JBC Origene Your Gene Company

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M104766200 on December 27, 2001

J. Biol. Chem., Vol. 277, Issue 10, 8572-8578, March 8, 2002
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
277/10/8572    most recent
M104766200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Woo, C.-H.
Right arrow Articles by Kim, J.-H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Woo, C.-H.
Right arrow Articles by Kim, J.-H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Leukotriene B4 Stimulates Rac-ERK Cascade to Generate Reactive Oxygen Species That Mediates Chemotaxis*

Chang-Hoon WooDagger , Hye-Jin YouDagger §, Sung-Hoon ChoDagger §, Young-Woo EomDagger , Jang-Soo Chun§, Yung-Joon Yoo§, and Jae-Hong KimDagger

From the Dagger  Graduate School of Biotechnology, Korea University, 5-1 Anam-dong, Seoul 136-701 and the § Department of Life Science, Kwangju Institute of Science and Technology, 1 Oryong-dong, Kwangju 500-712, Korea

Received for publication, May 24, 2001, and in revised form, November 26, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Leukotriene B4 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 B4 induces reactive oxygen species (ROS) generation via a Rac-dependent pathway, and that stable expression of RacN17, a dominant negative Rac1 mutant, completely blocks leukotriene B4-induced ROS generation. In addition, leukotriene B4-induced ROS generation is selectively blocked by inhibition of ERK or cytosolic phospholipase A2, but not p38 kinase, which is indicative of its dependence on ERK activation and synthesis of arachidonic acid. Consistent with those findings, leukotriene B4 Rac-dependently stimulates ERK and cytosolic phospholipase A2 activity, and transient transfection with plasmid expressing RacV12, a constitutively activated Rac1 mutant, also dose-dependently stimulates ERK activity. Our findings suggest that ERK and cytosolic phospholipase A2 are situated downstream of Rac, and we conclude that Rac, ERK, and cytosolic phospholipase A2 all play pivotal roles in mediating the ROS generation that appears to be a prerequisite for leukotriene B4-induced chemotaxis and cell proliferation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

LTs1 are potent biological mediators of inflammation generated from arachidonic acid via the 5-LO pathway (1, 2). Among them, LTB4 is one of the most potent chemoattractants known, acting mainly on neutrophils and eosinophils, but also on mast cells and endothelial cells (3-5). LTB4 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 LTB4-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 LTB4, the exact signaling pathway along which its biological activities are transduced remains largely unknown. It is known, however, that LTB4 acts via two G protein-coupled receptors, BLT1 and BLT2 (15-20). The former is a high affinity LTB4 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, LTB4-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 LTB4 signaling to chemotaxis (16). LTB4 also elicits increases in intracellular free Ca2+ and inositol 1,4,5-triphosphate, but these are apparently not involved in the chemotactic response by LTB4 (15). In addition to the BLTs, LTB4 can also bind to and activate the intranuclear transcription factor peroxisome proliferator-activated receptor-alpha , resulting in the activation of genes that terminate inflammatory processes (21, 22).

We previously observed that LTB4 plays a role in mediating TNF-alpha -induced ROS generation in Rat-2 fibroblasts (23). LTB4 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 LTB4 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 (23-25). Additionally, the generation of ROS by Rac was shown to be mediated mainly by cPLA2-linked cascade (24), suggesting a possible role of cPLA2 as a downstream mediator of Rac in the signaling to ROS generation. Therefore, in an effort to broaden our understanding of LTB4-induced signaling, we studied the pathway via which exogenous LTB4 induces the generation of ROS in Rat-2 fibroblasts. Our results suggest that Rac, ERK, and cPLA2 all play pivotal roles in the LTB4-induced generation of ROS required for the chemotactic activity and proliferation elicited by exogenous LTB4.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals and Plasmids-- 2',7'-Dichlorofluorescein diacetate was purchased from Molecular Probes. MK-886, genistein, herbimycin, and AACOCF3 were from BIOMOL. LTB4 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-RacV12 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 µg/ml) at 37 °C under a humidified 95%, 5% (v/v) mixture of air and CO2. Stable Rat2-RacN17 clones expressing RacN17, a dominant negative Rac1 mutant, were described previously (23, 26). Transient transfection was carried out by plating ~5 × 105 cells in 100-mm dishes for 24 h and then adding calcium phosphate:DNA precipitates prepared with 20 µg of DNA/dish. To control for variations in cell number and transfection efficiency, all clones were cotransfected with 1 µg of pCMV-beta GAL, a eukaryotic expression vector containing the Escherichia coli beta -galactosidase (lacZ) structural gene under the transcriptional control of the cytomegalovirus promoter. The quantity of DNA used in each transfection was held constant (20 µg) 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 beta -galactosidase activity.

Measurement of Intracellular H2O2-- Intracellular H2O2 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 (LTB4 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 H2O2, cells were then incubated for 10 min with the H2O2-sensitive fluorophore 2',7'-dichlorofluorescein diacetate (5 µg/ml), which when taken up fluorescently labels intracellular H2O2 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-beta -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 LTB4 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 Na2VO3), centrifuged for 20 s at 12,000 × 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 cPLA2) 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 cPLA2; 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 cPLA2, p38, and ERKs on XAR-5 film (Eastman Kodak Co.) were measured by densitometry.

Translocation of cPLA2-- To visualize the localization of endogenous cPLA2, 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-cPLA2 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 serum-starved 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 Na2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4, 2.5 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/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 × 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 µl 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-RacN17 cells was assayed using Transwell chambers with 6.5-mm diameter polycarbonate filters (8-µm pore size). Briefly, the lower surfaces of the filters were coated with 10 µg/ml gelatin (Collaborative Biomedicals) in HEPES-buffered RPMI 1640 medium for 2 h at 37 °C. Dry coated filters containing various amounts of LTB4 were placed in the lower wells of the Transwell chambers, after which 100 µl of Rat-2 or Rat2-RacN17 cells in DMEM containing 1% FBS were loaded into the top wells, yielding a final concentration of 1×106 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% CO2 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, ×200). Ten fields were counted in each assay; each sample was assayed in duplicate, and the assays were repeated twice.

Cell Growth Assay-- To assess cell growth, Rat-2 or Rat2-RacN17 cells were plated onto 60-mm plates (105 cells/plate) in DMEM containing 10% FBS. The next day, the medium was replaced with serum-free medium or serum-free medium containing LTB4 or LPA. The number of viable cells was then counted after an additional 48 h.

Data Analysis and Statistics-- Data are expressed as means ± S.D. or as percentages ± S.D. of control. Statistical comparisons between groups were made using Student's t tests. Values of p < 0.01 were considered significant.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

LTB4 Induces ROS Generation in Rat-2 Fibroblasts-- To assess whether LTB4 induces ROS generation, Rat-2 fibroblasts were serum-starved for 48 h and then exposed to exogenous LTB4 for 3 min. The resultant ROS generation was monitored as a function of H2O2-sensitive DCF fluorescence. As shown in Fig. 1A, 0.3 µM LTB4 elicited a significant (~2.5-fold) increase in the levels of ROS; no further increases were seen at LTB4 concentrations up to 1 µM (data not shown). This effect was completely inhibited by ZK158252, a specific BLT antagonist (17, 29), whereas ROS generated in response to exogenous cysLTs (LTC4/D4/E4 mixture) were unaffected by ZK158252, confirming that LTB4 generates ROS via a specific BLT-linked pathway (Fig. 1B). Likewise, the ROS response elicited by TNF-alpha (~2.6-fold increase) was completely abolished by ZK158252 (Fig. 1B), which is consistent with the previous report suggesting the mediatory role of BLTR in TNF-alpha signaling to ROS generation (23).


View larger version (36K):
[in this window]
[in a new window]
  		Fig. 1.   Exogenous LTB4 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 µM LTB4 (3 min), 1 µM cysLTs (3 min) or 20 ng/ml TNF-alpha (10 min). To assess the effects of BLT inhibition, cells were pretreated with ZK158252 (3 µM) for 30 min before the addition of LTB4. 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 ± S.D. (n = 30 cells) from three independent experiments. Statistical significance of ROS measurements was assessed with unpaired t tests (p < 0.01).

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 LTB4-induced ROS generation (Fig. 2). By contrast, LTB4-induced ROS generation was little affected by EGTA, U73122, or calphostin C, suggesting that Ca2+ mobilization, phospholipase C, and protein kinase C are not involved.


View larger version (42K):
[in this window]
[in a new window]
  		Fig. 2.   PTX-sensitive generation of ROS by LTB4. Serum-starved Rat-2 cells were exposed to 0.3 µM LTB4 for 3 min in the presence or absence of PTX (100 ng/ml), EGTA (2 mM), U73122 (0.1 µM), or calphostin C (30 nM). Inhibitors were added 20 min prior to the addition of LTB4, with the exception of PTX, which was added 18 h earlier. DCF fluorescence was quantified as described under "Experimental Procedures". Data are expressed as means ± S.D. (n = 30 cells) from three independent experiments. Statistical significance of ROS measurements was assessed using unpaired t tests (p < 0.01).

Essential Roles of Rac and cPLA2 in the LTB4 Signaling to ROS Generation-- Previously, we and others have reported that Rac1 plays a crucial role in ROS generation in fibroblasts (23-25). To examine whether Rac1 is involved in LTB4 signaling to ROS generation, we compared the effects of LTB4 in Rat-2 and Rat2-RacN17 cells, which express a dominant negative Rac1 mutant (26). Although substantial ROS generation (a ~2.3-fold increase over control) was observed within 3 min of exposing Rat-2 cells to 0.3 µM LTB4, little effect was observed in Rat2-RacN17 cells under the same conditions (Fig. 3, A and B). This result prompted us to test directly the extent to which exposure to LTB4 alters cellular Rac1 activity; we found that indeed LTB4 induced significant, time-dependent increase in Rac1 activity, consistent with the proposed mediatory role of LTB4 signaling (Fig. 3C).


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 3.   LTB4 stimulates ROS generation via a Rac-dependent pathway. A, serum starved Rat-2 or Rat2-RacN17 cells were exposed to LTB4 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 ± S.D. (n = 30 cells) from three independent experiments. Statistical significance of ROS measurements was assessed with unpaired t tests (p < 0.01). C, Rat-2 cells were serum-starved for 36 h prior to exposure to 0.3 µM LTB4 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.

We recently reported that cPLA2 serves as a key downstream mediator of Rac in Rat-2 cells (23, 24, 30, 31). We therefore assessed the effects of AACOCF3, a specific cPLA2 inhibitor, on LTB4-induced ROS generation and found that pretreatment with 10 µM AACOCF3 almost completely blocked LTB4-induced ROS generation (>90% 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 cPLA2, without subsequent metabolism of arachidonic acid by 5-LO or COX, is required for LTB4-induced ROS generation.


View larger version (26K):
[in this window]
[in a new window]
  		Fig. 4.   Rac-mediated cPLA2 activation is critical for LTB4 signaling to ROS generation. A, serum-starved Rat-2 cells were pretreated with 10 µM AACOCF3 or 50 nM MK886 and then exposed to 0.3 µM LTB4. DCF fluorescence was quantified as described under "Experimental Procedures." Data are expressed as means ± S.D. (n = 30 cells) from three independent experiments. Statistical significance of ROS measurements was assessed with unpaired t tests (p < 0.01). B, images of cells exposed to 0.3 µM LTB4 for 10 min and then labeled first with anti-cPLA2 antibody and then with a tetramethylrhodamine B isothiocyanate-conjugated anti-mouse secondary antibody. C, Rat-2 cells were exposed to 0.3 µM LTB4 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 cPLA2 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 ± S.D. of control from three independent experiments.

The role of cPLA2 in LTB4 signaling was further confirmed by our observations that LTB4 evoked translocation of cPLA2 to the membrane compartment (e.g. nuclear envelope area) in Rat-2, but not Rat2-RacN17 cells (Fig. 4B), as well as time-dependent increases (up to 10 min) in levels of cPLA2 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 LTB4 treatment), the level of cPLA2 in the particulate fraction was significantly diminished, whereas that in the soluble fraction increased (Fig. 4C).

LTB4-induced ROS Generation Requires ERK-- To investigate the possible involvement of MAP kinases in LTB4 signaling to ROS generation, we tested the effects of 10 µM PD098059, a specific MEK inhibitor, and 10 µM SB203580, a specific p38 kinase inhibitor, on the production of intracellular ROS. We found that, although pretreatment with the former inhibited LTB4-induced ROS generation, pretreatment with the latter did not (Fig. 5A). This finding suggested the presence of ERK in the LTB4 signaling pathway, which prompted us to test whether ERK is indeed activated in cells exposed to LTB4. We found that exposing serum-starved Rat-2 cells to 0.3 µM LTB4 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-RacN17 cells (Fig. 5B). Addition of epidermal growth factor, by contrast, evoked virtually the same level of ERK activation in both Rat-2 and Rat2-RacN17 cells. ERK activation was also dose-dependently elevated by transient transfection with plasmid expressing RacV12, a constitutively active form of Rac1 (Fig. 5C). Similarly, RacV12 elevated Elk-luciferase activity in a dose-dependent manner (Fig. 5D), confirming the ERK activation by Rac1.


View larger version (45K):
[in this window]
[in a new window]
  		Fig. 5.   LTB4 induces ROS generation via Rac-ERK-linked pathway. A, Serum-starved Rat-2 cells were exposed to 0.3 µM LTB4 for 3 min in the presence or absence of 10 µM PD098059 or 10 µM SB203580. Inhibitors were added 20 min prior to the addition of LTB4. DCF fluorescence was imaged and quantified as described under "Experimental Procedures". Data are expressed as means ± S.D. (n = 30 cells) from three independent experiments. Statistical significance of ROS measurements was assessed with unpaired t tests (p < 0.01). B, Rat-2 or Rat2-RacN17 cells were serum-starved and then incubated for 5 min with control buffer or 0.3 µM LTB4 or for 10 min with 50 ng/ml epidermal growth factor. 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 µg of a RacV12 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 µg of a RacV12 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 ± S.D. of control from three independent experiments.

PI 3-Kinase Activity Is Required for the LTB4 Signaling to ROS-- To investigate further mediators involved in the LTB4-induced ROS generation, we assessed the effects of inhibitors of PI 3-kinase on production of intracellular ROS. As shown in Fig. 6A, LTB4-evoked ROS generation was completely blocked by specific PI 3-kinase inhibitor LY294002 (10 µM) or wortmannin (50 nM). Besides PI 3-kinase, we observed that tyrosine kinase inhibitors, herbimycin and genistein, also dramatically diminished LTB4-evoked ROS generation. Moreover, when we tested whether LTB4-induced ERK activation is affected by PI 3-kinase inhibition, we found the effect of LTB4 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 µM LTB4APA, a specific BLT antagonist, confirmed that LTB4 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 LTB4 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 LTB4 (8, 16).


View larger version (24K):
[in this window]
[in a new window]
  		Fig. 6.   PI 3-kinase activity is required for LTB4 signaling to ROS. A, Rat-2 cells were exposed to 0.3 µM LTB4 for 3 min in the presence or absence of 10 µM LY294002, 50 nM wortmannin, 2 µM genistein or 2 µM herbimycin. Inhibitors were added 20 min prior to the addition of LTB4. DCF fluorescence was quantified as described under "Experimental Procedures". Data are expressed as means ± S.D. (n = 30 cells) from three independent experiments. Statistical significance of ROS measurements was assessed with unpaired t tests (p < 0.01). B, Serum-starved Rat-2 cells were pretreated with 10 µM LY294002, 50 nM wortmannin or 1 µM LTB4APA and then exposed to 0.3 µM LTB4 for 5 min. The cell lysates were then probed for levels of phospho-ERK and total ERK. The results shown are representative of at least three independent experiments.

ROS Generation Is Essential for the Chemotaxis and Proliferation by LTB4-- As LTB4 is known to be a potent chemotactic agent, we examined whether ROS generation could mediate the chemotaxis evoked by LTB4. As shown in Fig. 7 (A and B), pretreatment with DPI (2 µM), an inhibitor of NADPH oxidase-like flavoproteins, or NAC (2 mM), a free radical scavenger, diminished chemotaxis elicited by LTB4, suggesting a role for ROS generation in mediating chemotactic activity. RacN17 expression also inhibited chemotaxis induced by LTB4 (Fig. 7C), supporting the involvement of the Rac-ROS cascade.


View larger version (32K):
[in this window]
[in a new window]
  		Fig. 7.   Essential role of ROS generation for chemotaxis by LTB4. A, LTB4-induced chemotactic motility was determined in the presence of 2 µM 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 LTB4 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 ± S.D. of control. Each sample was assayed in duplicate, and the assays were repeated twice (p < 0.01). C, LTB4-induced chemotactic motility was analyzed in Rat-2 and Rat2-RacN17 cells as described above.

The role of ROS generation in LTB4-induced cell proliferation was similarly tested. As shown in Fig. 8A, LTB4 elicited significant cell proliferation, which was inhibited by pretreatment with 2 mM NAC or 2 µM DPI, and likewise LTB4-induced cell proliferation was not detected in Rat2-RacN17, although LPA-induced cell proliferation remained intact (Fig. 8B). Together, these results suggest a potential, mediatory role of Rac-ROS cascade for the LTB4-evoked chemotaxis and cell proliferation.


View larger version (23K):
[in this window]
[in a new window]
  		Fig. 8.   Essential role of ROS generation for cell proliferation by LTB4. A, Rat-2 were plated on 60 mm plates for 24 h and then incubated with serum-free medium (buffer) alone or containing 0.3 µM LTB4 in the presence and absence of 2 µM DPI or 2 mM NAC. Numbers of viable cells were counted 48 h later. The results are expressed as percentages ± S.D. of control from three independent experiments. Statistical significance of growth measurements was assessed using unpaired t tests (p < 0.01). B, Rat-2 or Rat2-RacN17 cells were plated on 60 mm plates for 24 h and then incubated with buffer alone or containing 0.3 µM LTB4 or 10 µM LPA. Numbers of viable cells were counted 48 h later. The results are expressed as percentages ± S.D. of control from three independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates the central role played by a Rac-linked cascade in LTB4-signaling to ROS generation in Rat-2 fibroblasts. Supporting that conclusion are the observations that exposure to LTB4 stimulates Rac activity and that stable expression of RacN17 dramatically inhibits LTB4-evoked ROS generation. In addition, our findings suggest that ERK and cPLA2 are situated downstream of Rac1, mediating LTB4 signaling to ROS generation.

Levels of ERK and cPLA2 activation were both Rac-dependently increased following treatment with LTB4, as demonstrated by ERK phosphorylation and translocation of cPLA2 to the nuclear envelope (Figs. 4B and 5B). It was previously shown that cPLA2 translocation from cytosol to particulate fraction represents an activation of cPLA2 (33, 34). Furthermore, expression of constitutively active RacV12 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 LTB4 and of ERK's involvement in the LTB4 signaling to eosinophil activation (7, 8, 35, 36). cPLA2 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 cPLA2 by Rac1 (23, 24, 30, 31). Most germane to the present study, Rac-dependent ROS generation was shown to be mediated largely by a cPLA2-linked cascade (24). The downstream signaling pathway via which cPLA2 activation leads to ROS generation is completely unknown, however. Nevertheless, because no detectable inhibition of LTB4-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 effect on LTB4-induced ROS generation (Fig. 2). Although LTB4 evokes rapid, transient increases in Ca2+, such Ca2+ mobilization is not involved in LTB4 signaling to ROS generation, as demonstrated by the failure of EGTA or SK&F 96365, a putative inhibitor of receptor-operated Ca2+ entry, to affect LTB4-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 LTB4 signaling cascade leading to ROS generation. On the other hand, PTX completely inhibited ROS generation in response to LTB4, which is consistent with LTB4 mediating chemotaxis via Gi protein-coupled receptors (15-19).

LTB4-induced ROS generation was completely abolished by ZK 158252, a potent BLT inhibitor (17). Several groups have cloned and characterized two distinct LTB4 receptors, BLT1 and BLT2 (15-19). 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, LTB4 showed biological activity at concentrations of 0.3-1 µM, which falls within the reported optimal range for BLT2 (e.g. ~0.1-1 µM) 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 LTB4 in Rat-2 fibroblasts, we observed that ROS generation by LTB4 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 LTB4 receptor, as ROS generation elicited by an LTC4/D4/E4 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 LTB4 and ROS generation. For example, Li et al. (6) and Lindsay et al. (7, 8) showed that LTB4 activates ROS generation in neutrophils and eosinophils (6-8), respectively. Further, NADPH oxidase appears to be involved in LTB4 signaling to H2O2 generation in guinea pig eosinophils (7), and, although details of the mechanism are still unclear, cPLA2 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 LTB4 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<UP><SUB>2</SUB><SUP>−</SUP></UP> as part of the body's defense against disease processes (42). Instead, our findings suggest that, at least in fibroblasts, ROS generation elicited by LTB4 functions in the regulation of intracellular signaling cascades leading to chemotaxis and cell proliferation. Future studies aimed at clarifying the linkage between cPLA2 and ROS will likely be pivotal to our complete understanding of LTB4-evoked intracellular signaling and inflammatory responses.

    ACKNOWLEDGEMENT

We thank Dr. Claudia Giesen for providing a BLT antagonist, ZK 158252.

    FOOTNOTES

* This work was supported by Grant R01-1999-00097 from the Interdisciplinary Research Program of Korea Science and Engineering Foundation, by a grant from the Life Phenomena and Function Research Group program of the Ministry of Science and Technology, by Grant 01-PJ2-PG4-J201PT01-0007 from the Korea Health 21 research and development project of the Ministry of Health & Welfare, and by a grant from the Brain Korea 21 program.

To whom correspondence should be addressed: Graduate School of Biotechnology, Korea University, 5-1 Anam-dong, Sungbuk-gu, Seoul 136-701, Korea. Tel.: 82-2-3290-3452; Fax: 82-2-927-9028; E-mail: jhongkim@korea.ac.kr.

Published, JBC Papers in Press, December 27, 2001, DOI 10.1074/jbc.M104766200

    ABBREVIATIONS

The abbreviations used are: LT, leukotriene; 5-LO, 5-lipoxygenase; LTB4, leukotriene B4; ROS, reactive oxygen species; BLT, leukotriene B4 receptor; PTX, pertussis toxin; TNF-alpha , tumor necrosis factor-alpha ; ERK, extracellular signal-regulated kinase; cPLA2, cytosolic phospholipase A2; DPI, diphenylene iodonium; NAC, N-acetylcysteine; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; BSA, bovine serum albumin; cysLT, cysteinyl leukotriene; COX, cyclooxygenase; PAK, p21-activated serine/threonine protein kinase; GST, glutathione S-transferase; PI 3-kinase, phosphatidylinositol 3-kinase; LPA, lysophosphatidic acid; DCF, 2',7'-dichlorofluorescein; PBD, Pak1-binding domain.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Brock, T. G., McNish, R. W., and Peters-Golden, M. (1995) J. Biol. Chem. 270, 21652-21658[Abstract/Free Full Text]
2. Leff, A. R. (2000) Am. J. Respir. Crit. Care Med. 161, S125-S132[Free Full Text] (review)
3. Mackarel, A. J., Russell, K. J., Brady, C. S., FitzGerald, M. X., and O'Connor, C. M. (2000) Am. J. Respir. Cell Mol. Biol. 23, 154-161[Abstract/Free Full Text]
4. Matsukawa, A., Hogaboam, C. M., Lukacs, N. W., Lincoln, P. M., Strieter, R. M., and Kunkel, S. L. (1999) J. Immunol. 163, 6148-6154[Abstract/Free Full Text]
5. Tager, A. M., Dufour, J. H., Goodarzi, K., Bercury, S. D., von Andrian, U. H., and Luster, A. D. (2000) J. Exp. Med. 192, 439-446[Abstract/Free Full Text]
6. Li, Y., Ferrante, A., Poulos, A., and Harvey, D. P. (1996) J. Clin. Invest. 97, 1605-1609[Medline] [Order article via Infotrieve]
7. Lindsay, M. A., Perkins, R. S., Barnes, P. J., and Giembycz, M. A. (1998) J. Immunol. 160, 4526-4534[Abstract/Free Full Text]
8. Lindsay, M. A., Haddad, E. B., Rousell, J., Teixeira, M. M., Hellewell, P. G., Barnes, P. J., and Giembycz, M. A. (1998) J. Leukocyte Biol. 64, 555-562[Abstract]
9. Turner, C. R., Breslow, R., Conklyn, M. J., Andresen, C. J., Patterson, D. K., Lopez-Anaya, A., Owens, B., Lee, P., Watson, J. W., and Showell, H. J. (1996) J. Clin. Invest. 97, 381-387[Medline] [Order article via Infotrieve]
10. Shindo, K., Koide, K., and Fukumura, M. (1997) Thorax 52, 1024-1029[Abstract]
11. Schmidt, C., Baumeister, B., Kipnowski, J., Schiermeyer-Dunkhase, B., and Vetter, H. (1996) Hepatogastroenterology 43, 1508-1512[Medline] [Order article via Infotrieve]
12. Endres, S., Lorenz, R., and Loeschke, K. (1999) Curr. Opin. Clin. Nutr. Care 2, 117-120
13. Iversen, L., Kragballe, K., and Ziboh, V. A. (1997) Skin Pharmacol. 10, 169-177[Medline] [Order article via Infotrieve]
14. Van Pelt, J. P., De, Jong, E. M., Seijger, M. M., Van Hooijdonk, C. A., De, Bakker, E. S., Van Vlijmen, I. M., Parker, G. L., Van Erp, P. E., and Van De Kerkhof, P. C. (1998) Br. J. Dermatol. 139, 396-402[CrossRef][Medline] [Order article via Infotrieve]
15. Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y., and Shimizu, T. (1997) Nature 387, 620-624[CrossRef][Medline] [Order article via Infotrieve]
16. Haribabu, B., Zhelev, D. V., Pridgen, B. C., Richardson, R. M., Ali, H., and Snyderman, R. (1999) J. Biol. Chem. 274, 37087-37092[Abstract/Free Full Text]
17. Yokomizo, T., Kato, K., Terawaki, K., Izumi, T., and Shimizu, T. (2000) J. Exp. Med. 192, 421-431[Abstract/Free Full Text]
18. Toda, A., Yokomizo, T., Masuda, K., Nakao, A., Izumi, T., and Shimizu, T. (1999) Biochem. Biophy. Res. Commun. 262, 806-812[CrossRef][Medline] [Order article via Infotrieve]
19. Kamohara, M., Takasaki, J., Matsumoto, M., Saito, T., Ohishi, T., Ishii, H., and Furuichi, K. (2000) J. Biol. Chem. 275, 27000-27004[Abstract/Free Full Text]
20. Martin, V., Ronde, P., Unett, D., Wong, A., Hoffman, T. L., Edinger, A. L., Doms, R. W., and Funk, C. D. (1999) J. Biol. Chem. 274, 8597-8603[Abstract/Free Full Text]
21. Devchand, P. R., Keller, H., Peters, J. M., Vazquez, M., Gonzalez, F. J., and Wahli, W. (1996) Nature 384, 39-43[CrossRef][Medline] [Order article via Infotrieve]
22. Chinetti, G., Fruchart, J.-C., and Staels, B. (2000) Inflamm. Res. 49, 479-505
23. Woo, C.-H., Eom, Y.-W., Yoo, M.-H., You, H.-J., Han, H.-J., Song, W.-K., Yoo, Y.-J., Chun, J.-S., and Kim, J.-H. (2000) J. Biol. Chem. 275, 32357-32362[Abstract/Free Full Text]
24. Woo, C-H., Lee, Z.-W., Kim, B.-C., Ha, K.-S., and Kim, J.-H. (2000) Biochem. J. 348, 525-530
25. Sundaresan, M., Yu, Z. X., Ferrans, V. J., Sulciner, D. J., Gutkind, J. S., Irani, K., Goldschmidt-Clermonst, P. J., and Finkel, T. (1996) Biochem. J. 318, 379-382
26. Kim, B.-C., Yi, Y.-J., Yi, S.-J., Shin, I.-C., Ha, K.-S., Jhun, B.-H., Hwang, S.-B., and Kim, J.-H. (1998) Mol. Cells 8, 90-95[Medline] [Order article via Infotrieve]
27. Ohba, M., Shibanuma, M., Kuroki, T., and Nose, K. (1994) J. Cell Biol. 26, 1079-1088[Abstract/Free Full Text]
28. Akasaki, T., Koga, H., and Sumimoto, H. (1999) J. Biol. Chem. 274, 18055-18059[Abstract/Free Full Text]
29. Devchand, P. R., Hihi, A. K., Perroud, M., Scheuning, W. D., Spiegelman, B. M., and Wahli, W. (1999) J. Biol. Chem. 274, 23341-23348[Abstract/Free Full Text]
30. Kim, B.-C., and Kim, J.-H. (1997) Biochem. J. 326, 333-337
31. Woo, C.-H., Kim, B.-C., Kim, K.-W., Yoo, M.-H., Eom, Y.-W., Choi, E.-J., Na, D.-S., and Kim, J.-H. (2000) Biochem. Biophy. Res. Commun. 268, 231-236[CrossRef][Medline] [Order article via Infotrieve]
32. Kim, B.-C., Lee, M.-N., Kim, J.-Y., Lee, S.-S., Chang, J.-D., Kim, S.-S., Lee, S.-Y., and Kim, J.-H. (1999) J. Biol. Chem. 274, 24372-24377[Abstract/Free Full Text]
33. Schievella, A. R., Regier, M. K., Smith, W. L., and Lin, L-L. (1996) J. Biol. Chem. 270, 30749-30754[Abstract/Free Full Text]
34. Schonhardt, T., and Ferber, E. (1987) Biochem. Biophys. Res. Commun. 149, 769-775[CrossRef][Medline] [Order article via Infotrieve]
35. O'Donnell, V. B., Spycher, S., and Azzi, A. (1995) Biochem. J. 310, 133-141
36. Araki, R., Komada, T., Nakatani, K., Naka, M., Shima, T., and Tanaka, T. (1995) Biochem. Biophys. Res. Commun. 210, 837-843[CrossRef][Medline] [Order article via Infotrieve]
37. Perkins, R. S., Lindsay, M. A., Barnes, P. J., and Giembycz, M. A. (1995) Biochem. J. 310, 795-806
38. Subramanian, N. (1992) Biochem. Biophys. Res. Commun. 187, 670-675[CrossRef][Medline] [Order article via Infotrieve]
39. Yokomizo, T., Izumi, T., and Shimizu, T. (2001) Life Sci. 68, 2207-2212[CrossRef][Medline] [Order article via Infotrieve]
40. Yokomizo, T., Kato, K., Hagiya, H., Izumi, T., and Shimizu, T. (2001) J. Biol. Chem. 276, 12454-12459[Abstract/Free Full Text]
41. Dana, R., Leto, T. L., Malech, H. L., and Levy, R. (1998) J. Biol. Chem. 273, 441-445[Abstract/Free Full Text]
42. Cross, A. R., and Jones, O. T. G. (1991) Biochim. Biophys. Acta 1057, 281-298[Medline] [Order article via Infotrieve]

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
M.-R. Ki, H.-R. Lee, M.-J. Goo, I.-H. Hong, S.-H. Do, D.-H. Jeong, H.-J. Yang, D.-W. Yuan, J.-K. Park, and K.-S. Jeong
Differential regulation of ERK1/2 and p38 MAP kinases in VacA-induced apoptosis of gastric epithelial cells
Am J Physiol Gastrointest Liver Physiol, March 1, 2008; 294(3): G635 - G647.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. A. Nardi, Y. Gor, S. J. Feinmark, F. Xu, and S. Karpatkin
Platelet particle formation by anti GPIIIa49-66 Ab, Ca2+ ionophore A23187, and phorbol myristate acetate is induced by reactive oxygen species and inhibited by dexamethasone blockade of platelet phospholipase A2, 12-lipoxygenase, and NADPH oxidase
Blood, September 15, 2007; 110(6): 1989 - 1996.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol SciHome page
B.-H. Liu, T.-S. Wu, F.-Y. Yu, and C.-C. Su
Induction of Oxidative Stress Response by the Mycotoxin Patulin in Mammalian Cells
Toxicol. Sci., February 1, 2007; 95(2): 340 - 347.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
Z. Sun, S. Sood, N. Li, D. Ramji, P. Yang, R. A. Newman, C. S. Yang, and X. Chen
Involvement of the 5-lipoxygenase/leukotriene A4 hydrolase pathway in 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in hamster cheek pouch, and inhibition of carcinogenesis by its inhibitors
Carcinogenesis, September 1, 2006; 27(9): 1902 - 1908.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
N. Dronadula, F. Rizvi, E. Blaskova, Q. Li, and G. N. Rao
Involvement of cAMP-response element binding protein-1 in arachidonic acid-induced vascular smooth muscle cell motility
J. Lipid Res., April 1, 2006; 47(4): 767 - 777.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
A.-C. Ceacareanu, B. Ceacareanu, D. Zhuang, Y. Chang, R. M. Ray, L. Desai, K. E. Chapman, C. M. Waters, and A. Hassid
Nitric oxide attenuates IGF-I-induced aortic smooth muscle cell motility by decreasing Rac1 activity: essential role of PTP-PEST and p130cas
Am J Physiol Cell Physiol, April 1, 2006; 290(4): C1263 - C1270.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
M. Edderkaoui, P. Hong, E. C. Vaquero, J. K. Lee, L. Fischer, H. Friess, M. W. Buchler, M. M. Lerch, S. J. Pandol, and A. S. Gukovskaya
Extracellular matrix stimulates reactive oxygen species production and increases pancreatic cancer cell survival through 5-lipoxygenase and NADPH oxidase
Am J Physiol Gastrointest Liver Physiol, December 1, 2005; 289(6): G1137 - G1147.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
C. H. C. Serezani, D. M. Aronoff, S. Jancar, P. Mancuso, and M. Peters-Golden
Leukotrienes enhance the bactericidal activity of alveolar macrophages against Klebsiella pneumoniae through the activation of NADPH oxidase
Blood, August 1, 2005; 106(