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Phosphatidylinositol 3-Kinase-independent Signal Transduction Pathway for Platelet-derived Growth Factor-induced Chemotaxis*

  • Masahide Higaki
    Affiliations
    National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565, Japan and the
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  • Hiroshi Sakaue
    Affiliations
    Second Department of Internal Medicine, Kobe University Medical School, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan
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  • Wataru Ogawa
    Affiliations
    Second Department of Internal Medicine, Kobe University Medical School, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan
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  • Masato Kasuga
    Affiliations
    Second Department of Internal Medicine, Kobe University Medical School, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan
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  • Kentaro Shimokado
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565, Japan and the
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  • Author Footnotes
    * This study was supported by grants from the Science and Technology Agency, the Ministry of Education, Science, and Culture, and Japan Cardiovascular Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:November 15, 1996DOI:https://doi.org/10.1074/jbc.271.46.29342
      Platelet-derived growth factor (PDGF)-BB is a potent chemoattractant for mesenchymal cells. Intracellular signal transduction for PDGF-induced chemotactic response has been reported to be dependent on phosphatidylinositol 3-kinase (PI3K) activation. Here, we report a PI3K-independent pathway operating for PDGF-induced chemotaxis in vascular smooth muscle cells and other cell types. Two different PI3K inhibitors, wortmannin (WT, 1 nM-1 μM) and LY294002 (100 nM-10 μM), did not inhibit PDGF-induced chemotaxis in smooth muscle cells and Swiss 3T3 cells, whereas WT inhibited activity of PI3K that were immunopurified from PDGF-stimulated cells as well as PI3K purified from cells that were stimulated with PDGF in the presence of the same concentrations of WT. Similarly, WT (100 nM) abolished the increase in intracellular phosphatidylinositol 3,4,5-triphosphate after PDGF stimulation. Furthermore, Chinese hamster ovary/Δp85 cells overexpressing a dominant negative p85 subunit of PI3K showed a chemotactic response comparable to that of parental cells while showing a remarkable decrease in PI3K activity. Rapamycin, a specific inhibitor of pp70 S6 kinase, which is one of the well characterized downstreams of PI3K, did not inhibit PDGF-induced chemotaxis. Both WT and LY294002 inhibited PDGF-induced amino acid uptake and actin-stress fiber reorganization and partly inhibited PDGF-induced glucose incorporation in Swiss 3T3 cells. Our findings indicate that, in vascular smooth muscle cells and other cell types, the signal transduction for PDGF-induced chemotaxis is independent of PI3K activity while the signal transduction for PDGF-induced amino acid uptake, glucose incorporation, and cytoskeletal reorganization is dependent on PI3K.

      INTRODUCTION

      Phosphatidylinositol 3-kinase (PI3K)
      The abbreviations used are: PI3K
      phosphatidylinositol 3-kinase
      PDGF
      platelet-derived growth factor
      SMCs
      smooth muscle cells
      WT
      wortmannin
      CHO
      Chinese hamster ovary
      PI
      phosphatidylinositol
      PI(3)P
      phosphatidylinositol 3-phosphate
      PI(3,4)P2
      phosphatidylinositol 3,4-bisphosphate
      PIP3
      phosphatidylinositol 3,4,5-triphosphate
      PDGFR
      platelet-derived growth factor receptor
      PLC-γ
      phospholipase C-γ
      DMEM
      Dulbecco's modified Eagle's medium
      BSA
      bovine serum albumin
      PBS
      phosphate-buffered saline.
      is an enzyme that phosphorylates the D-3 position of the inositol ring in phosphoinositides, resulting in formation of PI(3)P, PI(3,4)P2, and PI(3,4,5)P3 (
      • Vlahos C.J.
      • Matter W.F.
      ). PI3K is a heterodimer of an 85-kDa regulatory subunit (
      • Otsu M.
      • Hiles I.
      • Gout I.
      • Fry M.J.
      • Ruiz-Larrea F.
      • Panayotou G.
      • Thompson A.
      • Dhand R.
      • Hsuan J.
      • Totty N.
      • Smith A.D.
      • Morgan S.J.
      • Courtneidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ) and a 110-kDa catalytic subunit (
      • Hiles I.D.
      • Otsu M.
      • Volinia S.
      • Fry M.J.
      • Gout I.
      • Dhand R.
      • Panayotou G.
      • Ruiz-Larrea F.
      • Thompson A.
      • Totty N.F.
      • Hsuan J.J.
      • Courtneidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ). PI3K is activated when it binds to phosphotyrosine residues of activated growth factor receptors by the two Src homology region 2 domains in the p85 subunit (
      • Claesson-Welsh L.
      ). Studies using specific inhibitors (
      • Okada T.
      • Sakuma L.
      • Fukui Y.
      • Hazeki O.
      • Ui M.
      ,
      • Vlahos C.J.
      • Matter W.F.
      • Hui K.Y.
      • Brown R.F.
      ,
      • Wennström S.
      • Hawkins P.
      • Cooke F.
      • Hara K.
      • Yonezawa K.
      • Kasuga M.
      • Jackson T.
      • Claesson-Welsh L.
      • Stephens L.
      ,
      • Kotani K.
      • Yonezawa K.
      • Hara K.
      • Ueda H.
      • Kitamura Y.
      • Sakaue H.
      • Ando A.
      • Chavanieu A.
      • Calas B.
      • Grigorescu F.
      • Nishiyama M.
      • Waterfield M.D.
      • Kasuga M.
      ,
      • Wymann M.
      • Arcaro A.
      ,
      • Joly M.
      • Kazlauskas A.
      • Corvera S.
      ,
      • Li G.
      • D'Souza-Schorey C.
      • Barbieri M.A.
      • Roberts R.L.
      • Klippel A.
      • Williams L.T.
      • Stahl P.D.
      ,
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ,
      • Ma Y-H.
      • Reusch H.P.
      • Wilson E.
      • Escobedo J.A.
      • Fantl W.J.
      • Williams L.T.
      • Ives H.E.
      ,
      • Kimura K.
      • Hattori S.
      • Kabuyama Y.
      • Shizawa Y.
      • Takayanagi J.
      • Nakamura S.
      • Toki S.
      • Matsuda Y.
      • Onodera K.
      • Fukui Y.
      ), a dominant negative construct for p85 (
      • Kotani K.
      • Yonezawa K.
      • Hara K.
      • Ueda H.
      • Kitamura Y.
      • Sakaue H.
      • Ando A.
      • Chavanieu A.
      • Calas B.
      • Grigorescu F.
      • Nishiyama M.
      • Waterfield M.D.
      • Kasuga M.
      ,
      • Dhand R.
      • Hara K.
      • Hiles I.
      • Bax B.
      • Gout I.
      • Panayotou G.
      • Fry M.J.
      • Yonezawa K.
      • Kasuga M.
      • Waterfield M.D.
      ,
      • Hara K.
      • Yonezawa K.
      • Sakaue H.
      • Ando A.
      • Kotani K.
      • Kitamura T.
      • Kitamura Y.
      • Ueda H.
      • Stephens L.
      • Jackson T.R.
      • Hawkins P.T.
      • Dhand R.
      • Clark A.E.
      • Holman G.D.
      • Waterfield M.D.
      • Kasuga M.
      ) and mutant PDGF receptors (
      • Wennström S.
      • Hawkins P.
      • Cooke F.
      • Hara K.
      • Yonezawa K.
      • Kasuga M.
      • Jackson T.
      • Claesson-Welsh L.
      • Stephens L.
      ,
      • Joly M.
      • Kazlauskas A.
      • Corvera S.
      ,
      • Ma Y-H.
      • Reusch H.P.
      • Wilson E.
      • Escobedo J.A.
      • Fantl W.J.
      • Williams L.T.
      • Ives H.E.
      ), revealed that PI3K is involved in growth factor-induced membrane ruffling and actin reorganization (
      • Wennström S.
      • Hawkins P.
      • Cooke F.
      • Hara K.
      • Yonezawa K.
      • Kasuga M.
      • Jackson T.
      • Claesson-Welsh L.
      • Stephens L.
      ,
      • Kotani K.
      • Yonezawa K.
      • Hara K.
      • Ueda H.
      • Kitamura Y.
      • Sakaue H.
      • Ando A.
      • Chavanieu A.
      • Calas B.
      • Grigorescu F.
      • Nishiyama M.
      • Waterfield M.D.
      • Kasuga M.
      ,
      • Wymann M.
      • Arcaro A.
      ), intracellular trafficking of growth factor receptor (
      • Joly M.
      • Kazlauskas A.
      • Corvera S.
      ), endocytosis (
      • Li G.
      • D'Souza-Schorey C.
      • Barbieri M.A.
      • Roberts R.L.
      • Klippel A.
      • Williams L.T.
      • Stahl P.D.
      ), protein sorting (
      • Schu P.V.
      • Takegawa K.
      • Fry M.J.
      • Stack J.H.
      • Waterfield M.D.
      • Emr S.D.
      ), translocation of glucose transporter (
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ), and glucose incorporation (
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ,
      • Hara K.
      • Yonezawa K.
      • Sakaue H.
      • Ando A.
      • Kotani K.
      • Kitamura T.
      • Kitamura Y.
      • Ueda H.
      • Stephens L.
      • Jackson T.R.
      • Hawkins P.T.
      • Dhand R.
      • Clark A.E.
      • Holman G.D.
      • Waterfield M.D.
      • Kasuga M.
      ), which are induced by insulin, DNA synthesis (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ), activation of Na+/H+ exchange (
      • Ma Y-H.
      • Reusch H.P.
      • Wilson E.
      • Escobedo J.A.
      • Fantl W.J.
      • Williams L.T.
      • Ives H.E.
      ), and nerve growth factor-induced neurite outgrowth (
      • Kimura K.
      • Hattori S.
      • Kabuyama Y.
      • Shizawa Y.
      • Takayanagi J.
      • Nakamura S.
      • Toki S.
      • Matsuda Y.
      • Onodera K.
      • Fukui Y.
      ).
      Recently, PI3K has been reported to be indispensable for PDGF-induced chemotaxis, which is mediated by PDGF receptor-β (PDGFR-β) (
      • Kundra V.
      • Escobedo J.A.
      • Kazlauskas A.
      • Kim H.K.
      • Rhee S.G.
      • Williams L.T.
      • Zetter B.R.
      ,
      • Wennström S.
      • Siegbahn A.
      • Yokote K.
      • Arvidsson A-K.
      • Heldin C-H.
      • Mori S.
      • Claesson-Welsh L.
      ). Replacement of two tyrosine residues within PI3K binding sites of PDGFR-β causes loss of chemotactic response to PDGF-BB in cells transfected with this mutant receptor. However, evidence suggests that there are cell-type specific variations in chemotactic signal transduction mediated by PDGFR; PDGFR-α mediates chemotaxis when transfected in a hematopoietic cell line (
      • Matsui T.
      • Pierce J.H.
      • Fleming T.P.
      • Greenberger J.S.
      • LaRochelle W.J.
      • Ruggiero M.
      • Aaronson S.A.
      ) but not in porcine aortic endothelial cells (
      • Eriksson A.
      • Siegbahn A.
      • Westermark B.
      • Heldin C-H.
      • Claesson-Welsh L.
      ); a mutant PDGFR that lacks binding sites for PLC-γ does not transduce chemotactic signal when it is expressed in NIH 3T3 cells (
      • Kundra V.
      • Escobedo J.A.
      • Kazlauskas A.
      • Kim H.K.
      • Rhee S.G.
      • Williams L.T.
      • Zetter B.R.
      ) but does transduce chemotactic signal in porcine aortic endothelial cells (
      • Wennström S.
      • Siegbahn A.
      • Yokote K.
      • Arvidsson A-K.
      • Heldin C-H.
      • Mori S.
      • Claesson-Welsh L.
      ). We therefore decided to evaluate the role of PI3K in the signal transduction for PDGF-induced chemotaxis and related phenomena in vascular smooth muscle cells (SMCs) and other cell types that express authentic PDGFR-β.

      RESULTS

      Contrary to previous reports that PI3K is indispensable for PDGF-induced chemotaxis (
      • Kundra V.
      • Escobedo J.A.
      • Kazlauskas A.
      • Kim H.K.
      • Rhee S.G.
      • Williams L.T.
      • Zetter B.R.
      ,
      • Wennström S.
      • Siegbahn A.
      • Yokote K.
      • Arvidsson A-K.
      • Heldin C-H.
      • Mori S.
      • Claesson-Welsh L.
      ), two specific inhibitors of different modes of action, wortmannin (WT) and LY294002, did not inhibit PDGF-induced chemotaxis in vascular SMCs and Swiss 3T3 cells at concentrations that were sufficient to inhibit PI3K (1 nM-1 μM for WT, 100 nM-10 μM for LY294002) (Fig. 1). Under the same conditions, tyrosine kinase inhibitors and colchicine inhibited chemotaxis (
      • Shimokado K.
      • Yokota T.
      • Umezawa K.
      • Sasaguri T.
      • Ogata J.
      ,
      • Yokota T.
      • Shimokado K.
      • Zen K.
      • Kosaka C.
      • Sasaguri T.
      • Masuda J.
      • Ogata J.
      ).
      Figure thumbnail gr1
      Fig. 1Effect of wortmannin and LY294002 on PDGF-induced chemotaxis. SMCs (•,×) and Swiss 3T3 cells (∘, ▪) preincubated in DMEM containing 0.1% BSA for 48 h were used for a chemotactic assay as described under “Experimental Procedures.” Wortmannin (•, ∘) and LY294002 (×, ▪) were added both in the upper and lower compartments of microchemotactic chambers. The number of cells that migrated onto the lower surface of the membrane was counted in 10 high power fields. Each point, expressed as a percentage of the cell number in the absence of inhibitors, represents the mean of three independent experiments that were conducted in quadruplicate. The standard error of the mean was within 10% of the mean.
      To confirm that the inhibitors inhibit PI3K activity in these cells, we measured the PI3K activity in two different ways. First, various doses of WT were incubated with PI3K immunoprecipitated from PDGF-stimulated cells with anti-phosphotyrosine antibody, and PI3K activity was measured in the presence of WT. In this assay, WT inhibited PI3K with an IC50 value (3 nM) comparable to those in previous reports (
      • Wymann M.
      • Arcaro A.
      ,
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ). Second, PI3K was immunoprecipitated from cells that had been treated with PDGF-BB in the presence of various concentrations of WT. The dose response curve of the latter assay overlapped reasonably well with that of the former assay, indicating that WT inhibited PDGF-activated PI3K activity under our experimental conditions (Fig. 2).
      Figure thumbnail gr2
      Fig. 2Dose-dependent inhibition of PI3K activity by wortmannin. PI3K was immunoprecipitated with anti-phosphotyrosine monoclonal antibody from Swiss 3T3 cells (•) that were stimulated with 10 ng/ml PDGF-BB for 10 min, and PI3K activity was then assayed in the presence of indicated concentrations of wortmannin as described under “Experimental Procedures.” PI3K was also immunoprecipitated from Swiss 3T3 cells (∘) and SMCs (×), which were preincubated with indicated concentrations of wortmannin for 10 min and then stimulated with PDGF-BB (10 ng/ml) for another 10 min. Values are expressed as the percentage of the value obtained in the absence of WT. Each point represents the mean of three or four determinants obtained from independent experiments conducted in duplicate. The standard error of the mean was within 10%.
      Although the PI3K mentioned above represents the isoform that is tyrosine phosphorylated and activated by PDGF-BB, an isoform of PI3K, which is regulated by G protein-coupled pathway, has been reported (
      • Stephens L.
      • Smrcka A.
      • Cooke F.T.
      • Jackson T.R.
      • Sternweis P.C.
      • Hawkins P.T.
      ,
      • Stoyanov B.
      • Volinia S.
      • Hanck T.
      • Rubio I.
      • Loubtchenkov M.
      • Malek D.
      • Stoyanova S.
      • Vanhaesebroeck B.
      • Dhand R.
      • Nürnberg B.
      • Gierschik P.
      • Seedorf K.
      • Hsuan J.J.
      • Waterfield M.D.
      • Wetzker R.
      ). To rule out the potential indirect stimulation of other isoforms of PI3K by PDGF, we measured the intracellular PI(3,4,5)P3 formation after PDGF stimulation. Although PI(3,4,5)P3 was hardly detectable in quiescent cells, it became clearly detectable after PDGF stimulation. Preincubation of cells with WT (100 nM) for 30 min prevented the formation of PI(3,4,5)P3 completely (Fig. 3).
      Figure thumbnail gr3
      Fig. 3Inhibition of PDGF-stimulated production of PI(3,4,5)P3 by wortmannin. SMCs and Swiss 3T3 cells were labeled with 32Pi for 1 h and then further treated with 100 nM wortmannin for 30 min. 10 min after PDGF-BB (30 ng/ml) was added to the culture, phospholipids were extracted and separated on an oxalate-impregnated TLC plate as described under “Experimental Procedures.” An autoradiogram of the TLC plate is shown. The position of spots corresponding to PI, PI(3)P, PI(3,4)P2, and PI(3,4,5)P3 are indicated on the left. The position of PI(3,4,5)P3 was identified with PI(3,4,5)P3 generated from PI(4,5)P2 with purified PI3K.
      To substantiate the findings obtained with specific inhibitors, we studied the effect of a dominant negative p85 subunit of PI3K on PDGF-induced chemotaxis. A stable cell line of CHO cells overexpressing a dominant negative p85 subunit of PI3K (CHO/Δp85 cells) was cloned as described under “Experimental Procedures.” These cells were suitable to test whether PI3K was indispensable for PDGF-induced chemotaxis. Both CHO/Δp85 cells and their parental cell line, CHO-K1 cells, expressed PDGFR-β that was tyrosine phosphorylated by PDGF-BB (Fig. 4A). In CHO/Δp85 cells, which expressed the dominant negative p85 subunit (Fig. 4B), association of native p85 subunit to activated PDGFR was inhibited as indicated by a lack of p110 subunit association to PDGFR (Fig. 4C). However, other downstream signaling, such as activation of PLC-γ or tyrosine phosphorylation of various proteins, was not impaired (Fig. 4, D and E). PI3K activity in CHO/Δp85 cells was suppressed to less than 5% of that in CHO-K1 cells (Fig. 5). CHO/Δp85 cells responded to PDGF-BB chemotactically to the same extent as the parental cell line, CHO-K1 cells (Fig. 6), confirming the findings obtained with the inhibitors.
      Figure thumbnail gr4
      Fig. 4PDGF-induced tyrosine phosphorylation in CHO/Δp85 cells. Quiescent cells were treated with PDGF-BB (10 ng/ml) or vehicle (PBS) for 5 min, and cleared cell lysate was incubated with anti-PDGFR-β antiserum (A) or anti-phosphotyrosine monoclonal antibody (B-E). The immunoprecipitate was separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with anti-phosphotyrosine antibody (A), anti-bovine PI3K p85α antibody (G12) (B), anti-PI3K p110 antibody (C), anti-PLC-γ antibody (D), or anti-phosphotyrosine antibody (E). The experiment was repeated twice with similar results.
      Figure thumbnail gr5
      Fig. 5Attenuation of PI3K activity in CHO/Δp85 cells. PI3K was immunoprecipitated with anti-phosphotyrosine monoclonal antibody from CHO-K1, CHO-IR, and CHO/Δp85 cells that were stimulated with 10 ng/ml PDGF-BB for 10 min, and PI3K activity was then assayed as described under “Experimental Procedures.”
      Figure thumbnail gr6
      Fig. 6Chemotactic response of CHO/Δp85 cells. Subconfluent cells preincubated in F-12 medium containing 0.1% BSA for 48 h were used for a chemotactic assay in the presence of 0.1% plasma-derived serum. PDGF-BB (20 ng/ml) was used as a chemoattractant. The number of cells that migrated onto the lower surface of the membrane was counted in 10 high power fields. Each column indicates the mean ± S.E. of three experiments.
      To obtain further support for the non-involvement of PI3K in chemotactic signal transduction, we studied the effect of rapamycin on PDGF-induced chemotaxis in Swiss 3T3 cells. Rapamycin is a specific inhibitor of pp70 S6 kinase, which is one of the well characterized downstreams of PI3K signaling pathways, and is activated by PDGF-BB (
      • Chung J.
      • Grammer T.C.
      • Lemon K.P.
      • Kazlauskas A.
      • Blenis J.
      ,
      • Burgering B.M. Th.
      • Coffer P.J.
      ). Rapamycin (1 nM-100 nM) did not inhibit PDGF-induced chemotaxis (data not shown).
      For the comparison with previous reports on the effect of PI3K inhibitors in other cell types (
      • Wennström S.
      • Hawkins P.
      • Cooke F.
      • Hara K.
      • Yonezawa K.
      • Kasuga M.
      • Jackson T.
      • Claesson-Welsh L.
      • Stephens L.
      ,
      • Kotani K.
      • Yonezawa K.
      • Hara K.
      • Ueda H.
      • Kitamura Y.
      • Sakaue H.
      • Ando A.
      • Chavanieu A.
      • Calas B.
      • Grigorescu F.
      • Nishiyama M.
      • Waterfield M.D.
      • Kasuga M.
      ,
      • Wymann M.
      • Arcaro A.
      ), we studied the effect of WT on PDGF-induced actin reorganization. In accordance with previous reports (
      • Wymann M.
      • Arcaro A.
      ), this PDGF-induced actin reorganization was completely abolished by WT (data not shown).
      To further elucidate the role of PI3K activity in PDGF-induced phenomena in these cells, we studied the effect of WT and LY294002 on PDGF-induced glucose incorporation and amino acid uptake in Swiss 3T3 cells. As reported with other cell types (
      • Owen III, A.J.
      • Geyer R.P.
      • Antoniades H.N.
      ), PDGF-BB significantly increased [3H]leucine uptake in 6 h. WT and LY294002 inhibited this PDGF-induced leucine uptake completely (Table I). PDGF-BB significantly increased glucose incorporation after a 6-h incubation. In contrast to their inhibition of amino acid uptake, WT and LY294002 inhibited PDGF-induced glucose incorporation only partially (Table I). Although WT was recently reported to inhibit phospholipase A2 activity at similar concentrations for inhibition of PI3K in Swiss 3T3 cells (
      • Cross M.J.
      • Stewart A.
      • Hodgkin M.N.
      • Kerr D.J.
      • Wakelam M.J.O.
      ), neither PDGF-stimulated amino acid uptake nor glucose incorporation was inhibited by a specific inhibitor of cytosolic phospholipase A2, AACOCF3 (data not shown) (
      • Riendeau D.
      • Guay J.
      • Weech P.K.
      • Laliberté F.
      • Yergey J.
      • Li C.
      • Desmarais S.
      • Perrier H.
      • Liu S.
      • Nicoll-Griffith D.
      • Street I.P.
      ). Rapamycin did not affect these PDGF-induced phenomena (Table I).
      Table I.Effect of various inhibitors on PDGF-BB-induced leucine uptake and glucose incorporation
      Leucine uptakeGlucose incorporation
      dpm% of controldpm% of control
      Control2925 ± 941007252 ± 257100
      PDGF (10 ng/ml)3884 ± 108
      p < 0.01 in comparison with control.
      1339979 ± 481
      p < 0.01 in comparison with control.
      138
      PDGF + wortmannin (100 nM)2865 ± 53
      p < 0.01 in comparison with the PDGF-treated group.
      988577 ± 307
      p < 0.05 in comparison with the PDGF-treated group.
      118
      PDGF + LY294002 (10 μM)3100 ± 131
      p < 0.01 in comparison with the PDGF-treated group.
      1068417 ± 391
      p < 0.05 in comparison with the PDGF-treated group.
      116
      PDGF + rapamycin (10 nM)3819 ± 1381319748 ± 459134
      PDGF + rapamycin (100 nM)3756 ± 1841289835 ± 407136
      a p < 0.01 in comparison with control.
      b p < 0.01 in comparison with the PDGF-treated group.
      c p < 0.05 in comparison with the PDGF-treated group.

      DISCUSSION

      In the present study, we demonstrate that PI3K is not involved in PDGF-induced chemotaxis in three cell types that express authentic PDGF-β receptors by using two different methods: specific inhibitors for PI3K and a stable cell line overexpressing a dominant negative p85 subunit of PI3K. Two PI3K inhibitors with different structures did not affect the PDGF-induced chemotaxis in vascular SMCs and Swiss 3T3 cells (Fig. 1). PI3K purified from those cells is inhibited by WT (Fig. 2), and the increase in the PI3K product after PDGF stimulation was completely inhibited by WT (Fig. 3), indicating that the lack of inhibition was not due to the general insensitivity of those cells to the inhibitors. The non-involvement of PI3K in PDGF-induced chemotaxis was further substantiated by the finding that CHO cells overexpressing a dominant negative p85 subunit of PI3K showed decreased PI3K activation by PDGF-BB (Fig. 5) but a chemotactic response comparable to parental CHO-K1 cells (Fig. 6).
      Our finding that PI3K is not involved in PDGF-induced chemotaxis is in contrast to previous reports. Two groups (
      • Kundra V.
      • Escobedo J.A.
      • Kazlauskas A.
      • Kim H.K.
      • Rhee S.G.
      • Williams L.T.
      • Zetter B.R.
      ,
      • Wennström S.
      • Siegbahn A.
      • Yokote K.
      • Arvidsson A-K.
      • Heldin C-H.
      • Mori S.
      • Claesson-Welsh L.
      ) reported independently that the replacement of PI3K binding sites in the cytoplasmic domain of PDGFR (Tyr-741 and Tyr-751 in human PDGFR-β) with phenylalanine caused a loss of chemotactic response to PDGF-BB in porcine aortic endothelial cells and NIH 3T3 cells transfected with this mutant receptor, while the replacement of only one of the two PI3K binding sites did not affect the chemotactic response. Their findings suggested an indispensable role of PI3K in PDGF-induced chemotaxis in those cells. There are two possible explanations for the discrepancy between their findings and ours. First, the chemotactic signal may be transduced by molecules that share the same binding sites in PDGFR with PI3K, such as Shc, which binds to Tyr-741 and Tyr-751, and Nck, which binds to Tyr-751 (
      • Claesson-Welsh L.
      ). This possibility is supported by the discrepancy between suppression of PI3K activity and suppression of chemotactic response in cells transfected with mutant receptor; replacement of either one of two tyrosine residues in PI3K binding sites decreased PDGF-induced PI3K activation by 90% (
      • Burgering B.M. Th.
      • Coffer P.J.
      ) without influencing the chemotactic response (
      • Wennström S.
      • Siegbahn A.
      • Yokote K.
      • Arvidsson A-K.
      • Heldin C-H.
      • Mori S.
      • Claesson-Welsh L.
      ), whereas the insulin-induced glucose uptake decreased in parallel with PI3K activity (
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ). However, there was no direct evidence for the involvement of Shc or Nck in chemotactic signal transduction in the present study. The second possible explanation is that there may be PI3K-dependent and -independent chemotactic signal transduction pathways, and different cell types use different pathways. This possibility does not exclude the first one and is supported by previous observations using similar approaches with conflicting results; mutant receptors lacking PI3K binding sites (
      • Kundra V.
      • Escobedo J.A.
      • Kazlauskas A.
      • Kim H.K.
      • Rhee S.G.
      • Williams L.T.
      • Zetter B.R.
      ,
      • Wennström S.
      • Siegbahn A.
      • Yokote K.
      • Arvidsson A-K.
      • Heldin C-H.
      • Mori S.
      • Claesson-Welsh L.
      ), wortmannin, and the dominant negative p85 subunit of PI3K
      R. Hooshmand-Rad, L. Claesson-Welsh, S. Wennström, K. Yokote, C.-H. Heldin, and A. Siegbahn, manuscript in preparation.
      all suppress the chemotactic response in porcine aortic endothelial cells expressing wild-type or mutant PDGF receptors, whereas wortmannin only partially suppresses PDGF-induced chemotaxis in murine inner medullary collecting duct cells expressing mutant PDGF receptors that have an 11-amino acid sequence from c-met (
      • Derman M.P.
      • Chen J.Y.
      • Spokes K.C.
      • Songyang Z.
      • Cantley L.G.
      ). Interestingly, wortmannin inhibits hepatocyte growth factor-induced chemotaxis to the same extent as the mutant receptor in these collecting duct cells (
      • Derman M.P.
      • Cunha M.J.
      • Barros E.J.G.
      • Nigam S.K.
      • Cantley L.G.
      ). Cell type-specific variations have been known for other signaling molecules in PDGF-induced chemotaxis; PLC-γ is not required for PDGF-induced chemotaxis in porcine aortic endothelial cells expressing PDGFR (
      • Wennström S.
      • Siegbahn A.
      • Yokote K.
      • Arvidsson A-K.
      • Heldin C-H.
      • Mori S.
      • Claesson-Welsh L.
      ) but partly required in canine kidney epithelial cells, NIH 3T3 cells (
      • Kundra V.
      • Escobedo J.A.
      • Kazlauskas A.
      • Kim H.K.
      • Rhee S.G.
      • Williams L.T.
      • Zetter B.R.
      ), and murine inner medullary collecting duct cells (
      • Derman M.P.
      • Chen J.Y.
      • Spokes K.C.
      • Songyang Z.
      • Cantley L.G.
      ) expressing PDGFR. Our present results, together with those of the reports cited above, strongly suggest the existence of PI3K-dependent and -independent pathways for chemotactic signaling. Our findings also indicate that the authentic PDGF receptor transduces a chemotactic signal by a PI3K-independent pathway in vascular SMCs and the other cell types we tested.
      The downstreams of PI3K are not yet entirely clear. One of the well characterized downstreams of PI3K is pp70 S6 kinase (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Chung J.
      • Grammer T.C.
      • Lemon K.P.
      • Kazlauskas A.
      • Blenis J.
      ). Although rapamycin, a specific inhibitor of pp70 S6 kinase, inhibits PDGF-induced DNA synthesis in SMCs (
      • Marx S.O.
      • Jayaraman T.
      • Go L.O.
      • Marks A.R.
      ), in the present experiments it did not inhibit the PDGF-induced chemotaxis (data not shown), in accordance with our findings that PI3K is not involved in chemotactic signal transduction in this cell type. Another two proteins downstream of PI3K are small G proteins Rho and Rac. These small G proteins as well as PI3K are involved in PDGF-induced stress-fiber reorganization (
      • Ridley A.J.
      • Hall A.
      ,
      • Ridley A.J.
      • Paterson H.F.
      • Johnston C.L.
      • Diekmann D.
      • Hall A.
      ,
      • Zhang J.
      • King W.G.
      • Dillon S.
      • Hall A.
      • Feig L.
      • Rittenhouse S.E.
      ). In agreement with these previous reports, a PI3K inhibitor inhibited the PDGF-induced disappearance of stress fibers in Swiss 3T3 cells in the present study (data not shown).
      Another unexpected and interesting finding in our study is the effect of PI3K inhibitors on PDGF-induced glucose incorporation and amino acid uptake in Swiss 3T3 cells. PI3K inhibitors have been reported to inhibit insulin-stimulated glucose incorporation in all cell types tested (
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ). However, the same inhibitors only partly inhibited the present PDGF-induced glucose incorporation (Table I), indicating that PDGF stimulates cellular glucose incorporation both by PI3K-dependent and -independent pathways. This interpretation is supported by previous reports (
      • Rollins B.J.
      • Morrison E.D.
      • Usher P.
      • Flier J.S.
      ,
      • Kamohara S.
      • Hayashi H.
      • Todaka M.
      • Kanai F.
      • Ishii K.
      • Imanaka T.
      • Escobedo J.A.
      • Williams L.T.
      • Ebina Y.
      ) that PDGF stimulated glucose incorporation by both translocation of the glucose transporter to the cell surface and increasing in the amount of the glucose transporter. PI3K inhibitors completely inhibited amino acid uptake that was stimulated by PDGF-BB (Table I), indicating that PDGF-induced amino acid uptake is dependent on PI3K activity. Rapamycin did not inhibit these PDGF-induced phenomena (Table I), indicating that PDGF-induced amino acid uptake and glucose incorporation were not mediated by the pp70 S6 kinase pathway. Although wortmannin has been reported to inhibit phospholipase A2 activity in Swiss 3T3 cells (
      • Cross M.J.
      • Stewart A.
      • Hodgkin M.N.
      • Kerr D.J.
      • Wakelam M.J.O.
      ), the phospholipase A2 inhibitor did not affect this PDGF-induced glucose incorporation and amino acid uptake (data not shown), and, therefore, the inhibition of amino acid uptake was not due to the effect of wortmannin on phospholipase A2.
      In summary, we have revealed a PI3K-independent signal transduction pathway for PDGF-induced chemotaxis. Our findings suggest a diversity in signaling pathways for growth factor-induced chemotaxis.

      Acknowledgments

      We thank Dr. Hisayuki Matsuo for encouragement during this project.

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