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Growth Factors Outside of the Platelet-derived Growth Factor (PDGF) Family Employ Reactive Oxygen Species/Src Family Kinases to Activate PDGF Receptor α and Thereby Promote Proliferation and Survival of Cells*

  • Hetian Lei
    Affiliations
    Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114
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  • Andrius Kazlauskas
    Correspondence
    To whom correspondence should be addressed: 20 Staniford St., Boston, MA 02114. Tel.: 617-912-2517; Fax: 617-912-0101
    Affiliations
    Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114
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  • Author Footnotes
    2 The abbreviations used are: PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PVR, proliferative vitreoretinopathy; DMEM, Dulbecco’s modified eagle’s medium; bFGF, basic fibroblast growth factor; FGFR1, fibroblast growth factor receptor 1; NAC, N-acetylcysteine; DCF, 2′,7′-dichlorofluorescein; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; ROS, reactive oxygen species; SFK, Src family kinase; RV, rabbit vitreous; DCFH, 2′,7′-dichlorofluorescence that is reduced and therefore not fluorescent.
    3 H. Lei, G. Velez, P. Hovland, T. Hirose, D. Gilbertson, and A. Kazlauskas (2008) submitted for publication.
    * This work was supported, in whole or in part, by National Institutes of Health Grant EY012509 (to A. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.
Open AccessPublished:January 06, 2009DOI:https://doi.org/10.1074/jbc.M808426200
      The vitreous contains a plethora of growth factors that are strongly implicated in the formation of fibroproliferative diseases such as proliferative vitreoretinopathy. Although platelet-derived growth factors (PDGFs) are present in the vitreous, vitreal growth factors outside of the PDGF family activated the PDGF α receptor (PDGFRα) and promoted disease progression in a rabbit model of proliferative vitreoretinopathy (H. Lei, G. Velez, P. Hovland, T. Hirose, D. Gilbertson, and A. Kazlauskas (2008) submitted for publication.) In this report we investigated the mechanism by which non-PDGFs activated PDGFRα. We found that non-PDGFs increased the cellular level of reactive oxygen species (ROS) and that this event was necessary and sufficient for phosphorylation of PDGFRα. We speculated that the underlying mechanism was ROS-mediated inhibition of phosphotyrosine phosphatases, which antagonize receptor auto-phosphorylation. However, this did not appear to be the case. Non-PDGFs promoted tyrosine phosphorylation of catalytically inactive PDGFRα, and thereby indicated that at least one additional tyrosine kinase was involved. Indeed, preventing expression or blocking the kinase activity of Src family kinases suppressed non-PDGF-dependent tyrosine phosphorylation of PDGFRα. Thus non-PDGFs increased the level of ROS, which activated Src family kinases and resulted in phosphorylation of PDGFRα. Finally, although non-PDGFs induced only modest phosphorylation of PDGFRα, proliferation and survival of cells in response to non-PDGFs was significantly enhanced by expression of PDGFRα. These studies reveal a novel mechanism for activation of PDGFRα that appears capable of enhancing the responsiveness of cells to growth factors outside of the PDGF family.
      The platelet-derived growth factor (PDGF)
      The abbreviations used are: PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PVR, proliferative vitreoretinopathy; DMEM, Dulbecco’s modified eagle’s medium; bFGF, basic fibroblast growth factor; FGFR1, fibroblast growth factor receptor 1; NAC, N-acetylcysteine; DCF, 2′,7′-dichlorofluorescein; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; ROS, reactive oxygen species; SFK, Src family kinase; RV, rabbit vitreous; DCFH, 2′,7′-dichlorofluorescence that is reduced and therefore not fluorescent.
      family consists of five ligands, which assemble dimeric receptors consisting of homo- or hetero-combinations of the two PDGF receptor (PDGFR) subunits (
      • Fredriksson L.
      • Li H.
      • Eriksson U.
      ,
      • Reigstad L.J.
      • Varhaug J.E.
      • Lillehaug J.R.
      ,
      • Andrae J.
      • Gallini R.
      • Betsholtz C.
      ). Analysis of cultured cells and/or mice lacking PDGFs or PDGFRs has provided vast and convincing evidence that PDGF/PDGFRs regulate many cellular responses and thereby contribute to a wide spectrum of physiological processes (
      • Andrae J.
      • Gallini R.
      • Betsholtz C.
      ).
      Misregulation of PDGF/PDGFR contributes to numerous diseases, including fibrosis in the lung, liver, skin, kidney, heart, and retina (
      • Andrae J.
      • Gallini R.
      • Betsholtz C.
      ,
      • Andrews A.
      • Balciunaite E.
      • Leong F.L.
      • Tallquist M.
      • Soriano P.
      • Refojo M.
      • Kazlauskas A.
      ). For instance, proliferative vitreoretinopathy (PVR) occurs as a complication in 3.9–13.7% of patients undergoing surgery to re-attach a detached retina (
      • Nagasaki H.
      • Ideta H.
      • Uemura A.
      • Morita H.
      • Ito K.
      • Yonemoto J.
      ,
      • Yoshino Y.
      • Ideta H.
      • Nagasaki H.
      • Uemura A.
      ). This is a blinding disease in which the retina re-detaches because of the contraction of a fibroproliferative membrane that forms on the surface of the retina (
      • Laqua H.
      • Machemer R.
      ,
      • Glaser B.M.
      • Cardin A.
      • Biscoe B.
      ,
      • Campochiaro P.A.
      ,
      • Ryan S.J.
      ). Vitreal growth factors are thought to promote both membrane formation and its contraction. PDGF is among the long list of vitreal growth factors implicated in contributing to PVR (
      • Campochiaro P.A.
      • Hackett S.F.
      • Vinores S.A.
      ,
      • Lashkari K.
      • Rahimi N.
      • Kazlauskas A.
      ,
      • Hinton D.R.
      • He S.
      • Jin M.L.
      • Barron E.
      • Ryan S.J.
      ,
      • Cui J.Z.
      • Chiu A.
      • Maberley D.
      • Ma P.
      • Samad A.
      • Matsubara J.A.
      ,
      • Lei H.
      • Hovland P.
      • Velez G.
      • Haran A.
      • Gilbertson D.
      • Hirose T.
      • Kazlauskas A.
      ,
      • Dieudonne S.C.
      • La Heij E.C.
      • Diederen R.
      • Kessels A.G.
      • Liem A.T.
      • Kijlstra A.
      • Hendrikse F.
      ,
      • Mukherjee S.
      • Guidry C.
      ,
      • Kita T.
      • Hata Y.
      • Miura M.
      • Kawahara S.
      • Nakao S.
      • Ishibashi T.
      ,
      • La Heij E.C.
      • van de Waarenburg M.P.
      • Blaauwgeers H.G.
      • Kessels A.G.
      • Liem A.T.
      • Theunissen C.
      • Steinbusch H.
      • Hendrikse F.
      ,
      • Oshima Y.
      • Sakamoto T.
      • Hisatomi T.
      • Tsutsumi C.
      • Ueno H.
      • Ishibashi T.
      ,
      • Liou G.I.
      • Pakalnis V.A.
      • Matragoon S.
      • Samuel S.
      • Behzadian M.A.
      • Baker J.
      • Khalil I.E.
      • Roon P.
      • Caldwell R.B.
      • Hunt R.C.
      • Marcus D.M.
      ,
      • Asaria R.H.
      • Kon C.H.
      • Bunce C.
      • Sethi C.S.
      • Limb G.A.
      • Khaw P.T.
      • Aylward G.W.
      • Charteris D.G.
      ,
      • Harada C.
      • Mitamura Y.
      • Harada T.
      ,
      • Banerjee S.
      • Savant V.
      • Scott R.A.
      • Curnow S.J.
      • Wallace G.R.
      • Murray P.I.
      ,
      • Baudouin C.
      • Fredj-Reygrobellet D.
      • Brignole F.
      • Negre F.
      • Lapalus P.
      • Gastaud P.
      ). Additional evidence for the role of PDGF/PDGFR in PVR includes the observations that cells within the fibroproliferative membrane isolated from patient donors express both PDGF and PDGFRs and that the PDGFRs are activated (
      • Cui J.Z.
      • Chiu A.
      • Maberley D.
      • Ma P.
      • Samad A.
      • Matsubara J.A.
      ,
      • Robbins S.G.
      • Mixon R.N.
      • Wilson D.J.
      • Hart C.E.
      • Robertson J.E.
      • Westra I.
      • Planck S.R.
      • Rosenbaum J.T.
      ,
      • Cui J.
      • Lei H.
      • Samad A.
      • Basavanthappa S.
      • Kwee M.
      • Aspinall A.
      • Boctzkes J.
      • Matsubara J.
      • Kazlauskas A.
      ).
      In a rabbit model of PVR, PDGFRs are essential for disease formation (
      • Andrews A.
      • Balciunaite E.
      • Leong F.L.
      • Tallquist M.
      • Soriano P.
      • Refojo M.
      • Kazlauskas A.
      ,
      • Ikuno Y.
      • Leong F.L.
      • Kazlauskas A.
      ,
      • Zheng Y.
      • Ikuno Y.
      • Ohj M.
      • Kusaka S.
      • Jiang R.
      • Cekic O.
      • Sawa M.
      • Tano Y.
      ), and the vitreous contains high levels of PDGFs (
      • Lei H.
      • Hovland P.
      • Velez G.
      • Haran A.
      • Gilbertson D.
      • Hirose T.
      • Kazlauskas A.
      ). These observations suggest that vitreal PDGFs activate PDGFRs and thereby facilitate the development of PVR. Surprisingly, neutralizing vitreal PDGFs only modestly attenuate experimental PVR,
      H. Lei, G. Velez, P. Hovland, T. Hirose, D. Gilbertson, and A. Kazlauskas (2008) submitted for publication.
      which suggests that PDGFRs are undergoing activation via a non-traditional route. Indeed, vitreal growth factors outside of the PDGF family promote modest tyrosine phosphorylation of PDGFRα.
      H. Lei, G. Velez, P. Hovland, T. Hirose, D. Gilbertson, and A. Kazlauskas (2008) submitted for publication.
      Other investigators have also reported increased tyrosine phosphorylation of PDGFRs by agents that are not PDGF. Certain agonists of G protein-coupled receptors, autoantibodies in the blood of scleroderma patients and agents that are within the bone marrow (but are probably not PDGFs) promoted tyrosine phosphorylation of PDGFR (
      • Linseman D.A.
      • Benjamin C.W.
      • Jones D.A.
      ,
      • Herrlich A.
      • Daub H.
      • Knebel A.
      • Herrlich P.
      • Ullrich A.
      • Schultz G.
      • Gudermann T.
      ,
      • Heeneman S.
      • Haendeler J.
      • Saito Y.
      • Ishida M.
      • Berk B.C.
      ,
      • Tanimoto T.
      • Lungu A.O.
      • Berk B.C.
      ,
      • Siegbahn A.
      • Johnell M.
      • Nordin A.
      • Aberg M.
      • Velling T.
      ,
      • Liu Y.
      • Li M.
      • Warburton R.R.
      • Hill N.S.
      • Fanburg B.L.
      ,
      • Dolloff N.G.
      • Russell M.R.
      • Loizos N.
      • Fatatis A.
      ,
      • Baroni S.S.
      • Santillo M.
      • Bevilacqua F.
      • Luchetti M.
      • Spadoni T.
      • Mancini M.
      • Fraticelli P.
      • Sambo P.
      • Funaro A.
      • Kazlauskas A.
      • Avvedimento E.V.
      • Gabrielli A.
      ). Thus there is a growing appreciation that the direct, PDGF-based mechanism is not the only route to induce tyrosine phosphorylation of PDGFRs. The goal of this study was to identify the intracellular events and mediators by which non-PDGFs induced tyrosine phosphorylation of PDGFRα and to assess whether indirect activation of PDGFRα contributed to cellular responses induced by non-PDGFs.

      EXPERIMENTAL PROCEDURES

      Cell Culture-F, Fα, and FαΔX cells were previously described (
      • Andrews A.
      • Balciunaite E.
      • Leong F.L.
      • Tallquist M.
      • Soriano P.
      • Refojo M.
      • Kazlauskas A.
      ).3 Briefly, they are mouse embryo fibroblasts derived from mice null for both pdgfr genes. They were immortalized with SV40 T antigen. Fα and FαΔX cells are F cells in which we re-expressed the human PDGFRα that was the full-length or the mutant lacking the majority of the extracellular domain, respectively.
      SYF and SYF + Src cells were previously described (
      • Klinghoffer R.A.
      • Sachsenmaier C.
      • Cooper J.A.
      • Soriano P.
      ). Briefly, they are mouse embryo fibroblasts derived from src, yes, and fyn knockout mice. SYFα and SYFα + Src cells were generated by expressing human PDGFRα in SYF and SYF + Src cells, respectively (
      • Avrov K.
      • Kazlauskas A.
      ). The SYFα and SYFα + Src cells were used in these studies and are designated as “-/-” and “Src” in Fig. 3.
      Figure thumbnail gr3
      FIGURE 3SFKs were required for phosphorylation of PDGFRα by non-PDGFs. A, RPE19α cells were serum-starved for 24 h and exposed to PDGF-A (50 ng/ml), bFGF (100 ng/ml), RV, or H2O2 (5 mm) for 10 min at 37 °C. Cleared lysates were subjected to Western blot using a phospho-Src (Tyr-416) antibody. The stripped membrane was reprobed with a pan-Src antibody. The ratio of the resulting signals is shown at the bottom. In three independent experiments the average ± S.D. was 3.0 ± 0.3, 2.5 ± 0.2, 2.7 ± 0.3, and 36.2 ± 3.5 for PDGF-A, bFGF, RV, and H2O2, respectively. These data show that all agents activated Src. Although the magnitude of the increase in phosphorylation was modest, it is consistent with what other groups report using this approach (
      • Matsuoka H.
      • Nada S.
      • Okada M.
      ,
      • Veracini L.
      • Franco M.
      • Boureux A.
      • Simon V.
      • Roche S.
      • Benistant C.
      ) and what we have found using a different approach to monitor activation of SFKs (
      • Gelderloos J.A.
      • Rosenkranz S.
      • Bazenet C.
      • Kazlauskas A.
      ). B, mouse embryo fibroblasts that lacked all SFKs (
      • Klinghoffer R.A.
      • Sachsenmaier C.
      • Cooper J.A.
      • Soriano P.
      ) were reconstituted with either an empty expression vector (-/-), or wild type c-Src (Src) (
      • Avrov K.
      • Kazlauskas A.
      ) and stimulated with the indicated agents as described in panel A. In three independent experiments the PDGF-A, bFGF, RV, and H2O2-dependent -fold increases in phosphorylation of PDGFRα in -/- and Src-expressing cells were 28.5 ± 2.5 and 29.2 ± 3.2, 0.99 ± 0.1 and 4.8 ± 0.5, 0.98 ± 0.2 and 6.2 ± 0.6, and 2.2 ± 0.3 and 38.5 ± 2.3, respectively. The results indicate that, although c-Src greatly increased the ability of non-PDGFs to increase the phosphotyrosine content of PDGFRα, PDGF-dependent phosphorylation of PDGFRα was independent of expression of SFKs. C, cleared lysates from the indicated cell lines were subjected to Western blotting using an anti-PDGFRα or anti-RasGAP antibody. The resulting data are representative of at least two independent experiments and show that the level of PDGFRα was similar in both cell lines.
      Phα and R627 cells were previously described (
      • Ikuno Y.
      • Leong F.-L.
      • Kazlauskas A.
      ). Briefly, they are spontaneously immortalized mouse embryo fibroblasts derived from mice harboring the Ph deletion that includes the pdgfra gene. The wild-type or kinase-inactive human pdgfra cDNA was expressed in parental Ph cells to generate Phα and R627, respectively.
      ARPE19 (RPE19) cells are a human retinal pigment epithelial cell line that was purchased from American Type Culture Collection. RPE19α cells were generated by expressing PDGFRα in the parental RPE19 cells (
      • Lei H.
      • Hovland P.
      • Velez G.
      • Haran A.
      • Gilbertson D.
      • Hirose T.
      • Kazlauskas A.
      ), which naturally express a very low level of PDGFRα.
      Primary rabbit conjunctiva fibroblasts were isolated from rabbit conjunctiva as previously described (
      • Nakagawa M.
      • Refojo M.F.
      • Marin J.F.
      • Doi M.
      • Tolentino F.I.
      ). F, Fα, FαΔX, SYFα, SYFα + Src, Phα, R627, and rabbit conjunctiva fibroblast cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, high glucose, Invitrogen) supplemented with 10% fetal bovine serum (Gemini Bio Products, Calabasas, CA), 500 units/ml penicillin, and 500 μg/ml streptomycin. RPE19 and RPE19α cells were cultured in a 1:1 mixture of DMEM and Ham’s F-12 medium (Invitrogen) supplemented with 10% fetal bovine serum, 500 units/ml penicillin, and 500 μg/ml streptomycin. All cells were cultured at 37 °C in a humidified 5% CO2 atmosphere.
      Major Reagents-The anti-PDGFRα (27P) antibody was produced and characterized as previously described (
      • Gelderloos J.A.
      • Rosenkranz S.
      • Bazenet C.
      • Kazlauskas A.
      ). The two anti-phosphotyrosine antibodies, 4G10 and PY20, were purchased from Upstate and BD Transduction Laboratories (Madison, WI), respectively. Recombinant human PDGF-A and mouse basic fibroblast growth factor (bFGF) were purchased from Peprotech Inc. (Rocky Hill, NJ). Antibodies that recognized PDGFRα, fibroblast growth factor receptor 1 (FGFR1), phospho-src (Tyr-416) were purchased from Cell Signaling (Danvers, MA). The c-Src-2 antibody, horseradish peroxidase-conjugated goat anti-rabbit IgG, and goat anti-mouse IgG secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Enhanced chemiluminescent substrate for detection of horseradish peroxidase was from Pierce Protein Research Products (Rockford, IL). N-Acetylcysteine (NAC), 30% H2O2, Tiron, catalase, and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma. SU6656 (C19H21N3O3S) was purchased from Calbiochem.
      Immunoprecipitation and Western Blot-Cells were grown to 90% confluence and then incubated for 24 h in DMEM or DMEM/F-12 without serum. The cells were exposed to the desired agents as follows: NAC, Tiron: 10 mm for 10 min; Catalase: 144 nm for 10 min; SU6656: 1 μm for 1 h; unless indicated otherwise growth factors were added for 10 min at a concentration of 50 ng/ml for PDGF and 100 ng/ml for bFGF. The cells were washed twice with ice-cold phosphate-buffered saline, and then lysed in extraction buffer (10 mm Tris-HCl, pH 7.4, 5 mm EDTA, 50 mm NaCl, 50 mm NaF, 1% Triton X-100, 20 μg/ml aprotinin, 2 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride). Lysates were centrifuged for 15 min at 13,000 × g, 4 °C, and PDGFRα was immunoprecipitated from clarified lysates as previously described (
      • Lei H.
      • Hovland P.
      • Velez G.
      • Haran A.
      • Gilbertson D.
      • Hirose T.
      • Kazlauskas A.
      ). The immunoprecipitating antibody was a crude rabbit polyclonal (27P). The blotting antibody was a 1:1 mixture of anti-phosphotyrosine antibodies (4G10/PY20). The primary blot membrane was stripped and reprobed with the PDGFRα antibody, 27P, or number 3164 from Cell Signaling. At least three independent experiments were performed. Signal intensity was determined by densitometry using Quantity One (Bio-Rad) and normalized for the amount of PDGFRα in each sample (
      • Lei H.
      • Hovland P.
      • Velez G.
      • Haran A.
      • Gilbertson D.
      • Hirose T.
      • Kazlauskas A.
      ).
      DCF Assay-Intracellular H2O2 generation was measured indirectly using the fluorescent dye, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), as described by several other groups (
      • Bae Y.S.
      • Sung J.Y.
      • Kim O.S.
      • Kim Y.J.
      • Hur K.C.
      • Kazlauskas A.
      • Rhee S.G.
      ,
      • Kang S.W.
      • Chae H.Z.
      • Seo M.S.
      • Kim K.
      • Baines I.C.
      • Rhee S.G.
      ,
      • Wan X.S.
      • Zhou Z.
      • Kennedy A.R.
      ). Briefly, serum-deprived RPE19α, rabbit conjunctiva fibroblast, and Fα were stimulated for 10 min with bFGF (100 ng/ml) in DMEM without phenol red or PVR rabbit vitreous. Subsequently the cells were rinsed twice with Krebs-Ringer solution and incubated in Krebs-Ringer solution containing DCFH-DA (5 μm). DCFH-DA is non-polar and readily diffuses into cells, where it is hydrolyzed to the non-fluorescent polar derivative DCFH and thereby trapped within the cells. In the presence of H2O2, DCFH is oxidized to the highly fluorescent DCF. Culture dishes were sealed with paraffin film and placed inaCO2 incubator at 37 °C for 5 min, after which DCF fluorescence was photographed using a Zeiss Axiovert 135 inverted immunofluorescence microscope equipped with a digital camera. To avoid photooxidation of DCF, we collected the fluorescent images at a 9.0-s exposure under green light and identical contrast and brightness parameters for all samples. Cells that were cultured in 96-well plates and treated in the same way were read with a Bio-Tek fluorescence plate reader at excitation and emission wavelengths of 485 and 528 nm, respectively.
      Cell Proliferation Assay-Cells (F, Fα, and FαΔX) were seeded into 6-well plates at a density of 60,000 cells/well in DMEM plus 10% fetal bovine serum. After 8 h the cells had attached; the medium was aspirated, the cells were rinsed twice with phosphate-buffered saline, and the cells were cultured in serum-free DMEM and treated with certain reagents as follows: 1) DMEM only; 2) bFGF (1, 4, 20, 100, and 200 ng/ml); 3) bFGF (20 ng/ml) plus NAC (10 mm); 4) PDGF-A (50 ng/ml); and 5) PDGF-A plus NAC (10 mm). The media were replaced every day. The cells were counted in a hemocytometer on days 1, 2, 3, and 4, and at least three independent experiments were performed.
      Apoptosis Assay-Cells were seeded into 10 cm-dishes at a density of 2 × 105 cells per dish in DMEM plus 10% fetal bovine serum. After the cells had attached the dishes, they were treated as described above under “Cell Proliferation Assay.” On Day 3, the cells were harvested and stained with fluorescein isothiocyanate-conjugated Annexin V and propidium iodide according to the instructions provided with the apoptosis kit (BD Biosciences). The cells were analyzed by flow cytometry in Coulter Beckman XL (
      • Lei H.
      • Romeo G.
      • Kazlauskas A.
      ). At least three independent experiments were performed. The raw data from a representative experiment are shown in supplemental Fig. S1.
      Statistics-All data were analyzed using the unpaired t test. p values of <0.05 were considered statistically significant.

      RESULTS

      Non-PDGF-triggered Tyrosine Phosphorylation of PDGFRα was Dependent on ROS-It has been demonstrated that increasing the level of reactive oxygen species (ROS) can lead to tyrosine phosphorylation of many proteins, including the PDGF β receptor (PDGFRβ) (
      • Sundaresan M.
      • Yu Z.X.
      • Ferrans V.J.
      • Irani K.
      • Finkel T.
      ). Consequently, we tested if non-PDGFs increased ROS, and whether this event was likely to be involved with tyrosine phosphorylation of the closely related PDGFRα. In these experiments we used vitreous from normal rabbits (instead of rabbits with PVR) to mimic the conditions at the start of the disease. Unlike the vitreous from rabbits with PVR, vitreous from normal rabbits had undetectable amounts of PDGFs, although it did have a variety of growth factors outside of the PDGF family (
      • Lei H.
      • Hovland P.
      • Velez G.
      • Haran A.
      • Gilbertson D.
      • Hirose T.
      • Kazlauskas A.
      ). Fig. 1 (A and B) shows that either rabbit vitreous, or bFGF (a representative vitreal non-PDGF), increased the level of ROS in three cell lines that are highly relevant to experimental and/or clinical PVR. Furthermore, exogenously added hydrogen peroxide strongly stimulated phosphorylation of PDGFRα (Fig. 1C), indicating that a rise in ROS was sufficient to increase the phosphotyrosine content of PDGFRα. Pretreating cells with anti-oxidants NAC or Tiron (10 mm for 10 min), or an enzyme that scavenges H2O2 (catalase, 144 nm, for 10 min) prevented non-PDGFs and H2O2 from phosphorylating PDGFRα (Fig. 1C). In contrast, direct activation of PDGFRα and FGFR1 was unaffected by antioxidants (Fig. 1, C and D). These results indicate that ROS was produced in response to non-PDGFs and was required for them to induce tyrosine phosphorylation of PDGFRα.
      Figure thumbnail gr1
      FIGURE 1Non-PDGFs increased intracellular ROS, and this event was necessary for phosphorylation of PDGFRα by non-PDGFs. A, RPE19α cells were exposed for 10 min to bFGF (100 ng/ml) or rabbit vitreous (RV). The cells were then incubated with DCFH-DA (5 μm) for 5 min at 37 °C and photographed using an inverted immunofluorescence microscope. Both RV and bFGF triggered an increase in ROS as indicated by the increase in green color. B, the indicated cell lines were treated as described in panel A, and instead of photographing them, they were scanned in a Bio-Tek fluorescence plate reader (excitation and emission wavelengths of 485 and 528 nm, respectively). The results from three independent experiments were pooled, and the mean ± S.D. values are shown in the bar graph. In all three cell lines bFGF and RV increased the level of ROS. C, serum-starved Fα cells were left resting or pretreated for 10 min with 10 mm NAC, 10 mm Tiron, or 144 nm catalase and then exposed for 10 min to PDGF-A (50 ng/ml), bFGF (100 ng/ml), or H2O2 (5 mm). The cells were lysed, and PDGFRα was immunoprecipitated and subjected to Western blot analysis with anti-phosphotyrosine (top panel) anti-PDGFRα (bottom panel). In three independent experiments PDGF-A, H2O2, and bFGF induced a 39.8 ± 2.9-, 28.2 ± 2.3-, and 3.1 ± 0.5-fold increase in phosphorylation of PDGFR. The results show that the bFGF- and H2O2-triggered increased in phosphorylation of PDGFRα was sensitive to antioxidants, whereas PDGF-dependent phosphorylation of PDGFRα was not. D, same as panel C except that F cells were used in place of Fα cells. Like direct activation of PDGFRα, direct activation of FGFR using bFGF was not sensitive to anti-oxidants such as NAC. In three independent experiments the phospho-FGFR1/FGFR1 ratio was comparable in cells treated with bFGF plus NAC versus bFGF alone. The data in A–D are representative of at least three independent experiments.
      One of the mechanisms by which ROS could increase tyrosine phosphorylation of PDGFRα is by inactivating phosphotyrosine phosphatases that dephosphorylate autophosphorylated PDGFRα (
      • Meng T.C.
      • Fukada T.
      • Tonks N.K.
      ,
      • Klinghoffer A.R.
      • Kazlauskas A.
      ). To test this ROS/phosphotyrosine phosphatase hypothesis we determined if non-PDGFs induced tyrosine phosphorylation of kinase-inactive PDGFRα, which is incapable of autophosphorylating (
      • Rosenkranz S.
      • Kazlauskas A.
      ). Using a previously characterized pair of cell lines that expressed either the wild-type or kinase-inactive (R627) PDGFRα (
      • Rosenkranz S.
      • Kazlauskas A.
      ), we found that there was no difference in the extent of tyrosine phosphorylation of the two receptors in response to any of the three agents that promoted receptor phosphorylation in an ROS-dependent manner (Fig. 2A). As expected, direct activation of PDGFRα via PDGF increased tyrosine phosphorylation of the wild-type, but not R627 PDGFRα (Fig. 2A). Western blot analysis of total cell lysates prepared from the two cell lines indicated that the wild-type and R627 PDGFRα were expressed to similar levels (Fig. 2B). These findings indicate that the ROS/phosphotyrosine phosphatase hypothesis was either incorrect or incomplete. Furthermore, the mechanism by which non-PDGFs increased phosphorylation of PDGFRα involved a kinase that was not PDGFRα itself.
      Figure thumbnail gr2
      FIGURE 2PDGFRα kinase activity is dispensable for its phosphorylation in response to non-PDGFs. A, mouse embryo fibroblasts expressing either the full length (α) or kinase inactive (R627) PDGFRα were treated as described in the legend of . PDGFRα was immunoprecipitated and subjected to an anti-phosphotyrosine Western blot. In three independent experiments bFGF, RV, and H2O2 induced a 3.0 ± 0.5-, 6.1 ± 0.6-, and 34.5 ± 3.1-fold increase, respectively, of the wild-type receptor; whereas the -fold increase for the kinase-dead receptor was 3.2 ± 0.4-, 6.2 ± 0.5-, and 36.5 ± 3.6-fold, respectively. The results show that phosphorylation of PDGFRα by non-PDGFs was independent of the receptor’s kinase activity. B, cleared lysates from the indicated cell lines were subjected to Western blotting using an anti-PDGFRα or anti-RasGAP antibody. The resulting data are representative of at least two independent experiments and show that the level of PDGFRα was similar in both cell lines.
      SFKs Were Required for Non-PDGF-dependent Tyrosine Phosphorylation of PDGFRα-We considered whether this kinase was an Src family kinase (SFK), because SFKs can be activated by hydrogen peroxide (
      • Hardwick J.S.
      • Sefton B.M.
      ,
      • Abe J.
      • Takahashi M.
      • Ishida M.
      • Lee J.D.
      • Berk B.C.
      ) and have many substrates, including PDGFRs (
      • Hansen K.
      • Johnell M.
      • Siegbahn A.
      • Rorsman C.
      • Engstrom U.
      • Wernstedt C.
      • Heldin C.H.
      • Ronnstrand L.
      ). All of the agents that promoted phosphorylation of PDGFRα activated c-Src (Fig. 3A). Furthermore, phosphorylation of PDGFRα induced by bFGF, rabbit vitreous (RV) or hydrogen peroxide was either undetectable or severely attenuated in mouse embryo fibroblasts that lacked SFKs (SYF cells, “-/-”) (Fig. 3B). Re-expression of c-Src restored phosphorylation of PDGFRα by all three agents without altering the expression level of PDGFRα (Fig. 3, B and C). In contrast (and consistent with previous observations (
      • Klinghoffer R.A.
      • Sachsenmaier C.
      • Cooper J.A.
      • Soriano P.
      )), direct activation of PDGFRα did not require expression of SFKs (Fig. 3B). Similarly, pharmacologically inhibiting SFKs had no effect on PDGF-dependent phosphorylation of PDGFRα, yet it blocked non-PDGF-dependent phosphorylation of PDGFRα (Fig. 4). These results identify SFKs as an intracellular mediator of non-PDGF-dependent tyrosine phosphorylation of PDGFRα.
      Figure thumbnail gr4
      FIGURE 4Pharmacologically inhibiting SFKs prevented phosphorylation of PDGFRα by non-PDGFs. RPE19α were pretreated with DMSO (-) or a SFK inhibitor SU6656 (1 μm)(+) for 1 h at 37 °C and then stimulated with the indicated agents as described in the legend of . In three independent experiments the PDGF-A-, bFGF-, RV-, and H2O2-dependent -fold increases in phosphorylation of PDGFRα in control and SU6656-treated cells were 34.5 ± 2.5 and 33.5 ± 3.2, 4.5 ± 0.6 and 0.5 ± 0.1, 4.2 ± 0.6 and 0.5 ± 0.1, and 20.9 ± 1.6 and 0.4 ± 0.2, respectively. The data show that blocking SFK activity substantially reduced bFGF-, RV-, and H2O2-dependent tyrosine phosphorylation of PDGFRα but had no effect on PDGF-dependent phosphorylation of PDGFRα. The data are representative of at least three independent experiments.
      Indirect Activation of PDGFRα Promoted Proliferation and Survival of Cells in Response to Non-PDGFs-Although direct activation of PDGFRs robustly induced cellular responses such as proliferation and survival, whether the modest increase in the phosphotyrosine content of PDGFRα induced by non-PDGFs was also capable of doing so was an open question. Previous investigators found that non-PDGF-triggered signaling events related to cell proliferation and survival (activation of Akt) were enhanced by expression of PDGFRα (
      • Zhang H.
      • Bajraszewski N.
      • Wu E.
      • Wang H.
      • Moseman A.P.
      • Dabora S.L.
      • Griffin J.D.
      • Kwiatkowski D.J.
      ). The increase in such signaling events may be sufficient to enhance cellular responses. To test this idea we considered if indirect activation of PDGFRα promoted cellular responses by comparing bFGF-induced cell proliferation and survival in cells that did or did not express PDGFRα. A dose response experiment indicated that both responses plateaued at 20 ng/ml bFGF (Fig. 5A and 6A). bFGF-induced proliferation was enhanced by expression of full-length PDGFRα (Fig. 5B and Table 1). When we assessed apoptosis in this panel of cell lines we observed enhanced survival (reduced apoptosis) under both basal and bFGF-stimulated conditions in the PDGFRα-expressing cells as compared with the non-expressing cells (Fig. 6B and Table 1). The enhanced survival in the absence of exogenously added growth factors may by due to indirect activation of PDGFRα by endogenously produced factors. Cultured cells produce growth factors (
      • Janes K.A.
      • Albeck J.G.
      • Gaudet S.
      • Sorger P.K.
      • Lauffenburger D.A.
      • Yaffe M.B.
      ), and PDGFRα can be indirectly activated by a variety of such factors.3 We concluded that expression of PDGFRα enabled enhanced proliferation and survival in response to non-PDGFs such as bFGF.
      Figure thumbnail gr5
      FIGURE 5Expression of PDGFRα promoted non-PDGF-driven cell proliferation. The indicated cells were seeded at a density of 60,000 cells per well of 6-well plates. The cells were permitted to attach, and after two rinses with phosphate-buffered saline, serum-free medium was added that was (+) or was not (-) supplemented with bFGF. The media were replaced every day, and the cell number was counted on day 3. A, a dose-response curve showing that 20 ng/ml bFGF induced the maximal response. B, the cells were treated with 20 ng/ml bFGF, 50 ng/ml PDGF-A, 10 mm NAC as indicated. The bars indicate the raw data (cell number), whereas the -fold change is presented in . The mean ± S.D. of three independent experiments is shown; *, p < 0.05 using an unpaired t test. Although bFGF promoted proliferation of all cell lines, the response was enhanced in cells expressing PDGFRα, and this phenomenon was blocked by NAC. C, cleared lysates from the indicated cell lines were subjected to Western blotting using an anti-PDGFRα or anti-RasGAP antibody. The resulting data are representative of at least two independent experiments and show that the level of PDGFRα was similar in both cell lines.
      Figure thumbnail gr6
      FIGURE 6Expression of PDGFRα promoted non-PDGF-driven survival. The indicated cells were seeded at a density of 200,000 cells/10-cm dish. The cells were permitted to attach, and after two rinses with phosphate-buffered saline, serum-free medium was added that was (+) or was not (-) supplemented with bFGF. The media were replaced every day. On day 3 the cells were harvested, stained with fluorescein isothiocyanate-conjugated Annexin V and propidium iodide, and analyzed by flow cytometry. Cells in early apoptosis were defined as the population that was positive for Annexin V, but negative for propidium iodide. A, a dose-response curve showing that 20 ng/ml bFGF induced the maximal response. B, the cells were treated with 20 ng/ml bFGF, 50 ng/ml PDGF-A, 10 mm NAC as indicated. The bars indicate the % of apoptotic cells (raw data from a representative experiment is in supplemental ), whereas the -fold change is presented in . The mean ± S.D. of three independent experiments is shown; *, p < 0.05 using an unpaired t test. Although bFGF protected all cell lines from apoptosis, it was more effective for cells that expressed PDGFRα, and this phenomenon was blocked by NAC. C, cleared lysates prepared from F and Fα cells were subjected to Western blot analysis using the indicated antibodies. The data show that the level of FGFR1 was not altered by expression of PDGFRα.
      TABLE 1Expression of PDGFRα promoted non-PDGF-driven cell proliferation and survival The indicated cellular responses were assayed as described in the legend of Figs. 5 and 6. The mean ± S.D. of the bFGF-induced-fold change from three independent experiments is shown.
      CellsWithout NAC treatmentWith NAC treatment
      -bFGFPDGF-A-bFGFPDGF-A
      foldfold
      Proliferation
      F1.01.46 ± 0.02NA1.01.75 ± 0.03NA
      FαΔx1.01.95 ± 0.06
      Statistically significant difference (p < 0.05) between F and Fα or FαΔX
      NA1.01.75 ± 0.03NA
      1.01.98 ± 0.03
      Statistically significant difference (p < 0.05) between F and Fα or FαΔX
      2.20 ± 0.031.01.75 ± 0.022.13 ± 0.08
      Apoptosis
      F1.00.68 ± 0.04NA1.00.73 ± 0.04NA
      FαΔx1.00.18 ± 0.04
      Statistically significant difference (p < 0.05) between F and Fα or FαΔX
      NA1.00.72 ± 0.02NA
      1.00.17 ± 0.03
      Statistically significant difference (p < 0.05) between F and Fα or FαΔX
      0.15 ± 0.021.00.70 ± 0.020.63 ± 0.04
      a Statistically significant difference (p < 0.05) between F and Fα or FαΔX
      Another way to consider these data is in the reverse order. The non-PDGF was more effective at driving proliferation and survival of PDGFRα-expressing cells as compared with the cells that did not express PDGFRα (Figs. 5 and 6 and Table 1). Because FGFR1 was expressed to comparable levels in both cell lines (Fig. 6C), the difference in the magnitude of the responses was not simply due to a lower level of FGFR1 in the less-responsive cells.
      The cells used in these studies secrete both PDGF-C and the proteases necessary to process it (
      • Lei H.
      • Hovland P.
      • Velez G.
      • Haran A.
      • Gilbertson D.
      • Hirose T.
      • Kazlauskas A.
      ), which raised the possibility that the enhanced responsiveness of the PDGFRα-expressing cells was the result of direct activation of PDGFRα via endogenously produced PDGF-C. To address this possibility we repeated the experiments with cells the expressed αΔX PDGFRα. This truncated receptor cannot be activated by PDGF (because it lacks the majority of the extracellular domain), yet is fully transactivatable.3 As shown in Figs. 5 and 6 and Table 1, the αΔX PDGFRα potentiated non-PDGF-driven cellular responses as well as the full-length receptors. These findings rule out the possibility that a PDGF-C/PDGFRα autocrine loop is responsible for the greater responses in PDGFRα-expressing cells.
      Finally, we tested if the enhanced responsiveness in PDGFR-expressing cells was sensitive to anti-oxidants. When assayed in the presence of NAC, the basal proliferation of cells decreased and apoptosis increased somewhat (Figs. 5B and 6B and Table 1). More importantly, while bFGF promoted proliferation and survival in NAC-treated cells, the responses were not greater in PDGFR-expressing cells as compared with the F cells. These observations indicated that ROS is essential for non-PDGFs to promote cellular response in PDGFRα-expressing cells.

      DISCUSSION

      The findings described herein begin to elucidate intracellular events and identify effectors that are required for growth factors outside of the PDGF family to induce tyrosine phosphorylation of PDGFRα (Fig. 7). Non-PDGFs bind and activate their own receptors and thereby increase the cellular level of ROS. This leads to a rise in SFK activity, which either directly or indirectly phosphorylates PDGFRα. This series of events is associated with increased proliferation and survival of cells exposed to non-PDGFs.
      Figure thumbnail gr7
      FIGURE 7A model for how non-PDGFs drive phosphorylation of PDGFRα and enhance cellular responses. Growth factors outside of the PDGF family engage their own receptors and increase the level of ROS. This leads to activation of SFKs, which directly or indirectly promote phosphorylation of PDGFRα. Although this results in a relatively modest increase in the phosphotyrosine content of PDGFRα, it appears sufficient to boost cellular response such as proliferation and survival. Potentiating these types of cellular responses may tip the balance in favor of disease progression in the case of PVR.
      Tyrosine phosphorylation of receptor tyrosine kinases by ligands other than their cognate receptors has been previously reported. For instance, activation of G protein-coupled receptors increases the activity of metalloproteases that release bound heparin-binding-EGF, which activates the EGF receptor (
      • Prenzel N.
      • Zwick E.
      • Daub H.
      • Leserer M.
      • Abraham R.
      • Wallasch C.
      • Ullrich A.
      ). This mechanism seems relevant to PDGFRα phosphorylation, because the cell lines under investigation secrete PDGF-C and the proteases necessary to activate it (
      • Lei H.
      • Hovland P.
      • Velez G.
      • Haran A.
      • Gilbertson D.
      • Hirose T.
      • Kazlauskas A.
      ). Thus non-PDGFs could promote either secretion, or processing of PDGF-C, and thereby drive tyrosine phosphorylation of PDGFRα. However, this mechanism seems unlikely in light of the fact that removing the extracellular domain of PDGFRα did not prevent its tyrosine phosphorylation by non-PDGFs.3
      Profoundly different mechanisms appear responsible for direct (PDGF-driven) and indirect (via non-PDGFs) phosphorylation of PDGFRα. Direct activation involves the extracellular ligand-binding domain, depends on activating the receptor’s intrinsic kinase activity and proceeds without any apparent input from SFKs. In contrast, indirect activation appears to involve coordinating events that result in activation of SFKs that either directly or indirectly phosphorylate the resting receptor. These findings reveal that PDGFRα can be phosphorylated by SFKs under certain circumstances and in this regard is similar to other receptor tyrosine kinases. For instance, c-Src phosphorylates EGFR and potentiates EGF-dependent cellular responses (
      • Maa M.-C.
      • Leu T.-H.
      • McCarley D.J.
      • Schatzman R.C.
      • Parsons S.J.
      ,
      • Tice D.A.
      • Biscardi J.S.
      • Nickles A.L.
      • Parsons S.J.
      ). Moreover, SFKs directly or indirectly promote tyrosine phosphorylation of the hepatocyte growth factor receptor (Met) in colon cancer cell lines (
      • Emaduddin M.
      • Bicknell D.C.
      • Bodmer W.F.
      • Feller S.M.
      ).
      Although direct activation of PDGFR is thought to proceed by the same mechanism, regardless of which PDGF isoform is used, there may be multiple mechanisms by which agents outside of the PDGF family promote phosphorylation of PDGFRs. Tyrosine phosphorylation of PDGFRα by angiotensin (an agonist of a G protein-coupled receptor) is independent of SFKs (
      • Heeneman S.
      • Haendeler J.
      • Saito Y.
      • Ishida M.
      • Berk B.C.
      ). In contrast, there was a strict requirement for SFKs in order for polypeptide growth factors to promote tyrosine phosphorylation of PDGFRα. Thus there appear to be at least two different routes by which resting PDGFRs can be engaged in signaling events triggered by agents outside of the PDGF family.
      In light of the promiscuity of ROS/SFKs, one may expect that all receptor tyrosine kinases would be phosphorylated when these intracellular agents are increased/activated. Yet this turned out not to be the case. PDGF promoted a rise in ROS and activated SFKs, yet there was no perceptible increase in the phosphotyrosine content of FGFR1.3 How the ROS/SFK-directed pathway chooses a substrate remains an open question at this point.
      Although we organized our findings to emphasize the differences between the direct and indirect mechanisms by which PDGFRα was activated, there may be some overlap. As noted above, direct activation of PDGFRα via PDGF increased the level of ROS, which was not required for tyrosine phosphorylation of the receptor. However, increasing the level of ROS appeared to be required for cell survival even when the PDGFRα was activated directly. PDGF protected Fα cells from apoptosis much more efficiently in the absence of NAC than it its presence (Fig. 6 and Table 1). In contrast, NAC treatment had little impact on PDGF-dependent proliferation of these cells. These findings suggest that ROS was required for some of the responses, even when the receptor was activated directly.
      Our data re-enforce the emerging concept that PDGFRs can be activated by more than just PDGFs, and that this can boost the responsiveness of cells to non-PDGFs. How do these tissue culture findings extend to physiology/pathology? Because knocking out either PDGFRβ or PDGF-B gene in mice resulted in very similar phenotypes (
      • Soriano P.
      ,
      • Lindahl P.
      • Johansson B.R.
      • Leveen P.
      • Betsholtz C.
      ), it appears that PDGF-B is the primary activator of PDGFRβ during development. In contrast, activation of PDGFRα by non-PDGFs appears to play an important role in the manifestation of fibroproliferative diseases such as PVR.3 Furthermore, there is a breach of traditional signaling specificity in the context of breast cancer. A subpopulation of lung cancer patients treated with EGFR inhibitors developed lung cancers that overexpressed the hepatocyte growth factor receptor, which acted via the kinase-independent ErbB3 to activate phosphatidylinositol 3-kinase (
      • Engelman J.A.
      • Zejnullahu K.
      • Mitsudomi T.
      • Song Y.
      • Hyland C.
      • Park J.O.
      • Lindeman N.
      • Gale C.M.
      • Zhao X.
      • Christensen J.
      • Kosaka T.
      • Holmes A.J.
      • Rogers A.M.
      • Cappuzzo F.
      • Mok T.
      • Lee C.
      • Johnson B.E.
      • Cantley L.C.
      • Janne P.A.
      ). Thus nontraditional activation of receptor tyrosine kinases appears to be more relevant to pathology than development. Identifying the mediators of these pathways and elucidating the mechanisms by which they drive cellular responses is likely to provide the conceptual foundation to confront endemic diseases.

      Acknowledgments

      We thank Randy Huang for facilitating studies involving fluorescence-activated cell sorting analysis and Steven Pennock, Ruta Motiejunaite, Jorge Aranda, and Magdalena Staniszewska for reading the manuscript and/or helpful discussions.

      Supplementary Material

      References

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