to the appropriate agonist for the indicated times. The medium was then removed, and the cells were washed twice with ice-cold PBS, scraped, harvested by microcentrifugation, and resuspended in 0.2 ml of buffer A (137 mM NaCl, 8.1 mM Na 2 HPO 4 , 2.7 mM KCl, 1.5 mM KH 2 PO 4 , 2.5 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, pH 7.5). The resuspended cells were lysed by sonicating them twice for 15 s each or by passing them 20 times through a 21.1-gauge needle on ice. The lysates were centrifuged at 100,000 ϫ g for 1 h to prepare cytosolic and total particulate fractions. The particulate fraction, containing the membrane fraction, was washed twice and resuspended in 50 l of buffer A. The supernatant fraction was precipitated with five volumes of acetone, incubated on ice for 5 min, centrifuged, and the pellet was resuspended in buffer A. Protein concentrations were routinely determined using the Bradford procedure with Bio-Rad dye reagent using BSA as a standard.
Chemotaxis Assay-The chemotactic motility of Rat-2 or Rat2-Rac N17 cells was assayed using Transwell chambers with 6.5-mm diameter polycarbonate filters (8-m pore size). Briefly, the lower surfaces of the filters were coated with 10 g/ml gelatin (Collaborative Biomedicals) in HEPES-buffered RPMI 1640 medium for 2 h at 37°C. Dry coated filters containing various amounts of LTB 4 were placed in the lower wells of the Transwell chambers, after which 100 l of Rat-2 or Rat2-Rac N17 cells in DMEM containing 1% FBS were loaded into the top wells, yielding a final concentration of 1ϫ10 6 cells/ml. If necessary, inhibitors were applied to the cells for 20 min at room temperature before seeding. After incubation at 37°C in 5% CO 2 for 3 h, the filters were disassembled, and the upper surface of each filter was scraped free of cells by wiping it with a cotton swab. Cells that had migrated to the underside of the filter were fixed for 1 min with methanol and stained for 20 min with hematoxylin and eosin. Chemotaxis was quantified by counting the cells on the lower side of the filter under an optical microscope (magnification, ϫ200). Ten fields were counted in each assay; each sample was assayed in duplicate, and the assays were repeated twice.
Cell Growth Assay-⌻ assess cell growth, Rat-2 or Rat2-Rac N17 cells were plated onto 60-mm plates (10 5 cells/plate) in DMEM containing 10% FBS. The next day, the medium was replaced with serum-free medium or serum-free medium containing LTB 4 or LPA. The number of viable cells was then counted after an additional 48 h.
Data Analysis and Statistics-Data are expressed as means Ϯ S.D. or as percentages Ϯ S.D. of control. Statistical comparisons between groups were made using Student's t tests. Values of p Ͻ 0.01 were considered significant.

LTB 4 Induces ROS Generation in Rat-2
Fibroblasts-To assess whether LTB 4 induces ROS generation, Rat-2 fibroblasts were serum-starved for 48 h and then exposed to exogenous LTB 4 for 3 min. The resultant ROS generation was monitored as a function of H 2 O 2 -sensitive DCF fluorescence. As shown in Fig. 1A, 0.3 M LTB 4 elicited a significant (ϳ2.5-fold) increase in the levels of ROS; no further increases were seen at LTB 4 concentrations up to 1 M (data not shown). This effect was completely inhibited by ZK158252, a specific BLT antagonist (17,29), whereas ROS generated in response to exogenous cysLTs (LTC 4 /D 4 /E 4 mixture) were unaffected by ZK158252, confirming that LTB 4 generates ROS via a specific BLT-linked pathway (Fig. 1B). Likewise, the ROS response elicited by TNF-␣ (ϳ2.6-fold increase) was completely abolished by ZK158252 (Fig. 1B), which is consistent with the previous report suggesting the mediatory role of BLTR in TNF-␣ signaling to ROS generation (23).
It has been reported that BLTs are closely coupled to a PTX-sensitive G protein (15,16). Consistent with that idea, pretreatment with PTX (100 ng/ml) also completely blocked LTB 4 -induced ROS generation (Fig. 2). By contrast, LTB 4 -induced ROS generation was little affected by EGTA, U73122, or calphostin C, suggesting that Ca 2ϩ mobilization, phospholipase C, and protein kinase C are not involved.
Essential Roles of Rac and cPLA 2 in the LTB 4 Signaling to ROS Generation-Previously, we and others have reported that Rac1 plays a crucial role in ROS generation in fibroblasts (23)(24)(25). To examine whether Rac1 is involved in LTB 4 signaling to ROS generation, we compared the effects of LTB 4 in Rat-2 and Rat2-Rac N17 cells, which express a dominant negative Rac1 mutant (26). Although substantial ROS generation (a ϳ2.3-fold increase over control) was observed within 3 min of exposing Rat-2 cells to 0.3 M LTB 4 , little effect was observed in Rat2-Rac N17 cells under the same conditions (Fig. 3, A and B). This result prompted us to test directly the extent to which exposure to LTB 4 alters cellular Rac1 activity; we found that indeed LTB 4 induced significant, time-dependent increase in Rac1 activity, consistent with the proposed mediatory role of LTB 4 signaling (Fig. 3C).
We recently reported that cPLA 2 serves as a key downstream mediator of Rac in Rat-2 cells (23,24,30,31). We therefore assessed the effects of AACOCF 3 , a specific cPLA 2 inhibitor, on LTB 4 -induced ROS generation and found that pretreatment with 10 M AACOCF 3 almost completely blocked LTB 4 -induced ROS generation (Ͼ90% inhibition) (Fig. 4A). By contrast, MK-886, a specific 5-LO inhibitor (Fig. 4A), and indomethacin, a nonspecific COX inhibitor, had little or no inhibitory effect (data not shown). It thus appears that activation of cPLA 2 , without subsequent metabolism of arachidonic acid by 5-LO or COX, is required for LTB 4 -induced ROS generation.
The role of cPLA 2 in LTB 4 signaling was further confirmed by our observations that LTB 4 evoked translocation of cPLA 2 to the membrane compartment (e.g. nuclear envelope area) in Rat-2, but not Rat2-Rac N17 cells (Fig. 4B), as well as time-dependent increases (up to 10 min) in levels of cPLA 2 in the particulate fraction of Rat-2 cell lysates (Fig. 4C). Interestingly, we also observed that, with more prolonged incubations (e.g. 30 min after LTB 4 treatment), the level of cPLA 2 in the particulate fraction was significantly diminished, whereas that in the soluble fraction increased (Fig. 4C).

LTB 4 -induced ROS Generation
Requires ERK-To investigate the possible involvement of MAP kinases in LTB 4 signaling to ROS generation, we tested the effects of 10 M PD098059, a specific MEK inhibitor, and 10 M SB203580, a specific p38 kinase inhibitor, on the production of intracellular ROS. We found that, although pretreatment with the former inhibited LTB 4 -induced ROS generation, pretreatment with the latter did not (Fig. 5A). This finding suggested the presence of ERK in the LTB 4 signaling pathway, which prompted us to test whether ERK is indeed activated in cells exposed to LTB 4 . We found that exposing serum-starved Rat-2 cells to 0.3 M LTB 4 for 5 min significantly elevated levels of the activated (phosphorylated) ERK form in manner that was entirely dependent on Rac activity, as the effect was absent in Rat2-Rac N17 cells (Fig. 5B). Addition of epidermal growth factor, by contrast, evoked virtually the same level of ERK activation in both Rat-2 and Rat2-Rac N17 cells. ERK activation was also dose-dependently elevated by transient transfection with plasmid expressing Rac V12 , a constitutively active form of Rac1 (Fig. 5C). Similarly, Rac V12 elevated Elk-luciferase activity in a dose-dependent manner (Fig. 5D), confirming the ERK activation by Rac1.
PI 3-Kinase Activity Is Required for the LTB 4 Signaling to ROS-To investigate further mediators involved in the LTB 4induced ROS generation, we assessed the effects of inhibitors of PI 3-kinase on production of intracellular ROS. As shown in Fig. 6A, LTB 4 -evoked ROS generation was completely blocked

FIG. 1. Exogenous LTB 4 induces ROS generation in Rat-2 fibroblasts.
A and B, Rat-2 cells were serum-starved for 2 days and then exposed to 0.3 M LTB 4 (3 min), 1 M cysLTs (3 min) or 20 ng/ml TNF-␣ (10 min). To assess the effects of BLT inhibition, cells were pretreated with ZK158252 (3 M) for 30 min before the addition of LTB 4 . DCF fluorescence, reflecting of the relative levels of ROS (arbitrary units), was imaged using a confocal laser scanning microscope (A) and then quantified as described under "Experimental Procedures" (B). Data are expressed as means Ϯ S.D. (n ϭ 30 cells) from three independent experiments. Statistical significance of ROS measurements was assessed with unpaired t tests (p Ͻ 0.01). by specific PI 3-kinase inhibitor LY294002 (10 M) or wortmannin (50 nM). Besides PI 3-kinase, we observed that tyrosine kinase inhibitors, herbimycin and genistein, also dramatically diminished LTB 4 -evoked ROS generation. Moreover, when we tested whether LTB 4 -induced ERK activation is affected by PI 3-kinase inhibition, we found the effect of LTB 4 on ERK to be highly dependent on PI 3-kinase and tyrosine kinase(s) activities, as ERK activation was clearly diminished by pretreatment with LY294002, wortmannin (Fig. 6B), herbimycin, or genistein (data not shown). Administration of 1 M LTB 4 APA, a specific BLT antagonist, confirmed that LTB 4 elicited ERK activation via BLT (Fig. 6B). Together, our results strongly suggest the mediatory roles of PI 3-kinase and tyrosine kinase(s) in the LTB 4 signaling pathway to ROS production, acting upstream of ERK. This result is consistent with our previous report (32), suggesting the role of PI 3-kinase acting upstream of Rac1 in Rat-2 fibroblasts. Additionally, PI 3-kinase or tyrosine kinase activities were shown to be essential for mediating the chemotaxis in response to LTB 4 (8,16).
ROS Generation Is Essential for the Chemotaxis and Proliferation by LTB 4 -As LTB 4 is known to be a potent chemotactic agent, we examined whether ROS generation could mediate the chemotaxis evoked by LTB 4 . As shown in Fig. 7 (A and B), pretreatment with DPI (2 M), an inhibitor of NADPH oxidaselike flavoproteins, or NAC (2 mM), a free radical scavenger, diminished chemotaxis elicited by LTB 4 , suggesting a role for ROS generation in mediating chemotactic activity. Rac N17 expression also inhibited chemotaxis induced by LTB 4 (Fig. 7C), supporting the involvement of the Rac-ROS cascade.
The role of ROS generation in LTB 4 -induced cell proliferation was similarly tested. As shown in Fig. 8A, LTB 4 elicited significant cell proliferation, which was inhibited by pretreatment with 2 mM NAC or 2 M DPI, and likewise LTB 4 -induced cell proliferation was not detected in Rat2-Rac N17 , although LPA-induced cell proliferation remained intact (Fig. 8B). Together, these results suggest a potential, mediatory role of Rac-ROS cascade for the LTB 4 -evoked chemotaxis and cell proliferation. DISCUSSION The present study demonstrates the central role played by a Rac-linked cascade in LTB 4 -signaling to ROS generation in Rat-2 fibroblasts. Supporting that conclusion are the observations that exposure to LTB 4 stimulates Rac activity and that stable expression of Rac N17 dramatically inhibits LTB 4 -evoked ROS generation. In addition, our findings suggest that ERK and cPLA 2 are situated downstream of Rac1, mediating LTB 4 signaling to ROS generation.
Levels of ERK and cPLA 2 activation were both Rac-dependently increased following treatment with LTB 4 , as demonstrated by ERK phosphorylation and translocation of cPLA 2 to the nuclear envelope (Figs. 4B and 5B). It was previously   FIG. 3. LTB 4 stimulates ROS generation via a Rac-dependent pathway. A, serum starved Rat-2 or Rat2-Rac N17 cells were exposed to LTB 4 for 3 min, after which DCF fluorescence was imaged using a confocal laser scanning microscope. B, DCF fluorescence levels reflecting of the relative levels of ROS (arbitrary units) quantified as described under "Experimental Procedures". Data are expressed as means Ϯ S.D. (n ϭ 30 cells) from three independent experiments. Statistical significance of ROS measurements was assessed with unpaired t tests (p Ͻ 0.01). C, Rat-2 cells were serum-starved for 36 h prior to exposure to 0.3 M LTB 4 for the indicated times. Cell lysates were incubated with GST-PAK-PBD coupled to glutathione-agarose beads. Bound Rac-GTPase was eluted, resolved by 15% SDS-PAGE, and transferred to a polyvinylidene difluoride membrane, which was then probed with an anti-Rac1 antibody. The results shown are representative of at least three independent experiments.

FIG. 4. Rac-mediated cPLA 2 activation is critical for LTB 4 signaling to ROS generation.
A, serum-starved Rat-2 cells were pretreated with 10 M AACOCF 3 or 50 nM MK886 and then exposed to 0.3 M LTB 4 . DCF fluorescence was quantified as described under "Experimental Procedures." Data are expressed as means Ϯ S.D. (n ϭ 30 cells) from three independent experiments. Statistical significance of ROS measurements was assessed with unpaired t tests (p Ͻ 0.01). B, images of cells exposed to 0.3 M LTB 4 for 10 min and then labeled first with anti-cPLA 2 antibody and then with a tetramethylrhodamine B isothiocyanate-conjugated anti-mouse secondary antibody. C, Rat-2 cells were exposed to 0.3 M LTB 4 for the indicated times (0, 5, 10, and 30 min) and lysed, after which the cytosolic and particulate fractions were prepared as described under "Experimental Procedures." Cell lysates were analyzed for the level of cPLA 2 by Western blotting of equal amounts of cellular protein. The results shown are representative of at least three independent experiments. The relative intensity was measured and expressed as percentages Ϯ S.D. of control from three independent experiments.
shown that cPLA 2 translocation from cytosol to particulate fraction represents an activation of cPLA 2 (33,34). Furthermore, expression of constitutively active Rac V12 also elicited dose-dependent activation of ERK (Fig. 5, C and D). Consistent with our findings are earlier reports of increased ERK activity in response to LTB 4 and of ERK's involvement in the LTB 4 signaling to eosinophil activation (7,8,35,36). cPLA 2 mediates a variety of cellular activities (e.g. stimulation of c-fos serum response element or c-Jun amino-terminal kinase, among others) induced by Rac activation, suggesting stimulation of cPLA 2 by Rac1 (23,24,30,31). Most germane to the present study, Rac-dependent ROS generation was shown to be mediated largely by a cPLA 2 -linked cascade (24). The downstream signaling pathway via which cPLA 2 activation leads to ROS generation is completely unknown, however. Nevertheless, because no detectable inhibition of LTB 4 -induced ROS generation was observed with treatment with MK-886 or indomethacin, we predict that eicosanoid synthesis by 5-LO or COX is likely not involved.
Our results clearly demonstrate that EGTA has no inhibitory The cell lysates were probed for levels of phospho-ERK and total ERK. The results shown are representative of at least three independent experiments. C, Rat-2 cells were transiently transfected with 0, 1, 2, or 5 g of a Rac V12 expression vector, after which the transfectants were serum-starved for 24 h and then lysed. The lysates were probed for levels of phospho-ERK and total ERK. The results shown are representative of at least three independent experiments. D, ERK kinase activity was measured using Elk-luciferase trans-reporter system as described under "Experimental Procedures". Rat-2 cells were transiently co-transfected with 0, 1, 3, or 5 g of a Rac V12 expression vector, after which the transfectants were serum-starved for 24 h and then lysed. The supernatants were assayed for Elk-luciferase activity. Data are expressed as means Ϯ S.D. of control from three independent experiments. effect on LTB 4 -induced ROS generation (Fig. 2). Although LTB 4 evokes rapid, transient increases in Ca 2ϩ , such Ca 2ϩ mobilization is not involved in LTB 4 signaling to ROS generation, as demonstrated by the failure of EGTA or SK&F 96365, a putative inhibitor of receptor-operated Ca 2ϩ entry, to affect LTB 4 -induced ROS generation (7,37,38). Furthermore, U73122, a specific inhibitor of phospholipase C, and calphostin C, a protein kinase C inhibitor, also had no effect, thereby excluding these enzymes from the LTB 4 signaling cascade leading to ROS generation. On the other hand, PTX completely inhibited ROS generation in response to LTB 4 , which is consistent with LTB 4 mediating chemotaxis via G i protein-coupled receptors (15)(16)(17)(18)(19). LTB 4 -induced ROS generation was completely abolished by ZK 158252, a potent BLT inhibitor (17). Several groups have cloned and characterized two distinct LTB 4 receptors, BLT1 and BLT2 (15)(16)(17)(18)(19). Although we do not yet precisely know which BLT receptor mediates ROS generation in Rat-2 fibroblasts, we suspect that BLT2 occupation is possibly involved. This is because, whereas BLT1 expression has not been detected in Rat-2 fibroblasts, expression of BLT2 was well detected in Rat-2 fibroblasts as well as other cell lines, including A549 epithelial cells using RT-PCR analysis with each BLT-specific primers (39) (data not shown). In addition, the pharmacological properties of BLT2 are distinct from those of BLT1 (17,40). Throughout this experiment, LTB 4 showed biological activity at concentrations of 0.3-1 M, which falls within the reported optimal range for BLT2 (e.g. ϳ0.1-1 M) and is 2 orders of magnitude higher than the optimal range for BLT1.
In any event, consistent with the proposed action of BLT as a receptor for LTB 4 in Rat-2 fibroblasts, we observed that ROS generation by LTB 4 is completely abolished by pretreatment with ZK 158252, a potent inhibitor antagonizing both BLT1 and BLT2 (17). The action of ZK 158252 was quite specific to the LTB 4 receptor, as ROS generation elicited by an LTC 4 / D 4 /E 4 mixture was not affected by this compound (Fig. 1). In support of our finding, increasing evidence points to the existence of a signaling link between LTB 4 and ROS generation. For example, Li et al. (6) and Lindsay et al. (7,8) showed that LTB 4 activates ROS generation in neutrophils and eosinophils (6 -8), respectively. Further, NADPH oxidase appears to be involved in LTB 4 signaling to H 2 O 2 generation in guinea pig eosinophils (7), and, although details of the mechanism are still unclear, cPLA 2 appears to play an essential role in the activation of NADPH oxidase in human phagocyte myeloid cells (41).
The generation of ROS in response to LTB 4 does not appear to cause cytotoxicity. Usually, production of ROS in non-phagocytes is only 1-2% of that seen in phagocytes, which produce large amounts of O 2 Ϫ as part of the body's defense against disease processes (42). Instead, our findings suggest that, at least in fibroblasts, ROS generation elicited by LTB 4 functions in the regulation of intracellular signaling cascades leading to chemotaxis and cell proliferation. Future studies aimed at clar- FIG. 7. Essential role of ROS generation for chemotaxis by LTB 4 . A, LTB 4 -induced chemotactic motility was determined in the presence of 2 M DPI or 2 mM NAC, an NADPH oxidase inhibitor and a free radical scavenger, respectively. Cells were preincubated for 20 min with or without these compounds prior to exposure to LTB 4 for 3 h. Migrating cells were fixed and stained with hematoxylin/eosin. The results shown are representative of two independent experiments. B, Cell migration, summarized and expressed as percentages Ϯ S.D. of control. Each sample was assayed in duplicate, and the assays were repeated twice (p Ͻ 0.01). C, LTB 4 -induced chemotactic motility was analyzed in Rat-2 and Rat2-Rac N17 cells as described above. ifying the linkage between cPLA 2 and ROS will likely be pivotal to our complete understanding of LTB 4 -evoked intracellular signaling and inflammatory responses.