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• W2061293090 abstract "Iba1 is a macrophage/microglia-specific calcium-binding protein that is involved in RacGTPase-dependent membrane ruffling and phagocytosis. In this study, we introduced Iba1 into Swiss 3T3 fibroblasts and demonstrated the enhancement of platelet-derived growth factor (PDGF)-induced membrane ruffling and chemotaxis. Wortmannin treatment did not completely suppressed this enhanced membrane ruffling in Iba1-expressing cells, whereas it did in Iba1-nonexpressing cells, suggesting that the enhancement is mediated through a phosphatidylinositol 3-kinase (PI3K)-independent signaling pathway. Porcine aorta endothelial cells transfected with expression constructs of Iba1 and PDGF receptor add-back mutants were used to analyze the signaling pathway responsible for the Iba1-induced enhancement of membrane ruffling. In the absence of Iba1 expression, PDGF did not induced membrane ruffling in cells expressing the Tyr-1021 receptor mutant, which is capable of activating phospholipase C-γ (PLC-γ) but not PI3K. In contrast, in the presence of Iba1 expression, membrane ruffling was formed in cells expressing the Tyr-1021 mutant. In addition, Rac was shown to be activated during membrane ruffling in cells expressing Iba1 and the Tyr-1021 mutant. Furthermore, dominant negative forms of PLC-γ completely suppressed PDGF-induced Iba1-dependent membrane ruffling and Rac activation. These results indicate the existence of a novel signaling pathway where PLC-γ activates Rac in a manner dependent on Iba1. Iba1 is a macrophage/microglia-specific calcium-binding protein that is involved in RacGTPase-dependent membrane ruffling and phagocytosis. In this study, we introduced Iba1 into Swiss 3T3 fibroblasts and demonstrated the enhancement of platelet-derived growth factor (PDGF)-induced membrane ruffling and chemotaxis. Wortmannin treatment did not completely suppressed this enhanced membrane ruffling in Iba1-expressing cells, whereas it did in Iba1-nonexpressing cells, suggesting that the enhancement is mediated through a phosphatidylinositol 3-kinase (PI3K)-independent signaling pathway. Porcine aorta endothelial cells transfected with expression constructs of Iba1 and PDGF receptor add-back mutants were used to analyze the signaling pathway responsible for the Iba1-induced enhancement of membrane ruffling. In the absence of Iba1 expression, PDGF did not induced membrane ruffling in cells expressing the Tyr-1021 receptor mutant, which is capable of activating phospholipase C-γ (PLC-γ) but not PI3K. In contrast, in the presence of Iba1 expression, membrane ruffling was formed in cells expressing the Tyr-1021 mutant. In addition, Rac was shown to be activated during membrane ruffling in cells expressing Iba1 and the Tyr-1021 mutant. Furthermore, dominant negative forms of PLC-γ completely suppressed PDGF-induced Iba1-dependent membrane ruffling and Rac activation. These results indicate the existence of a novel signaling pathway where PLC-γ activates Rac in a manner dependent on Iba1. Cell motility is a dynamic process driven by structurally and functionally coordinated reorganization of the actin cytoskeleton (1Stossel T.P. Science. 1993; 260: 1086-1094Crossref PubMed Scopus (905) Google Scholar,2Mitchison T.J. Cramer L.P. Cell. 1996; 84: 371-379Abstract Full Text Full Text PDF PubMed Scopus (1303) Google Scholar). Among various types of cells, macrophages are extremely motile to migrate rapidly to sites of infection or inflammation, suggesting that highly integrated systems should exist to regulate the actin cytoskeleton in macrophages (3Hartwig J.H. Yin H.L. Cell Motil. Cytoskeleton. 1988; 10: 117-125Crossref PubMed Scopus (35) Google Scholar, 4Downey G.P. Curr. Opin. Immunol. 1994; 6: 113-124Crossref PubMed Scopus (127) Google Scholar). In addition to circulating monocytes/macrophages, there are many types of tissue-resident macrophages, including Langerhans cells, Kupffer cells, dendritic cells, splenocytes, and microglia. In response to various pathological phenomena, microglia are activated to exhibit drastic changes in shape and the abilities to become locomotive and to phagocytose (5Nakajima K. Kohsaka S. Neurosci. Res. 1993; 7: 187-203Crossref Scopus (192) Google Scholar, 6Kreutzberg G.W. Trends Neurosci. 1996; 19: 312-318Abstract Full Text Full Text PDF PubMed Scopus (3723) Google Scholar). These cellular reactions are also profoundly underlaid by dynamic remodeling of the actin cytoskeleton. The Rho family GTPases, Cdc42, Rac, and Rho, are known to be molecular switches that organize remodeling of the actin cytoskeleton (7Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5200) Google Scholar). Among them, in fibroblasts, Rac is activated by receptor tyrosine kinases such as platelet-derived growth factor receptor (PDGFR), 1The abbreviations used are: PDGFRplatelet-derived growth factor receptorM-CSFmacrophage colony-stimulating factorPAEporcine aorta endothelialCHOChinese hamster ovaryPI3Kphosphatidylinositol-3 kinasePLCphospholipase CFCSfetal bovine serumWTwild typeGSTglutathione S-transferaseDMEMDulbecco's modified Eagle's mediumHRPhorseradish peroxidaseLPAlysophosphatidic acidPBSphosphate-buffered salineBSAbovine serum albuminFITCfluorescein isothiocyanatePKCprotein kinase C[Ca2+]iintracellular calciumPAKp21-activated kinase1The abbreviations used are: PDGFRplatelet-derived growth factor receptorM-CSFmacrophage colony-stimulating factorPAEporcine aorta endothelialCHOChinese hamster ovaryPI3Kphosphatidylinositol-3 kinasePLCphospholipase CFCSfetal bovine serumWTwild typeGSTglutathione S-transferaseDMEMDulbecco's modified Eagle's mediumHRPhorseradish peroxidaseLPAlysophosphatidic acidPBSphosphate-buffered salineBSAbovine serum albuminFITCfluorescein isothiocyanatePKCprotein kinase C[Ca2+]iintracellular calciumPAKp21-activated kinase leading to the formation of lamellipodia and membrane ruffles (8Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3066) Google Scholar). Dominant active RacV12 induces remarkable membrane ruffling, and dominant negative RacN17 completely inhibits peptide growth factor-induced membrane ruffling; therefore, Rac is recognized to be an essential component in this type of membrane ruffling (8Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3066) Google Scholar). Some studies describe signaling molecules capable of interacting with Rac; however, the processes by which receptor tyrosine kinases activate Rac are not fully understood. platelet-derived growth factor receptor macrophage colony-stimulating factor porcine aorta endothelial Chinese hamster ovary phosphatidylinositol-3 kinase phospholipase C fetal bovine serum wild type glutathione S-transferase Dulbecco's modified Eagle's medium horseradish peroxidase lysophosphatidic acid phosphate-buffered saline bovine serum albumin fluorescein isothiocyanate protein kinase C intracellular calcium p21-activated kinase platelet-derived growth factor receptor macrophage colony-stimulating factor porcine aorta endothelial Chinese hamster ovary phosphatidylinositol-3 kinase phospholipase C fetal bovine serum wild type glutathione S-transferase Dulbecco's modified Eagle's medium horseradish peroxidase lysophosphatidic acid phosphate-buffered saline bovine serum albumin fluorescein isothiocyanate protein kinase C intracellular calcium p21-activated kinase Previously, we identified a calcium-binding protein, Iba1, which is restrictedly expressed in macrophages/microglia (9Imai Y. Ibata I. Ito D. Ohsawa K. Kohsaka S. Biochem. Biophys. Res. Commun. 1996; 224: 855-862Crossref PubMed Scopus (670) Google Scholar), and showed that the expression of Iba1 is up-regulated in activated microglia following facial nerve axotomy (10Ito D. Imai Y. Ohsawa K. Nakajima K. Fukuuchi Y. Kohsaka S. Brain Res. Mol. Brain Res. 1998; 57: 1-9Crossref PubMed Scopus (1044) Google Scholar). In our recent study, Iba1 was further characterized by using a microglial cell line MG5 (11Ohsawa K. Imai Y. Nakajima K. Kohsaka S. Glia. 1997; 21: 285-298Crossref PubMed Scopus (71) Google Scholar) and loss of function Iba1 mutants, and it was demonstrated that mutant Iba1 effectively suppresses the membrane ruffling produced by stimulation with macrophage colony-stimulating factor (M-CSF) or by expression of dominant active RacV12 (12Ohsawa K. Imai Y. Kanazawa H. Sasaki Y. Kohsaka S. J. Cell Sci. 2000; 113: 3073-3084PubMed Google Scholar). These observations suggested that Iba1 was involved in the molecular basis of membrane ruffling of macrophages/microglia and interacted with the signaling of Rac, which is a key molecule in controlling membrane ruffling also in macrophages (13Allen W.E. Jones G.E. Pollard J.W. Ridley A.J. J. Cell Sci. 1997; 110: 707-720Crossref PubMed Google Scholar). Iba1 is therefore considered to be one of the candidate molecules underlying the extremely motile property of macrophages/microglia. In this study, to address this hypothesis, we introduced Iba1 in Swiss 3T3 fibroblasts, porcine aorta endothelial (PAE) cells, and Chinese hamster ovary (CHO) cells, none of which expresses endogenous Iba1, and examined the formation of membrane ruffles, chemotaxis, and profiles of intracellular signaling molecules, including PDGFR, phosphatidylinositol-3 kinase (PI3K), phospholipase C-γ (PLC-γ), and Rac. Swiss 3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). Mouse iba1 cDNA (9Imai Y. Ibata I. Ito D. Ohsawa K. Kohsaka S. Biochem. Biophys. Res. Commun. 1996; 224: 855-862Crossref PubMed Scopus (670) Google Scholar) was inserted into the tetracycline-regulated expression vector pTet-Splice (Invitrogen) to construct pTet-iba1. The cells were transfected with pTet-iba1, transactivator pTet-tTAK, and pSV2-neo by calcium phosphate coprecipitation, and stably transfected clones were isolated by selection with 400 μg/ml G418 (Invitrogen). PAE cells (14Westermark B. Siegbahn A. Heldin C.-H. Claesson-Welsh L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 128-132Crossref PubMed Scopus (132) Google Scholar), kindly provided by Dr. C.-H. Heldin (Ludwig Institute for Cancer Research, Sweden) and Dr. Koutaro Yokote (Chiba University, Japan) were cultured in Ham's F12 medium (Invitrogen) supplemented with 10% FCS. pLXSN plasmids carrying wild type (WT) and a series of mutant human β-PDGFRs (15Valius M. Kazlauskas A. Cell. 1993; 73: 321-334Abstract Full Text PDF PubMed Scopus (570) Google Scholar) were kindly provided by Dr. A. Kazlauskas (Schepens Eye Research Institute, Harvard Medical School, Boston, MA). F5 mutant PDGFR, which was constructed by the substitution of phenylalanines for five tyrosine residues that are required for the binding of PI3K, RasGAP, SHP-2, and PLC-γ1, is unable to associate with any of these proteins. Add-back mutants of PDGFR were generated by restoring tyrosine residues at individual binding sites for each of the receptor-associated proteins (15Valius M. Kazlauskas A. Cell. 1993; 73: 321-334Abstract Full Text PDF PubMed Scopus (570) Google Scholar). PAE cells were transfected with the tetracycline-regulated Iba1-expressing system and cloned as described above. Subsequently, Iba1-expressing cells were transfected with WT PDGFR or the add-back series of PDGFR mutants by the FuGENE6 transfection reagent (Roche Molecular Biochemicals, Germany) and selected by 5 μg/ml of blasticidin S (Funakoshi, Japan). PAE transfectants were incubated in Ham's F12 containing 0.5% FCS for 8 h before microinjection of 0.6 μg/μl pFLAG-CMV2 carrying WT and Y771F/Y783F plc-γ1 cDNAs, which were kindly provided by Dr. P.-G. Suh (16Kim H.K. Kim J.W. Zilberstein A. Margolis B. Kim J.G. Schlessinger J. Rhee S.G. Cell. 1991; 65: 435-441Abstract Full Text PDF PubMed Scopus (444) Google Scholar) (Pohang University of Science and Technology, Korea). Injected PAE cells were maintained for 3 h at 37 °C to induce protein expression. Expression plasmids for glutathione S-transferase (GST)-PLC-γ1–2SH2 and GST-PI3-K SH2 (N) (17Homma Y. Takenawa T. J. Biol. Chem. 1992; 267: 21844-21849Abstract Full Text PDF PubMed Google Scholar) were kindly provided by Dr. T. Takenawa and Dr. K. Fukami (Institute of Medical Science, University of Tokyo, Japan). The purified GST fusion proteins were microinjected into the cytosol and incubated for 10 min at 37 °C. The cells were then stimulated with PDGF (50 ng/ml) for 5 min. CHO cells were maintained in RPMI 1640/Ham's F12/DMEM (2:1:1) medium supplemented with 10% FCS. CHO cells were transiently transfected using LipofectAMINE Plus reagent (Invitrogen) with pLXSN carrying WT PDGFR, pFLAG-CMV2 carrying WT or mutant plc-γ1, and pEGFP-C1 (CLONTECH, Palo Alto, CA) carrying WT-iba1 or mutant iba1-(1–115) (12Ohsawa K. Imai Y. Kanazawa H. Sasaki Y. Kohsaka S. J. Cell Sci. 2000; 113: 3073-3084PubMed Google Scholar). A microglial cell line, MG5, was maintained as described previously (12Ohsawa K. Imai Y. Kanazawa H. Sasaki Y. Kohsaka S. J. Cell Sci. 2000; 113: 3073-3084PubMed Google Scholar). Cells were lysed in radioimmune precipitation buffer containing 20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 10% glycerol, 5 mm EDTA, 1% Triton-X100, 1% sodium deoxycholate, 0.1% SDS, 1 mmphenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 20 μm leupeptin, 1 mm sodium vanadate, and 10 μm pepstatin. The lysate was clarified by centrifugation, normalized for protein concentration, and subjected to SDS-PAGE. Separated proteins were electrotransferred to an Immobilon (Millipore, MA) membrane, which was then blocked with 25 mm Tris-HCl, pH 7.5, containing 125 mm NaCl, 0.1% Tween 20, and 4% skim milk. The membrane was incubated with anti-Iba1 antibody (1 μg/ml) (12Ohsawa K. Imai Y. Kanazawa H. Sasaki Y. Kohsaka S. J. Cell Sci. 2000; 113: 3073-3084PubMed Google Scholar), then subsequently with a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (1:1500 dilution) (Bio-Rad, CA), and visualized using an ECL Western blotting detection system (Amersham Biosciences, Inc., UK). Swiss 3T3 or PAE transfectants (1 × 104) were plated on a 13-mm poly-d-lysine (Sigma Chemical Co., St. Louis, MO)-coated glass coverslip, cultured for 2 days, then serum-starved for 13 h in DMEM without FCS (Swiss 3T3) or in Ham's F12 with 0.5% FCS (PAE) in the presence or absence of 0.5 μg/ml tetracycline. The cells were stimulated with human PDGF-BB (UBI, NY), bradykinin (Sigma), or lysophosphatidic acid (LPA) (Sigma), then fixed for 60 min at room temperature with 3% paraformaldehyde in phosphate-buffered saline (PBS). The cells were treated for 5 min with PBS containing 1 mg/ml sodium borohydride, permeabilized for 20 min with PBS containing 0.1% Triton-X100, and blocked for 2 h with PBS containing 3% normal goat serum and 3% bovine serum albumin (BSA) (blocking buffer). The cells were incubated for 13 h at 4 °C in blocking buffer containing 8 μg/ml rabbit anti-Iba1 antibody, then washed with PBS and incubated for 2 h in blocking buffer containing 5 μg/ml fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibody (BioSource, Camarillo, CA) and 6 units/ml Texas Red-conjugated phalloidin (Molecular Probes, Eugene, OR). The cells were then observed with fluorescence microscope AX70 (Olympus, Japan) or confocal laser scanning microscope CLSM2010 (Amersham Biosciences, Inc.). Cell migration was assayed by a modified Boyden chamber method (18Yokote K. Mori S. Siegbahn A. Rönnstrand L. Wernstedt C. Heldin C.-H. Claesson-Welsh L. J. Biol. Chem. 1996; 271: 5101-5111Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) using a 96-well chemotaxis chamber (Neuro Probe, Cabin John, MD). The lower wells that were filled with 25 μl of DMEM containing 1% BSA and PDGF were overlaid with an 8-μm pore polycarbonate filter (Neuro Probe) pre-coated with 100 μg/ml type I collagen (Cohesion, Palo Alto, CA). Above the membrane, the upper wells were set and loaded with 50 μl of DMEM with 1% BSA containing 3 × 105 trypsin-dispersed cells per milliliter. The chamber was then incubated at 37 °C for 8 h. The cells on the upper side of the filter were scraped off, and the cells that had migrated to the lower side of the filter were fixed and stained in PBS containing 12% formaldehyde, 10% ethanol, and 0.05% crystal violet. Cell numbers were counted in four fields per well. Values are means of triplicate experiments. The PAK pull-down assay was performed mainly as described previously (12Ohsawa K. Imai Y. Kanazawa H. Sasaki Y. Kohsaka S. J. Cell Sci. 2000; 113: 3073-3084PubMed Google Scholar, 19Manser E. Loo T.-H. Koh C.-G. Zhao Z.-S. Chen X.-Q. Tan L. Tan I. Leung T. Lim L. Mol. Cell. 1998; 1: 183-192Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar). Cells (106) were lysed in 500 μl of Hepes-buffered saline (25 mm Hepes-NaOH, pH 7.3, 150 mm NaCl, 5 mm MgCl2, 0.5 mm EGTA, 20 mm β-glycerophosphate, 0.5% Triton-X100, 4% glycerol, 10 mm NaF, 2 mm sodium vanadate, 5 mm phenylmethylsulfonyl fluoride, 10 μmleupeptin, 10 μm pepstatin, and 0.5 mmdithiothreitol). The lysates were cleared by centrifugation, incubated with GST-PAK fusion protein (12Ohsawa K. Imai Y. Kanazawa H. Sasaki Y. Kohsaka S. J. Cell Sci. 2000; 113: 3073-3084PubMed Google Scholar, 19Manser E. Loo T.-H. Koh C.-G. Zhao Z.-S. Chen X.-Q. Tan L. Tan I. Leung T. Lim L. Mol. Cell. 1998; 1: 183-192Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar) and glutathione-Sepharose 4B (Amersham Biosciences, Inc.) for 30 min at 4 °C, and subsequently washed in Hepes-buffered saline. Bound activated Rac was visualized by Western blotting with an anti-Rac1 antibody (0.5 μg/ml) and HRP-conjugated anti-mouse goat IgG (1:1500 dilution) using the ECL system. Serum-starved quiescent PAE and M-CSF-starved MG5 cells were stimulated with 50 ng/ml PDGF and 100 ng/ml mouse M-CSF (R&D Systems, Minneapolis, MN), respectively. The cells were lysed in radioimmune precipitation buffer at 4 °C for 20 min. Insoluble material was removed by centrifugation, and the cell lysates were normalized for protein concentration before immunoprecipitation. The lysates were incubated with 1.7 μg/ml anti-PLC-γ1 (UBI) or anti-PLC-γ2 (Santa Cruz Biotechnology, Santa Cruz, CA) antibody and with protein G-Sepharose beads (AmershamBiosciences, Inc.). The precipitated proteins were then subjected to Western blotting with an anti-phosphotyrosine antibody, 4G10 (Seikagaku, Japan), and HRP-conjugated anti-mouse goat IgG (AmershamBiosciences, Inc.) using the ECL system. In this study, to analyze the functions of intact Iba1, we transfected a tetracycline-inducible Iba1-expression construct into Swiss 3T3, a fibroblast cell line expressing no endogenous Iba1. As a result, we established five clones of stable transfectants exhibiting inducible Iba1 expression. Immunoblotting with the anti-Iba1 antibody demonstrated that the expression of Iba1 was tightly inhibited under the presence of tetracycline whereas 13 or 22 h after the removal of tetracycline, strong expression of Iba1 was induced (Fig. 1A). In Swiss 3T3 cells, bradykinin, LPA, and PDGF are known to specifically activate Cdc42, Rho, and Rac, respectively, and lead the cells to form filopodia, stress fibers, and membrane ruffles (7Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5200) Google Scholar). To examine the effects of Iba1 on these structures, the Iba1-inducible cells were serum-starved, stimulated with bradykinin, LPA, and PDGF, and stained with phalloidin to visualize the actin cytoskeleton. When Iba1 expression was suppressed in the presence of tetracycline, the cells formed filopodia, stress fibers, and membrane ruffles in response to bradykinin, LPA, and PDGF, respectively (Fig. 1B), as reported for parent Swiss 3T3 cells (8Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3066) Google Scholar, 20Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3809) Google Scholar, 21Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (881) Google Scholar, 22Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3714) Google Scholar). When Iba1 expression was induced by tetracycline removal, the cells also formed filopodia and stress fibers indistinguishable from those shown in the absence of Iba1 expression after stimulation with bradykinin and LPA (Fig. 1B). By contrast, in response to PDGF, the Iba1-expressing cells formed apparently enhanced membrane ruffles in comparison with the Iba1-nonexpressing cells (Fig. 1B). When the cells were doubly stained with phalloidin and the anti-Iba1 antibody after PDGF stimulation, Iba1 was shown to be localized at the sites of membrane ruffles, together with F-actin (Fig. 1C), but Iba1 did not colocalize with F-actin in filopodia or stress fibers induced by bradykinin or LPA stimulation (data not shown). All other clones of the transfectants exhibited similar enhanced membrane ruffling (data not shown), indicating that Iba1 definitely enhances PDGF-dependent membrane ruffling in Swiss 3T3 transfectants. Because membrane ruffling is considered to be related to cell motility (23Sánchez-Madrid F. del Pozo M.A. EMBO J. 1999; 18: 501-511Crossref PubMed Scopus (519) Google Scholar), we determined the chemotaxis of Iba1-expressing cells by the Boyden chamber method (18Yokote K. Mori S. Siegbahn A. Rönnstrand L. Wernstedt C. Heldin C.-H. Claesson-Welsh L. J. Biol. Chem. 1996; 271: 5101-5111Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) using PDGF as a chemoattractant. As shown in Fig. 2, Swiss 3T3 parent cells and the Iba1-nonexpressing transfectants showed similar motile responses toward PDGF in a dose-dependent manner, whereas the Iba1-expressing cells exhibited about a 2-fold increase in chemotactic response. Similar results were obtained in all clones of Iba1 transfectants. Tetracycline itself had no effect on PDGF-induced migration in Swiss 3T3 cells (data not shown). These results indicate that Iba1 is also able to enhance the chemotaxis of Swiss 3T3 cells. The PI3K signaling pathway is reported to be necessary for PDGF-induced membrane ruffling of Swiss 3T3 cells (24Nobes C.D. Hawkins P. Stephens L. Hall A. J. Cell Sci. 1995; 108: 225-233Crossref PubMed Google Scholar). To investigate whether this pathway is also required for the Iba1-dependent enhancement of membrane ruffling, the effect of PI3K inhibitors, wortmannin and LY294002, on PDGF-induced membrane ruffling was examined in both Iba1-nonexpressing and -expressing cells. Without treatment with the inhibitors, both transfectants formed membrane ruffles as a result of PDGF stimulation, but the extent of ruffle formation was greater in Iba1-expressing cells than in Iba1-nonexpressing cells (Fig. 3). With the wortmannin treatment, membrane ruffling of Iba1-nonexpressing transfectants was completely abolished, indicating that the formation of membrane ruffles of Iba1-nonexpressing Swiss 3T3 cells depends totally on the PI3K signaling pathway. By contrast, the Iba1-expressing cells formed obvious membrane ruffles even after wortmannin treatment, indicating that membrane ruffling of Iba1-expressing cells does depend on a certain signaling pathway in addition to PI3K. The same results were obtained using another PI3K inhibitor, LY294002 (Fig. 3). These observations led us to speculate that the enhanced membrane ruffling associated with Iba1 is transduced by a PI3K-independent pathway. To elucidate the possibility that Iba1 is involved in the PI3K-independent signaling pathway, we utilized the add-back mutants of PDGFR, which were transfected into PAE cells lacking endogenous PDGFR (14Westermark B. Siegbahn A. Heldin C.-H. Claesson-Welsh L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 128-132Crossref PubMed Scopus (132) Google Scholar). PDGFR associates with various signaling molecules via its autophosphorylated tyrosines. PI3K selectively targets tyrosine at amino acid positions 740 (Tyr-740) and Tyr-751, whereas RasGAP, SHP-2, and PLC-γ recognize Tyr-771, Tyr-1009, and Tyr-1021, respectively. These signaling molecules are unable to bind to the PDGFR F5 mutant, in which all of the five tyrosines were replaced by phenylalanines (15Valius M. Kazlauskas A. Cell. 1993; 73: 321-334Abstract Full Text PDF PubMed Scopus (570) Google Scholar). PAE cells, which did not express Iba1, were co-transfected with the Iba1-inducible construct and PDGFR mutant-expression vectors. Similar expression levels were seen for all the PDGFRs in the stable transfectants, as measured by fluorescence-activate cell sorting analysis (data not shown). Without stimulation with PDGF, the morphology of the cells was identical in the presence and absence of Iba1 expression (data not shown). When the Iba1-nonexpressing cells were stimulated with PDGF, obvious membrane ruffles were formed in the cells co-transfected with WT PDGFR receptor or Y740/51 mutant, which is capable of PI3K activation, in agreement with a previous report (25Wennström S. Siegbahn A. Yokote K. Arvidsson A.-K. Heldin C.-H. Mori S. Claesson-Welsh L. Oncogene. 1994; 9: 651-660PubMed Google Scholar). In contrast, cells expressing F5 or Tyr-1021 receptor did not respond to PDGF (Fig. 4). These observations indicate the necessity of PI3K signaling for PDGF-induced membrane ruffling in the absence of Iba1. On the other hand, after the induction of Iba1, apparent membrane ruffles were formed in the cells transfected with Tyr-1021 mutant, capable of activation of PLC-γ. Membrane ruffling was also detected in the Iba1-expressing cells with the WT or Y740/51 mutant, as in the Iba1-nonexpressing cells (Fig. 4). Cells expressing Tyr-771, Tyr-1009, or kinase inactive receptor (Arg-635) did not show PDGF-induced membrane ruffling regardless of Iba1 expression (data not shown). These observations strongly suggest that PLC-γ is the key signaling molecule in Iba1-dependent and wortmannin-resistant membrane ruffling. Iba1 was recently demonstrated to function together with Rac in the membrane ruffling of microglia, and Rac was shown to be activated during their membrane ruffling (12Ohsawa K. Imai Y. Kanazawa H. Sasaki Y. Kohsaka S. J. Cell Sci. 2000; 113: 3073-3084PubMed Google Scholar). To investigate whether Rac is also activated in Iba1- and PLC-γ-dependent membrane ruffling, the activation of Rac was monitored by pull-down assay with the Cdc42/Rac interactive binding domain of PAK (19Manser E. Loo T.-H. Koh C.-G. Zhao Z.-S. Chen X.-Q. Tan L. Tan I. Leung T. Lim L. Mol. Cell. 1998; 1: 183-192Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar) using PAE transfectants. In the absence of Iba1, PDGF stimulation efficiently converted Rac into the GTP-bound form in the cells expressing WT and the Y740/51 mutant but not in the cells expressing the Tyr-1021 (Fig. 5A), Tyr-771, or Tyr-1009 (data not shown) mutant. All lysates contained equal amounts of total Rac. These results indicate that Rac was activated through the PI3K-dependent pathway in the absence of Iba1. However, in the presence of Iba1, in addition to WT- or Y740/51-expressing cells, Tyr-1021-expressing cells also showed Rac activation in response to PDGF (Fig. 5B). These observations indicate the existence of an Iba1- and PLC-γ-dependent Rac-activating pathway that triggers the formation of membrane ruffles. To confirm the involvement of PLC-γ in Iba1-dependent and PI3K-independent Rac activation, we investigated the effects of PLC-γ mutants that act as dominant negative forms against endogenous PLC-γ. PLC-γ1-Y771F/Y783F had phenylalanines substituted for tyrosines 771 and 783 and obtained the ability to specifically suppress the activity of endogenous PLC-γ1 (16Kim H.K. Kim J.W. Zilberstein A. Margolis B. Kim J.G. Schlessinger J. Rhee S.G. Cell. 1991; 65: 435-441Abstract Full Text PDF PubMed Scopus (444) Google Scholar). FLAG-tagged PLC-γ1 WT or Y771F/Y783F mutant was expressed in the PAE cells expressing both PDGFR Tyr-1021 and Iba1. Subsequently, the cells were stimulated with PDGF and stained with phalloidin. Typical membrane ruffles were formed in WT PLC-γ-expressing cells that were located by the marker signal of co-expressed EGFP (Fig. 6A, upper panel). By contrast, PDGF-induced membrane ruffling was inhibited in PLC-γ1-Y771F/Y783F-expressing cells (Fig. 6A, lower panel). When the PLC-γ1-expressing cells were located with an anti-FLAG antibody, the cells that were recognized corresponded perfectly to the EGFP-expressing cells (data not shown). GST-PLC-γ1–2SH2 is a fusion protein of GST and a PLC-γ1 fragment containing two SH2 domains but no catalytic domain and is able to specifically inhibit PLC-γ1 signaling, whereas GST-PI3K SH2 (N) contains the PI3K N-terminal SH2 domain but does not suppress PLC-γ signaling (17Homma Y. Takenawa T. J. Biol. Chem. 1992; 267: 21844-21849Abstract Full Text PDF PubMed Google Scholar). Into the PAE cells expressing both PDGFR Tyr-1021 and Iba1, we microinjected GST-PLC-γ1–2SH2 or GST-PI3K SH2(N) fusion protein together with FITC-conjugated dextran to mark the cells that were injected. The cells injected with GST-PLC-γ1–2SH2 fusion protein were stimulated with PDGF and stained with phalloidin. PDGF-induced membrane ruffling was inhibited in the GST-PLC-γ1–2SH2- injected cells, whereas the cells injected with GST-PI3K SH2 (N) were not inhibited. Next we investigated whether PLC-γ1-Y771F/Y783F mutant blocked Iba1-dependent Rac activation. We induced the transient expression of WT PDGFR, and WT or mutant Iba1 and PLC-γ1 in CHO cells, which lacked PDGFR expression (26Ma Y.-H. Reusch H.P. Wilson E. Escobedo J.A. Fantl W.J. Williams L.T. Ives H.E. J. Biol. Chem. 1994; 269: 30734-30739Abstract Full Text PDF PubMed Google Scholar), and then exam" @default.
• W2061293090 created "2016-06-24" @default.
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• W2061293090 date "2002-05-01" @default.
• W2061293090 modified "2023-10-10" @default.
• W2061293090 title "Macrophage/Microglia-specific Protein Iba1 Enhances Membrane Ruffling and Rac Activation via Phospholipase C-γ-dependent Pathway" @default.
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• W2061293090 doi "https://doi.org/10.1074/jbc.m109218200" @default.
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