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Endocytic Function of von Hippel-Lindau Tumor Suppressor Protein Regulates Surface Localization of Fibroblast Growth Factor Receptor 1 and Cell Motility

2006; Elsevier BV; Volume: 281; Issue: 17 Linguagem: Inglês

10.1074/jbc.m511621200

ISSN

1083-351X

Autores

Tien Hsu, Yair Adereth, Nurgun Kose, Vincent Dammai,

Tópico(s)

Metabolism, Diabetes, and Cancer

Resumo

The tumor suppressor VHL (von Hippel-Lindau protein) serves as a negative regulator of hypoxia-inducible factor-α subunits. However, accumulated evidence indicates that VHL may play additional roles in other cellular functions. We report here a novel hypoxia-inducible factor-independent function of VHL in cell motility control via regulation of fibroblast growth factor receptor 1 (FGFR1) endocytosis. In VHL null tumor cells or VHL knock-down cells, FGFR1 internalization is defective, leading to surface accumulation and abnormal activation of FGFR1. The enhanced FGFR1 activity directly correlates with increased cell migration. VHL disease mutants, in two of the mutation hot spots favoring development of renal cell carcinoma, failed to rescue the above phenotype. Interestingly, surface accumulation of the chemotactic receptor appears to be selective in VHL mutant cells, since other surface proteins such as epidermal growth factor receptor, platelet-derived growth factor receptor, IGFR1, and c-Met are not affected. We demonstrate that 1) FGFR1 endocytosis is defective in the VHL mutant and is rescued by reexpression of wild-type VHL, 2) VHL is recruited to FGFR1-containing, but not EGFR-containing, endosomal vesicles, 3) VHL exhibits a functional relationship with Rab5a and dynamin 2 in FGFR1 internalization, and 4) the endocytic function of VHL is mediated through the metastasis suppressor Nm23, a protein known to regulate dynamin-dependent endocytosis. The tumor suppressor VHL (von Hippel-Lindau protein) serves as a negative regulator of hypoxia-inducible factor-α subunits. However, accumulated evidence indicates that VHL may play additional roles in other cellular functions. We report here a novel hypoxia-inducible factor-independent function of VHL in cell motility control via regulation of fibroblast growth factor receptor 1 (FGFR1) endocytosis. In VHL null tumor cells or VHL knock-down cells, FGFR1 internalization is defective, leading to surface accumulation and abnormal activation of FGFR1. The enhanced FGFR1 activity directly correlates with increased cell migration. VHL disease mutants, in two of the mutation hot spots favoring development of renal cell carcinoma, failed to rescue the above phenotype. Interestingly, surface accumulation of the chemotactic receptor appears to be selective in VHL mutant cells, since other surface proteins such as epidermal growth factor receptor, platelet-derived growth factor receptor, IGFR1, and c-Met are not affected. We demonstrate that 1) FGFR1 endocytosis is defective in the VHL mutant and is rescued by reexpression of wild-type VHL, 2) VHL is recruited to FGFR1-containing, but not EGFR-containing, endosomal vesicles, 3) VHL exhibits a functional relationship with Rab5a and dynamin 2 in FGFR1 internalization, and 4) the endocytic function of VHL is mediated through the metastasis suppressor Nm23, a protein known to regulate dynamin-dependent endocytosis. The von Hippel-Lindau disease is an inherited disorder that manifests in tumor formation in multiple organs (1Lonser R.R. Glenn G.M. Walther M. Chew E.Y. Libutti S.K. Linehan W.M. Oldfield E.H. Lancet. 2003; 361: 2059-2067Abstract Full Text Full Text PDF PubMed Scopus (1116) Google Scholar, 2Kim W.Y. Kaelin W.G. J. Clin. Oncol. 2004; 22: 4991-5004Crossref PubMed Scopus (800) Google Scholar). The disease is characterized by highly vascularized tumors mainly due to overproduction of angiogenic factors. The underlying genetic defect was identified as mutations in the VHL tumor suppressor gene (3Latif F. Tory K. Gnarra J. Yao M. Duh F.M. Orcutt M.L. Stackhouse T. Kuzmin I. Modi W. Geil L. Science. 1993; 260: 1317-1320Crossref PubMed Scopus (2500) Google Scholar). The biological role of VHL is prominently linked to its E3 3The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; FGF, fibroblast growth factor; FGFR, FGF receptor; HIF, hypoxia-inducible factor; RCC, renal cell carcinoma; PDGFR III, 4-(6,7-dimethoxy-4-quinazolinyl)-N-(4-phenoxyphenyl)-1-piperazinecarboxamide; DMEM, Dulbecco's modified Eagle's medium; CMV, cytomegalovirus; shRNA, short hairpin RNA; EGFP, enhanced green fluorescent protein; GST, glutathione S-transferase; MTC, mitomycin C; PBS, phosphate-buffered saline; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; PDGFR, platelet-derived growth factor receptor; p-FGFR, phospho-FGFR; bFGF, basic fibroblast growth factor; DMBI, 3-(4-dimethylaminobenzylidenyl)-2-indolinone; DN-FGFR1, dominant negative FGFR1; TfR, transferrin receptor. ubiquitin ligase activity toward a subset of cellular proteins, thus promoting their ubiquitination and degradation (4Iwai K. Yamanaka K. Kamura T. Minato N. Conaway R.C. Conaway J.W. Klausner R.D. Pause A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12436-12441Crossref PubMed Scopus (430) Google Scholar, 5Kamura T. Conrad M.N. Yan Q. Conaway R.C. Conaway J.W. Genes Dev. 1999; 13: 2928-2933Crossref PubMed Scopus (237) Google Scholar). Prominent among its cellular targets are the α subunits (1α, 2α, and 3α) of the key transcription factor, hypoxia-inducible factor (HIF), involved in the cellular oxygen-sensing mechanism (6Kamura T. Sato S. Iwai K. Czyzyk-Krzeska M. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10430-10435Crossref PubMed Scopus (554) Google Scholar, 7Krieg M. Haas R. Brauch H. Acker T. Flamme I. Plate K.H. Oncogene. 2000; 19: 5435-5443Crossref PubMed Scopus (325) Google Scholar, 8Maynard M.A. Qi H. Chung J. Lee E.H. Kondo Y. Hara S. Conaway R.C. Conaway J.W. Ohh M. J. Biol. Chem. 2003; 278: 11032-11040Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Tumorigenic mutations that affect E3 ligase function of VHL result in constitutive stabilization of HIF-α, leading to transcriptional activation of several target genes of HIF (9Maynard M.A. Ohh M. Am. J. Nephrol. 2004; 24: 1-13Crossref PubMed Scopus (101) Google Scholar), including those encoding critical angiogenic factors such as vascular endothelial growth factor and enzymes involved in glucose metabolism (10Bruick R.K. Genes Dev. 2003; 17: 2614-2623Crossref PubMed Scopus (375) Google Scholar). Renal cell carcinomas (RCCs) harboring VHL mutations are often metastatic, and reexpression of wild-type VHL suppresses the metastatic behavior in RCC-derived cell lines (11Kamada M. Suzuki K. Kato Y. Okuda H. Shuin T. Cancer Res. 2001; 61: 4184-4189PubMed Google Scholar, 12Esteban-Barragan M.A. Avila P. Alvarez-Tejado M. Gutierrez M.D. GarciaPardo A. Sanchez-Madrid F. Landazuri M.O. Cancer Res. 2002; 62: 2929-2936PubMed Google Scholar), although the mechanisms remain unclear. VHL mutant cells exhibit increased scattering upon hepatocyte growth factor treatment (13Koochekpour S. Jeffers M. Wang P.H. Gong C. Taylor G.A. Roessler L.M. Stearman R. Vasselli J.R. Stetler-Stevenson W.G. Kaelin Jr., W.G. Linehan W.M. Klausner R.D. Gnarra J.R. Vande Woude G.F. Mol. Cell. Biol. 1999; 19: 5902-5912Crossref PubMed Scopus (181) Google Scholar). Enhanced response to SDF-1 (stromal cell-derived factor-1), due to overexpression of chemokine receptor CXCR4, was also recently identified as a possible mechanism by which VHL tumors might disseminate to distant organs (14Staller P. Sulitkova J. Lisztwan J. Moch H. Oakeley E.J. Krek W. Nature. 2003; 425: 307-311Crossref PubMed Scopus (749) Google Scholar). On the other hand, accumulated evidence suggests that VHL null RCC cells exhibit intrinsically elevated migratory potential under normal serum conditions without chemokine or hepatocyte growth factor/scatter factor induction (11Kamada M. Suzuki K. Kato Y. Okuda H. Shuin T. Cancer Res. 2001; 61: 4184-4189PubMed Google Scholar, 15Lieubeau-Teillet B. Rak J. Jothy S. Iliopoulos O. Kaelin W. Kerbel R.S. Cancer Res. 1998; 58: 4957-4962PubMed Google Scholar, 16Davidowitz E.J. Schoenfeld A.R. Burk R.D. Mol. Cell. Biol. 2001; 21: 865-874Crossref PubMed Scopus (95) Google Scholar, 17Lewis M.D. Roberts B.J. Oncogene. 2003; 22: 3992-3997Crossref PubMed Scopus (25) Google Scholar). In this report, we sought to understand the function of VHL in control of cell motility. Our data reveal an intriguing VHL mutant phenotype of selective FGFR1 accumulation on the cell surface. Surface accumulation of FGFR1 leads to elevated FGFR1 signaling via ERK1/2 and influences the migratory potential of VHL mutant cells toward serum. Further, we show that this phenotype is the result of a defect in VHL-mediated endocytosis of FGFR1. Interestingly, this VHL function required partnership with Nm23H1, a metastasis suppressor protein known to regulate dynamin-mediated endocytosis (18Dammai V. Adryan B. Lavenburg K.R. Hsu T. Genes Dev. 2003; 17: 2812-2824Crossref PubMed Scopus (72) Google Scholar, 19Palacios F. Schweitzer J.K. Boshans R.L. D'Souza-Schorey C. Nat. Cell Biol. 2002; 4: 929-936Crossref PubMed Scopus (268) Google Scholar, 20Deitcher D. Trends Neurosci. 2001; 24: 625-626Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar, 21Krishnan K.S. Rikhy R. Rao S. Shivalkar M. Mosko M. Narayanan R. Etter P. Estes P.S. Ramaswami M. Neuron. 2001; 30: 197-210Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Cell Lines and Reagents—VHL null RCC line 786-O and VHL+ human embryonic kidney HEK293 cells were from the American Type Culture Collection (ATCC). Other cell lines from ATCC were Caki-1, Caki-2, ACHN, and HK-2. 786-Vec, 786-EGFP, and 786-VHL cells were generated by stable transfection of 786-O cells with pCDNA3.1, pCMV-EGFP, and pCMV-VHL (see below), respectively, and polyclonal selection by G418 (Invitrogen). Cells were maintained in DMEM (high glucose) supplemented with 10% dialyzed fetal bovine serum (Invitrogen) and used within eight passages. G418 was excluded in all assay conditions. Transfection of HEK293 with Lipofectamine 2000 (∼85% efficiency) was performed in a 6-well format according to the supplier's protocol (Invitrogen). Electroporation using Nucleofector (Amaxa Biosystems) was performed with Solution T and program T-01 supplied by the vendor, which consistently achieved ∼75% transfection efficiency. All inhibitors (PD98059, 3-(4-dimethylaminobenzylidenyl)-2-indolinone (DMBI), and PDGFR III (Calbiochem)), dissolved in Me2SO (Sigma), were added to prewarmed (37 °C) culture medium and used at the indicated concentrations (see below). Plasmid Constructs—pCMV-EGFP and pCMV-VHL were constructed by PCR, cloning EGFP and human VHL into the EcoRV site of pCDNA3.1 (Invitrogen). For EGFP-VHL, VHL was PCR-cloned into XhoI-KpnI sites of pEGFP-C1 (BD Biosciences Clontech). Plasmid-based short hairpin RNAs (shRNAs) were constructed as follows. Target sequences (VHL-shRNA1, gagaactgggacgaggccg; VHL-shRNA2, gctgcccgtatggctcaac; VHL-shRNA3, gagcctagtcaagcctgag; Nm23H1-shRNA1, gtgagcgtaccttcattgc; Nm23H1-shRNA2, ggtgaaatacatgcactca) and control (random sequence ctactcagtatgcacgtcg) were cloned into pSuppressor-Neo according to instructions (Imgenex). The U6 promoter-shRNA cassette (BamHI-BglII fragment) was subcloned into the BglII site of pCMV-EGFP and screened for promoters (U6 and CMV) oriented in the opposite direction. To express shRNAs without EGFP, the constructs in pSuppressor-Neo were used. DN-FGFR1-RFP was created by PCR cloning of the FGFR1 coding sequence encompassing amino acids 1-468 into XhoI-HindIII sites of pDsRed-N1 (BD Biosciences Clontech). GST fusions of VHL were prepared by first cloning VHL open reading frames into pGEX3T (BamHI-EcoRI), followed by PCR amplification of GST or GST-VHL and subcloning into EcoRV-EcoRI of pIRES-Neo (3Latif F. Tory K. Gnarra J. Yao M. Duh F.M. Orcutt M.L. Stackhouse T. Kuzmin I. Modi W. Geil L. Science. 1993; 260: 1317-1320Crossref PubMed Scopus (2500) Google Scholar) (BD Biosciences Clontech). EGFP fusions of Nm23H1 and Nm23H2 were constructed by reverse transcriptase-PCR amplification of the open reading frames and cloning into pEGFP-C1 (XhoI-KpnI). VHL mutants were generated by a PCR-based method (22Adereth Y. Champion K.J. Hsu T. Dammai V. BioTechniques. 2005; 38: 864-868Crossref PubMed Scopus (27) Google Scholar). The constitutively active version of HIF-2α (HIF-2α (P/A)) is an amino acid substitution of the proline residue at position 531 (P531A) that is the target of hydroxylation in normoxia. Both wild type and the constitutive HIF-2α coding sequences are cloned in the pCDNA3.0 vector and are gifts from W. Kaelin of Harvard Medical School. Wild-type and dominant negative dynamin 2-GFP and Rab5a-GFP constructs are gifts from S. Schmid of the Scripps Institute and N. Bunnet of the University of California San Francisco, respectively. Boyden Chamber and Wound-healing Assays—Cell migration was assayed in the presence of 10 μg/ml mitomycin C (MTC). Cells grown to 90% confluence in 6-well plates were scratched with a 1000-μl pipette tip, and the wound was rinsed thoroughly. Fresh 1% serum and MTC was added to follow healing for 24 h. For inhibitor treatment, cells were treated with either mock (Me2SO) or the indicated inhibitors for 1 h prior to the addition of 1% serum. The medium mix (inhibitors or mock plus 1% serum and MTC) was replaced every 6-8 h. The assays were performed in triplicate. Trans-well Boyden chamber (8-μm pore size; Corning) assays were performed using 15,000 cells/well seeded into the upper chamber (1% serum), and migration toward high serum (10%) was followed for 12 h in the presence of MTC. Inserts were rinsed in PBS, fixed in methanol for 10 min, and stained with crystal violet for 5 min. Cells on the upper side were removed with a cotton swab, and migrated cells attached to the bottom side were counted using a × 10 objective focused at the center. For inhibitor treatment, cells were serum-starved for 12 h, trypsinized, counted, and seeded into the upper chamber (mock or inhibitors plus 1% serum and MTC). The lower chamber contained the same mix except for a 10% serum concentration. The medium mix was replaced every 6-8 h. Boyden chamber results of six independent experiments done in triplicate were analyzed using Student's t test. DMBI and PD98056 were used at 25 μm. Cell Surface Biotinylation—Cells grown in 6-well plates were washed with ice-cold PBS (pH 7.4) and incubated with 0.5 ml/well PBS containing 0.5 mg/ml sulfo-NHS-LC-biotin (Pierce) for 30 min at 4 °C. Following rinsing, cells were lysed with 300 μl/well 1% Triton-radioimmune precipitation buffer containing protease inhibitors (Roche Applied Science). After protein estimation (BCA; Pierce), 1.5 mg of precleared extracts were incubated with respective rabbit polyclonal primary antibodies for 12 h at 4 °C in 1 ml of 1% Triton-radioimmune precipitation buffer. Protein A-agarose binding and washing was in 0.5% Triton-radioimmune precipitation buffer, and elution was in low pH buffer (50 mm glycine-HCl, pH 2.5). For β-actin control, 1.5-mg extracts were bound to streptavidin-agarose for 12 h at 4 °C, eluted in 1× SDS sample buffer, and blotted for β-actin. Immunoprecipitated receptors were detected using specific mouse monoclonal antibodies (total levels) and streptavidin-horseradish peroxidase (surface fraction). Northern Blotting—Total RNA was extracted (RNAeasy Protect; Qiagen), and 20 μg/lane of total RNA were used for Northern blotting. Primeit II (Stratagene) was used to prepare FGFR1 (PCR product corresponding to amino acids 1-468 of human FGFR1) and β-actin probes (Amersham Biosciences). Northern blotting followed standard procedures. Western Blotting and Antibodies—Cells starved for 12 h (90% confluent in 6-well plates) were stimulated with prewarmed DMEM containing 20% serum for 15 min, washed with cold DMEM, and lysed using 200 μl of 2× SDS sample buffer/well. Lysates were collected with a cell scraper and diluted to 1× SDS sample buffer concentration. GST pulldown assays were performed according to the standard protocol provided by the manufacturer (Invitrogen). For final elution, 1× SDS sample buffer was directly added to the glutathione beads. Western blotting was according to standard procedures. All antibodies were used at recommended dilutions. Mouse monoclonal antibodies were p-p38MAPK (Cell Signaling), doubly phosphorylated ERK1/2 (Sigma), β-actin (Sigma), VHL (BD Biosciences, Santa Cruz Biotechnology, and Neomarkers), GFP (Santa Cruz Biotechnology), EGFR (Neomarkers), HIF-1α (cross-reacted with HIF-2α; Novus Biologicals), FGFR1 (Upstate Biotechnology, Inc., Lake Placid, NY), p-FGFR1 (Cell Signaling), LAMP1 (Developmental Studies Hybridoma Bank, University of Iowa), Nm23H1 (Biomeda), Nm23H2 (Santa Cruz Biotechnology), and GST (Santa Cruz Biotechnology). Rabbit polyclonal antibodies were FGFR1 (Abcam, Sigma), p-FGFR1 (Cell Signaling), ERK1/2 (Sigma), p38MAPK (Cell Signaling), AKT (Cell Signaling), p-AKT (Cell Signaling), CXCR4 (Zymed Laboratories Inc.), c-Met (Cell Signaling), IGFR1 (Cell Signaling), and EGFR (Santa Cruz Biotechnology). Horseradish peroxidase-conjugated anti-mouse IgG (Sigma), anti-rabbit IgG (Sigma), streptavidin (Pierce), and Tyr(P) (Zymed Laboratories Inc.) were used as secondary conjugates. Indirect Immunofluorescence—Cells were fixed in PBS plus 3.7% formaldehyde for 20 min, quenched with PBS plus 10 mm Tris-HCl (pH 7.4), and permeabilized with 0.15% Saponin (Sigma) for 10 min at room temperature. Incubation with respective primary and secondary antibodies was in PBS plus 1% bovine serum albumin (1 h at room temperature). Primary antibodies were used at 1:100 dilution. Secondary antibodies are highly cross-absorbed goat anti-rabbit Alexa 546, goat anti-mouse Alexa 546, goat anti-rabbit Alexa 488; and goat anti-mouse Alexa 488 (Molecular Probes, Inc., Eugene, OR), which were used at 1:150 dilution. Confocal images were acquired with an Olympus IX70 microscope (Fluoview 300). Activated Receptor Chase and Endocytosis Studies—200 ng/ml recombinant human bFGF (Promega) was mixed with 2.5 μg/ml heparin (Sigma) in serum-free medium (with 1% bovine serum albumin) and added to serum-starved cells (for 24 h) grown on coverslips. Cells were incubated at 4 °C for 2 h to allow ligand-receptor engagement and washed with cold DMEM, and chase was initiated in prewarmed DMEM (37 °C). Coverslips were lifted and directly fixed in PBS plus 3.7% formaldehyde. 500 ng/ml recombinant human EGF (Clonetics) was used in EGFR endocytosis studies. Cells were fixed as above at the end of chase. For transferrin internalization, serum-starved cells were incubated at 4 °C with filter-sterilized 1% bovine serum albumin in DMEM for 30 min to block nonspecific binding. Cells were incubated at 4 °C with 5 μg/ml transferrin-Alexa 546 (Molecular Probes) for 30 min, washed in cold DMEM, and chased with prewarmed DMEM containing 1% bovine serum albumin (37 °C). Cells were washed once in cold DMEM, fixed as above, and directly mounted in Prolong Antifade (Molecular Probes). VHL Mutant Cells Exhibit Increased Cell Motility—"Wound-healing" and Boyden chamber assays were employed to quantify the migratory properties of the human 786-O RCC cell line (23Wykoff C.C. Sotiriou C. Cockman M.E. Ratcliffe P.J. Maxwell P. Liu E. Harris A.L. Br. J. Cancer. 2004; 90: 1235-1243Crossref PubMed Scopus (86) Google Scholar) polyclonally selected for stable expression of either enhanced green fluorescent protein (786-EGFP) or wild-type human VHL (786-VHL). EGFP is widely used as an inert control protein (e.g. see Refs. 24De Corte V. Van Impe K. Bruyneel E. Boucherie C. Mareel M. Vandekerckhove J. Gettemans J. J. Cell Sci. 2004; 117: 5283-5292Crossref PubMed Scopus (71) Google Scholar and 25Fuchs M. Hutzler P. Handschuh G. Hermannstadter C. Brunner I. Hofler H. Luber B. Cell Motil. Cytoskeleton. 2004; 59: 50-61Crossref PubMed Scopus (13) Google Scholar). Accordingly, we do not observe overt differences in gene expression between 786-O parental and 786-EGFP cells (supplementary Fig. S1B; see below). We have also determined that the expression level of VHL (in the 786-VHL stable cell line) is comparable with the endogenous levels in several known VHL+ kidney cell lines, such as HK-2, HEK293, and ACHN (supplemental Fig. S1A). To account for any difference in cell proliferation, all migration assays (Figs. 1, 4, and 5) were performed in the presence of 10 μg/ml MTC, a chemical that inhibits cell proliferation (Fig. 1A) (26Miura Y. Yanagihara N. Imamura H. Kaida M. Moriwaki M. Shiraki K. Miki T. Jpn. J. Ophthalmol. 2003; 47: 268-275Crossref PubMed Scopus (29) Google Scholar) but not motility (26Miura Y. Yanagihara N. Imamura H. Kaida M. Moriwaki M. Shiraki K. Miki T. Jpn. J. Ophthalmol. 2003; 47: 268-275Crossref PubMed Scopus (29) Google Scholar). In wound-healing experiments, 786-EGFP cells completely filled a ∼500-μm wound within 24 h, whereas 786-VHL exhibited significantly reduced two-dimensional planar motility, with an estimated 78% wound remaining unhealed (Fig. 1B). Quantitative analysis of three-dimensional and chemotactic migration toward serum, assayed using Boyden chambers, showed that compared with 786-VHL cells, 786-EGFP cells exhibit a ∼6-fold increased cell migration (p < 0.005) within a 12-h assay period (Fig. 1C). Thus, VHL null cells displayed an elevated migratory property that is inhibited by expression of VHL.FIGURE 4FGFR1 signaling is the major contributor to elevated ERK1/2 activity and cell migration in VHL mutant cells. A, 786-EGFP cells were serum-starved overnight before being seeded into the upper chamber of a transwell. The cells were then incubated with medium containing 1% serum, the indicated reagents (DMBI at 25 μm), and 10 μg/ml MTC. The lower chamber contains the same mix except for 10% serum concentration. Medium/inhibitor mix was replenished every 6 h. Cells that migrated to the underside of the membrane were counted after 12 h. DMBI significantly reduces the transwell migration of the VHL(-) cells. The cell numbers presented are the total cell counts within a field of view at the center of the filter taken with a × 10 objective. Cell numbers are averages of four independent assays done in triplicate. The error bars indicate S.D. (n = 12). B, 786-EGFP cells were serum-starved overnight and then treated with serum-free medium containing the indicated reagents (solvent Me2SO, 25 μm for DMBI or PD98056) and 10 μg/ml MTC for 1 h. The scratch was then made for the wound-healing assays, and the cells were incubated in medium containing 1% serum, the inhibitors, or Me2SO and MTC. Medium/inhibitor mix was replenished every 6 h. 786-EGFP cell migration into the wound over 24 h is inhibited by DMBI and the ERK inhibitor PD98056. C, untransfected 786-O cells ((-) plasmid) or 786-O cells transfected with the control vector (RFP) or plasmid containing dominant-negative FGFR1-RFP fusion (DN-FGFR1-RFP) were stimulated with 20% serum for 15 min, and the protein extracts were subjected to Western blot. The DN-FGFR1-RFP fusion protein is detected as a faster migrating band (∼12 kDa smaller; lower arrowhead) than the endogenous FGFR1 (upper arrowhead). There was no change in the endogenous FGFR1 levels. Expression of DN-FGFR1-RFP inhibited the activities of FGFR1 (p-FGFR) and ERK1/2 (doubly phosphorylated ERK1/2; dp-ERK1/2). There were no changes in AKT or p38MAPK pathways upon the expression of DN-FGFR1-RFP or RFP. Loading controls for cytosolic protein (β-actin) and membrane protein (EGFR) are shown. D, transwell assays showing expression of DN-FGFR1-RFP, but not RFP, inhibits chemotactic migration of 786-O cells (∼3-fold inhibition, p < 0.005; n = 18).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Loss of VHL leads to elevated FGFR1 activity and increased cell migration in HEK293 cells. A, HEK293 cells were transfected with vectors expressing control shRNA (lane 1), three independent VHL shRNA constructs (lanes 2-4), wild-type HIF-2α (HIF-2α (wt); lane 5), or constitutive HIF-2a (HIF-2α (P/A); lane 6). Cell lysates were subjected to Western blot using the antibodies indicated on the left. GFP co-expressed from the shRNA-containing plasmid was used as an expression level control. VHL protein levels are significantly knocked down by the VHL-specific shRNAs. HIF-α levels are increased in VHL knock-down cells (lanes 2-4) and in HIF-2α ectopic expressing cells (lanes 5 and 6). CXCR4 levels are increased in VHL knockdown and in HIF-expressing cells, as expected. Controls of a cytosolic protein (β-actin), a membrane protein (EGFR), and EGFP (GFP) for shRNAs are shown. For FGFR1, equal amounts of lysates were immunoprecipitated with rabbit FGFR1 antibody and detected with mouse FGFR1 antibody or with mouse Tyr(P)-horseradish peroxidase antibody. B, Boyden chamber assays showing effects of loss of VHL and HIF-2α overexpression on cell migration in HEK293 cells. Numbers of cells that migrated to the underside of the membrane (scale on the left) were counted. The number for each graph corresponds to the sample shown in A. ∼2.5-Fold elevated cell migration (p < 0.005) is seen in VHL-shRNA-treated cells compared with control shRNA-treated cells. Expression of HIF-2α (wt) or HIF-2α (P/A) led to decreased cell migration compared with the control. The insets show representative images of Boyden chamber assays of six independent experiments done in triplicate. The error bars indicate S.D. (n = 18). -Fold FGFR1 activation (scale on the right) was calculated as follows. Signal densities from Western blotting of total immunoprecipitated FGFR1 (A) and from reprobing for activated FGFR (Tyr(P)) were measured by densitometric analysis using ImageJ. The ratio of activated versus total FGFR1 (FGFR1 activation level) for control shRNA was arbitrarily set at 1. Relative FGFR1 activation levels were then plotted (line graph).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cell Surface Accumulation of FGFR1 and Cellular Changes in VHL Null Cells—In the Drosophila tracheal tubule system, epithelial cell migration is mainly regulated by the FGF chemotactic signaling. It has been shown that Drosophila VHL negatively regulates tracheal cell migration (27Adryan B. Decker H.J. Papas T.S. Hsu T. Oncogene. 2000; 19: 2803-2811Crossref PubMed Scopus (50) Google Scholar). We have also demonstrated that abnormal accumulation of FGFR on tracheal cell surface, a result of defective endocytosis, can lead to aberrant cell migration (18Dammai V. Adryan B. Lavenburg K.R. Hsu T. Genes Dev. 2003; 17: 2812-2824Crossref PubMed Scopus (72) Google Scholar). In tracheal cells, the ERK1-type mitogen-activated protein kinase is the major downstream mediator of the FGFR signaling pathway (18Dammai V. Adryan B. Lavenburg K.R. Hsu T. Genes Dev. 2003; 17: 2812-2824Crossref PubMed Scopus (72) Google Scholar). We therefore examined whether the FGFR-ERK signaling function is evolutionarily conserved in Drosophila trachea and in the renal proximal tubule-derived tumor cells such as 786-O. We indeed observed surface FGFR1 accumulation in the VHL mutant cells (arrowheads in Fig. 2A). Reexpression of VHL in the 786-O cells dramatically reduced the cell membrane accumulation of FGFR1. We did not observe increased surface accumulation in VHL mutant cells in other surface proteins studied, namely EGFR, IGFR1, c-Met, PDGFR, and N-cadherin (Fig. 2B). The differential membrane distribution of FGFR1 was confirmed by surface biotinylation assays to measure the fraction of surface versus total cellular levels. In this assay, surface proteins were selectively labeled with biotin using a membrane-impermeable cross-linker, sulfo-NHS-LC-biotin. The surface protein levels were identified, after immunoprecipitation, by Western blotting with horseradish peroxidase-streptavidin. As shown in Fig. 2C, whereas the total cellular levels of FGFR1 remained unchanged with or without VHL, reexpression of VHL led to a dramatic reduction of surface FGFR1. Consistent with our immunofluorescence data (Fig. 2B), surface levels of PDGFR were unchanged, whereas EGFR were slightly higher on the surface of 786-VHL cells compared with 786-EGFP cells (Fig. 2C). Reduced surface EGFR in VHL null cells may be due to overexpression of the autocrine tumor growth factor-α in these cells (28Knebelmann B. Ananth S. Cohen H.T. Sukhatme V.P. Cancer Res. 1998; 58: 226-231PubMed Google Scholar), which can result in increased ligand engagement of EGFR and consequently increased receptor internalization. To verify that the membrane-localized FGFR1 is indeed active, we immunostained with an antibody that recognized the phosphotyrosine moieties in FGFR. Since FGFR2 is not expressed in these cells (data not shown), the phospho-FGFR (p-FGFR) antibody staining should correlate well with the activation status of FGFR1. We found a significant increase in tyrosine-phosphorylated FGFR1 at the cell surface in VHL mutant cells even after serum starvation for 6 h (Fig. 3, A and B). This indicated that high levels of surface FGFR1 lead to persistent and ligand-independent activation of the receptors as has been suggested for receptor tyrosine kinases (29Verveer P.J. Wouters F.S. Reynolds A.R. Bastiaens P.I. Science. 2000; 290: 1567-1570Crossref PubMed Scopus (306) Google Scholar, 30Sawano A. Takayama S. Matsuda M. Miyawaki A. Dev. Cell. 2002; 3: 245-257Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). On the other hand, VHL-positive cells showed no surface and very low overall p-FGFR staining under identical conditions (Fig. 3C). Stimulation of cells with bFGF and heparin leads to further increased p-FGFR staining in VHL mutant cells (Fig.

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