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Phosphatidylinositol 3-Kinase Activity Is Required for Hepatocyte Growth Factor-induced Mitogenic Signals in Epithelial Cells*

  • Nader Rahimi
    Footnotes
    Affiliations
    Department of Pathology, Cancer Research Laboratories, Queen's University, Kingston, Ontario K7L 3N6, Canada
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  • Eric Tremblay
    Affiliations
    Department of Pathology, Cancer Research Laboratories, Queen's University, Kingston, Ontario K7L 3N6, Canada
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  • Bruce Elliott
    Correspondence
    To whom reprint requests/correspondence should be addressed.
    Affiliations
    Department of Pathology, Cancer Research Laboratories, Queen's University, Kingston, Ontario K7L 3N6, Canada
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  • Author Footnotes
    * This work was supported by the National Cancer Institute of Canada and U.S. Army Medical Research and Materiel Command Grant DAMD 17-94-J-4407. 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.
    ‡ Present address: Eye Research Institute, Harvard Medical School, 20 Staniford St., Boston, MA 02114.
Open AccessPublished:October 04, 1996DOI:https://doi.org/10.1074/jbc.271.40.24850
      Phosphatidylinositol (PI) 3-kinase is an important enzyme implicated in growth factor-stimulated intracellular signaling. In this study we have shown that hepatocyte growth factor (HGF) induces a rapid tyrosine phosphorylation of PI 3-kinase and association with HGF receptor/Met in Mv1Lu epithelial cells. Murine mammary carcinoma (SP1) cells, which co-express HGF and HGF receptor/Met, showed sustained phosphorylation of PI 3-kinase. Wortmannin, a potent inhibitor of PI 3-kinase, inhibited HGF-induced PI 3-kinase activity, proliferation of Mv1Lu cells, and spontaneous growth of SP1 cells in a dose-, and time-dependent manner. Transfection of a dominant negative mutant p85 (Δp85) subunit of PI 3-kinase into SP1 cells strongly inhibited HGF-stimulated proliferation and PI 3-kinase activity. However, wortmannin did not influence HGF-induced c-Jun expression. Furthermore, HGF stimulated S6 kinase activity, but its activity was not required for HGF-induced proliferation. Overall, these results suggest that HGF-induced PI 3-kinase activity is important for the mitogenic action of HGF in epithelial cells and further demonstrate that expression of c-Jun is not influenced by inhibition of PI 3-kinase activity.

      INTRODUCTION

      Hepatocyte growth factor (HGF)
      The abbreviations used are: HGF
      hepatocyte growth factor
      PI
      phosphatidylinositol; PIP2; PI 4,5-bisphosphate
      PIP3
      PI 4,5-triphosphate
      PKC
      protein kinase C.
      is a protein expressed in a variety of cell types and tissues (
      • Pepper M.S.
      • Matsumoto K.
      • Nakamura T.
      • Orci L.
      • Montesano R.
      ,
      • Zarnegar R.
      • DeFrances M.C.
      ,
      • DeFrances M.C.
      • Wolf H.K.
      • Michalopoulos G.K.
      • Zarnegar R.
      ,
      • Noji S.
      • Tashiro K.
      • Koyama E.
      • Nohno T.
      • Ohyama K.
      • Taniguchi S.
      • Nakamura T.
      ). HGF is mitogenic, motogenic, and morphogenic for epithelial cells in culture (
      • Matsumoto K.
      • Nakamura T.
      ,
      • Santos O.F.
      • Moura L.A.
      • Rosen E.M.
      • Nigam S.K.
      ,
      • Rosen E.M.
      • Grant D.S.
      • Kleinman H.K.
      • Goldberg I.D.
      • Bhargava M.M.
      • Nickoloff B.J.
      • Kinsella J.L.
      • Polverini P.
      ,
      • Rahimi N.
      • Saulnier R.
      • Nakamura T.
      • Park M.
      • Elliott B.
      ). All of these responses are believed to be mediated by HGF receptor, identified as the product of the met protooncogene (
      • Bottaro D.P.
      • Rubin J.S.
      • Faletto D.L.
      • Chan A.M.
      • Kmiecik T.E.
      • Vande Woude G.F.
      • Aaronson S.A.
      ). The HGF receptor/Met is a heterodimeric protein of 190 kDa, which consists of a 45-kDa α-subunit linked by disulfide bonds to a 145-kDa β-subunit (
      • Lehtola L.
      • Sistonen L.
      • Koskinen P.
      • Lehvaslaiho H.
      • Di Renzo M.F.
      • Comoglio P.M.
      • Alitalo K.
      ). The β-subunit spans the membrane and shows tyrosine kinase activity (
      • Naldini L.
      • Vigna E.
      • Narsimhan R.P.
      • Gaudino G.
      • Zarnegar R.
      • Michalopoulos G.K.
      • Comoglio P.M.
      ).
      It has been shown that HGF receptor/Met is autophosphorylated on Tyr1234 and Tyr1235 (
      • Ferracini R.
      • Longati P.
      • Naldini L.
      • Vigna E.
      • Comoglio P.M.
      ) and that substitution of these residues with Phe significantly reduces kinase activity of the HGF receptor/Met (
      • Longati P.
      • Bardelli A.
      • Ponzetto C.
      • Naldini L.
      • Comoglio P.M.
      ,
      • Zhen Z.
      • Giordano S.
      • Longati P.
      • Medico E.
      • Campiglio M.
      • Comoglio P.M.
      ). Over the past few years, much effort has been focused to identify the tyrosine kinase(s) that are associated with, and/or are phosphorylated by, HGF receptor/Met following stimulation by HGF. In this context, GAP, Src, mitogen-activated protein kinase, phospholipase C-γ, phosphatidylinositol (PI) 3-kinase, and Ras have been identified as being activated by HGF stimulation (
      • Ponzetto C.
      • Bardelli A.
      • Maina F.
      • Longati P.
      • Panayotou G.
      • Dhand R.
      • Waterfield M.D.
      • Comoglio P.M.
      ,
      • Ponzetto C.
      • Bardelli A.
      • Zhen Z.
      • Maina F.
      • dalla Zonca P.
      • Giordano S.A.U.
      • Panayotou G.
      • Comoglio P.M.
      ,
      • Graziani A.
      • Gramaglia D.
      • dalla Zonca P.
      • Comoglio P.M.
      ). Two phosphorylation sites, Tyr1349 and Tyr1356, located in the carboxyl terminus of HGF receptor/Met, have been identified as docking sites responsible for binding of Src, Shc, Grb2, and PI 3-kinase (
      • Ponzetto C.
      • Bardelli A.
      • Maina F.
      • Longati P.
      • Panayotou G.
      • Dhand R.
      • Waterfield M.D.
      • Comoglio P.M.
      ,
      • Ponzetto C.
      • Bardelli A.
      • Zhen Z.
      • Maina F.
      • dalla Zonca P.
      • Giordano S.A.U.
      • Panayotou G.
      • Comoglio P.M.
      ,
      • Fixman E.D.
      • Naujokas M.A.
      • Rodrigues G.A.
      • Moran M.F.
      • Park M.
      ).
      PI 3-kinase is one of the key enzymes activated in HGF-induced signal transduction. It catalyzes phosphorylation of PI to PI 3-phosphate, PI 4-phosphate to PI 3,4-bisphosphate, and PI 4,5-bisphosphate (PIP2) to PI 3,4,5-triphosphate (PIP3) (
      • Auger K.R.
      • Serunian L.A.
      • Soltoff S.P.
      • Libby P.
      • Cantley L.C.
      ,
      • Carpenter C.L.
      • Duckworth B.C.
      • Auger K.R.
      • Cohen B.
      • Schaffhausen B.S.
      • Cantley L.C.
      ,
      • Whitman M.
      • Downes C.P.
      • Keeler M.
      • Keller T.
      • Cantley L.
      ). Recent studies support the involvement of PI 3-kinase in the action of S6 kinase (
      • Chung J.
      • Grammer T.C.
      • Lemon K.P.
      • Kazlauskas A.
      • Blenis J.
      ), activation of protein kinase C-ζ (PKC-ζ) (
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ), membrane ruffling (
      • Wymann M.
      • Arcaro A.
      ), and actin polymerization (
      • Zhang J.
      • Fry M.J.
      • Waterfield M.D.
      • Jaken S.
      • Liao L.
      • Fox J.E.B.
      • Rittenhouse S.E.
      ). Although a role for PI 3-kinase in mitogenesis has been suggested (
      • Sjolander A.
      • Yamamoto K.
      • Huber B.E.
      • Lapetina E.G.
      ,
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ,
      • Fantl W.J.
      • Escobedo J.A.
      • Martin G.A.
      • Turck C.W.
      • del Rosario M.
      • McCormick F.
      • Williams L.T.
      ), the direct role of PI 3-kinase or its lipid products in mitogenesis remains unclear.
      Evidence that PI 3-kinase is associated with HGF receptor/Met (
      • Ponzetto C.
      • Bardelli A.
      • Maina F.
      • Longati P.
      • Panayotou G.
      • Dhand R.
      • Waterfield M.D.
      • Comoglio P.M.
      ) suggested that this enzyme may be necessary for the growth-promoting action of HGF. In this study, we investigated the involvement of PI 3-kinase in HGF-induced mitogenic signals in two epithelial derived cell lines. Our results suggest that PI 3-kinase is physically associated with HGF receptor/Met and is activated upon HGF stimulation. Transfection of a dominant negative mutant p85 subunit of PI 3-kinase into murine mammary carcinoma (SP1) cells and treatment of these cells with wortmannin inhibited HGF-induced proliferation and PI 3-kinase activity. Inhibition of PI 3-kinase activity also caused a pronounced reduction in tyrosine phosphorylation of S6 kinase. However, the involvement of PI 3-kinase in HGF-induced mitogenic signaling appears not to be mediated by regulating the expression of the transcription factor, c-Jun.

      DISCUSSION

      PI 3-kinase is an important enzyme implicated in growth factor-stimulated intracellular signaling. Recent studies support the involvement of PI 3-kinase in the action of S6 kinase (
      • Chung J.
      • Grammer T.C.
      • Lemon K.P.
      • Kazlauskas A.
      • Blenis J.
      ), activation of PKC (
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ), membrane ruffling (
      • Wymann M.
      • Arcaro A.
      ), and actin polymerization (
      • Zhang J.
      • Fry M.J.
      • Waterfield M.D.
      • Jaken S.
      • Liao L.
      • Fox J.E.B.
      • Rittenhouse S.E.
      ). Evidence supports the involvement of differential signal transduction pathways in HGF-induced functions. For example, Ras activation by HGF is required for HGF-induced cell motility (
      • Hartmann G.
      • Weidner K.M.
      • Schwarz H.
      • Birchmeier W.
      ). In addition to Ras, other signaling molecules activated by HGF include phospholipase C-γ and PI 3-kinase (
      • Ponzetto C.
      • Bardelli A.
      • Maina F.
      • Longati P.
      • Panayotou G.
      • Dhand R.
      • Waterfield M.D.
      • Comoglio P.M.
      ,
      • Ponzetto C.
      • Bardelli A.
      • Zhen Z.
      • Maina F.
      • dalla Zonca P.
      • Giordano S.A.U.
      • Panayotou G.
      • Comoglio P.M.
      ,
      • Graziani A.
      • Gramaglia D.
      • dalla Zonca P.
      • Comoglio P.M.
      ). Phospholipase C-γ catalyzes the hydrolysis of (PIP2), which yields diacylglycerol and inositol 1,4,5-triphosphate, resulting in the activation of PKC. Activation of PKC negatively regulates HGF receptor/Met kinase activity (
      • Gandino L.
      • Di Renzo M.F.
      • Giordano S.
      • Bussolino F.
      • Comoglio P.M.
      ). On the other hand, phosphorylation of PIP2 by PI 3-kinase yields PIP3, the function of which has yet not been established.
      In the present study we have shown that HGF induces a rapid tyrosine phosphorylation of PI 3-kinase and association with HGF receptor/Met in Mv1Lu cells. SP1 cells, which co-express HGF and HGF receptor/Met (
      • Rahimi N.
      • Saulnier R.
      • Nakamura T.
      • Park M.
      • Elliott B.
      ) showed sustained phosphorylation of PI 3-kinase. The expression of the dominant negative mutant Δp85 in SP1 cells strongly inhibited HGF-induced cell proliferation. In addition, wortmannin, a potent inhibitor of PI 3-kinase, inhibited HGF-induced PI 3-kinase activity and proliferation of Mv1Lu cells and spontaneous growth of SP1 cells in a dose-, and time-dependent manner. Maximal inhibition of PI 3-kinase and proliferation was observed with 40 ng/ml of wortmannin. In addition, wortmannin also inhibited HGF-induced tyrosine phosphorylation of S6 kinase, suggesting that S6 kinase may act downstream of PI 3-kinase.
      Recent studies support the involvement of S6 kinase in cell growth of some cell types. Microinjection of antibodies that inhibit S6 kinase and the use of rapamycin, which blocks S6 kinase activity, showed that S6 kinase is important for G1/S phase transition in some cells (
      • Lane H.A.
      • Fernandez A.
      • Lamb N.J.
      • Thomas G.
      ). We therefore examined whether S6 kinase stimulation by HGF is required for HGF-induced cell proliferation. Incubation of SP1 cells and Mv1Lu cells with different concentrations of rapamycin did not influence HGF-induced cell growth. Our results indicate that although S6 kinase is stimulated by HGF, activity of this enzyme is not required for HGF-induced mitogenic signals. To gain insight into the regulatory mechanisms and downstream targets of PI 3-kinase, we therefore evaluated the effect of inhibition of PI 3-kinase on the expression of c-Jun. Our results demonstrate that HGF stimulates a rapid induction of c-Jun protein; however, inhibition of PI 3-kinase, does not affect HGF-induced c-Jun expression.
      The mechanism by which the HGF receptor/Met stimulates c-Jun expression is not known; however, recent reports indicate that c-Jun expression is regulated in a PKC-dependent manner. 12-O-Tetradecanoylphorbol-13-acetate, a potent activator of PKC (
      • Kikkawa U.
      • Takai Y.
      • Tanaka Y.
      • Miyake R.
      • Nishizuka Y.
      ), and other agents that lead to PKC activation, such as serum and growth factors, also induce expression of c-Jun (
      • Chung J.
      • Kuo C.J.
      • Crabtree G.R.
      • Blenis J.
      ). Inhibitors of PKC block these induction responses (
      • Angel P.
      • Imagawa M.
      • Chiu R.
      • Stein B.
      • Imbra R.J.
      • Rahmsdorf H.J.
      • Jonat C.
      • Herrlich P.
      • Karin M.
      ,
      • Brenner D.A.
      • O'Hara M.
      • Angel P.
      • Chojkier M.
      • Karin M.
      ). PKC is also activated in response to HGF (
      • Gandino L.
      • Di Renzo M.F.
      • Giordano S.
      • Bussolino F.
      • Comoglio P.M.
      ); it is therefore possible that HGF-induced c-Jun expression is regulated by stimulation of PKC by HGF receptor/Met, and thus PI 3-kinase activity may not be required for induction of this gene.
      Overall, these results suggest that HGF-induced PI 3-kinase activity is important for the mitogenic action of HGF in epithelial cells and further demonstrate that expression of c-Jun is not influenced by inhibition of PI 3-kinase activity. During the preparation of this work, Royal and Park (
      • Royal I.
      • Park M.
      ) also reported that PI 3-kinase activity is required for HGF-induced scatter activity of Madin-Darby canine kidney cells. Collectively, these results suggest that PI 3-kinase plays a critical role in many HGF-induced cellular functions.
      Taken together, our results indicate that PI 3-kinase is activated in response to HGF and is associated with HGF receptor/Met, and they further suggest that PI 3-kinase plays a major role in HGF-induced cell proliferation. However, the involvement of PI 3-kinase in HGF-induced mitogenic signaling appears not to be mediated by regulating the expression of the transcription factor, c-Jun. Further work will determine whether PI 3-kinase-independent pathways, e.g. PKC that stimulates c-Jun expression (
      • Kikkawa U.
      • Takai Y.
      • Tanaka Y.
      • Miyake R.
      • Nishizuka Y.
      ,
      • Angel P.
      • Imagawa M.
      • Chiu R.
      • Stein B.
      • Imbra R.J.
      • Rahmsdorf H.J.
      • Jonat C.
      • Herrlich P.
      • Karin M.
      ,
      • Brenner D.A.
      • O'Hara M.
      • Angel P.
      • Chojkier M.
      • Karin M.
      ), are also involved in HGF-stimulated proliferation.

      Acknowledgments

      We thank Marian Arnold and Stephanie Etchells for excellent technical assistance and Dr. M. Kasuga for providing the Δp85 mutant of PI 3-kinase. Dr. R. Schwall provided recombinant human HGF.

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