Notch Activation Suppresses Fibroblast Growth Factor-dependent Cellular Transformation
2003; Elsevier BV; Volume: 278; Issue: 18 Linguagem: Inglês
10.1074/jbc.m300464200
ISSN1083-351X
AutoresDeena Small, Д. В. Коваленко, Raffaella Soldi, Anna Mandinova, Vihren N. Kolev, Radiana Trifonova, Cinzia Bagalà, Doreen Kacer, Chiara Battelli, Lucy Liaw, Igor Prudovsky, Thomas Maciag,
Tópico(s)Developmental Biology and Gene Regulation
ResumoAberrant activations of the Notch and fibroblast growth factor receptor (FGFR) signaling pathways have been correlated with neoplastic growth in humans and other mammals. Here we report that the suppression of Notch signaling in NIH 3T3 cells by the expression of either the extracellular domain of the Notch ligand Jagged1 or dominant-negative forms of Notch1 and Notch2 results in the appearance of an exaggerated fibroblast growth factor (FGF)-dependent transformed phenotype characterized by anchorage-independent growth in soft agar. Anchorage-independent growth exhibited by Notch-repressed NIH 3T3 cells may result from prolonged FGFR stimulation caused by both an increase in the expression of prototypic and oncogenic FGF gene family members and the nonclassical export of FGF1 into the extracellular compartment. Interestingly, FGF exerts a negative effect on Notch by suppressing CSL (CBF-1/RBP-Jk/KBF2 in mammals, Su(H) inDrosophila and Xenopus, and Lag-2 in Caenorhabditis elegans)-dependent transcription, and the ectopic expression of constitutively active forms of Notch1 or Notch2 abrogates FGF1 release and the phenotypic effects of FGFR stimulation. These data suggest that communication between the Notch and FGFR pathways may represent an important reciprocal autoregulatory mechanism for the regulation of normal cell growth. Aberrant activations of the Notch and fibroblast growth factor receptor (FGFR) signaling pathways have been correlated with neoplastic growth in humans and other mammals. Here we report that the suppression of Notch signaling in NIH 3T3 cells by the expression of either the extracellular domain of the Notch ligand Jagged1 or dominant-negative forms of Notch1 and Notch2 results in the appearance of an exaggerated fibroblast growth factor (FGF)-dependent transformed phenotype characterized by anchorage-independent growth in soft agar. Anchorage-independent growth exhibited by Notch-repressed NIH 3T3 cells may result from prolonged FGFR stimulation caused by both an increase in the expression of prototypic and oncogenic FGF gene family members and the nonclassical export of FGF1 into the extracellular compartment. Interestingly, FGF exerts a negative effect on Notch by suppressing CSL (CBF-1/RBP-Jk/KBF2 in mammals, Su(H) inDrosophila and Xenopus, and Lag-2 in Caenorhabditis elegans)-dependent transcription, and the ectopic expression of constitutively active forms of Notch1 or Notch2 abrogates FGF1 release and the phenotypic effects of FGFR stimulation. These data suggest that communication between the Notch and FGFR pathways may represent an important reciprocal autoregulatory mechanism for the regulation of normal cell growth. soluble Jagged1 bovine calf serum constitutively active Notch dominant-negative Notch fibroblast growth factor fibroblast growth factor receptor reverse transcription-PCR Notch receptors and their ligands are components of an evolutionarily conserved signaling pathway that regulate cell proliferation, differentiation, and survival in a cell- and tissue-specific manner (for reviews, see Refs. 1Artavanis-Tsakonas S. Rand M.D. Lake R.J. Science. 1999; 284: 770-773Crossref PubMed Scopus (4951) Google Scholar and 2Miele L. Osborne B. J. Cell. Physiol. 1999; 181: 393-409Crossref PubMed Scopus (204) Google Scholar). Notch receptors and their ligands are structurally conserved transmembrane polypeptides, and four Notch receptors (Notch1–4) and six ligands (Delta1–4, Jagged1, and Jagged2) have been identified in vertebrates to date (3del Amo F.F. Gendron-Maguire M. Swiatek P.J. Jenkins N.A. Copeland N.G. Gridley T. Genomics. 1993; 15: 259-264Crossref PubMed Scopus (92) Google Scholar, 4Gray G.E. Mann R.S. Mitsiadis E. Henrique D. Carcangiu M.L. Banks A. Leiman J. Ward D. Ish-Horowitz D. Artavanis-Tsakonas S. Am. J. Pathol. 1999; 154: 785-794Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 5Lardelli M. Lendahl U. Exp. Cell Res. 1993; 204: 364-372Crossref PubMed Scopus (121) Google Scholar, 6Lindsell C.E. Shawber C.J. Boulter J. Weinmaster G. Cell. 1995; 80: 909-917Abstract Full Text PDF PubMed Scopus (540) Google Scholar, 7Shutter J.R. Scully S. Fan W. Richards W.G. Kitajewski J. Deblandre G.A. Kintner C.R. Stark K.L. Genes Dev. 2000; 14: 1313-1318PubMed Google Scholar, 8Uyttendaele H. Marazzi G. Wu G. Yan Q. Sassoon D. Kitajewski J. Development. 1996; 122: 2251-2259Crossref PubMed Google Scholar). Upon ligand binding, the intracellular domain of the Notch receptor is released by proteolytic cleavage and becomes a nuclear transcriptional regulator by interacting with members of the CSL (CBF-1/RBP-Jk/KBF2 in mammals, Su(H) inDrosophila and Xenopus, and Lag-2 inCaenorhabditis elegans) family of transcription factors (9Jarriault S. Brou C. Logeat F. Schroeter E.H. Kopan R. Israel A. Nature. 1995; 377: 355-358Crossref PubMed Scopus (1218) Google Scholar). Notch receptors have also been reported to regulate cellular processes through CSL-independent pathways that may involve interactions with other signaling molecules such as nuclear factor κB and Src (10Nofziger D. Miyamoto A. Lyons K.M. Weinmaster G. Development. 1999; 126: 1689-1702Crossref PubMed Google Scholar, 11Small D. Kovalenko D. Kacer D. Liaw L. Landriscina M. Di Serio C. Prudovsky I. Maciag T. J. Biol. Chem. 2001; 276: 32022-32030Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 12Shawber C. Nofziger D. Hsieh J.J. Lindsell C. Bogler O. Hayward D. Weinmaster G. Development. 1996; 122: 3765-3773Crossref PubMed Google Scholar). Phenotypic analysis of mice null for Notch receptors or their ligands emphasizes the requirement for proper Notch signaling not only during development but also in the adult (13Conlon R.A. Reaume A.G. Rossant J. Development. 1995; 121: 1533-1545Crossref PubMed Google Scholar, 14Hamada Y. Kadokawa Y. Okabe M. Ikawa M. Coleman J.R. Tsujimoto Y. Development. 1999; 126: 3415-3424Crossref PubMed Google Scholar, 15Jiang R. Lan Y. Chapman H.D. Shawber C. Norton C.R. Serreze D.V. Weinmaster G. Gridley T. Genes Dev. 1998; 12: 1046-1057Crossref PubMed Scopus (348) Google Scholar, 16Krebs L.T. Xue Y. Norton C.R. Shutter J.R. Maguire M. Sundberg J.P. Gallahan D. Closson V. Kitajewski J. Callahan R. Smith G.H. Stark K.L. Gridley T. Genes Dev. 2000; 14: 1343-1352PubMed Google Scholar, 17Xue Y. Gao X. Lindsell C.E. Norton C.R. Chang B. Hicks C. Gendron-Maguire M. Rand E.B. Weinmaster G. Gridley T. Hum. Mol. Genet. 1999; 8: 723-730Crossref PubMed Scopus (641) Google Scholar). Indeed, aberrant Notch signaling has been implicated in several human pathological conditions including the development of the CADASIL (18Joutel A. Corpechot C. Ducros A. Vahedi K. Chabriat H. Mouton P. Alamowitch S. Domenga V. Cecillion M. Marechal E. Maciazek J. Vayssiere C. Cruaud C. Cabanis E.-A. Ruchoux M.M. Weissenbach J. Bach J.F. Bousser M.G. Tournier-Lasserve E. Nature. 1996; 383: 707-710Crossref PubMed Scopus (1711) Google Scholar) and Alagille syndromes (19Li L. Krantz I.D. Deng Y. Genin A. Banta A.B. Collins C.C. Qi M. Trask B.J. Kuo W.L. Cochran J. Costa T. Pierpont M.E. Rand E.B. Piccoli D.A. Hood L. Spinner N.B. Nat. Genet. 1997; 16: 243-251Crossref PubMed Scopus (1022) Google Scholar, 20Oda T. Elkahloun A. Pike B. Okajima K. Krantz I. Genin A. Piccoli D. Meltzer P. Spinner N. Collins F. Chandrasekharappa S. Nat. Genet. 1997; 16: 235-242Crossref PubMed Scopus (944) Google Scholar) and the formation of neoplasias in mice and humans (21Rae F.K. Stephenson S.A. Nicol D.L. Clements J.A. Int. J. Cancer. 2000; 88: 726-732Crossref PubMed Scopus (114) Google Scholar, 22Rohn J.L. Lauring A.S. Linenberger M.L. Overbaugh J. J. Virol. 1996; 70: 8071-8080Crossref PubMed Google Scholar, 23Zagouras P. Stifani S. Blaumueller C.M. Carcangiu M.L. Artavanis-Tsakonas S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6414-6418Crossref PubMed Scopus (345) Google Scholar). We have reported previously that suppression of endogenous Notch signaling mediated by the ectopic expression of either an extracellular and soluble form of Jagged1 (sJ1)1 or dominant-negative mutants of Notch1 (dnN1) or Notch2 (dnN2) induces dramatic changes in the NIH 3T3 cellular phenotype in comparison with NIH 3T3 cells stably transfected with the empty vector (vector control). These changes in cellular phenotype include chord formation on collagen matrices; increased Src activation and enhanced phosphorylation of the Src substrate, cortactin; a decrease in the formation of actin filaments and focal adhesion sites; impaired migratory ability; and increased survival at high cell densities (11Small D. Kovalenko D. Kacer D. Liaw L. Landriscina M. Di Serio C. Prudovsky I. Maciag T. J. Biol. Chem. 2001; 276: 32022-32030Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 24Lindner V. Booth C. Prudovsky I. Small D. Maciag T. Liaw L. Am. J. Pathol. 2001; 159: 875-883Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 25Wong M.K. Prudovsky I. Vary C. Booth C. Liaw L. Mousa S. Small D. Maciag T. Biochem. Biophys. Res. Commun. 2000; 268: 853-859Crossref PubMed Scopus (27) Google Scholar). In contrast, NIH 3T3 cells stably expressing either constitutively active mutants of Notch1 (caN1) or Notch2 (caN2) display a phenotype similar to that exhibited by the vector control cells (11Small D. Kovalenko D. Kacer D. Liaw L. Landriscina M. Di Serio C. Prudovsky I. Maciag T. J. Biol. Chem. 2001; 276: 32022-32030Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Our initial interest in Notch originated from our observation that Jagged1 was an FGF response gene in human endothelial cells undergoing differentiation on fibrin clots (26Zimrin A.B. Pepper M.S. McMahon G.A. Nguyen F. Montesano R. Maciag T. J. Biol. Chem. 1996; 271: 32499-32502Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Because several of the phenotypic characteristics displayed by Notch-repressed cells were dependent on the activity of the FGFR effector molecule Src (11Small D. Kovalenko D. Kacer D. Liaw L. Landriscina M. Di Serio C. Prudovsky I. Maciag T. J. Biol. Chem. 2001; 276: 32022-32030Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), we anticipated that communication between Notch and FGFR signaling pathways may also represent an important mechanism regulating cellular behavior in fibroblasts. Whereas several studies report that the expression of Notch and/or its ligands is correlated with activation of the FGFR signaling pathway (27Bongarzone E.R. Byravan S. Givogri M.I. Schonmann V. Campagnoni A.T. J. Neurosci. Res. 2000; 62: 319-328Crossref PubMed Scopus (24) Google Scholar, 28Faux C.H. Turnley A.M. Epa R. Cappai R. Bartlett P.F. J. Neurosci. 2001; 21: 5587-5596Crossref PubMed Google Scholar, 29Matsumoto T. Turesson I. Book M. Gerwins P. Claesson-Welsh L. J. Cell Biol. 2002; 156: 149-160Crossref PubMed Scopus (177) Google Scholar) and vice versa (30Ikeya T. Hayashi S. Development. 1999; 126: 4455-4463Crossref PubMed Google Scholar), little is known about how interactions between these two important and ubiquitous pathways influence cellular phenotype, including growth. To address this question, we examined the effects of FGFR stimulation in combination with Notch repression or activation in NIH 3T3 cells. We decided to study these interactions in the NIH 3T3 cell because we had already identified phenotypic characteristics associated with the down-regulation of Notch signaling (11Small D. Kovalenko D. Kacer D. Liaw L. Landriscina M. Di Serio C. Prudovsky I. Maciag T. J. Biol. Chem. 2001; 276: 32022-32030Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), and the NIH 3T3 cell represents a relatively simple system because it primarily expresses transcripts encoding Notch1 and Notch2, but not Notch3 and Notch4. 2D. Small, R. Trifonova, and T. Maciag, unpublished observations. In addition, aberrant activation of both Notch (21Rae F.K. Stephenson S.A. Nicol D.L. Clements J.A. Int. J. Cancer. 2000; 88: 726-732Crossref PubMed Scopus (114) Google Scholar, 22Rohn J.L. Lauring A.S. Linenberger M.L. Overbaugh J. J. Virol. 1996; 70: 8071-8080Crossref PubMed Google Scholar, 23Zagouras P. Stifani S. Blaumueller C.M. Carcangiu M.L. Artavanis-Tsakonas S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6414-6418Crossref PubMed Scopus (345) Google Scholar) and FGFR (31Forough R. Xi Z. MacPhee M. Friedman S. Engleka K.A. Sayers T. Wiltrout R.H. Maciag T. J. Biol. Chem. 1993; 268: 2960-2968Abstract Full Text PDF PubMed Google Scholar, 32Maerz W.J. Baselga J. Reuter V.E. Mellado B. Myers M.L. Bosl G.J. Spinella M.J. Dmitrovsky E. Oncogene. 1998; 17: 761-767Crossref PubMed Scopus (28) Google Scholar, 33Yan G. Fukabori Y. McBride G. Nikolaropolous S. McKeehan W.L. Mol. Cell. Biol. 1993; 13: 4513-4522Crossref PubMed Scopus (376) Google Scholar) signaling pathways have been found to be associated with neoplastic growth, and the NIH 3T3 cell is uniquely sensitive to oncogene-mediated transformation (34Shih C. Shilo B.Z. Goldfarb M.P. Dannenberg A. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 5714-5718Crossref PubMed Scopus (314) Google Scholar). We report that antagonistic interactions between the Notch and FGFR signaling pathways regulate anchorage-independent growth in murine fibroblasts. Stimulation of the FGFR pathway by exogenous FGF1 causes Notch-repressed cells to grow as detached spheroids in tissue culture and to aggressively form colonies in soft agar, phenotypic characteristics associated with cellular transformation (reviewed in Ref. 35Danen E.H. Yamada K.M. J. Cell. Physiol. 2001; 189: 1-13Crossref PubMed Scopus (395) Google Scholar). The transformed phenotype exhibited by Notch-repressed cells may be attributed to their maintenance of FGFR-generated signals generated by an increase in both the expression and release of FGF family members. Furthermore, FGF1 has an inhibitory effect on Notch/CSL-dependent transcription. In contrast, the expression of caN1 or caN2 protects the NIH 3T3 cell from FGF-induced anchorage-independent growth and suppresses the release of FGF1 under normal growth conditions. These results suggest that cross-talk between the Notch and FGFR signaling pathways may represent an important autoregulatory mechanism that is involved in the regulation of cell growth. Stable NIH 3T3 transfectants for vector control, sJ1, dnN1, dnN2, caN1, and caN2 were obtained and screened for expression as described previously (11Small D. Kovalenko D. Kacer D. Liaw L. Landriscina M. Di Serio C. Prudovsky I. Maciag T. J. Biol. Chem. 2001; 276: 32022-32030Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The FGF1 mutant containing the 3′ signal peptide sequence of FGF4 (hst-β(FGF4):FGF1) and FGF1 (pXZ38) stable transfectants were obtained and screened for expression as described in Ref. 31Forough R. Xi Z. MacPhee M. Friedman S. Engleka K.A. Sayers T. Wiltrout R.H. Maciag T. J. Biol. Chem. 1993; 268: 2960-2968Abstract Full Text PDF PubMed Google Scholar. Stably transfected constitutively active Ras (caRas) clonal populations were obtained by previously described methods (11Small D. Kovalenko D. Kacer D. Liaw L. Landriscina M. Di Serio C. Prudovsky I. Maciag T. J. Biol. Chem. 2001; 276: 32022-32030Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) using the activated H-Ras pUSEamp plasmid (Upstate Biotechnology). Clonal populations of NIH 3T3 cells stably transfected with vector control, sJ1, caN1, caN2, dnN1, dnN2, hst-β(FGF4):FGF1, or caRas were plated on 6-cm tissue culture dishes with 0.5% agar in an overlay containing Dulbecco's modified Eagle's medium (Invitrogen), 10% bovine calf serum (BCS; Hyclone), and 0.33% agar at 1.5 × 103 cells/dish. As indicated, some dishes were also treated with 1 μm of the FGFR1-specific inhibitor, PD166866 (Ref. 36Panek R.L. Lu G.H. Dahring T.K. Batley B.L. Connolly C. Hamby J.M. Brown K.J. J. Pharmacol. Exp. Ther. 1998; 286: 569-577PubMed Google Scholar; a generous gift from R. L. Panek, Park-Davis), and/or 10 ng/ml recombinant human FGF1 and 10 units/ml heparin (Sigma). Cells were fed with 0.5 ml of media with or without FGF1 and/or the FGFR1-specific inhibitor every 3 days as indicated. Twenty days after plating, colonies were stained with p-iodonitrotetrazolium violet (Sigma) for visualization. Quantitation of colony formation was achieved by counting all p-iodonitrotetrazolium violet-stained colonies consisting of more than 4 cells under a Zeiss Stemi SVII Apo dissecting microscope. Clonal populations of NIH 3T3 stable transfectants were resuspended and grown in media containing 10% BCS, 10% BCS plus 1 μmPD166866, 10% BCS plus 10 ng/ml recombinant human FGF1 and 10 units/ml heparin, or 10% BCS plus 10 ng/ml recombinant human FGF1, 10 units/ml heparin, and 1 μm PD166866 as indicated in the figure legend. Approximately 2 × 105 cells were plated per well (6-well dish), and 3 days after plating, phase-contrast micrographs of the cells were taken. hst-β(FGF4):FGF1 stable transfectants were transduced with either adenovirus expressing lacZ, dominant negative FGFR, caN1, or caN2 as described below. After 24 h, the cells were plated on 6-well dishes at a concentration of 105cells/well, and 3 days after plating, phase-contrast micrographs of the cells were taken. For colony formation in the soft agar assay, the hst-β(FGF4):FGF1 transfectants were transduced with the indicated recombinant adenoviral vectors and plated into 0.33% agar 24 h after the transduction. Two weeks after plating, colonies were visualized by staining with p-iodonitrotetrazolium violet. Total RNA from vector control, sJ1, caN1, and dnN1 stable transfectants was isolated using Tri ReagentTM(Sigma) according to the manufacturer's protocol. cDNA was obtained from 5 μg of total RNA with SuperScriptTM(Invitrogen) reverse transcriptase using an oligo(dT) primer (Invitrogen). The following specific primers were purchased from IDT and used for RT-PCR analysis (sense primers are indicated by (s); antisense primers are indicated by (as)): FGF1(s), 5′-ATGGCTGAAGGGGAGATCACAACC-3′; FGF1(as), 5′-CGCGCTTACAGCTCCCGTTC-3′; FGF2(s), 5′-ATGGCTGCCAGCGGCATCAC-3′; FGF2(as), 5′-GAAGAAACAGTATGGCCTTCTGTCC-3′; FGF3(s), 5′-GCCTGATCTGGCTTCTGCTGC-3′; FGF3(as), 5′-GCAGCTGGGTGCTTGGAGGTGG-3′; FGF4(s), 5′-ACCACAGGGACGACTG-3′; FGF4(as), 5′-CATACCGGGGTACGCGTAGG-3′; FGF6(s), 5′-GGGCCATTAATTCTGACCACGTGCCTG-3′; FGF6(as), 5′-GGTCCTTATATCCTGGGGAGGAAGTGAGTG-3′; FGF7(s), 5′-CACGGATCCTGCCAACTCTGC-3′; FGF7(as), 5′-CCACAATTCCAACTGCCACGGTC-3′; FGF8(s), 5′-CTCTGCCTCCAAGCCAGGTAAG-3′; FGF8(as), 5′-GCTGATGCTGGCGCGTCTTGGAG-3′; FGF9(s), 5′-GGTGAAGTTGGGAGCTATTTCG-3′; FGF9(as), 5′-CATAGTATCTCCTTCCGGTGTCCAC-3′; FGF10(s), 5′-CACATTGTGCCTCAGCCTTTC-3′; FGF10(as), 5′-CCTCTATTCTCTCTTTCAGCTTAC-3′; FGFR1(s), 5′-AGGCCAGCCCCAACCTTG-3′; FGFR1(VT + as), 5′-GGAGTCAGCTGACACTGTTAC-3′; FGFR1(VT −as), 5′-CACTGGAGTCAGCTGACACC-3′; FGFR2(s), 5′-TCCTTCAGTTTAGTTGAGGATAC-3′; FGFR2(as), 5′-GCAGCTTTCAGAACCTTGAGG-3′; FGFR3(s), 5′-CAAGTGCTAAATGCCTCCCAC-3′; and FGFR3(as), 5′-GCAGAGTATCACAGCTGC-3′. PCR amplification was performed for 45 cycles as follows: 40 s at 94 °C, 40 s at 50 °C (for FGF2, FGF3, FGF4, and FGFR3) or at 55 °C (for FGF1, FGF6, FGF7, FGF8, FGF9, FGF10, FGFR1, and FGFR2), and 1 min at 72 °C. For FGF5, RT-PCR analysis was performed as described previously (37Ozawa K. Suzuki S. Asada M. Tomooka Y. Li A.J. Yoneda A. Komi A. Imamura T. J. Biol. Chem. 1998; 273: 29262-29271Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), and all amplified DNA was visualized with ethidium bromide on 1.5% agarose gels. Adenovirus vector expressing lacZ, FGF1, caN1, dnN1, caN2, or dnN2 was prepared as described previously (38Hardy S. Kitamura M. Harris-Stansil T. Dai Y. Phipps M.L. J. Virol. 1997; 71: 1842-1849Crossref PubMed Google Scholar) at a titer of ∼1012 viral particles/ml. For adenoviral transduction, NIH 3T3 stable transfectants were incubated in serum-free medium with ∼103 viral particles/cell in the presence of poly-d-lysine hydrobromide (Sigma) (5 × 103 molecules/viral particle) for 2 h at 37 °C, after which the adenovirus-containing medium was removed and replaced with serum-containing medium (10% BCS) for an additional 24 h. The transduced cells were harvested by trypsin digestion and seeded for the heat shock experiments as described previously (39Jackson A. Friedman S. Zhan X. Engleka K.A. Forough R. Maciag T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10691-10695Crossref PubMed Scopus (226) Google Scholar). After heat shock, the conditioned media from cells exposed to either 37 °C (normal conditions) or 42 °C (heat shock conditions) were treated with 0.1% dithiothreitol for 2 h at 37 °C, adsorbed to heparin-Sepharose, and eluted from the column with 1.5 mNaCl. The eluants were resolved by 15% (w/v) SDS-PAGE and evaluated by FGF1 immunoblot analysis as described previously (39Jackson A. Friedman S. Zhan X. Engleka K.A. Forough R. Maciag T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10691-10695Crossref PubMed Scopus (226) Google Scholar). NIH 3T3 cells were plated onto fibronectin-coated (10 μg/cm2) 12-well tissue culture dishes and transiently transfected at ∼80% confluency with 500 ng of a luciferase construct activated by four tandem copies of the CSL (CBF1) response element (40Hsieh J.J. Henkel T. Salmon P. Robey E. Peterson M.G. Hayward S.D. Mol. Cell. Biol. 1996; 16: 952-959Crossref PubMed Scopus (395) Google Scholar), 100 ng of the TK Renilla (Promega) construct as an internal control for transfection efficiency, and 500 ng of either vector control, sJ1, or caN1 constructs using FuGENE 6 (Roche Molecular Biochemicals) per the manufacturer's instructions. For analysis of caN1 activity in the background of NIH 3T3 stable lines, vector control, caN1, sJ1, and hst-β(FGF4):FGF1 stable transfectants were plated onto fibronectin-coated (10 μg/cm2) 12-well tissue culture dishes and transiently transfected with 500 ng of the CSL-luciferase construct, 100 ng of the TK Renilla (Promega) construct as an internal control for transfection efficiency, and 200 ng of caN1. For all experiments, the medium was replaced 24 h after transfection with fresh Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% BCS or with 10% BCS containing 1, 2.5, 5, or 10 ng/ml recombinant human FGF1 and 10 units/ml heparin as indicated in the figure legend. The cells were harvested 48 h after the media change, and luciferase/Renilla activity was measured using Promega's Dual-Luciferase Reporter Assay System. The efficiency of transcription was measured and normalized in relationship to the activity of pRL-TK Renilla, and the activity is reported as the ratio of luciferase/Renilla activity. Each experiment was done in triplicate, and error bars represent the S.E. Vector control, sJ1, caN1, Jagged1, hst-β(FGF4):FGF1, and sJ1:caN1 stable NIH 3T3 cell transfectants were plated on tissue culture dishes in either normal growth media (Dulbecco's modified Eagle's medium; Invitrogen), 10% BCS (Hyclone), or normal growth media containing 10 ng/ml recombinant FGF1 and 10 units/ml heparin. Cells were harvested at confluence (based on the status of vector control cells grown in normal growth media). Because hst-β(FGF4):FGF1 stable transfectants and the sJ1 transfectants grown in FGF1-containing media form spheroids, all cells were harvested by scraping in the presence of the growth media, and then the cells/growth media were collected into sterile 15-ml conical tubes. Cells were pelleted by centrifugation at 800 ×g for 5 min. After centrifugation, the supernatant was removed, and the cell pellets were washed three times with 1× phosphate-buffered saline. Cells were lysed in 500 μl of 20 mm Tris, pH 7.5, containing 300 mm sucrose, 60 mm KCl, 15 mm NaCl, 0.5 mm EDTA, 0.5% (v/v) Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 0.1% SDS, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mm sodium orthovanadate for 15 min on ice. Cell lysates were pelleted, and the supernatant containing soluble proteins was removed. Samples were normalized for protein concentration using the BCA Protein Assay Kit (Pierce). Equal protein loads were resolved by 8% acrylamide (w/v) SDS-PAGE, transferred to Hybond C (Amersham Biosciences), and immunoblotted with the Jagged1 antibody (Santa Cruz Biotechnology). Jagged1 was visualized using a horseradish peroxidase-conjugated antibody against goat IgG (Sigma) and the ECL detection system (Amersham Biosciences). In an effort to further our understanding of how interactions between the Notch and FGFR signaling pathways regulate cellular processes, we examined the response of Notch-activated and Notch-repressed NIH 3T3 cells to the addition of recombinant FGF1 to the growth media (Fig.1). Surprisingly, NIH 3T3 cells in which endogenous Notch signaling was repressed (sJ1 and dnN1) formed multicellular, spheroid-like structures similar to those observed in NIH 3T3 cells stably expressing an oncogenic mutant of FGF1 engineered with the FGF4 signal peptide sequence (hst-β(FGF4):FGF1) to force constitutive secretion of FGF1 through the conventional ER-Golgi pathway (31Forough R. Xi Z. MacPhee M. Friedman S. Engleka K.A. Sayers T. Wiltrout R.H. Maciag T. J. Biol. Chem. 1993; 268: 2960-2968Abstract Full Text PDF PubMed Google Scholar). The cells contained within the spheroid structures were viable because they continued to proliferate over time and also grew as a monolayer when replated onto fresh tissue culture dishes in the absence of recombinant FGF1 in the growth media (data not shown). In contrast, vector control and caN1 NIH 3T3 cell stable transfectants did not form spheroids but instead continued to grow as a monolayer in the presence of recombinant FGF1. Spheroid formation was a specific response to FGF1 because treatment with the FGFR1-specific inhibitor PD166866 (36Panek R.L. Lu G.H. Dahring T.K. Batley B.L. Connolly C. Hamby J.M. Brown K.J. J. Pharmacol. Exp. Ther. 1998; 286: 569-577PubMed Google Scholar) completely abolished FGF1-induced spheroid formation in sJ1, dnN1, and hst-β(FGF4):FGF1 stable transfectants. Cellular proliferation despite detachment from the extracellular matrix is indicative of anchorage-independent growth in most cell types and is an in vitro signature of the NIH 3T3 cell transformed phenotype (34Shih C. Shilo B.Z. Goldfarb M.P. Dannenberg A. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 5714-5718Crossref PubMed Scopus (314) Google Scholar). Therefore, we examined our Notch-activated and Notch-repressed cell lines for anchorage-independent growth in soft agar in both the presence and absence of exogenously added recombinant FGF1 (Fig. 2, A andB). Although we have previously reported (25Wong M.K. Prudovsky I. Vary C. Booth C. Liaw L. Mousa S. Small D. Maciag T. Biochem. Biophys. Res. Commun. 2000; 268: 853-859Crossref PubMed Scopus (27) Google Scholar) that sJ1 NIH 3T3 stable transfectants do not form colonies in soft agar when plated at low seed densities (100 cells/6-cm dish) in growth media containing 10% BCS, we have found during the course of these studies that sJ1and dnN1 transfectants do form small, pinpoint-sized colonies in soft agar when plated at seed densities greater than 1,500 cells/6-cm dish. The addition of FGF1 to the growth media greatly exaggerated the size of the colonies formed by sJ1 and dnN1 transfectants so that they were clearly visible to the eye, although the number of colonies did not significantly increase. Indeed, the intensity of the transformed phenotype induced by FGF1 in the Notch-repressed NIH 3T3 cells resembled that exhibited by NIH 3T3 cells stably expressing either an oncogenic Ras construct (caRas) or an oncogenic mutant of FGF1 (hst-β(FGF4):FGF1). Interestingly, the size of the colonies also increased at seed densities of 5,000–10,000 cells/6-cm dish, even in the absence of exogenously added recombinant FGF1 (data not shown). In contrast, the addition of FGF1 had either no effect or only resulted in the formation of sparse and very small colonies in the caN1 and vector control transfectants. These transfectants also do not form colonies, regardless of plating concentration, in the absence of FGF1. NIH 3T3 cells stably expressing dnN2 but not caN2 also formed small colonies whose size was dramatically increased in the presence of FGF1 (data not shown). Similar to spheroid formation, FGF potentiation of soft agar growth in NIH 3T3 cells was a specific response to FGFR stimulation because the addition of the FGFR1 inhibitor PD166866 substantially reduced FGF1-mediated colony formation in sJ1, dnN1, and the hst-β(FGF4):FGF1 transfectants but had no effect on colony formation in the caRas cells. Treatment with PD166866 also inhibited small colony growth exhibited by sJ1 and dnN1 in the absence of FGF1. These data suggest that repression of endogenous Notch signaling sensitizes the NIH 3T3 cell to FGFR-mediated cellular transformation and that activation of the Notch signaling pathway may protect the NIH 3T3 cell from abnormal growth. To further explore the possibility that Notch signaling may protect the NIH 3T3 cell from FGF-mediated anchorage-independent growth, we assayed the ability of sJ1 NIH 3T3 stable transfectants cotransfected with caN1 to form colonies in both the presence and absence of FGF1. Expression of caN1 in the sJ1 NIH 3T3 background dramatically inhibited, in terms of both number and size, colony formation that occurred in the presence or absence of FGF1. Indeed, the number of colonies formed by the sJ1:caN1 cotransfectants was similar to that observed in vector control and caN1 stable lines (Fig. 2, A and B). Unlike sJ1 single transfectants, sJ1:caN1 cotransfectants did not form spheroids in the presence of FGF1. However, these cells were less adherent to the tissue culture dish than the vector control or caN1 cells (Fig. 1). Altho
Referência(s)