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The Pro-inflammatory Mediator Leukotriene D4 Induces Phosphatidylinositol 3-Kinase and Rac-dependent Migration of Intestinal Epithelial Cells*

  • Sailaja Paruchuri
    Affiliations
    Experimental Pathology, Department of Laboratory Medicine, Lund University, University Hospital Malmö, SE-205 02 Malmö, Sweden
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  • Oliver Broom
    Affiliations
    Experimental Pathology, Department of Laboratory Medicine, Lund University, University Hospital Malmö, SE-205 02 Malmö, Sweden
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  • Karim Dib
    Affiliations
    Experimental Pathology, Department of Laboratory Medicine, Lund University, University Hospital Malmö, SE-205 02 Malmö, Sweden
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  • Anita Sjölander
    Correspondence
    To whom correspondence should be addressed: Dept. of Laboratory Medicine, Experimental Pathology, Lund University, Malmö University Hospital, Entrance 78, SE-205 02 Malmö, Sweden. Tel.: 46-40-337223; Fax: 46-40-337353
    Affiliations
    Experimental Pathology, Department of Laboratory Medicine, Lund University, University Hospital Malmö, SE-205 02 Malmö, Sweden
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  • Author Footnotes
    * This work was supported by the grants from the Swedish Cancer Foundation, the Swedish Medical Research Council, the Foundations at Malmö University Hospital, the Ruth and Richard Julins Foundation, Magnus Bergvalls Foundation, Gunnar Nilssons Foundation, and the Österlund Foundation (to A. S.) and from the Royal Physiographic Society in Lund and the Swedish Society for Medical Research (to S. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:January 18, 2005DOI:https://doi.org/10.1074/jbc.M409811200
      Inflammatory bowel diseases are associated with increased risk of developing colon cancer. A possible role of the pro-inflammatory leukotriene D4 (LTD4) in this process has been implicated by the findings that LTD4 can signal increased proliferation and survival, both hallmarks of a cancer cell, in non-transformed intestinal epithelial cells. Here we make the novel finding that LTD4 can also signal increased motility in these cells. In parallel, we found that LTD4 induced a simultaneous transient 10-fold increase in Rac but not Cdc42 activity. These data were also supported by the ability of LTD4 to activate the Rac GDP/GTP exchange factor Vav2. Further, LTD4 triggered a 3-fold transient increase in phosphatidylinositol 3-kinase (PI3K) phosphorylation, a possible upstream activator of the Vav2/Rac signaling pathway. The activation of Rac was blocked by the PI3K inhibitors LY294002 and wortmannin and by transfection of a kinase-negative mutant of PI3K or a dominant-negative form of Vav2. Furthermore, Rac was found to co-localize with actin in LTD4-generated membrane ruffles that were formed by a PI3K-dependent mechanism. In accordance, the inhibition of the PI3K and Rac signaling pathway also blocked the LTD4-induced migration of the intestinal cells. The present data reveal that an inflammatory mediator such as LTD4 cannot only increase proliferation and survival of non-transformed intestinal epithelial cells but also, via a PI3K/Rac signaling pathway, trigger a motile response in such cells. These data demonstrate the capacity of inflammatory mediators to participate in the process by which inflammatory bowel conditions increase the risk for colon cancer development.
      Migration of epithelial cells is essential during the development of the gut and during different pathological situations such as wound healing and tumor metastasis (
      • Schmitz A.A.
      • Govek E.E.
      • Bottner B.
      • Van Aelst L.
      ). The conversion from a sessile to a migratory phenotype requires an extensive remodeling of the actin cytoskeleton (
      • Mitchison T.J.
      • Cramer L.P.
      ). In response to different chemotactic substances, cell migration is initiated by the formation of lamellipodia or membrane ruffles at the leading front of a migrating cell (
      • Jaffe A.B.
      • Hall A.
      ,
      • Small J.V.
      • Stradal T.
      • Vignal E.
      • Rottner K.
      ).
      It is well established that epithelial cell migration is stimulated by activation of receptor tyrosine kinases (
      • Royal I.
      • Lamarche-Vane N.
      • Lamorte L.
      • Kaibuchi K.
      • Park M.
      ,
      • Boyer B.
      • Valles A.M.
      • Edme N.
      ). The intracellular signaling pathways activated by such receptors and responsible for the coordinated changes in the actin cytoskeleton seen during cell motility generally include the activation of different members of the Rho family of GTPases (
      • Jaffe A.B.
      • Hall A.
      ). The Rho family consists of three main members: Rho; Rac; and Cdc42. In most cell types, these proteins execute specific functions, i.e. Rho promotes the formation of stress fibers and focal adhesion complexes and Rac initiates actin polymerization at the cell membrane and is responsible for the generation of lamellipodia and membrane ruffles, whereas Cdc42 promotes the formation of filopodia and microspikes at the cell periphery (
      • Raftopoulou M.
      • Hall A.
      ). These monomeric GTPases cycle between a GDP-bound inactive state and a GTP-bound active state. In their inactive state, Rho GTPases are bound to proteins called guanine nucleotide dissociation inhibitors. Guanine nucleotide exchange factors (GEFs),
      The abbreviations used are: GEF, guanine nucleotide exchange factor; PI3K, phosphatidylinositol 3-kinase; PH, pleckstrin homology; LTD4, leukotriene D4; PTX, pertussis toxin; EGF, epidermal growth factor; DN, dominant-negative; GST, glutathione S-transferase; PBS, phosphate-buffered saline; Int 407, intestinal epithelial 407 cell line.
      1The abbreviations used are: GEF, guanine nucleotide exchange factor; PI3K, phosphatidylinositol 3-kinase; PH, pleckstrin homology; LTD4, leukotriene D4; PTX, pertussis toxin; EGF, epidermal growth factor; DN, dominant-negative; GST, glutathione S-transferase; PBS, phosphate-buffered saline; Int 407, intestinal epithelial 407 cell line.
      activated by extracellular stimuli, catalyze the exchange of GDP for GTP on Rho GTPases and thereby activate these proteins. In their GTP-bound state, these proteins interact with specific effectors to initiate downstream signals and functions (
      • Raftopoulou M.
      • Hall A.
      ). The subsequent hydrolysis of bound GTP to GDP is catalyzed by the family of GTPase-activating proteins (
      • Tapon N.
      • Hall A.
      ).
      The Dbl family constitutes the main group of GEFs acting on Rho GTPases (
      • Schmidt A.
      • Hall A.
      ). A hallmark of these exchange factors is that they contain two key conserved domains, a Dbl homology domain that is believed to be responsible for catalyzing GDP/GTP exchange on Rho GTPases and a pleckstrin homology (PH) domain that is important for cellular localization through its interaction with lipids and/or proteins (
      • Cerione R.A.
      • Zheng Y.
      ). Notably, the PH domain on GEFs are known to bind phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, a substrate and a product of the PI3K, respectively. Such a lipid/protein interaction offers a reasonable explanation for how the PI3K can enable activation of Rac GTPase. It has been suggested that the association between GEF and phosphatidylinositol 3,4,5-trisphosphate may cause dissociation of the GEF from its binding to guanine nucleotide dissociation inhibitor (
      • Schmidt A.
      • Hall A.
      ). Possibly, such a mechanism could explain how agonist-induced activation of PI3K and generation of phosphatidylinositol 3,4,5-trisphosphate could enable a downstream phosphorylation and activation of Vav (
      • Han J.
      • Luby-Phelps K.
      • Das B.
      • Shu X.
      • Xia Y.
      • Mosteller R.D.
      • Krishna U.M.
      • Falck J.R.
      • White M.A.
      • Broek D.
      ). Src tyrosine kinase, a kinase known to be activated by LTD4 (
      • Thodeti C.K.
      • Adolfsson J.
      • Juhas M.
      • Sjölander A.
      ), has been shown to phosphorylate and activate Vav2 and subsequently activate Rac (
      • Marignani P.A.
      • Carpenter C.L.
      ). PI3Ks are composed of a catalytic subunit (p110) and a regulatory (p85/p55) subunit (
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.C.
      ). Phosphorylation of the p85 subunit is essential for the translocation and activation of the p110/85 complex from the cytosol to the plasma membrane where it interacts with its effectors (
      • Cantrell D.A.
      ). Interestingly, the inhibition of PI3K by both molecular and pharmacological approaches blocks membrane ruffling and inhibits cell migration (
      • Hawkins P.T.
      • Eguinoa A.
      • Qiu R.G.
      • Stokoe D.
      • Cooke F.T.
      • Walters R.
      • Wennstrom S.
      • Claesson-Welsh L.
      • Evans T.
      • Symons M.
      ).
      Leukotrienes belong to an important group of pro-inflammatory mediators derived from arachidonic acid via the 5-lipoxygenase pathway and have been implicated in many inflammatory conditions (
      • Claesson H.E.
      • Dahlen S.E.
      ,
      • Serhan C.N.
      • Haeggström J.Z.
      • Leslie C.C.
      ). LTD4 is the most potent of the cysteinyl leukotrienes (LTC4, LTD4, and LTE4), and it has been implicated in conditions such as asthma and inflammatory bowel diseases (
      • Chan C.C.
      • McKee K.
      • Tagari P.
      • Chee P.
      • Ford-Hutchinson A.W.
      ). LTD4 mediates its effects by the binding to two different G protein-coupled receptors, CysLT1 and CysLT2 (
      • Brink C.
      • Dahlen S.E.
      • Drazen J.
      • Evans J.F.
      • Hay D.W.
      • Nicosia S.
      • Serhan C.N.
      • Shimizu T.
      • Yokomizo T.
      ). The CysLT1 receptor has a much higher affinity for its ligand than the CysLT2-receptor and has been shown to couple to both a pertussis toxin (PTX)-sensitive and a PTX-insensitive G protein, indicating that different signal transduction pathways can be initiated and possibly mediate distinct downstream functions (
      • Thodeti C.K.
      • Adolfsson J.
      • Juhas M.
      • Sjölander A.
      ,
      • Paruchuri S.
      • Hallberg B.
      • Juhas M.
      • Larsson C.
      • Sjolander A.
      ,
      • Hoshino M.
      • Izumi T.
      • Shimizu T.
      ). We have previously shown that prolonged exposure of non-transformed intestinal epithelial cells to LTD4 resulted in up-regulation of factors related to colon carcinogenesis such as cyclooxygenase-2, β-catenin, and Bcl-2 (
      • Öhd J.F.
      • Wikström K.
      • Sjölander A.
      ). More recently, we have shown that LTD4 induces proliferative and survival responses, hallmarks of a cancer cell, in non-transformed intestinal epithelial cells (
      • Paruchuri S.
      • Hallberg B.
      • Juhas M.
      • Larsson C.
      • Sjolander A.
      ,
      • Paruchuri S.
      • Sjölander A.
      ). Taken together with the fact that ulcerative colitis is associated with an increased incidence of neoplastic transformation (
      • Ekbom A.
      • Helmick C.
      • Zack M.
      • Adami H.O.
      ), the indications showing that there exists a link between inflammation and cancer (
      • Sheng H.
      • Shao J.
      • Kirkland S.C.
      • Isakson P.
      • Coffey R.J.
      • Morrow J.
      • Beauchamp R.D.
      • DuBois R.N.
      ) and earlier studies demonstrating that colon cancer is underrepresented in a population of patients with ulcerative colitis treated with non-steroidal anti-inflammatory drugs (
      • Smalley W.E.
      • Du Bois R.N.
      ) suggest that LTD4 could be an essential factor in mediating the coupling between inflammatory bowel disease and colon cancer.
      Transformed cancer cells exhibit distinct properties including increased proliferation, survival, and migration. Interestingly enough, we have previously shown that LTD4 triggered both a proliferative and a survival response in non-transformed intestinal epithelial cells (
      • Smalley W.E.
      • Du Bois R.N.
      ). In this study, we explored whether and how the pro-inflammatory LTD4 affects migration in these intestinal epithelial cells.

      EXPERIMENTAL PROCEDURES

      Materials—Antibodies for Rac and p85 subunit of PI3K were purchased from Upstate Biotechnology. Akt antibodies (phospho-Akt, Ser473; total Akt) were from Cell Signaling technology (Beverly, MA). LTD4 was purchased from Cayman Chemical Company (Ann Arbor, MI). ZM198615 was a gift from (AstraZeneca, R&D Lund). ECL Western blot detection reagents and Hyperfilms were from Amersham Biosciences (Buckinghamshire, United Kingdom). Wortmannin and LY294002 were from Calbiochem. PTX was obtained from Speywood Pharma Ltd. (Maidenhead, United Kingdom). Peroxidase-linked goat anti-rabbit and mouse IgG originated from Dako A/S (Copenhagen, Denmark). Alexa 546 phalloidin is from Molecular Probes Inc. (Eugene, OR). All other chemicals were of analytical grade and obtained from Sigma.
      Cell Culture—The Intestine 407 cell line was isolated from the intestine of a human embryo of ∼2-month gestation and was successfully maintained in culture without any immortalizing transfections (
      • Henle G.
      • Deinhardt F.
      ). These human intestinal epithelial cells (Int 407; Flow Laboratory), which exhibit typical epithelial morphology and growth (
      • Henle G.
      • Deinhardt F.
      ), were cultured as a monolayer to ∼80% confluence for 5 days. Cell cultures were kept at 37 °C in a humidified atmosphere of 5% CO2 and 95% air in basal medium Earle's supplemented with 15% newborn calf serum, 55 IU/ml penicillin, and 55 μg/ml streptomycin. The cells were regularly tested to ensure the absence of mycoplasma contamination.
      cDNAs and Transfections—Cells were transfected with dominant negative p85 of PI3K or dominant-negative (DN) form of Vav2, which lacks an active dbl domain (L342R/L343S) for 6 h and were allowed to grow in serum containing medium for another 24 h. The DN-p85 construct was generously provided by Dr. Arthur Mercurio (Beth Israel Deaconess Medical Center, Boston, MA), whereas the DN-Vav2 construct was generously provided by Dr. Christopher Carpenter (Beth Israel Deaconess Medical Center). Control cells were transfected with empty pEGFP-N1 vector (Clontech). Transient transfections of the cells were achieved using 3.5 μl of Lipofectamine (Invitrogen) and 1.8 μg of plasmid DNA/ml and were performed in serum-free medium, essentially according to the protocol provided by the supplier. In all of the transfection experiments, it was routinely confirmed that the empty vector had no effect.
      GST Pull-down Assays—The cDNA clone encoding the GST fusion protein of the PAK1B binding domain of Rac and Cdc-42 (PAKcrib; amino acids 56–267) was cloned into the bacterial expression vector pGEX-2T and was expressed in Escherichia coli and cultured at 30 °C (
      • Dib K.
      • Melander F.
      • Axelsson L.
      • Dagher M.C.
      • Aspenstrom P.
      • Andersson T.
      ). The expression of GST fusion proteins was induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside, and the E. coli were subsequently collected by centrifugation at 3500 × g for 15 min followed by sonication in phosphate-buffered saline. Triton X-100 (final concentration 1%) was added to lysate, and particulate matter was removed by centrifuging at 5000 × g for 15 min. The cleared lysate was incubated with glutathione-Sepharose beads (Sigma) for 1 h at 4°C, and the beads were subsequently washed three times with ice-cold PBS. Lysates of unstimulated or stimulated cells were prepared in 1.0 ml of the lysis buffer supplemented with 10 mm MgCl2. GST fusion protein pre-bound to Sepharose beads was incubated with 1.0 ml of the cell lysates (1 mg/ml total cell protein) for 1 h at 4°C. Thereafter, the beads were washed once with ice-cold lysis buffer supplemented with 0.5 m NaCl and twice with buffer A (50 mm Tris, pH 7.5, 1 mm EDTA, 1 mm EGTA, 1 mm Na3VO4, 1% Triton X-100, 50 mm NaF, 5 mm sodium pyrophosphate, 10 mm sodium glycerophosphate, 4 μg/ml leupeptin, and 30 μg/ml phenylmethanesulfonyl-fluoride).
      Cell Lysis—Cells were preincubated with inhibitors (Gi protein inhibitor PTX (500 ng/ml) for 2 h and PI3K inhibitors wortmannin and LY294002 for 30 min), and stimulations (80 nm LTD4) were terminated by adding ice-cold buffer A. Thereafter, the cells were kept on ice for 30 min in the lysis buffer, and remaining cell debris was scraped loose into the buffer. The lysates were homogenized 10 times on ice in a glass tissue grinder (Dounce) and then centrifuged at 10,000 × g for 15 min. The protein content of each supernatant was measured and compensated for to ensure equal protein loading.
      Immunoprecipitation and Immunoblotting—For immunoprecipitation, lysates containing 1 mg/ml protein were incubated with 4 μgofthe anti-p85, anti-Vav2, or anti-phosphotyrosine antibodies at 4 °C for 2 h, after which 20 μl of protein G plus agarose was added and incubated for an additional 1 h. The beads then were washed three times in the lysis buffer mentioned above, mixed with sample buffer, boiled for 10 min, and centrifuged, and the proteins are separated on SDS-polyacrylamide gels. The separated proteins were electrophoretically transferred to a polyvinylidene difluoride membrane and were blocked for 1 h with 5% nonfat dried milk at room temperature and then incubated with a primary antibody for 1 h at room temperature or overnight at 4 °C. Phospho-Akt 1:500 and 1:1000 dilutions were used for all of the other antibodies. Subsequently, all of the membranes were washed extensively and incubated with a horseradish peroxidase-linked goat antirabbit, anti-sheep, or anti-mouse antibody (1:5000) for 1 h at room temperature. Thereafter, the membrane was washed again extensively, incubated with ECL Western blot detection reagents, and finally exposed to hyperfilm-ECL to visualize immunoreactive proteins.
      Immunofluorescence—The cells were seeded on glass coverslips and grown for 5 days. Thereafter, the cells were serum-starved for 2 h and stimulated with LTD4. The stimulation was terminated by fixing for 10 min at room temperature in a 3.7% paraformaldehyde and PBS solution, after which the cells were permeabilized in a 0.5% Triton X-100 and PBS solution for 15 min. The coverslips were subsequently washed twice in PBS and incubated at room temperature in a 5% goat serum albumin, 0.39 Triton X-100, and PBS solution for 40 min, and the cells were stained for 1 h with an antibody against Rac. The coverslips then were washed six times in PBS and incubated with Alexa Fluor 568 goat anti-rabbit secondary antibody (diluted 1:200 in 1% goat serum and PBS solution for 1 h). The coverslips were washed thereafter six times in PBS, counterstained with Alexa phalloidin 546, washed, and mounted in fluorescent-mounting medium (Dako A/S). Samples were examined and photographed with a Nikon Eclipse 800 microscope using a Plan-Apo ×60 objective. Images were recorded with a scientific grade CCD camera (Hamamatsu, Japan) and subsequently analyzed with HazeBuster deconvolution software (Vay Tek, Inc., Fairland, CT).
      Migration Assay—Migration of intestinal epithelial cells was assayed using Boyden chamber with polycarbonate filters of 8-μm pore size. The LTD4 was added to the lower well of the chamber along with serum-free medium, and the filter was placed above. 100 μl of cell suspension (1 × 105 cells/ml) in serum-free basal medium Earle's was added to the top chamber. In the case of inhibitor treatments, the cells were pretreated with the indicated inhibitors and the respective inhibitors were also added at the concentrations indicated to the upper chamber during the process of cell migration. After incubation at 37 °C in 5% CO2 for 18 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 outer surface of the filter were fixed with 3.7% paraformaldehyde and stained for 20 min with 0.1% crystal violet in 10% methanol. The dye was eluted with 100 ml of 1% SDS. The absorbance was measured in Nunc Polysorp 96-well plates, and the absorbance at 590 nm was measured using a Fluostar plate reader (BMG Labtechnologies GmbH, Offenberg, Germany).

      RESULTS

      LTD4 Induces Cell Migration and Activation of Vav and Rac in the Intestinal Epithelial Cell Line, Int 407—As shown in Fig. 1A, LTD4 could induce migration of intestinal epithelial cells in a concentration-dependent manner, although less prominent than that seen in a colon cancer cell line, Caco-2 (
      • Massoumi R.
      • Nielsen C.K.
      • Azemovic D.
      • Sjölander A.
      ). The Rac and Cdc42 GTPase have both been implicated in the regulation of migration of different cell types (
      • Raftopoulou M.
      • Hall A.
      ). To examine the ability of LTD4 to activate Rac and Cdc42, we measured the activity of Rac and Cdc42 in cells using the affinity precipitation assay in which the GST-PAKcrib fusion protein binds the active GTP-bound form of Rac or Cdc42. We found that the amount of active Rac-GTP increased in a time-dependent manner upon LTD4 stimulation of Int 407 cells with a maximum after 15 min (Fig. 1B). Densitometric analysis confirmed the transient activation of Rac with a maximum of 942 ± 118%, 15 min after the addition of LTD4. Under similar conditions, we were unable to detect any activation of Cdc42 (Fig. 1B). Whole cell lysates were used to ensure that the total amounts of Rac and Cdc42 did not change upon LTD4 stimulation (Fig. 1B). As a positive control for the activation of Rac and Cdc42, the cells were stimulated with the epidermal growth factor (EGF) (Fig. 1B). Rac is known to be activated by GEFs such as Vav and Sos. Therefore, we investigated whether Vav2 or Sos was activated by LTD4. We could not detect any activation of Sos by LTD4 in these cells (data not shown); however, Vav2 immunoprecipitated from cells stimulated with LTD4 for 15 min, the time for maximal activation of Rac, exhibited a distinctly increased tyrosine phosphorylation (Fig. 1C). This tyrosine phosphorylation of Vav2 is indicative of its activation (
      • Schuebel K.E.
      • Movilla N.
      • Rosa J.L.
      • Bustelo X.R.
      ). We transfected cells thereafter with a previously described (
      • Marignani P.A.
      • Carpenter C.L.
      ) DN form of Vav2 (DN-Vav2) and noted that, in such transfected cells, the LTD4-induced activation of Rac was abolished (Fig. 1D). These results clearly show that the LTD4-induced activation of Rac is mediated through Vav2.
      Figure thumbnail gr1
      Fig. 1LTD4 induces cell migration as well as activation of Vav2 and Rac in the non-transformed intestinal epithelial cell line, Int 407. The migration of Int 407 cells was evaluated in a Boyden chamber as described under “Experimental Procedures.” A, the cells incubated in the absence or presence of the indicated LTD4 concentration for 18 h. B, cells were incubated in the absence or presence of 80 nm LTD4 for indicated periods of time, after which cells were lysed and analyzed with a GST-PAKcrib pull-down assay as described under “Experimental Procedures.” Proteins bound to GST-PAKcrib and also the corresponding whole lysates from samples used for the GST-PAKcrib pull-down assays were separated on 12% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and immunoblotted with antibodies specific for Rac or Cdc42 (1:1000). Representative blots of the activity of Rac and Cdc42 are shown together with the accumulated results from the densitometric analysis of the LTD4-induced Rac activation. C, cells were incubated in the absence or presence of 80 nm LTD4 for 15 min or 100 ng/ml EGF for 15 min. The cells were lysed, and Vav2 was immunoprecipitated (IP) from the different samples. The precipitates were immunoblotted subsequently with an anti-phosphotyrosine (PTyr) antibody (1:5000) and then reprobed with an anti-Vav2 antibody (1:1000) as a loading control. Representative blots and the accumulated results of the densitometric analysis of LTD4-induced tyrosine phosphorylation of Vav2 are shown. The degree of Vav2 phosphorylation was calculated as the percentage of unstimulated cells and given as the means ± S.E. of four separate experiments. D, this blot shows Rac activity (analyzed as in B) in cells transfected with either empty vector (lanes 1–2) or DN-Vav2 vector (lanes 3–4) and then incubated in the absence or presence of LTD4. Representative blots and the accumulated results of the densitometric analysis of Rac activities are shown. The degree of Rac activity was calculated as the percentage of unstimulated cells transfected with an empty vector and given as the means ± S.E. of four separate experiments. All of the statistical significances were evaluated using the unpaired Student's t test. *, p < 0.05; **, p < 0.01.
      To further analyze the LTD4-induced Rac signaling pathway, we investigated whether LTD4 could activate PI3K in these cells. The activity of this enzyme was first assessed by investigating the degree of tyrosine phosphorylation of p85 subunit of PI3K (
      • Cantrell D.A.
      ). The cells were stimulated with LTD4 for indicated periods of time, and immunoprecipitates of tyrosine phosphorylated proteins were then blotted with an anti-p85 antibody (Fig. 2A) or vice versa (Fig. 2B). As seen in Fig. 2, A and B, LTD4 caused an increased tyrosine phosphorylation of the p85 regulatory subunit of PI3K. The time kinetics of this activation of PI3K was similar to the LTD4-induced activation Rac (Fig. 1B). Secondly, we also investigated the ability of LTD4 to induce activation of the serine/threonine kinase Akt, a well known downstream target of PI3K activity. Cells stimulated with LTD4 exhibited a clear activation of Akt that was also confirmed to be sensitive to the PI3K inhibitor LY294002 (Fig. 2C). These results clearly argue for the ability of LTD4 to cause activation of PI3K in intestinal epithelial Int 407 cells.
      Figure thumbnail gr2
      Fig. 2LTD4 induces phosphorylation of the p85 regulatory subunit of PI3K and Akt in Int 407 cells. Cells were incubated in the absence or presence of 80 nm LTD4 for indicated periods of time, after which the cells were lysed. A, the cell lysates were immunoprecipitated (IP) with an anti-phosphotyrosine (PTyr) antibody (4 μg) and immunoblotted with an anti-p85 subunit antibody (1:1000). B, the cells were immunoprecipitated with an anti-p85 subunit antibody (5 μg) and immunoblotted with an anti-phosphotyrosine antibody (1:5000). These blots were then reprobed with an anti-p85 antibody to confirm equal loading. Representative blots and the accumulated results of a densitometric analysis of the LTD4-induced phosphorylation of the p85 subunit are shown. The p85 tyrosine phosphorylation values were calculated as percentages of those seen in unstimulated cells and are given as the means ± S.E. of three separate experiments. C, the cells were preincubated in the absence or presence of 50 μm LY294002 (LY) for 30 min before LTD4 stimulation. The cells then were lysed, and the proteins were separated by SDS-PAGE and immunoblotted with an antibody specific for phosphorylated Akt (Ser473). Thereafter, the blots were stripped and reprobed with an antibody for total Akt. The accumulated results of densitometric analysis of the LTD4-induced phosphorylation of Akt are shown. The illustrated blots are representative of at least three separate experiments. All of the statistical significances were evaluated using the unpaired Student's t test. *, p < 0.05; **, p < 0.01.
      Involvement of Gi and PI3K in the LTD4-induced Activation of Rac—To explore whether Gi PI3K signals are involved in the LTD4-induced activation of Rac, we pretreated the cells with the Gi protein inhibitor PTX (500 ng/ml for 2 h) and either of two different PI3K inhibitors, LY294002 (50 μm for 30 min) or wortmannin (100 nm for 30 min) (
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Ahmadi K.
      • Timms J.
      • Katso R.
      • Driscoll P.C.
      • Woscholski R.
      • Parker P.J.
      • Waterfield M.D.
      ). The cells then were stimulated with LTD4 for 15 min, after which they were lysed. Thereafter, the different lysates were analyzed in the GST-PAKcrib binding assay to assess the activity of Rac. We have reported earlier that LTD4 can signal via a PTX-sensitive G protein (
      • Paruchuri S.
      • Hallberg B.
      • Juhas M.
      • Larsson C.
      • Sjolander A.
      ,
      • Paruchuri S.
      • Sjölander A.
      ). Consistent with those results, preincubation with PTX significantly inhibited the LTD4-mediated activation of Rac (Fig. 3A). We also observed that preincubation with either of the PI3K inhibitors led to an almost complete block of the LTD4-mediated activation of Rac, suggesting that a PI3K signals upstream of Rac in these cells (Fig. 3A). This conclusion was confirmed by transfecting the cells with a DN p85 expressing vector (DN-p85). As shown in Fig. 3B, the expression of DN-p85 in these cells also blocked the LTD4-induced activation of Rac.
      Figure thumbnail gr3
      Fig. 3Intracellular signals involved in the LTD4-induced activation of Rac. A, cells were preincubated for 2 h in the absence or presence PTX (500 ng/ml) or for 30 min with the PI3K inhibitors LY294002 (50 μm) or wortmannin (100 nm) and then stimulated or not as indicated in the figure with 80 nm LTD4. IP, immunoprecipitation; PTyr, phosphotyrosine. B, cells were transfected with either an empty vector (lanes 1–2) or a dominant-negative p85 expressing vector (lanes 3–4) and then stimulated or not as indicated in the figure with 80 nm LTD4 for 15 min. Thereafter, the cells in all of the panels were lysed and the GST-PAKcrib pull-down assay was used to analyze the samples as described under “Experimental Procedures.” Proteins bound to GST-PAKcrib and also the corresponding whole lysates from samples used for the GST-PAKcrib pull-down assay were separated by 12% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and immunoblotted with an anti-Rac-specific antibody (1:1000). Shown in the figure are representative blots as well as a densitometric analysis of the LTD4-induced Rac activation in the absence or presence of the indicated inhibitors. The blots shown are representative of three separate experiments. The densitometric data are expressed as percent of unstimulated control cells and given as the means ± S.E. of three separate experiments. Statistical significance versus control cells and LTD4-treated cells were evaluated using the unpaired Student's t test. *, p < 0.05; **, p < 0.01.
      LTD4 Mediates PI3K- and Rac-dependent Formation of Membrane Ruffles in Intestinal Epithelial Cells—The Rac GTPase has previously been shown to induce the formation of lamellipodia and membrane ruffles in different cell types (
      • Fenteany G.
      • Janmey P.A.
      • Stossel T.P.
      ,
      • Nobes C.D.
      • Hall A.
      ). Here we show that cells stimulated with LTD4 formed more membrane ruffles when compared with unstimulated control cells (Fig. 4). The formation of these membrane ruffles was blocked effectively by inhibiting the PI3K/Rac signaling pathway with either wortmannin or LY294002 (Fig. 4). We also examined whether and how LTD4 influenced the distribution of Rac during the formation of membrane ruffles. Unstimulated control cells immunostained with an anti-Rac antibody exhibited a fairly homogenous staining of Rac throughout the cell (Fig. 5, top left). In contrast, LTD4 stimulation caused a redistribution of Rac to the actin-containing membrane ruffles (Fig. 5, bottom left and middle). Merge pictures more clearly show the LTD4-induced co-localization of Rac and actin in the membrane ruffles (Fig. 5, the two panels to the right). To ascertain that the LTD4-induced membrane ruffles are indeed mediated via the CysLT1 receptor, we also preincubated cells with the CysLT1 receptor antagonist ZM198615. Alone this antagonist did not affect the number of ruffles of unstimulated cells, 17 ± 2 ruffles/cell in unexposed control cells and 20 ± 2 ruffles/cell in cells incubated with ZM198615 alone. The obtained results clearly show that, in the presence of ZM198615, all of the LTD4-induced membrane ruffles were abolished (Fig. 5).
      Figure thumbnail gr4
      Fig. 4LTD4 generates the formation of membrane ruffles via a PI3K signaling pathway. The cells were pretreated or not for 30 min with PI3K inhibitors LY294002 (50 μm) or wortmannin (100 nm) and stimulated or not with 80 nm LTD4 for 30 min. Cells then were fixed, permeabilized, and stained for actin with Alexa 488 phalloidin (1:200) and examined with a Nikon Eclipse 800 microscope. A, the panels show, from the left to the right, unstimulated control cells, cells stimulated with LTD4, and then cells preincubated with either LY294002 (LY) or wortmannin (Wort) before stimulation with LTD4. The results illustrated in panel A are representative of at least four separate experiments. B, the diagram shows the accumulated results of LTD4-induced membrane ruffles, calculated as number of ruffles per cell. A minimum of 100 cells was investigated in each experimental group. The data are given as the means ± S.E. of four separate experiments. Statistical significance versus control cells and LTD4-treated cells were evaluated using the unpaired Student's t test. *, p < 0.05.
      Figure thumbnail gr5
      Fig. 5Co-localization of Rac and actin in the LTD4-generated membrane ruffles. A, cells were stimulated or not with 80 nm LTD4 for 30 min. The cells then were fixed, permeabilized, and stained for with Alexa 488 phalloidin (1:200) for F-actin and immunostained for Rac as described under “Experimental Procedures.” The different samples then were examined in a Nikon Eclipse 800 microscope. The top and middle three panels show, from left to right, Rac immunostaining, F-actin staining, and an overlay image of the two. B, the cells were preincubated with 30 μm CysLT1 antagonist ZM198615 for 15 min. Thereafter, the cells were stimulated or not with 80 nm LTD4 for 30 min. The two panels show F-actin staining of unstimulated and LTD4-stimulated cells. The results illustrated in the figure are representative of four separate experiments. C, the diagram shows the accumulated results of LTD4-induced membrane ruffles in the absence of presence of ZM198615, unstimulated control cells (C), calculated as number of ruffles per cell. A minimum of 100 cells was investigated in each experimental group. The data are given as the means ± S.E. of four separate experiments. Statistical significances were evaluated using the unpaired Student's t test. *, p < 0.05.
      The LTD4-induced Migration Is Dependent on the PI3K/Rac Signaling Pathway—Based on the finding that LTD4 generates the formation of membrane ruffles via a PI3K/Rac signaling pathway, we investigated whether the same pathway was also involved in the LTD4-induced migration of intestinal epithelial cells. As shown in Fig. 6A, the intestinal epithelial cell motility initiated by LTD4 was effectively impaired by PTX and both of the PI3K inhibitors, LY294002 and wortmannin. Finally, a simplified schematic model of the signaling pathway participating in the regulation of LTD4-induced motility in intestinal epithelial cell is outlined in Fig. 6B.
      Figure thumbnail gr6
      Fig. 6The LTD4-induced migration of Int 407 cells is mediated via a Gi protein and PI3K signaling pathway. A, the migration of Int 407 cells was evaluated in a Boyden chamber as described under “Experimental Procedures.” The cells were preincubated for 2 h in the absence or presence of PTX (500 ng/ml) or for 30 min with the PI3K inhibitors LY294002 (50 μm) or wortmannin (100 nm) and then allowed to migrate in the chamber for 18 h in the absence or presence of 80 nm LTD4. The data are given as the means ± S.E. of three independent experiments, each performed in duplicate. Statistical significances of the effects of LTD4 and the inhibitors were evaluated using the unpaired Student's t test. *, p < 0.05. B, a simplified schematic model of the LTD4-induced signaling pathway involved in intestinal epithelial 407 cell migration.

      DISCUSSION

      The CysLT1-receptor has been shown previously to activate both a PTX-sensitive and a PTX-insensitive heterotrimeric G protein signaling pathway (
      • Thodeti C.K.
      • Adolfsson J.
      • Juhas M.
      • Sjölander A.
      ,
      • Paruchuri S.
      • Hallberg B.
      • Juhas M.
      • Larsson C.
      • Sjolander A.
      ,
      • Hoshino M.
      • Izumi T.
      • Shimizu T.
      ). This receptor has also previously been shown to mediate the activation of the small RhoA GTPase via a PTX-insensitive signaling pathway (
      • Thodeti C.K.
      • Adolfsson J.
      • Juhas M.
      • Sjölander A.
      ). In contrast, here we show that LTD4-triggered activation of the small GTPase Rac was inhibited by PTX similar to the LTD4-induced increase in survival and proliferation of intestinal epithelial cells (
      • Paruchuri S.
      • Hallberg B.
      • Juhas M.
      • Larsson C.
      • Sjolander A.
      ,
      • Paruchuri S.
      • Sjölander A.
      ). Investigation of leukotriene-mediated activation of Rac in other cell types supports the present observation. In HL-60 cells, LTB4-induced Rac activation and chemotaxis were blocked by pretreatment with PTX (
      • Yokomizo T.
      • Izumi T.
      • Chang K.
      • Takuwa Y.
      • Shimizu T.
      ), as was LTD4-mediated Rac activation in THP-1 cells (
      • Hoshino M.
      • Izumi T.
      • Shimizu T.
      ). The notion that two different signaling pathways regulate the activity of RhoA and Rac in intestinal epithelial cell probably provides the CysLT1-receptor with a more sophisticated control of epithelial cell motility. Subsequent to these findings, we then sought to identify the signaling molecules downstream of CysLT1-receptor activation of a PTX-sensitive heterotrimeric G protein that could be implicated in the activation of Rac. One obvious candidate to look for is the PI3K. Indeed, it has been reported that lipids generated by the PI3K can participate in the regulation of small G proteins as well as cell motility (
      • Han J.
      • Luby-Phelps K.
      • Das B.
      • Shu X.
      • Xia Y.
      • Mosteller R.D.
      • Krishna U.M.
      • Falck J.R.
      • White M.A.
      • Broek D.
      ,
      • Marcoux N.
      • Vuori K.
      ). We not only found that LTD4 induced tyrosine phosphorylation of the p85 subunit of PI3K, which was used here as an index of its activation, but that the time kinetics of LTD4-induced phosphorylation of the p85 subunit of PI3K was also parallel to the activation of Rac in intestinal epithelial cells. To further investigate the possibility that PI3K has a role in the LTD4-induced activation of Rac, we blocked the PI3K activity by different means and tested the effects of such treatments on the Rac activity. We found that pretreatment of Int 407 cells either with wortmannin or LY294002, two structurally unrelated PI3K inhibitors, and also transfection of these cells with a DN p85 construct halted the LTD4-induced activation of Rac. The fact that we also found that LTD4 had the ability to cause activation of Akt, a well known downstream target of PI3K, further supports our conclusion that LTD4 causes the activation of PI3K in intestinal epithelial Int 407 cells. In keeping with these results, a requirement of PI3K activity for the agonist-induced activation of Rac has also been reported in other cell systems (
      • Royal I.
      • Lamarche-Vane N.
      • Lamorte L.
      • Kaibuchi K.
      • Park M.
      ). It is likely that the PI3K dependence in these experiments is related to the fact that the PI3K-generated phosphatidylinositol 3,4,5-trisphosphate lipid enables the activation of a number of Rac-specific GEFs (
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.C.
      ). Vav2 seems to be the GEF involved in the activation of Rac by LTD4, because not only does it contain a PH domain and is therefore a target of the PI3K lipid product, we also found that Vav2 was tyrosinephosphorylated, indicative of its activation. In addition, the LTD4-induced activation of Rac was totally blocked in intestinal epithelial 407 cells transfected with a dominant-negative form of Vav2.
      In this study, we found that stimulation of Int 407 cells with LTD4 led to the formation of circular membrane ruffles rather than the classical Rac-induced lamellipodia (
      • Mohri T.
      • Adachi Y.
      • Ikehara S.
      • Hioki K.
      • Tokunaga R.
      • Taketani S.
      ). This observation was somehow surprising, because circular membrane ruffle formation has mainly been described in response to different growth factors and we have previously shown that stimulation of Int 407 cells with LTD4 does not result in a transactivation of either EGF or platelet-derived growth factor receptors (
      • Massoumi R.
      • Nielsen C.K.
      • Azemovic D.
      • Sjölander A.
      ). Although we cannot totally rule out the possibility of a cross-talk between the Cyst-LT1-receptor and alternative growth factor receptors, we do believe that our present results reflect a direct effect of the Cys-LT1-receptor on the cytoskeleton. Several findings support the view that the activation of PI3K and its downstream effector Rac mediate the LTD4-stimulated membrane ruffling in Int 407 cells. First, decreased formation of membrane ruffles was observed in LTD4-stimulated Int 407 cells pretreated with LY294002 or wortmannin as well as in cells transiently transfected with a dominant-negative (N17) mutant of Rac. Secondly, we observed that Rac redistributed to a membrane-enriched fraction upon stimulation of the cells with LTD4. Thirdly, we found that Rac co-localized with filamentous actin in the membrane ruffles of cells stimulated with LTD4. Furthermore, our conclusion is also indirectly supported by the observation that expression of constitutively active Rac1 results in the formation of membrane ruffles in colon cancer cells (
      • Mohri T.
      • Adachi Y.
      • Ikehara S.
      • Hioki K.
      • Tokunaga R.
      • Taketani S.
      ). Furthermore, microinjection of dominant-negative Rac1 constructs into epithelial cells (
      • Fenteany G.
      • Janmey P.A.
      • Stossel T.P.
      ) or rat embryo fibroblasts (
      • Nobes C.D.
      • Hall A.
      ) has been shown to inhibit both the formation of lamellipodia and migration of cells into the wound. Activation of distinct Rho family proteins constitutes key signaling events that link ligation of membrane receptors to cytoskeletal reorganization. For the first time, we show here that stimulation of human intestinal epithelial cells with LTD4 leads to a transient and time-dependent activation of Rac but not Cdc42. This finding is similar to the observation in vascular smooth muscle cells where angiotensin and thrombin stimulation have been shown to result in the activation of Rac but not Cdc42 (
      • Pelletier S.
      • Duhamel F.
      • Coulombe P.
      • Popoff M.R.
      • Meloche S.
      ). In contrast, the majority of agonists that cause activation of Rac also cause a simultaneous activation of Cdc42 in their target cells (
      • Benard V.
      • Bohl B.P.
      • Bokoch G.M.
      ,
      • Paik J.H.
      • Chae S.S.
      • Lee M.J.
      • Thangada S.
      • Hla T.
      ). In the present study, we could clearly show that the LTD4-induced migratory response in intestinal epithelial Int 407 cells was significantly impaired by PTX, LY294002, and wortmannin, all of which have been shown to also block the LTD4-mediated activation of Rac. Taken together, our results support the notion that LTD4 generates the formation of circular membrane ruffles via a PI3K and Rac signaling pathway and that the formation of these ruffles serves as an important driving force for the LTD4-induced migration of intestinal epithelial cells.
      Cell migration is central to several important biological processes including tissue morphogenesis, wound healing, and cancer cell metastasis (). Our previous results demonstrating that the pro-inflammatory mediator LTD4 can induce significant increases in proliferation and survival (
      • Smalley W.E.
      • Du Bois R.N.
      ) can now be extended by the present finding that it also increases the motility of these cells, all hallmarks of tumor cell behavior. Consequently, the present results add further support for an important role of LTD4 as a link between inflammatory bowel diseases and subsequent development of colon cancer.

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