Urokinase Signaling through Its Receptor Protects against Anoikis by Increasing BCL-xL Expression Levels
2006; Elsevier BV; Volume: 281; Issue: 26 Linguagem: Inglês
10.1074/jbc.m601812200
ISSN1083-351X
AutoresDaniela Alfano, Ingram Iaccarino, Maria Patrizia Stoppelli,
Tópico(s)Signaling Pathways in Disease
ResumoThe acquired capabilities of resistance to apoptotic cell death and tissue invasion are considered to be obligate steps in tumor progression. The binding of the serine protease urokinase (uPA) to its receptor (uPAR) plays a central role in the molecular events coordinating tumor cell adhesion, migration, and invasion. Here we investigate whether uPAR signaling may also prevent apoptosis following loss of anchorage (anoikis) or DNA damage. If nontransformed human retinal pigment epithelial cells are pre-exposed to uPA or to its noncatalytic amino-terminal region (residues 1–135), they exhibit a markedly reduced susceptibility to anoikis as well as to UV-induced apoptosis. This anti-apoptotic effect is retained by a uPA-derived synthetic peptide corresponding to the receptor binding domain and is inhibited by anti-uPAR polyclonal antibodies. Furthermore, the stable reduction of uPA or uPAR expression by RNA interference leads to an increased susceptibility to UV-, cisplatin-, and detachment-induced apoptosis. In particular, the level of uPAR expression positively correlates with cell resistance to anoikis. The protective ability of uPA is prevented by UO126, LY294002, by an MAPK targeting small interference RNA, and by a dominant negative Akt variant. Accordingly, incubation of retinal pigment epithelial cells with uPA elicits a time-dependent enhancement of MAPK and phosphatidylinositol 3-kinase activities as well as the transcriptional activation of Bcl-xL anti-apoptotic factor. Vice versa, the silencing of Bcl-xL expression prevents uPA protection from anoikis. In conclusion, the data show that ligand engagement of uPAR promotes cell survival by activating Bcl-xL transcription through the MEK/ERK- and phosphatidylinositol 3-kinase/Akt-dependent pathways. The acquired capabilities of resistance to apoptotic cell death and tissue invasion are considered to be obligate steps in tumor progression. The binding of the serine protease urokinase (uPA) to its receptor (uPAR) plays a central role in the molecular events coordinating tumor cell adhesion, migration, and invasion. Here we investigate whether uPAR signaling may also prevent apoptosis following loss of anchorage (anoikis) or DNA damage. If nontransformed human retinal pigment epithelial cells are pre-exposed to uPA or to its noncatalytic amino-terminal region (residues 1–135), they exhibit a markedly reduced susceptibility to anoikis as well as to UV-induced apoptosis. This anti-apoptotic effect is retained by a uPA-derived synthetic peptide corresponding to the receptor binding domain and is inhibited by anti-uPAR polyclonal antibodies. Furthermore, the stable reduction of uPA or uPAR expression by RNA interference leads to an increased susceptibility to UV-, cisplatin-, and detachment-induced apoptosis. In particular, the level of uPAR expression positively correlates with cell resistance to anoikis. The protective ability of uPA is prevented by UO126, LY294002, by an MAPK targeting small interference RNA, and by a dominant negative Akt variant. Accordingly, incubation of retinal pigment epithelial cells with uPA elicits a time-dependent enhancement of MAPK and phosphatidylinositol 3-kinase activities as well as the transcriptional activation of Bcl-xL anti-apoptotic factor. Vice versa, the silencing of Bcl-xL expression prevents uPA protection from anoikis. In conclusion, the data show that ligand engagement of uPAR promotes cell survival by activating Bcl-xL transcription through the MEK/ERK- and phosphatidylinositol 3-kinase/Akt-dependent pathways. Oncogenic cell transformation is currently viewed as a multistep process in which a series of genetic lesions change cellular physiology leading to the acquisition of new capabilities, such as an enhanced ability to proliferate, migrate, and escape apoptotic cell death (1Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (22370) Google Scholar). Apoptosis can be viewed as a safe-lock mechanism that could prevent the establishment of a fully transformed phenotype. For instance, it is currently accepted that uncontrolled proliferation could by itself prime the transforming cell to apoptotic cell death (2Hood J.D. Cheresh D.A. Nat. Rev. Cancer. 2002; 2: 91-100Crossref PubMed Scopus (1484) Google Scholar, 3Pelengaris S. Khan M. Evan G. Nat. Rev. Cancer. 2002; 2: 764-776Crossref PubMed Scopus (922) Google Scholar). Similarly, the ability to migrate and invade through the basement membrane into surrounding tissues is one of the essential hallmarks of cancer and a prerequisite for both local tumor progression and metastatic spread, but it can also lead to apoptosis if not counterbalanced by survival signals (1Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (22370) Google Scholar). In particular, apoptosis induced by loss of anchorage, a phenomenon also known as anoikis (from the Greek for "homelessness"), has been shown to limit the spread of epithelial cells outside of the tissue environment. Therefore, successful cellular transformation requires an increased cell resistance to death through the overexpression of anti-apoptotic factors or the activation of survival kinases (4Fanidi A. Harrington E.A. Evan G.I. Nature. 1992; 359: 554-556Crossref PubMed Scopus (702) Google Scholar, 5Schulze A. Lehmann K. Jefferies H.B. McMahon M. Downward J. Genes Dev. 2001; 15: 981-994Crossref PubMed Scopus (222) Google Scholar). Many cell types derived from human malignancies, such as gastric cancers, mammary tumors, colon cancers, osteosarcomas, and lung carcinomas, are resistant to anoikis (6Khwaja A. Rodriguez-Viciana P. Wennstrom S. Warne P.H. Downward J. EMBO J. 1997; 16: 2783-2793Crossref PubMed Scopus (939) Google Scholar, 7Wei L. Yang Y. Yu Q. Cancer Res. 2001; 61: 2439-2444PubMed Google Scholar). Among the many genes involved in the regulation of cell spreading and migration, those coding for the serine protease urokinase (uPA) 2The abbreviations used are: uPA, urokinase-type plasminogen activator; uPAR, uPA receptor; ATF, amino-terminal fragment (residues 1–135); GFD, uPA growth factor-like domain (residues 1–49); GFDp, peptide corresponding to uPA residues 12–32; RPE, retinal pigment epithelial cells; HEK, human embryonic kidney cells; ECM, extracellular matrix; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; poly-HEMA, poly (2-hydroxyethyl methacrylate); MEK, MAPK/ERK kinase; siRNA, small interference RNA; FBS, fetal bovine serum; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; FAK, focal adhesion kinase; PARP, poly(ADP-ribose) polymerase; PBS, phosphate-buffered saline. and its cognate receptor (uPAR) play a central role in tumor development because of their clear-cut ability to regulate cytoskeleton dynamics, cell adhesion, and matrix integrity (8Blasi F. Carmeliet P. Nat. Rev. Mol. Cell Biol. 2002; 3: 932-943Crossref PubMed Scopus (1070) Google Scholar). High levels of uPA and uPAR have been found in many human malignant tumors, and they strongly correlate with poor prognosis and unfavorable clinical outcome (9Dano K. Behrendt N. Hoyer-Hansen G. Johnsen M. Lund L.R. Ploug M. Romer J. Thromb. Haemostasis. 2005; 93: 676-681Crossref PubMed Scopus (388) Google Scholar, 10Harbeck N. Kates R.E. Look M.P. Meijer-Van Gelder M.E. Klijn J.G. Kruger A. Kiechle M. Janicke F. Schmitt M. Foekens J.A. Cancer Res. 2002; 62: 4617-4622PubMed Google Scholar). Urokinase converts the pro-enzyme plasminogen into plasmin, a wide spectrum serine protease able to degrade most of the extracellular matrix (ECM) components and activate latent collagenases. The uPA is secreted in the pro-enzyme form (pro-uPA), which can be activated in the extracellular milieu by a single proteolytic cleavage occurring between Lys158 and Ile159, thus generating a two-chain enzyme. The uPA is a multidomain protein that includes an amino-terminal "growth factor-like" domain (GFD, residues 1–49) followed by a "kringle" region (residues 50–131) linked by the "connecting peptide" (residues 135–158) to a large catalytic moiety (residues 158–411). High affinity binding to uPAR occurs through the GFD of uPA and neither involves nor prevents the protease catalytic activity (11Vassalli J.D. Baccino D. Belin D. J. Cell Biol. 1985; 100: 86-92Crossref PubMed Scopus (590) Google Scholar, 12Stoppelli M.P. Corti A. Soffientini A. Cassani G. Blasi F. Assoian R.K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4939-4943Crossref PubMed Scopus (373) Google Scholar). The urokinase-type plasminogen activator receptor is a three-domain (D1, D2, and D3) glycosylphosphatidylinositol-anchored protein with a high affinity for uPA and the amino-terminal fragment ATF (residues 1–135), mainly interacting with the external D1 domain (13Llinas P. Le Du M.H. Gardsvoll H. Dano K. Ploug M. Gilquin B. Stura E.A. Menez A. EMBO J. 2005; 24: 1655-1663Crossref PubMed Scopus (206) Google Scholar). Enzymatic cleavage of uPAR between D1 and D2 unmasks a region, corresponding to residues 88–92 of the human sequence (SRSRY), that is able to elicit intracellular signaling, even as a synthetic peptide (14Fazioli F. Resnati M. Sidenius N. Higashimoto Y. Appella E. Blasi F. EMBO J. 1997; 16: 7279-7286Crossref PubMed Scopus (230) Google Scholar, 15Gargiulo L. Longanesi-Cattani I. Bifulco K. Franco P. Raiola R. Campiglia P. Grieco P. Peluso G. Stoppelli M.P. Carriero M.V. J. Biol. Chem. 2005; 280: 25225-25232Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Mechanistically, signal transduction is activated through the physical and functional interaction of uPAR with transmembrane receptors, such as the integrins, FPRL1, or the EGF receptor (16Resnati M. Pallavicini I. Wang J.M. Oppenheim J. Serhan C.N. Romano M. Blasi F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1359-1364Crossref PubMed Scopus (323) Google Scholar, 17Carriero M.V. Del Vecchio S. Capozzoli M. Franco P. Fontana L. Zannetti A. Botti G. D'Aiuto G. Salvatore M. Stoppelli M.P. Cancer Res. 1999; 59: 5307-5314PubMed Google Scholar, 18Aguirre Ghiso J.A. Kovalski K. Ossowski L. J. Cell Biol. 1999; 147: 89-104Crossref PubMed Scopus (466) Google Scholar). Considerable effort has been directed to the analysis of uPAR-dependent signaling effects together with the relative responses, mainly related to cell motility, adhesion, and cytoskeletal status (19Preissner K.T. Kanse S.M. May A.E. Curr. Opin. Cell Biol. 2000; 12: 621-628Crossref PubMed Scopus (206) Google Scholar). In most cases, these effects are clearly proteolytically independent but uPAR-dependent. One example is provided by the uPA-dependent regulation of the ratio between cell motility and adhesion through the control of p56/59 hck activity in U937 monocyte-like cells (20Chiaradonna F. Fontana L. Iavarone C. Carriero M.V. Scholz G. Barone M.V. Stoppelli M.P. EMBO J. 1999; 18: 3013-3023Crossref PubMed Scopus (58) Google Scholar). In the HEp3 human carcinoma cells, association of uPA to uPAR regulates the ERK1/2/p38 activity ratio, thus modulating the balance between tumor cell proliferation and dormancy (21Ossowski L. Aguirre-Ghiso J.A. Curr. Opin. Cell Biol. 2000; 12: 613-620Crossref PubMed Scopus (357) Google Scholar). To date, increasing evidence supports a role for uPA also in the regulation of cell proliferation (22Alfano D. Franco P. Vocca I. Gambi N. Pisa V. Mancini A. Caputi M. Carriero M.V. Iaccarino I. Stoppelli M.P. Thromb. Haemostasis. 2005; 93: 205-211Crossref PubMed Scopus (144) Google Scholar). First of all, endogenous uPA is an autocrine mitogen for the human melanoma cell line GUBSB, as inhibition of receptor binding by anti-uPA antibodies in the culture medium causes a strong reduction of cell proliferation (23Kirchheimer J.C. Wojta J. Christ G. Binder B.R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5424-5428Crossref PubMed Scopus (92) Google Scholar). If the expression of uPA and uPAR is reduced by antisense or interference RNA, prostate cancer tumorigenicity and invasion is prevented (24Pulukuri S.M. Gondi C.S. Lakka S.S. Jutla A. Estes N. Gujrati M. Rao J.S. J. Biol. Chem. 2005; 280: 36529-36540Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Moreover, unlike the wild-type mice, the uPA–/– animals do not allow progression of chemically induced melanocytic neoplasms to melanomas, indicating that uPA contributes to malignant growth (25Shapiro R.L. Duquette J.G. Roses D.F. Nunes I. Harris M.N. Kamino H. Wilson E.L. Rifkin D.B. Cancer Res. 1996; 56: 3597-3604PubMed Google Scholar). Furthermore, T241 fibrosarcoma cells implanted in uPA–/– mice exhibit decreased proliferation and increased apoptosis, suggesting that alterations in host expression of uPA may affect the balance between tumor cell death and proliferation (26Gutierrez L.S. Schulman A. Brito-Robinson T. Noria F. Ploplis V.A. Castellino F.J. Cancer Res. 2000; 60: 5839-5847PubMed Google Scholar). Interestingly, uPAR-deficient HEp3 human carcinoma cells inoculated in the modified chorioallantoic membrane enter a state of dormancy in which cells survive but do not proliferate (27Yu W. Kim J. Ossowski L. J. Cell Biol. 1997; 137: 767-777Crossref PubMed Scopus (149) Google Scholar). Recent findings indicate that a decreased uPAR expression may promote apoptosis. This is the case of SNB19 glioblastoma cells expressing antisense uPAR constructs that are less invasive than parental cells when injected in vivo and undergo loss of mitochondrial transmembrane potential, release of cytochrome c, caspase-9 activation, and apoptosis (28Yanamandra N. Konduri S.D. Mohanam S. Dinh D.H. Olivero W.C. Gujrati M. Nicolson G.L. Obeyeseke M. Rao J.S. Clin. Exp. Metastasis. 2000; 18: 611-615Crossref PubMed Scopus (24) Google Scholar). Furthermore, glioma cells bearing a reduced uPAR number are more susceptible to tumor necrosis factor-α-related apoptosis-inducing ligand-induced apoptosis than parental cells (29Krishnamoorthy B. Darnay B. Aggarwal B. Dinh D.H. Kouraklis G. Olivero W.C. Gujrati M. Rao J.S. Clin. Cancer Res. 2001; 7: 4195-4201PubMed Google Scholar). Limited information about the pathways involved in the uPA-dependent control of cell growth and survival is available to date; in MDA-MB-231 breast cancer cells cultured in the presence of anti-uPA blocking antibodies, the level of phosphorylated ERK decreases substantially and apoptosis is promoted, showing that endogenous uPA is a major determinant of ERK activation and protection from apoptosis (30Ma Z. Webb D.J. Jo M. Gonias S.L. J. Cell Sci. 2001; 114: 3387-3396Crossref PubMed Google Scholar). Furthermore, the ability of the uPA/uPAR interaction to stimulate PI3K/Akt signaling through β5 integrin has been described, although it seems to be dispensable for chemotaxis (31Sturge J. Hamelin J. Jones G.E. J. Cell Sci. 2002; 115: 699-711Crossref PubMed Google Scholar). The aim of this study is to investigate whether uPA, in addition to its ability to degrade ECM, to drive migration, and to elicit proliferation, may contribute to tumorigenesis by preventing apoptotic cell death. In particular, we analyzed the effects of uPA on detachment-induced apoptosis of retinal pigment epithelial (RPE) cells immortalized with human TERT. These cells undergo anoikis, as well as UV light- or cis-platin-induced apoptosis, unless they are pre-exposed to uPA or to its noncatalytic amino-terminal region. The relevance of the uPA/uPAR system to the RPE survival is further indicated by the finding that the reduction of uPA or uPAR by an RNAi approach enhances cell sensitivity to anoikis. Finally, the present work sheds light on the uPAR-dependent anti-apoptotic mediators, indicating the involvement of PI3K/Akt- and MEK/ERK-dependent pathways impinging on the transcriptional activation of Bcl-xL anti-apoptotic factor. Reagents—Cell death detection ELISA kit, FuGENE 6 transfection reagent, and Complete™ protease inhibitor mixture were from Roche Applied Science. All cell culture reagents were purchased from Invitrogen. RNAi human/mouse starter kit was from Qiagen (Valencia, CA). The poly(2-hydroxyethyl methacrylate) (poly-HEMA), MEK inhibitors PD98059 and UO126, the PI3K inhibitors LY294002, cycloheximide, cisplatin, 12-O-tetradecanoylphorbol-13-acetate, and EGF were from Sigma. The Src kinase inhibitor PP2 was from Calbiochem. 399 rabbit (399R) anti-uPAR polyclonal antibody was from American Diagnostica (Greenwich, CT). The 5B4 anti-uPA monoclonal antibody was a gift of M. L. Nolli, Areta International, Milan, Italy. The anti-αvβ5 monoclonal antibody was from Chemicon (Temecula, CA). The methylcellulose was from Stem Cell Technologies (Vancouver, British Columbia, Canada). The pPIC9 vector and Pichia strain GS115 were obtained from Invitrogen. The two uPA variants, uPA 1–158 or ATF (corresponding to the first 158 amino acids or 135 amino acids of human uPA, respectively), have been expressed as secreted products in the methylotrophic yeast Pichia pastoris and purified by 5B4-agarose chromatography, as described (12Stoppelli M.P. Corti A. Soffientini A. Cassani G. Blasi F. Assoian R.K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4939-4943Crossref PubMed Scopus (373) Google Scholar). The GFDp (DCLNGGTAVSNKYFSNIHWCN) and the 5Ala-GFDp (DCLNGGTAVSAAAAANIHWCN) peptides are a gift of P. Grieco, Naples, Italy. Plasmids—To obtain the pPIC9 vector expressing ATF or uPA 1–158, the region encoding residues 1–130 of human uPA, included in the 1414-bp SacI/FspI fragment, was excised from pPIC9-His-uPA. This fragment was either ligated into the SacI/EcoRI sites of the pPIC9 multicloning site, together with a double strand oligonucleotide coding for residues 131–135 followed by three stop codons (5′-gcagatggaaaataatgatcaagtactg) to encode ATF, or ligated to a double strand oligonucleotide coding for residues 131–158 (5′-gcagatggaaaaaagccctcctctcctccggaagaattaaaatttcagtgtggccaaaagactctgaggcctcgctttaagtgag) to encode uPA 1–158. The uPAR expression vector pcDNA3.1-uPAR contains the entire human uPAR cDNA (17Carriero M.V. Del Vecchio S. Capozzoli M. Franco P. Fontana L. Zannetti A. Botti G. D'Aiuto G. Salvatore M. Stoppelli M.P. Cancer Res. 1999; 59: 5307-5314PubMed Google Scholar). The pUSE/AktK179M was from Upstate (Charlottesville, VA). Gene Silencing—The mammalian expression vector, pSUPER (Oligo-Engine, Seattle, WA), was used for siRNA expression in RPE cells. The gene-specific insert includes a 19-nucleotide sequence corresponding to nucleotides 1124–1142 downstream of the transcription start site (tgactgttgtgaagctgat) of uPA or to nucleotides 565–583 (gccgttacctcgaatgcat) of uPAR. In both cases, this insert is separated by a 9-nucleotide noncomplementary spacer (tctcttgaa) from the reverse complement of the same 19-nucleotide sequence. These constructs are referred to as pSUPER/uPAi or pSUPER/uPARi. A control vector (pSUPER/GFPi) was constructed using a 19-nucleotide sequence (gagcgcaccatcttcttca) with no significant homology to any mammalian gene sequence and based on the gene sequence of the green fluorescent protein (GFP) gene. These sequences were inserted into the pSUPER vector digested with BglII and HindIII (New England Biolabs). The siRNA oligonucleotides against MAPK1 and Bcl-xL were from Qiagen. Cell Culture and Generation of Stable and Transient Transfectants—hTert-RPE (Clontech) and HEK-293 were cultured in Dulbecco's modified Eagle's medium supplemented with 100 μg/ml streptomycin, 100 units/ml penicillin, and 10% fetal bovine serum (FBS) (pH 7.2–7.4) in a humidified atmosphere containing 5% CO2 at 37 °C. For stable RPE transfectants, a semi-confluent 10-cm dish was incubated with 18 μl of FuGENE 6 and 6 μg of DNA according to the manufacturer's protocol. The RPE cells were then diluted, passaged, and selected with the neomycin analogue G418 (800 μg/ml). Stable HEK-293 transfectants were obtained by electroporating 1 × 107 subconfluent HEK-293 cells with 80 μg of plasmid DNA, in 0.9 ml of culture medium. The expression of uPAR by HEK-293/uPAR clones was quantitated by Western blotting with anti-uPAR RII antibody from total cell lysates. For transient RPE transfections, 1 × 105 cells were incubated in serum-free DMEM with 3 μl of FuGENE 6 transfection reagent with 1 μg of the relevant plasmids. A transfection efficiency of 60% was obtained. Transient transfectants were analyzed after 48 h. For the transfection of siRNA oligonucleotides, we used the "fast-forward" protocol; 3.5 × 104 cells/well were seeded in a 24-well plates and incubated with the transfection complexes (5 nm siRNA with 3 μl of HiPerFect Transfection Reagent), according to the manufacturer's protocols. Transfection efficiency (nearly 90%) and functional specificity were monitored with the RNAi starter kit by using an Alexa Fluor 488 labeled nonsilencing siRNA (Qiagen). Western Blot Analysis—Cells, treated as specified in the figure legends, were lysed (1.5 × 105/100 μl) for 30 min on ice in a buffer containing 50 mm Tris-HCl (pH 8.0), 120 mm NaCl, 100 mm NaF, 1% Triton X-100, supplemented with 0.5 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 1× Complete™ protease inhibitor mixture. The debris was then removed by centrifugation at 10,000 × g for 10 min at 4 °C, and protein content was assessed by the Bradford protein assay. Equal protein amounts were separated on 10 or 12.5% SDS-PAGE and transferred to an Immobilon-P membrane (Millipore Corp., Billerica, MA). The membrane was subsequently incubated for 1 h at room temperature in a TBST buffer (125 mm Tris-HCl (pH 8.0), 625 mm NaCl, 0.5% Tween 20) containing 4% skim milk and further incubated with 1 μg/ml of the following antibodies: anti-uPAR RII (a gift of G. Hoyer-Hansen, Finsen Institute, Copenhagen, Denmark); anti-uPA polyclonal (kindly provided by P.A. Andreasen, Aarhus, Denmark); anti-Bcl-x (BD Biosciences); anti-β-actin (Sigma); anti-phospho-p44/p42 MAPK, anti-phospho-Akt (Ser-473), anti-Akt, and anti-PARP (Cell Signaling, Beverly, MA). Reaction was detected using the chemiluminescence system ECL Plus (Amersham Biosciences). Cell Sensitivity to Detachment-induced Apoptosis—RPE cells were grown to subconfluence in 24-well plates and serum-starved for 24 h. To remove membrane-bound ligands, cells were treated with an acidic buffer (50 mm glycine, 100 mm NaCl (pH 3)) for 2 min (32Stoppelli M.P. Tacchetti C. Cubellis M.V. Corti A. Hearing V.J. Cassani G. Appella E. Blasi F. Cell. 1986; 45: 675-684Abstract Full Text PDF PubMed Scopus (275) Google Scholar), washed with DMEM, and then incubated in serum-free medium with uPA-related proteins or EGF at the indicated concentrations for 8 h. When indicated, cells were pretreated with 399R anti-uPAR antibody (10 μg/ml) or with different pharmacological inhibitors (20 μm LY294002, 50 μm PD98059, 10 μm UO126, or 10 μm PP2) or with diluents for 30 min; treated cells were then collected by mild trypsinization and seeded onto 24-well plates coated or not with poly-HEMA in the presence of the indicated molecules for 8 h. To coat 24-well culture plates, 0.5 ml of a 10 mg/ml solution of poly-HEMA in ethanol was applied twice to each dish, dried, and extensively washed with PBS. To avoid potential anti-apoptotic effects caused by clumping, cells were seeded at 5 × 104cells/ml in DMEM, 10% FBS in the presence of 0.6% methylcellulose and plated on poly-HEMA-coated 24-well plates. After 8 h, cells were collected, washed with PBS, and then analyzed for the extent of apoptosis by the cell death ELISA, according to the manufacturer's instructions. Induction of UV- and Cisplatin-induced Apoptosis—RPE/Vec, RPE/SiuPA, or RPE/SiuPAR cells (5 × 104cells/sample) were grown in 24-well plates for 16 h, cultured for 24 h with or without 10% serum, and then UV-irradiated with 20 J/cm2 using a Stratalinker 2400 (Stratagene) in PBS. Immediately after UV irradiation, PBS was removed, and the original medium was put back into plates. When indicated, cells were treated with an acidic buffer for 2 min, washed with DMEM, and then exposed, in serum-free conditions, to the indicated effectors or antibodies for 30 min prior to UV irradiation. After 24 h, both attached and floating cells were collected and subjected to cell death ELISA. A sample of cells not exposed to UV light was included as negative control. For cisplatin-induced apoptosis, RPE/Vec, RPE/SiuPA, or RPE/SiuPAR cells (5 × 104 cells/sample) were plated into 24-well plates and after 16 h were serum-starved or not, and after 24 h the cell culture medium was replaced with fresh DMEM, 10% FBS either containing 100 μm cisplatin or diluents for 24 h. mRNA Quantification by Real Time PCR—RPE cells were grown to subconfluence and, 16 h later, serum-starved for 24 h; 4 × 106 cells were incubated for 5 h with or without 10 nm uPA 1–158 and washed with phosphate-buffered saline; the total RNA was isolated by acid-phenol extraction using TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. 1 μg of total RNA was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, MI), and 2 μl of a 1:10 dilution of reverse transcription reaction were analyzed by quantitative real time PCR with a DNA Engine Opticon 2 System (MJ Research, Boston), using DyNAmo HS SYBR Green qPCR kit (Finnzyme). Bcl-xL mRNA quantitation was normalized to the internal glyceraldehyde-3-phosphate dehydrogenase mRNA. Primers, designed using Primer3 software and used at 0.25 μm, were as follows: for Bcl-xL amplification, 198-bp product, forward primer 5′-AAGGATACAGCTGGAGTCAG-3′ and reverse primer 5′-GAGTTCATTCACTACCTGTTC-3′; for glyceraldehyde-3-phosphate dehydrogenase amplification, 161-bp product, forward primer 5′-ACATGTTCCAATATGATTCCA-3′ and reverse primer 5′-TGGACTCCACGACGTACTCAG-3′. The sample from cells not exposed to uPA 1–158 was chosen as reference value. The relative level of expression was calculated with the formula 2–ΔΔCt. Statistics—Data are shown as mean ± S.D. and represent one of at least three separate experiments undertaken in triplicate, unless stated otherwise. Differences between data sets were determined by the Student's t test. Differences described as significant in the text correspond to p < 0.05. The Amino-terminal Region of uPA Protects Retinal Pigment Epithelial Cells from Anoikis—The catalytically independent association of urokinase (uPA) with its receptor (uPAR) has an established role in cell mobilization. Because several motogens have been shown to exert an intrinsic survival activity on cells deprived of ECM attachment (33Frisch S.M. Francis H. J. Cell Biol. 1994; 124: 619-626Crossref PubMed Scopus (2774) Google Scholar), we sought to investigate if uPAR engagement with uPA could have a similar protective effect. To study the effect of uPA on detachment-induced apoptosis (anoikis), we made use of a telomerase-immortalized, nontransformed retinal epithelial cell line (hTert-RPE or RPE). Nontransformed epithelial cells can undergo anoikis if seeded in dishes coated with poly-HEMA, a compound that inhibits cell adhesion by preventing matrix deposition (33Frisch S.M. Francis H. J. Cell Biol. 1994; 124: 619-626Crossref PubMed Scopus (2774) Google Scholar). In agreement with pre-existing literature, we observed that as early as 4–6 h after plating RPE cells onto poly-HEMA-coated dishes, a significant fraction (∼40%) of cells become positive to annexin V and subsequently exhibit caspase-3 and PARP activation (not shown) (34Porstmann T. Griffiths B. Chung Y.L. Delpuech O. Griffiths J.R. Downward J. Schulze A. Oncogene. 2005; 24: 6465-6481Crossref PubMed Scopus (342) Google Scholar). To test the effect of human uPA on cells undergoing anoikis, recombinant pro-uPA and catalytically inactive uPA-related products, including the ATF (residues 1–135), uPA 1–158, and GFD peptide (GFDp, DCLNGGTAVSNKYFSNIHWCN) were employed (Fig. 1A). In agreement with Elner et al. (35Elner S.G. Elner V.M. Kindzelskii A.L. Horino K. Davis H.R. Todd R.F. III Glagov Petty S.H.R. Exp. Eye Res. 2003; 76: 585-595Crossref PubMed Scopus (25) Google Scholar), we found that RPE express about 8,000 surface receptors per cell. 3P. Franco, D. Alfano, and M. P. Stoppelli, unpublished data. When RPE cells were plated onto poly-HEMA-coated dishes, the extent of anoikis increased by 10-fold with respect to cells seeded in uncoated dishes, as assessed by quantitating the cytosolic DNA-histone complexes (Fig. 1B). However, if RPE cells deprived of cell attachment were incubated with recombinant prourokinase for 8 h, the extent of anoikis was reduced by ∼50%. To exclude any effect due to catalytic activity, cells were exposed to ATF under the same conditions. As a result, the protective ability of full-length pro-uPA is fully retained by ATF, showing that proteolytic activity is dispensable to the anti-apoptotic effect of uPA. uPA-mediated Protection from Anoikis Depends on uPAR Binding—Because ATF retains the receptor binding ability of uPA, we tested whether uPAR engagement is needed to achieve protection from anoikis. To answer this question we employed either a peptide corresponding to part of the GFD domain, shown to be the minimal requirement for receptor binding (GFDp, residues 12–32) (36Appella E. Robinson E.A. Ullrich S.J. Stoppelli M.P. Corti A. Cassani G. Blasi F. J. Biol. Chem. 1987; 262: 4437-4440Abstract Full Text PDF PubMed Google Scholar), or a specific anti-uPAR polyclonal antibody (399R) that prevents uPAR binding (Fig. 1A) (17Carriero M.V. Del Vecchio S. Capozzoli M. Franco P. Fontana L. Zannetti A. Botti G. D'Aiuto G. Salvatore M. Stoppelli M.P. Cancer Res. 1999; 59: 5307-5314PubMed Google Scholar). As shown in Fig. 1B, unlike the effect of the control peptide (5Ala-GFDp, in which the residues critical to uPAR bin
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