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The Osteopontin-CD44 Survival Signal Involves Activation of the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway*

  • Yi-Hung Lin
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  • Hsin-Fang Yang-Yen
    Correspondence
    To whom correspondence should be addressed: Inst. of Molecular Biology, Academia Sinica, 128 Yen-Jiou Yuan Road, Section 2, Nankang, Taipei 11529, Taiwan, Republic of China. Tel.: 886-2-2789-9228; Fax: 886-2-2782-6085;
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  • Author Footnotes
    * This work was supported by Grants NSC89-2316-B-001-006-M46 and NSC90-2316-B-001-004-M46 from the National Science Council of Taiwan (to H.-F. Y.-Y.).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:December 07, 2001DOI:https://doi.org/10.1074/jbc.M105132200
      We have recently demonstrated that the gene encoding the osteopontin (OPN) protein is activated both by interleukin-3 and granulocyte-macrophage colony-stimulating factor signaling pathways and that, through binding to the cell surface receptor CD44, OPN contributes to the survival activities of interleukin (IL)-3 and GM-CSF (Lin, Y.-H., Huang, C.-J., Chao, J.-R., Chen, S.-T., Lee, S.-F., Yen, J. J.-Y., and Yang-Yen, H.-F. (2000)Mol. Cell. Biol. 20, 2734–2742). In this report, we demonstrate that the CD44-binding domain of OPN involves a region containing amino acid residues from 121 to 140 and that both threonine and serine at positions 137 and 147, respectively, are essential for the survival stimulatory effect of OPN. Substitution of either residue with alanine results into a dominant negative mutant that overrides the survival effect of IL-3. Upon binding to the CD44 receptor, the wild-type OPN but not the inactive mutant induces activation of phosphatidylinositol 3-kinase and Akt. Last, we demonstrate that two waves of Akt activation are detected in IL-3-treated cells and that the survival promoting effect of OPN is mediated predominantly through the phosphatidylinositol 3-kinase/Akt signaling pathway. Together, our results suggest that a positive autoregulatory loop is involved in the survival pathway of IL-3.
      IL-3
      interleukin-3
      GM-CSF
      granulocyte macrophage-colony stimulating factor
      OPN
      osteopontin
      PCR
      polymerase chain reaction
      PI3K
      phosphatidylinositol 3-kinase
      CM
      conditioned medium
      ELISA
      enzyme-linked immunosorbent assay
      Both interleukin-3 (IL-3)1 and granulocyte-macrophage colony-stimulating factor (GM-CSF) belong to a family of cytokine growth factors that regulate the proliferation, differentiation, viability, and function of multipotential hematopoietic progenitors as well as of various other hematopoietic cells (
      • Arai K.
      • Lee F.
      • Miyajima A.
      • Arai N.
      • Yokota T.
      ). The high-affinity receptors for these two cytokines consist of two subunits, cytokine-specific α subunit and a common β subunit, which is also shared by the IL-5 receptor. The α subunit is primarily responsible for cytokine binding, whereas the β subunit is mainly used for signal transduction (
      • Arai K.
      • Lee F.
      • Miyajima A.
      • Arai N.
      • Yokota T.
      ).
      Ligand binding to the IL-3 or GM-CSF receptor induces tyrosine phosphorylation of various signaling molecules including the receptor β chain itself, JAK2, Shc, Vav, Fps, STAT5A, and STAT5B (
      • Azam M.
      • Erdjument-Bromage H.
      • Kreider B.L.
      • Xia M.
      • Quelle F.
      • Basu R.
      • Saris C.
      • Tempst P.
      • Ihle J.N.
      • Schindler C.
      ,
      • Duronio V.
      • Clark-Lewis I.
      • Federsppiel B.
      • Wieler J.S.
      • Schrader J.W.
      ,
      • Hanazono Y.
      • Chiba S.
      • Sasaki K.
      • Mano H.
      • Miyajima A.
      • Arai K.
      • Yazaki Y.
      • Hirai H.
      ,
      • Miyajima A.
      • Mui A.L.-F.
      • Ogorochi T.
      • Sakamaki K.
      ,
      • Mui A.L.-F.
      • Wakao H.
      • O'Farrell M.
      • Harada A.M.
      • Miyajima A.
      ,
      • Silvennoinen O.
      • Witthuhn B.
      • Quelle F.W.
      • Cleveland J.L.
      • Yi T.
      • Ihle J.N.
      ); activation of phosphatidylinositol (PI) 3-kinase and the Ras-Raf-mitogen-activated protein (MAP) kinase pathway (
      • Corey S.
      • Eguinoa A.
      • Puyana-Theall K.
      • Bolen J.B.
      • Cantley L.
      • Mollinedo F.
      • Jackson T.R.
      • Hawkins P.T.
      • Stephens L.R.
      ,
      • Gold M.R.
      • Duronio V.
      • Saxena S.P.
      • Schrader J.W.
      • Aebersold R.
      ,
      • Kinoshita T.
      • Yokota T.
      • Arai K.
      • Miyajima A.
      ,
      • Sato N.
      • Sakamaki K.
      • Terada N.
      • Arai K.
      • Miyajima A.
      ,
      • Scheid M.P.
      • Lauener R.W.
      • Duronio V.
      ); and transcriptional activation of immediate-early genes such as c-jun, c-fos, c-myc, cis, andmcl-1 (
      • Chao J.-R.
      • Wang J.-M.
      • Lee S.-F.
      • Peng H.-W.
      • Lin Y.-H.
      • Chou C.-H.
      • Li J.-C.
      • Huang H.-M.
      • Chou C.-K.
      • Kuo M.-L.
      • Yen J.J.-Y.
      • Yang-Yen H.-F.
      ,
      • Conscience J.F.
      • Verrier B.
      • Martin G.
      ,
      • Yoshimura A.
      • Ohkubo T.
      • Kiguchi T.
      • Jenkins N.A.
      • Gilbert D.J.
      • Copeland N.G.
      • Hara T.
      • Miyajima A.
      ). Deletion mapping analysis has revealed that while membrane-proximal domain of the receptor β subunit is essential for transducing mitogenic signals, the membrane-distal domain is required for transducing anti-apoptotic signals (
      • Sato N.
      • Sakamaki K.
      • Terada N.
      • Arai K.
      • Miyajima A.
      ,
      • Chao J.-R.
      • Wang J.-M.
      • Lee S.-F.
      • Peng H.-W.
      • Lin Y.-H.
      • Chou C.-H.
      • Li J.-C.
      • Huang H.-M.
      • Chou C.-K.
      • Kuo M.-L.
      • Yen J.J.-Y.
      • Yang-Yen H.-F.
      ).
      Osteopontin (OPN) is an acidic phosphoprotein that is secreted by osteoblasts, macrophages, cardiac fibroblasts, activated T cells, and many other cell types (
      • Denhardt D.T.
      • Guo X.
      ,
      • Rodan G.A.
      ). OPN binds to a variety of cell surface receptors including integrins αvβ1, αvβ3, and αvβ5(
      • Hu D.D.
      • Lin E.C.
      • Kovach N.L.
      • Hoyer J.R.
      • Smith J.W.
      ,
      • Miyauchi A.
      • Alvarez J.
      • Greenfield E.M.
      • Teti A.
      • Grano M.
      • Colucci S.
      • Zambonin-Zallone A.
      • Ross F.P.
      • Teitelbaum S.L.
      • Cheresh D.
      ,
      • Ross F.P.
      • Chappel J.
      • Alvarez J.I.
      • Sander D.
      • Butler W.T.
      • Farach-Carson M.C.
      • Mintz K.A.
      • Robey P.G.
      • Teitelbaum S.L.
      • Cheresh D.A.
      ) and CD44 (
      • Weber G.F.
      • Ashkar S.
      • Glimcher M.J.
      • Cantor H.
      ). Many cellular processes, including cell attachment, spreading and migration, homing of lymphocytes, and other hematopoietic cells and vascular remodeling are thought to involve OPN binding to its cell surface receptors (
      • Denhardt D.T.
      • Guo X.
      ,
      • Denhardt D.T.
      • Giachelli C.M.
      • Rittling S.R.
      ). Using OPN-null mutant mice as a model system, OPN was demonstrated to have a role in the growth or survival of metastatic cells (
      • Crawford H.C.
      • Matrisian L.M.
      • Liaw L.
      ), tissue remodeling (
      • Liaw L.
      • Birk D.E.
      • Ballas C.B.
      • Whitsitt J.S.
      • Davidson J.M.
      • Hogan B.L.
      ), and the type-1 immune response (
      • Ashkar S.
      • Weber G.F.
      • Panoutsakopoulou V.
      • Sanchirico M.E.
      • Jansson M.
      • Zawaideh S.
      • Rittling S.R.
      • Denhardt D.T.
      • Glimcher M.J.
      • Cantor H.
      ). However, the molecular mechanisms that underlie these activities of OPN remain largely unclear. By use of a PCR-based subtraction cloning approach, we have previously identifiedosteopontin to be a gene induced by the membrane-distal region of the β subunit between amino acids 573 and 755 of the human GM-CSF receptor (
      • Lin Y.-H.
      • Huang C.-J.
      • Chao J.-R.
      • Chen S.-T.
      • Lee S.-F.
      • Yen J.J.-Y.
      • Yang-Yen H.-F.
      ). We have also demonstrated that, in response to stimulation with IL-3 or GM-CSF, OPN is induced and released into the medium of cultured cells and that, through binding to the cell surface receptor CD44, it contributes to the survival activities of these two cytokines (
      • Lin Y.-H.
      • Huang C.-J.
      • Chao J.-R.
      • Chen S.-T.
      • Lee S.-F.
      • Yen J.J.-Y.
      • Yang-Yen H.-F.
      ).
      To gain further insight into OPN's mechanism of action in cytokine-triggered cell survival response, in this report, we have undertaken a structure-function study of the OPN molecule. We demonstrate that both threonine and serine at positions 137 and 147, respectively, are essential for the survival promoting effect of OPN and that substitution of either amino acid residue with alanine results in a dominant negative mutant that overrides the survival effect of IL-3 on IL-3-dependent cells. We further show that, via binding to the CD44 receptor, the wild-type OPN but not the inactive mutant activates the PI3K/Akt (PKB) kinase cascade and activation of the latter signaling pathway plays a major role in the survival promoting effect of OPN in IL-3-dependent cells.

      DISCUSSION

      The PI3K/Akt (PKB) signaling pathway plays an important role in the survival response induced by a variety of growth factors, matrix adhesion, and oncogene transformation (
      • Kulik G.
      • Klippel A.
      • Weber M.J.
      ,
      • Wang J.-M.
      • Chao J.-R.
      • Chen W.
      • Kuo M.-L.
      • Yen J.J.-Y.
      • Yang-Yen H.-F.
      ,
      • Ahmed N.N.
      • Grimes H.L.
      • Bellacosa A.
      • Chan T.O.
      • Tsichlis P.N.
      ,
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ,
      • Khwaja A.
      • Rodriguez-Viciana P.
      • Wennstrom S.
      • Warne P.H.
      • Downward J.
      ,
      • Minshall C.
      • Arkins S.
      • Freund G.G.
      • Kelley K.W.
      ,
      • Skorski T.
      • Bellacosa A.
      • Nieborowska-Skorska M.
      • Majewski M.
      • Martinez R.
      • Choi J.K.
      • Trotta R.
      • Wlodarski P.
      • Perrotti D.
      • Chan T.O.
      • Wasik M.A.
      • Tsichlis P.N.
      • Calabretta B.
      ,
      • Songyang Z.
      • Baltimore D.
      • Cantley L.C.
      • Kaplan D.R.
      • Franke T.F.
      ). In this report, we demonstrate that the survival promoting effect of OPN in IL-3-dependent cells also involves activation of the PI3K/Akt signaling pathway. OPN protects endothelial cells from serum withdrawal-induced apoptosis via interaction with integrin αVβ3 and activation of nuclear factor-κB (NF-κB) (
      • Scatena M.
      • Almeida M.
      • Chaisson M.L.
      • Fausto N.
      • Nicosia R.F.
      • Giachelli C.M.
      ). In contrast, the anti-apoptotic activity of OPN in IL-3-dependent cells is mediated predominantly through interaction with the CD44 receptor (
      • Lin Y.-H.
      • Huang C.-J.
      • Chao J.-R.
      • Chen S.-T.
      • Lee S.-F.
      • Yen J.J.-Y.
      • Yang-Yen H.-F.
      ) and activation of the PI3K/Akt signaling pathway. These results suggest that OPN exert its survival activity in a cell type-specific mechanism.
      The PI3K/Akt pathway is activated by IL-3 (39 and this study) as well as by OPN, a downstream effector gene product of IL-3 (
      • Lin Y.-H.
      • Huang C.-J.
      • Chao J.-R.
      • Chen S.-T.
      • Lee S.-F.
      • Yen J.J.-Y.
      • Yang-Yen H.-F.
      ). Interestingly, the activation kinetics of PI3-K and Akt by OPN (within 5–10 min stimulation) is very similar to that stimulated by IL-3. The weaker activation effect of OPN (Fig. 4) could be either due to an intrinsic property of the OPN-CD44 interaction or due to the limitation of our experimental conditions where the maximal effect of OPN might not yet be achieved with the amounts of conditioned medium (50%) used in the assay. Given that IL-3 induction of OPN expression occurs at a much later time point (∼3 h, see Ref.
      • Lin Y.-H.
      • Huang C.-J.
      • Chao J.-R.
      • Chen S.-T.
      • Lee S.-F.
      • Yen J.J.-Y.
      • Yang-Yen H.-F.
      ), it is unlikely that IL-3-triggered early activation of the PI3K/Akt kinase cascade is mediated through induction of OPN expression. In fact, following cytokine treatment of cells, two waves of Akt activation were observed in cells under conditions where OPN was produced (e.g.αβ755 cells stimulated with IL-3 or hGM-CSF, and αβ573 cells stimulated with IL-3), but not in the same cells when OPN expression was not induced (e.g. αβ573 cells stimulated with hGM-CSF). Our results strongly suggest that the early wave of Akt activation is mediated through the interaction between IL-3 and its receptor, whereas the second wave of Akt activation is mediated mainly through the OPN-CD44 pathway. Activation of the PI3K/Akt pathway is crucial to the survival activity of IL-3 (
      • Wang J.-M.
      • Chao J.-R.
      • Chen W.
      • Kuo M.-L.
      • Yen J.J.-Y.
      • Yang-Yen H.-F.
      ,
      • Songyang Z.
      • Baltimore D.
      • Cantley L.C.
      • Kaplan D.R.
      • Franke T.F.
      ). Our results suggest that IL-3-dependent cells (at least in the Ba/F3 cell system) may have evolved to adopt a positive autoregulatory mechanism by which the PI3K/Akt survival pathway can be first activated from the IL-3 receptor itself and later from its downstream effector gene product such as OPN (Fig. 10). In this way, the crucial survival signal can thus be effectively activated.
      Figure thumbnail gr10
      Figure 10Model of the positive autoregulatory loop involving OPN-CD44 and the PI3K/Akt kinase cascade in the IL-3 signaling pathway (see text).
      Mutational studies revealed that both Thr137 and Ser147 residues are critical for OPN to assume a conformation that can activate the CD44 receptor. However, it is not clear why S147A has a prominent dominant negative effect, whereas the T137A mutant only exerts a partially inhibitory effect on OPN. One possibility is that these two mutants may differ in their ability to compete with the wild-type protein for binding to the CD44 receptor. However, more experiments including a detailed structural analysis of the wild-type OPN molecule are required to reveal the exact mechanism(s) responsible for differential properties of these two mutants. On the other hand, several post-translational variants of OPN have been identified (
      • Rodan G.A.
      ) and the phosphorylation status affects OPNs ability to interact with the integrin receptor (
      • Ashkar S.
      • Weber G.F.
      • Panoutsakopoulou V.
      • Sanchirico M.E.
      • Jansson M.
      • Zawaideh S.
      • Rittling S.R.
      • Denhardt D.T.
      • Glimcher M.J.
      • Cantor H.
      ). It would be interesting to determine whether post-translational modification of either Thr137 or Ser147 residue (or both) is required for OPNs anti-apoptotic activity.
      OPN expression is increased in the blood of patients with metastatic disease (
      • Senger D.R.
      • Perruzzi C.A.
      • Gracey C.F.
      • Papadopoulos A.
      • Tenen D.G.
      ). Whereas overexpression of OPN in benign cells leads to increased metastasis (
      • Oates A.J.
      • Barraclough R.
      • Rudland P.S.
      ), reduced production of OPN by an antisense approach inhibits the tumorigenicity of transformed cell lines (
      • Behrend E.I.
      • Craig A.M.
      • Wilson S.M.
      • Denhardt D.T.
      • Chambers A.F.
      ,
      • Gardner H.A.
      • Berse B.
      • Senger D.R.
      ,
      • Su L.
      • Mukherjee A.B.
      • Mukherjee B.B.
      ). Our finding that OPN has a survival promoting activity (
      • Lin Y.-H.
      • Huang C.-J.
      • Chao J.-R.
      • Chen S.-T.
      • Lee S.-F.
      • Yen J.J.-Y.
      • Yang-Yen H.-F.
      ) further helps us interpret why a large variety of malignant cells have evolved to produce an increased level of OPN and have a growth advantage in vitro and in vivo. Moreover, the identification of S147A to be an effective dominant negative mutant of OPN implies that S147A may have a useful application in the therapy of certain types of metastatic tumor that involve deregulated expression of OPN.

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