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Hepatocyte Growth Factor-induced Differential Activation of Phospholipase Cγ1 and Phosphatidylinositol 3-Kinase Is Regulated by Tyrosine Phosphatase SHP-1 in Astrocytes*

  • Mitsuru Machide
    Affiliations
    Department of Neurochemistry, National Institute of Neuroscience, Tokyo 187-8502, Japan
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  • Kazuyo Kamitori
    Affiliations
    Department of Neurochemistry, National Institute of Neuroscience, Tokyo 187-8502, Japan
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  • Shinichi Kohsaka
    Correspondence
    To whom correspondence should be addressed: Dept. of Neurochemistry, National Inst. of Neuroscience, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan. Tel.: 81-423-46-1721; Fax: 81-423-46-1751
    Affiliations
    Department of Neurochemistry, National Institute of Neuroscience, Tokyo 187-8502, Japan
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  • Author Footnotes
    * This work was supported by grants from the Japanese Ministry of Health and Welfare and the Science and Technology Agency of Japan, and by a grant-in-aid for Scientific Research on Priority Areas from the Japanese Ministry of Education, Science, Sports and Culture.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:October 06, 2000DOI:https://doi.org/10.1074/jbc.M002817200
      Hepatocyte growth factor (HGF) elicits pleiotropic effects on various types of cells through the c-Met receptor tyrosine kinase. However, the mechanisms underlying the diverse responses of cells remain unknown. We show here that HGF promoted chemokinesis of rat primary astrocytes through the activation of phosphatidylinositol 3 (PI3)-kinase without any influence on mitogenesis of the cells. Under the same condition, phospholipase Cγ1 (PLCγ1), which is another signal mediator of c-Met, was not tyrosine-phosphorylated during HGF stimulation. However, treatment of the cells with orthovanadate, a tyrosine phosphatase inhibitor, restored the HGF-induced tyrosine phosphorylation of PLCγ1. A tyrosine phosphatase, SHP-1, was associated with both PI3-kinase and PLCγ1 before HGF stimulation, but it was dissociated only from PI3-kinase after the stimulation. Furthermore, transfectants of catalytically inactive mutant of SHP-1 showed tyrosine phosphorylation of PLCγ1 and mitogenic responses to HGF, and the mitogenic response was blocked with U73122, an inhibitor of phosphatidylinositol-specific PLC, and calphostin C, an inhibitor of protein kinase C downstream of the PLCγ1. These results indicate that PLCγ1 is selectively prevented from being a signal mediator by constitutive association of SHP-1, and that this selective inhibition of PLCγ1 may determine the cellular response of astrocytes to HGF.
      HGF
      hepatocyte growth factor
      PLC
      phospholipase C
      PI
      phosphatidylinositol
      BrdUrd
      bromodeoxyuridine
      DMEM
      Dulbecco's modified Eagle's medium
      GFAP
      glial fibrillary acidic protein
      Hepatocyte growth factor/scatter factor (HGF)1 exerts mitogenic, morphogenic and motogenic activities in various types of cells (
      • Matsumoto K.
      • Nakamura T.
      ,
      • Brinkmann V.
      • Foroutan H.
      • Sachs M.
      • Weidner K.M.
      • Birchmeier W.
      ). All these physiological activities are initially mediated by c-Met tyrosine kinase, the receptor for HGF (
      • Naldini L.
      • Vigna E.
      • Narshiman R.P.
      • Guadino Gs.
      • Zarnegar R.
      • Michalopoulos G.
      • Comoglio P.M.
      ,
      • Bottaro D.P.
      • Rubin J.S.
      • Faletto D.L.
      • Chan A.M. -L
      • Kmiecik T.E.
      • Vande Woude G.F.
      • Aaronson S.A.
      ). Previous studies have shown that, upon tyrosine phosphorylation, c-Met is associated with a number of SH2-containing signal mediators, such as GTPase-activating protein for Ras, p85 subunit of phosphatidylinositol (PI) 3-kinase, phospholipase C (PLC)γ1, and cytoplasmic tyrosine kinases of the Src family (
      • Bardelli A.
      • Maina F.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Comoglio P.M.
      • Ponzetto C.
      ,
      • Ponzetto C.
      • Bardelli A.
      • Zhen Z.
      • Maina F.
      • dalla Zonca P.
      • Giordano S.
      • Graziani A.
      • Panayotou G.
      • Comoglio P.M.
      ).
      It has been revealed that PI3-kinase is involved in HGF-induced migration of mIMCD cells (
      • Derman M.P.
      • Cunha M.J.
      • Barros E.J.G.
      • Nigam S.K.
      • Cantley L.G.
      ) and Madin-Darby canine kidney cells (
      • Royal I.
      • Park M.
      ), and that PLCγ1 mediates an intracellular signal for the HGF-enhanced mitogenesis of rat primary hepatocytes (
      • Okano Y.
      • Mizuno K.
      • Osada S.
      • Nakamura T.
      • Nozawa Y.
      ). Furthermore, PLCγ1 is considered to participate in cell migration, since activation of protein kinase C, the downstream effector of PLCγ1, mimics HGF-induced membrane ruffling in KB cells (
      • Nishiyama T.
      • Sasaki T.
      • Takaishi K.
      • Kato M.
      • Yaku H.
      • Araki K.
      • Matsuura Y.
      • Takai Y.
      ) and R308 cells (
      • Takaishi K.
      • Sasaki T.
      • Kato M.
      • Yamochi W.
      • Kuroda S.
      • Nakamura T.
      • Takeichi M.
      • Takai Y.
      ).
      In addition to the biological significance of HGF in peripheral organs and cells, various effects of HGF on cells of the central nervous system have also been reported (
      • Honda S.
      • Kagoshima M.
      • Wanaka A.
      • Tohyama M.
      • Matsumoto K.
      • Nakamura T.
      ,
      • Hamanoue M.
      • Takemoto N.
      • Matsumoto K.
      • Nakamura T.
      • Nakajima K.
      • Kohsaka S.
      ,
      • Ebens A.
      • Brose K.
      • Leonardo E.D.
      • Hanson Jr, M.G.
      • Bladt F.
      • Birchmeier C.
      • Barres B.A.
      • Tessier-Lavigne M.
      ,
      • Maina F.
      • Klein R.
      ). We have previously found that HGF promotes neurite outgrowth of cultured rat embryonic neocortical explants (
      • Hamanoue M.
      • Takemoto N.
      • Matsumoto K.
      • Nakamura T.
      • Nakajima K.
      • Kohsaka S.
      ). In this system, tyrosine phosphorylation of PLCγ1 was critical for the neurite outgrowth, whereas PI3-kinase was not phosphorylated during the stimulation (
      • Machide M.
      • Kamitori K.
      • Nakamura Y.
      • Kohsaka S.
      ). These findings suggested that two major downstream effectors of c-Met, PI3-kinase, and PLCγ1 were not necessarily co-activated, but rather differentially regulated in neuronal cells.
      In the present study, we analyzed the activation of PI3-kinase and PLCγ1 by HGF stimulation in rat primary astrocytes, a species of glial cells of the central nervous system. We found that HGF specifically stimulated tyrosine phosphorylation of PI3-kinase, not PLCγ1 in the cells. Furthermore, HGF caused rapid dissociation of a tyrosine phosphatase, SHP-1, a mammalian homologue ofDrosophila Csw (
      • Freeman Jr., R.M.
      • Plutzky J.
      • Neel B.G.
      ), from PI3-kinase, while the phosphatase still bound to PLCγ1, which may be a biochemical mechanism accounting for the selective activation of PI3-kinase with HGF. Furthermore, PLCγ1 was phosphorylated with tyrosine in the cells expressing a dominant negative mutant of SHP-1, and the cells showed mitogenic responses to HGF. Our study revealed that SHP-1 plays an important role in both selective activation of PI3-kinase and prevention of phosphorylation of PLCγ1 during the stimulation with HGF, and contributes to induction of the novel neurotrophic functions of HGF to the glial cells.

      DISCUSSION

      This article describes how HGF promotes actin reorganization and chemokinetic migration of astrocytes (Fig. 2), which is the first demonstration of the effects of HGF on glial cells as well as neuronal cells of the central nervous system. Promotion of cell motility is a typical activity of HGF observed in various types of cells of peripheral organs (
      • Matsumoto K.
      • Nakamura T.
      ). With regard to the intracellular signaling underlying the biological effect, PLCγ1 and its downstream effector(s), PKC(s) (
      • Nishiyama T.
      • Sasaki T.
      • Takaishi K.
      • Kato M.
      • Yaku H.
      • Araki K.
      • Matsuura Y.
      • Takai Y.
      ,
      • Takaishi K.
      • Sasaki T.
      • Kato M.
      • Yamochi W.
      • Kuroda S.
      • Nakamura T.
      • Takeichi M.
      • Takai Y.
      ,
      • Derman M.P.
      • Chen J.Y.
      • Spokes K.C.
      • Songyang Z.
      • Cantley L.G.
      ), and PI3-kinase (
      • Derman M.P.
      • Cunha M.J.
      • Barros E.J.G.
      • Nigam S.K.
      • Cantley L.G.
      ,
      • Royal I.
      • Park M.
      ) have been reported to function as mediators in this process. However, it has not been clarified whether the activation of both PLCγ1 and PI3-kinase is necessary for HGF-induced cell migration. Our results clearly indicated that HGF selectively induced tyrosine phosphorylation and activation of PI3-kinase and not PLCγ1 in astrocytes, and that the activation of PI3-kinase was essential for the HGF-induced cell migration. This selective activation of PI3-kinase by HGF is unique to astrocytes, since both PI3-kinase and PLCγ1 are activated in most of the cell types (
      • Ponzetto C.
      • Bardelli A.
      • Zhen Z.
      • Maina F.
      • dalla Zonca P.
      • Giordano S.
      • Graziani A.
      • Panayotou G.
      • Comoglio P.M.
      ,
      • Okano Y.
      • Mizuno K.
      • Osada S.
      • Nakamura T.
      • Nozawa Y.
      ,
      • Derman M.P.
      • Chen J.Y.
      • Spokes K.C.
      • Songyang Z.
      • Cantley L.G.
      ) or only PLCγ1 is activated by HGF in neurons (
      • Machide M.
      • Kamitori K.
      • Nakamura Y.
      • Kohsaka S.
      ). These results suggest that the selective activation of PI3-kinase determines the cellular response of astrocytes to HGF.
      PLCγ1 is also an essential signal mediator for the mitogenesis promoted by various growth factors (
      • Wang Z.
      • Gluck S.
      • Zhang L.
      • Moran M.F.
      ). Through the experiment, tyrosine phosphorylation of PLCγ1 or mitogenic response could not be detected in astrocytes stimulated with HGF (Figs. 2 A and4 A). In the cells treated with Na3VO4, an inhibitor of protein-tyrosine phosphatases, however, PLCγ1 was significantly tyrosine-phosphorylated as in other types of cells (Fig.4 B). This observation led us to identify SHP-1 as a PLCγ1-associated protein-tyrosine phosphatase (Figs. 5 and6 A). The association was sustained during HGF stimulation up to 10 min of examination (data not shown). Furthermore, transfection of a catalytically inactive mutant of SHP-1 resulted in tyrosine phosphorylation of PLCγ1 by HGF (Fig. 7), and the cells showed a mitogenic response to HGF (Fig. 8). This mitogenic response was inhibited by an inhibitor of PLC. The inhibitor could not suppress astrocytic migration stimulated by HGF (Fig. 2 D), indicating that the cells were viable under the treatment. These results indicated that PLCγ1 was prevented from being activated by SHP-1 during HGF stimulation, and the prevention was considered to have been responsible for the lack of mitogenic response to HGF in astrocytes.
      Furthermore, astrocytes showed enhanced mitogenic response even 5 h after stimulation with 10% serum (Fig. 2 A), which is much shorter than that generally observed in many other cell types. The same profile of mitogenic response was also observed in astrocytes carrying the mutant SHP-1 by the stimulation of HGF (Fig. 8). The rapid mitogenic response of astrocytes may be explained by the fact that primary astrocytes continue to proliferate slowly even in the serum-deprived condition. Thus, it is possible that molecules, except for PLCγ1, involved in progression of cell cycle may be constitutively active in the serum-starved astrocytes and activation of PLCγ1 may led the cells to immediate response to the mitogenic stimuli.
      In contrast to PLCγ1, PI3-kinase was activated by HGF, which in turn induced cell chemokinesis. In the process, activation of PLCγ1 is not essential, since the cells treated by U73122 still showed chemokinetic response to HGF (Fig. 2 D). SHP-1 was also associated with PI3-kinase before HGF stimulation, but was dissociated from PI3-kinase immediately after HGF stimulation (Fig. 6 B), suggesting that the dissociation of SHP-1 is responsible for the selective activation of PI3-kinase. The activation of PI3-kinase may also contribute to cell proliferation as well as chemokinesis, since wortmannin treatment also inhibited HGF-promoted mitogenic response emerged by mutant SHP-1 (Fig.8 A). These results indicated for the first time that SHP-1 selectively regulates PI3-kinase and PLCγ1 and may determine the astrocyte-specific response to HGF. The rapid dissociation of SHP-1 from PI3-kinase after HGF stimulation contrasts with general observations showing that their association is promoted after stimulation with various cytokines or growth factors (
      • Imani F.
      • Rager K.J.
      • Catipovic B.
      • Marsh D.G.
      ,
      • Yu Z.
      • Su L.
      • Hoglinger O.
      • Jaramillo M.L.
      • Banville D.
      • Shen S.-H.
      ).
      SHP-2, the structurally related molecule of SHP-1 (
      • Freeman Jr., R.M.
      • Plutzky J.
      • Neel B.G.
      ), has been reported to be associated with PI3-kinase (
      • Craddock B.L.
      • Welham M.J.
      ,
      • Gesbert F.
      • Guenzi C.
      • Bertoglio J.
      ,
      • Yamada M.
      • Ohnishi H.
      • Sano S.
      • Araki T.
      • Nakatani A.
      • Ikeuchi T.
      • Hatanaka H.
      ), and the expression of SHP-2 predominated over that of SHP-1 in astrocytes (data not shown). However, SHP-2 may not be involved in the selective inhibition of PLCγ1, because that SHP-2 was not associated with PLCγ1 or PI3-kinase in astrocytes (Fig. 6, E and F), and that Cys → Ser mutant of SHP-2 did not induce tyrosine phosphorylation of PLCγ1 (Fig. 7 A).
      Our results suggest that the selective activation (or suppression) of universal signal mediators, such as PI3-kinase and PLCγ1, is responsible for cell type-specific responses to HGF. As another example of the selective activation of the signal mediators, we have previously reported that HGF induces tyrosine phosphorylation of PLCγ1 and not PI3-kinase in rat primary neocortical neurons (
      • Machide M.
      • Kamitori K.
      • Nakamura Y.
      • Kohsaka S.
      ). Since PI3-kinase was not tyrosine-phosphorylated even in the cells treated with sodium orthovanadate (data not shown), the tyrosine phosphorylation of PI3-kinase was prevented in a manner independent of tyrosine phosphatase. With regard to the differential activation of PI3-kinase by c-Met in COS-7 cells, it has been reported that serine phosphorylation in the juxtamembrane domain of c-Met abolished the binding of PI3-kinase to the receptors and that spliced variant of c-Met lacking this domain facilitated the recruitment of PI3-kinase to c-Met (
      • Lee C.C.
      • Yamada K.M.
      ,
      • Lee C.C.
      • Yamada K.M.
      ). However, only the full-length form was detected in astrocytes and neurons (data not shown). Multiple mechanisms may contribute to the selective activation of PLCγ1 and PI3-kinase according to the cell types.
      Our observations revealed the biochemical bases for the selective activation of PI3-kinase and the constitutive suppression of PLCγ1 in astrocytes. However, multiple, cell type-specific mechanisms accounting for the selective activation of diverse subsets of signal mediators may function to exert the pleiotropic effects of HGF. Further studies in this regard may shed light on the understanding of diverse biological responses of cells to HGF.

      Acknowledgement

      We are grateful to Dr. Seisuke Hattori, Department of Biochemistry and Cellular Biology, National Institute of Neuroscience, for valuable discussion and comments.

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