Extracellular Signal-regulated Kinase (ERK)-dependent Gene Expression Contributes to L1 Cell Adhesion Molecule-dependent Motility and Invasion
2004; Elsevier BV; Volume: 279; Issue: 28 Linguagem: Inglês
10.1074/jbc.m404075200
ISSN1083-351X
AutoresSteve Silletti, Mayra Yebra, Brandon Perez, Vincenzo Cirulli, Martin McMahon, Anthony M.P. Montgomery,
Tópico(s)Cellular Mechanics and Interactions
ResumoThe cell adhesion molecule L1 has been implicated in a variety of motile processes, including neurite extension, cerebellar cell migration, extravasation, and metastasis. Homophilic or heterophilic L1 binding and concomitant signaling have been shown to promote cell motility in the short term. In this report, L1 is also shown to induce and maintain a motile and invasive phenotype by promoting gene transcription. In the presence of serum or platelet-derived growth factor, L1 promotes heightened and sustained activation of the extracellular signal-regulated kinase pathway. Activation of this pathway then induces the expression of motility- and invasion-associated gene products, including the β3-integrin subunit, small GTPases, and the cysteine proteases cathepsin-L and -B. Induction of integrin αvβ3 and rac-1 is shown to contribute directly to L1-dependent haptotaxis, whereas induction of cathepsins-L and -B promotes matrix invasion. This study provides a novel translational mechanism to account for the association between L1 expression and motile processes involved in metastasis and development. The cell adhesion molecule L1 has been implicated in a variety of motile processes, including neurite extension, cerebellar cell migration, extravasation, and metastasis. Homophilic or heterophilic L1 binding and concomitant signaling have been shown to promote cell motility in the short term. In this report, L1 is also shown to induce and maintain a motile and invasive phenotype by promoting gene transcription. In the presence of serum or platelet-derived growth factor, L1 promotes heightened and sustained activation of the extracellular signal-regulated kinase pathway. Activation of this pathway then induces the expression of motility- and invasion-associated gene products, including the β3-integrin subunit, small GTPases, and the cysteine proteases cathepsin-L and -B. Induction of integrin αvβ3 and rac-1 is shown to contribute directly to L1-dependent haptotaxis, whereas induction of cathepsins-L and -B promotes matrix invasion. This study provides a novel translational mechanism to account for the association between L1 expression and motile processes involved in metastasis and development. The L1 cell adhesion molecule is a phylogenetically conserved neural recognition molecule that belongs to the immunoglobulin superfamily. Structurally, L1 is a transmembrane (type 1) glycoprotein with a complex ectodomain consisting of multiple immunoglobulin and fibronectin-like repeats (1Brummendorf T. Rathjen F.G. J. Neurochem. 1993; 61: 1207-1219Crossref PubMed Scopus (122) Google Scholar, 2Hortsch M. Neuron. 1996; 17: 587-593Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). L1 was first described as a neural cell adhesion molecule based upon a restricted distribution that included post-mitotic neurons (3Lindner J. Rathjen F.G. Schachner M. Nature. 1983; 305: 427-430Crossref PubMed Scopus (391) Google Scholar). However, both murine and human L1 homologues have now also been described for other cell types of diverse origin, including endothelial cells, epithelial cells, reticular fibroblasts, and cells of lymphoid and myelomonocytic origin (4Thor G. Probstmeier R. Schachner M. EMBO J. 1987; 6: 2581-2586Crossref PubMed Scopus (84) Google Scholar, 5Ebeling O. Duczmal A. Aigner S. Geiger C. Schollhammer S. Kemshead J.T. Moller P. Schwartz-Albiez R. Altevogt P. Eur. J. Immunol. 1996; 26: 2508-2516Crossref PubMed Scopus (95) Google Scholar, 6Montgomery A.M.P. Becker J.C. Siu C.-H. Lemmon V.P. Cheresh D.A. Pancook J.D. Zhao X. Reisfeld R.A. J. Cell Biol. 1996; 132: 475-485Crossref PubMed Scopus (207) Google Scholar, 7Pancook J.D. Reisfeld R.A. Varki N. Vitiello A. Fox R.I. Montgomery A.M.P. J. Immunol. 1997; 158: 4413-4421PubMed Google Scholar, 8Debiec H. Christensen E.I. Ronco P.M. J. Cell Biol. 1998; 143: 2067-2079Crossref PubMed Scopus (88) Google Scholar, 9Di Sciullo G. Donahue T. Schachner M. Bogen S.A. J. Exp. Med. 1998; 187: 1953-1963Crossref PubMed Scopus (24) Google Scholar, 10Nolte C. Moos M. Schachner M. Cell Tissue Res. 1999; 298: 261-273Crossref PubMed Scopus (50) Google Scholar). Although L1 serves to promote cell-cell interactions, the functional outcome of such interactions is rarely static cell-cell adhesion. In the central nervous system, for example, L1 has been shown to initiate a variety of dynamic motile processes that include cerebellar cell migration and neurite extension (3Lindner J. Rathjen F.G. Schachner M. Nature. 1983; 305: 427-430Crossref PubMed Scopus (391) Google Scholar, 11Miura M. Asou H. Kobayashi M. Uyemura K. J. Biol. Chem. 1992; 267: 10752-10758Abstract Full Text PDF PubMed Google Scholar, 12Dahme M. Bartsch U. Martini R. Anliker B. Schachner M. Mantei N. Nat. Genet. 1997; 17: 346-349Crossref PubMed Scopus (416) Google Scholar, 13Demyanenko G.P. Tsai A.Y. Maness P.F. J. Neurosci. 1999; 19: 4907-4920Crossref PubMed Google Scholar). Function-blocking antibodies to L1 have been shown to abrogate the migration of granule precursors in cerebellar explant cultures (3Lindner J. Rathjen F.G. Schachner M. Nature. 1983; 305: 427-430Crossref PubMed Scopus (391) Google Scholar, 14Crossin K.L. Prieto A.L. Hoffman S. Jones F.S. Friedlander D.R. Exp. Neurol. 1990; 109: 6-18Crossref PubMed Scopus (78) Google Scholar), and the migration and positioning of dopaminergic neuronal precursors is disrupted in L1-deficient mice (13Demyanenko G.P. Tsai A.Y. Maness P.F. J. Neurosci. 1999; 19: 4907-4920Crossref PubMed Google Scholar). Additional studies have linked L1 to motile processes involved in tumor cell extravasation and glioma dissemination in the brain (15Izumoto S. Ohnishi T. Arita N. Hirage S. Taki T. Hayakawa T. Cancer Res. 1996; 56: 1440-1444PubMed Google Scholar, 16Voura E.B. Ramjeesingh R.A. Montgomery A.M.P. Siu C.-H. Mol. Biol. Cell. 2001; 12: 2699-2710Crossref PubMed Scopus (166) Google Scholar). Several recent reports have also now linked L1 expression to melanoma and prostate metastasis (17Linnemann D. Raz A. Bock E. Int. J. Cancer. 1989; 43: 709-712Crossref PubMed Scopus (70) Google Scholar, 18Thies A. Schachner M. Moll I. Berger J. Schulze H.-J. Brunner G. Schumacher U. Eur. J. Cancer. 2002; 38: 1708-1716Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 19Calvo A. Xiao N. Kang J. Best C.J.M. Leiva I. Emmert-Buck M.R. Jorcyk C. Green J.E. Cancer Res. 2002; 62: 5325-5335PubMed Google Scholar, 20Fogel M. Mechtersheimer S. Huszar M. Smirnov A. Abu-Dahi A. Tilgen W. Reichrath J. Georg T. Altevogt P. Gutwein P. Cancer Lett. 2003; 189: 237-247Crossref PubMed Scopus (104) Google Scholar). For example, Thies et al. (18Thies A. Schachner M. Moll I. Berger J. Schulze H.-J. Brunner G. Schumacher U. Eur. J. Cancer. 2002; 38: 1708-1716Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) demonstrated that L1 expression in primary melanoma is a highly significant indicator for subsequent metastasis and reduced patient survival. Cell-cell interaction and homophilic L1-ligation has been shown to be an important stimulus for cell motility (11Miura M. Asou H. Kobayashi M. Uyemura K. J. Biol. Chem. 1992; 267: 10752-10758Abstract Full Text PDF PubMed Google Scholar). However, L1 is also now known to have a significant impact upon cell-matrix interactions required for haptotaxis and matrix remodeling (9Di Sciullo G. Donahue T. Schachner M. Bogen S.A. J. Exp. Med. 1998; 187: 1953-1963Crossref PubMed Scopus (24) Google Scholar, 21Mechtersheimer S. Gutwein P. Agmon-Levin N. Stoeck A. Oleszewski M. Riedle S. Fogel M. Lemmon V. Altevogt P. J. Cell Biol. 2001; 155: 661-673Crossref PubMed Scopus (28) Google Scholar, 22Thelen K. Keder V. Panicker A.K. Schmid R.-S. Midkiff B.R. Maness P.F. J. Neurosci. 2002; 22: 4918-4931Crossref PubMed Google Scholar). Two recent reports (21Mechtersheimer S. Gutwein P. Agmon-Levin N. Stoeck A. Oleszewski M. Riedle S. Fogel M. Lemmon V. Altevogt P. J. Cell Biol. 2001; 155: 661-673Crossref PubMed Scopus (28) Google Scholar, 22Thelen K. Keder V. Panicker A.K. Schmid R.-S. Midkiff B.R. Maness P.F. J. Neurosci. 2002; 22: 4918-4931Crossref PubMed Google Scholar) have shown that ectopic L1 expression strongly potentiates α5β1-mediated haptotaxis. In both of these studies, initiation of haptotaxis is proposed to involve direct L1-integrin interaction via an Arg-Gly-Asp integrin-recognition motif in the L1 ectodomain. Such an association may depend upon a transient cis-association at the cell surface (22Thelen K. Keder V. Panicker A.K. Schmid R.-S. Midkiff B.R. Maness P.F. J. Neurosci. 2002; 22: 4918-4931Crossref PubMed Google Scholar) or involve L1-shedding and autocrinal binding via αvβ5 (21Mechtersheimer S. Gutwein P. Agmon-Levin N. Stoeck A. Oleszewski M. Riedle S. Fogel M. Lemmon V. Altevogt P. J. Cell Biol. 2001; 155: 661-673Crossref PubMed Scopus (28) Google Scholar). In both studies, it is suggested that direct L1-integrin ligation potentiates haptotaxis via non-translational mechanisms that involve the induction of integrin-mediated signaling events (21Mechtersheimer S. Gutwein P. Agmon-Levin N. Stoeck A. Oleszewski M. Riedle S. Fogel M. Lemmon V. Altevogt P. J. Cell Biol. 2001; 155: 661-673Crossref PubMed Scopus (28) Google Scholar, 22Thelen K. Keder V. Panicker A.K. Schmid R.-S. Midkiff B.R. Maness P.F. J. Neurosci. 2002; 22: 4918-4931Crossref PubMed Google Scholar). In this report, we present evidence for a translational mechanism that can further account for the association between L1 expression and cell motility and invasion. Ectopic L1 expression in two different cell lines is shown to induce sustained activation of the extracellular signal-regulated kinase (ERK) 1The abbreviations used are: ERK, extracellular signal-regulated kinase; mAb, monoclonal antibody; pAb, polyclonal antibody; FACS, fluorescence-activated cell sorting; PDGF, platelet-derived growth factor; 4HT, 4-hydroxytamoxifen; RBM, reconstituted basement membrane. pathway and the concomitant induction of ERK-regulated gene products intimately associated with cell motility and invasion. Several of these gene products are shown to contribute directly to L1-mediated migration and matrix invasion. Such a mechanism may allow L1 to induce and maintain a motile cellular phenotype that facilitates both normal developmental repositioning and metastasis. Reagents—Anti-mouse integrin antibodies to β1 (Ha2/5), α1 (Ha31/8), α2 (HMa2), α5 (5H10–27), α6 (GoH3), β3 (2C9.G2), and αv (H9.2b8) were purchased from BD Biosciences. A goat polyclonal antibody (pAb) to α5β1 (FNR) was obtained from Chemicon, Inc. (Temecula, CA). Anti-phospho-ERK monoclonal antibody (mAb) (E10) and anti-ERK-2 pAb (C-14) were purchased from Cell Signaling Technology (Beverly, MA) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Anti-L1 mAb (UJ127) was from Neomarkers (Fremont, CA) and an anti-L1 pAb (L1-ECD) was kindly provided by Dr. William Stallcup (Burnham Institute, La Jolla, CA). Anti-thrombospondin-2 mAb Cl.4 and anti-Rac1 mAb (Cl.102) and anti-S100A4 pAb were obtained from BD Transduction Labs and DAKO (Glostrup, Denmark), respectively. The mitogen-activated protein/ERK kinase inhibitor U0126 was purchased from Promega (Madison, WI). Purified vitronectin was obtained from Chemicon; laminin from BD Biosciences; purified type I collagen from Upstate Biotechnology (Lake Placid, NY); and human type IV collagen from Sigma. Cell Models and Transfections—NIH-3T3 cells used for stable and transient transfections were obtained from the American Type Culture Collection (Rockville, MD). 3T3 cells stably expressing L1 were provided by Dr. V. Lemmon (University of Miami) and were generated by using LipofectAMINE and a pcDNA3 vector containing full-length L1 cDNA (23Kamiguchi H. Long K.E. Pendergast M. Schaefer A.W. Rapoport I. Kirchhausen T. Lemmon V. J. Neurosci. 1998; 18: 5311-5321Crossref PubMed Google Scholar). Mock-transfected 3T3 cells were generated using LipofectAMINE and an empty pcDNA3.1/neo/lacZ vector. Drug-resistant L1- and mock-transfected 3T3 cell populations were cultured in Dulbecco's modified Eagle's medium containing G418 (800 μg/ml) and 10% fetal calf serum and were routinely passaged to avoid overgrowth. The clonal melanoma cell line K1735-C11 was provided by Dr. Avi Raz (Wayne State University). To produce stable transfectants, the cells were transfected using LipofectAMINE Plus reagent (Invitrogen) and PvuI-linearized pcDNA3.1(zeo) vector encoding full-length human L1. Transfected cells were selected and maintained in Dulbecco's modified Eagle's medium containing Zeocin (800 μg/ml) and 10% fetal calf serum. L1-expressing cells were sorted by repeated fluorescence-activated cell sorter (FACS) analysis with an anti-L1 pAb. Transfected, non-expressing mock control cells were maintained under identical culture conditions and were used after an equal number of passages. For the transient transfection of L1, subconfluent 3T3 cells were incubated with LipofectAMINE Plus reagent and a pcDNA3.1 expression vector (Invitrogen) incorporating a Zeocin selection marker and the full-length L1 complementary DNA sequence. Empty vector was used for mock transfection. After 5 h, the cells were washed twice and replenished with fresh serum-containing media. Cells were lysed for Western blot analysis after an additional 72 h. L1-transfected 3T3 cells were also transiently transfected with a dominant-negative hemagglutinin-tagged Rac1 expression construct containing an asparagine mutation of Thr17 (Rac-N17) (24Minden A. Lin A. Claret F. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1447) Google Scholar). Control cells received empty vector, and all cells were cotransfected with a β-galactosidase reporter vector (pEF4-lacZ). Cells were transfected with LipofectAMINE Plus reagent as described above. After 48 h, cells were harvested and assessed for migration; 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) staining was used to identify transfected cells. Protein production by the RacN17 construct was verified by immunoblotting with an anti-hemagglutinin mAb (HA-7, Sigma). Migration Assays—Migration assays were performed with 24-well Transwell migration plates (Costar). The undersides of the insert membranes (8.0-μm pore size) were precoated overnight at 4 °C with matrix components diluted into phosphate-buffered saline including vitronectin (8 μg/ml), laminin-1 (40 μg/ml), native type I collagen (40 μg/ml), or denatured collagen, types I and IV (40 μg/ml). The collagens were denatured by heating at 100 °C for 30 min. Cells were harvested from cultures at 70–80% confluence using versene (1:5000, Invitrogen) and were added at 5 × 104 cells per upper chamber in fibroblast basal media (BioWhittaker) supplemented with 0.5% bovine serum albumin and 0.4 mm MnCl2 (pH 7.4). Cells that had migrated to the undersides of the insert membranes were stained with 1% Toluidine blue and were counted based upon the number of cells per 20× field using an inverted stereomicroscope. Eight fields per insert were scored, and treatments were performed in duplicate. For inhibition studies, anti-integrin antibodies were added to the upper and lower chambers at 40 μg/ml. Invasion Assays—Matrigel invasion assays were performed using BD BioCoat growth factor reduced Matrigel invasion chambers as recommended by the manufacturer (BD Biosciences). Cells were added at 5 × 104 cells per upper chamber in fibroblast basal media supplemented with 0.5% bovine serum albumin and 0.4 mm MnCl2. These cells were given fresh culture media 18 h prior to use and were harvested from cultures at 70–80% confluence using versene. Invasion of cells from upper to lower chambers was determined after 22–42 h and quantified by counting cells on the undersides of the insert membranes as described for migration assays. PDGF (50 ng/ml) was added to lower chambers as a chemoattractant. The contribution of cathepsins to invasion was assessed using peptides CA-064 Me (selective inhibitor of procathepsin-B) and E-64-d (inhibitor of cathepsins-L and -B) (Peptide International Inc, Louisville, KY). These peptides were added to upper and lower chambers at concentrations ranging from 1.5 to 25 μm. To optimize cathepsin activity, the invasion fibroblast basal medium was adjusted to pH 6.8. Flow Cytometry—Surface integrin expression was assessed by FACS analysis. Sub-confluent cultures were harvested using versene and stained with the following anti-integrin antibodies: anti-β1 (CD29,Ha2/5), anti-α1 (CD49a, Ha31/8), anti-α2 (CD49b, HMa2), anti-α6 (CD49f, GoH3), anti-β3 (CD61, 2C9.G2), and anti-αv (CD51, H9.2b8). Anti-α2, α6, and β3 antibodies were all conjugated directly to phycoerythrin. Phycoerythrin-conjugated non-binding isotype-matched antibodies were used as controls. Unlabelled anti-integrin antibodies to β1, α1, and αv were detected using a fluorescein isothiocyanate-labeled anti-Armenian hamster IgG secondary. Cells were stained in 70 μl of ice-cold FACS buffer (phosphate-buffered saline, 0.5% bovine serum albumin, 0.05% azide) and were analyzed using a FACScan flow cytometer (BD Biosciences). Conjugated and unconjugated anti-integrin antibodies were used at 10 μg/ml. Some L1-transfected 3T3 cells were treated with pharmacological inhibitors of ERK-1/2 (U0126, 25 μm), as indicated in the text. Mitogen-activated Protein Kinase Phosphorylation—Levels of ERK-1/2 phosphorylation were determined using lysates from mock- or L1-transfected 3T3 or K1735-C11 cells. To assess the effect of serum starvation, transfected 3T3 cells were seeded at 5 × 105 or 1 × 106 cells/well. After 24 h, the cells seeded at 1 × 106/well were cultured in 0.5% serum for 48 h followed by 1 h without serum. Cells at 5 × 105/well were maintained in 10% serum. Cells were extracted with a lysis buffer consisting of 1% Nonidet P-40, 150 mm NaCl, 50 mm Tris, pH 8.0, containing Complete protease inhibitor mixture (EDTA-free, Roche Applied Science) as well as 10 mm sodium orthovanadate and 1 mm sodium fluoride to prevent phosphatase activity. Lysates were clarified by centrifugation at 14,000 × g for 20 min, and equal quantities of protein were separated under reducing conditions by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and detected with an anti-phospho-ERK mAb (E10, 1:2000). Membranes were then stripped and re-probed to confirm equal loading using an anti-ERK2 pAb (C14, 1:100). Cathepsin Assays—Intracellular cathepsin activity was assessed using Magic Red™ cathepsin detection kits for cathepsin-L and -B (Immunochemistry Technologies, LLC). Mock- or L1-transfected 3T3 cells were grown to 90% confluence, harvested, and then incubated with Magic Red fluorogenic substrates specific for each cathepsin. The cells were incubated for 60 min according to the manufacturer's instructions (Immunochemistry Technologies, LLC). Because of the high levels of intracellular cathepsin-B activity in 3T3 cells, the fluorogenic substrate for cathepsin-B was diluted 1:780, which is below the manufacturer's recommendation of 1:260. Red fluorescence, generated as a result of intracellular enzymatic cleavage, was assessed by flow cytometry using a FACScan flow cytometer (BD Biosciences). To assess the contribution of the ERK-pathway to intracellular cathepsin activity, L1-transfected 3T3 cells were treated with U0126 (25 μm) or Me2SO vehicle alone for 72 h prior to harvesting. Reverse Transcription-PCR Analysis for Integrin β3 Expression— Mock- or L1-transfected 3T3 cells were grown to 90% confluence, and total RNA was extracted using TRIzol reagent following the manufacturer's recommendations (Invitrogen). Ten percent of the cDNA synthesized from 5 μg of total cellular RNA was utilized for PCR of both the integrin-β3 and β-actin gene products. For analysis of mouse integrin β3 expression, the following primer set was used: forward, 5′-GTGCTGACGCTAACCGACCAG-3′, reverse, 5-CATGGTAGTGGAGGCAGAGTAGTGG-3′. Actin primers were purchased from Stratagene. Amplification was performed for 35 cycles with an annealing temperature of 60 °C using the HotStarTaq Mastermix Kit (Qiagen). Equal volumes of products were separated by agarose electrophoresis and documented with a Bio-Rad imaging system. Gene Array Analysis—Total RNA was harvested from sub-confluent cultures of stable mock- and L1-expressing 3T3 or K1735-C11 cells according to the manufacturer's instructions for TRIzol reagent (Invitrogen). Biotinylated probes were then synthesized according to the manufacturer's instructions for the SuperArray mouse tumor metastasis Q10 kit (SuperArray, Inc., Bethesda, MD). Briefly, equal quantities (5 μg) of total RNA were hybridized with kit-specific primers, and reverse transcription was performed in the presence of biotin-16-dUTP. Labeled probe was denatured and then hybridized to the prehybridized membranes overnight at 60 °C. The next day, the membranes were washed and incubated with a SuperArray blocking solution. Membranes were blocked for 45 min prior to application of an horseradish peroxidase-labeled anti-biotin mAb (BN34, Sigma) in SuperArray wash buffer. The membranes were incubated for 30 min and washed vigorously with SuperArray wash buffer prior to rinsing with phosphate-buffered saline and chemiluminescent detection of bound probe with the horseradish peroxidase substrate PS-3 (Lumigen, Inc., Southfield, MI). Densitometric quantification of images was performed using both ScanAlyze and NIH Image software with similar results. L1 Co-operates with Serum or Serum-growth Factors to Induce Sustained ERK Activation—Levels of constitutive ERK activation were assessed in 3T3 or K1735-C11 cells stably transfected with full-length human L1. L1 levels achieved in these cell lines are shown in Fig. 1A and are within the range of endogenous L1 expression displayed by a variety of cell lines (6Montgomery A.M.P. Becker J.C. Siu C.-H. Lemmon V.P. Cheresh D.A. Pancook J.D. Zhao X. Reisfeld R.A. J. Cell Biol. 1996; 132: 475-485Crossref PubMed Scopus (207) Google Scholar, 25Nayeem N. Silletti S. Yang X.-M. Lemmon V.P. Reisfeld R.A. Stallcup W.B. Montgomery A.M.P. J. Cell Sci. 1999; 112: 4739-4749PubMed Google Scholar, 26Balaian L. Moelher T. Montgomery A.M.P. Eur. J. Immunol. 2000; 30: 938-943Crossref PubMed Scopus (14) Google Scholar, 27Primiano T. Baig M. Maliyekkkel A. Cahng B.D. Fellars S. Sadhu J. Axenovich S.A. Holzayer T.A. Roninson I.B. Cancer Cells. 2003; 4: 415-423Abstract Full Text Full Text PDF Scopus (1) Google Scholar). Levels of ERK activation were determined in mock- or L1-transfected cells cultured in growth media and harvested at equivalent cell densities. Significantly higher levels of ERK-1/2 phosphorylation were observed as a result of stable ectopic L1 expression in both 3T3 and K1735-C11 cells (Fig. 1B). To ensure that the differences in ERK activation observed are due to L1 expression, rather than a drift in phenotype between mock- and L1-transfected populations, it was further confirmed that transient transfection of full-length L1 cDNA into wild-type 3T3 cells also induces higher levels of ERK activation (Fig. 1C). The L1-mediated ERK activation observed was found to be critically dependent upon cell density. Thus, no evidence of L1-mediated ERK activation was observed when transfected 3T3 cells were seeded at a low cell density and were harvested as a dispersed or a sparse population (Fig. 1, D and E; 0.6 × 105/well). Differences in ERK activation were only evident at higher seeding densities (e.g. 2.5 × 105/well), which allowed for more contiguous monolayer formation and cell-cell contact (Fig. 1, D and E). After seeding at an optimal cell density, L1-dependent ERK activation was maintained for at least 24 h (Fig. 1F). After this time, the 3T3 cell cultures became excessively overgrown and ERK activation declined. The ability of L1 to support sustained elevated ERK activation was also found to be critically dependent upon the presence of serum. Accordingly, no difference in ERK activation was found when the mock- or L1-transfected 3T3 cells were maintained in the absence of serum (Fig. 2A). One explanation for this finding is that sustained L1-dependent ERK activation requires co-operation with one or more serum growth factors. Because platelet-derived growth factor (PDGF) is a major mitogenic factor in serum, it was further determined whether L1 specifically co-operates with this growth factor to induce heightened levels of ERK activation. Importantly, levels of ERK activation resulting from stimulation with PDGF were significantly higher in 3T3 cells expressing L1 (Fig. 2, B and C). Together these data indicate that L1 can support sustained ERK activation; such a sustained response seems to require both cell-cell interaction and growth factor co-operation. Ectopic L1 Expression and Sustained ERK Activation Induce Expression of Integrin αvβ3—A prior report (28Woods D. Cherwinski H. Venetsanakos E. Bhat A. Gysin S. Humbert M. Bray P.F. Saylor V.L. McMahon M. Mol. Cell. Biol. 2001; 21: 3192-3205Crossref PubMed Scopus (111) Google Scholar) has shown that sustained activation of the ERK pathway induces the expression of select integrins, including α5β1 and αvβ3. To determine whether L1 induces changes in integrin expression, we compared the integrin profiles of mock- or L1-transfected 3T3 cells. Cells were stained with antibodies to αv, β3, β1, α6, α5, α2, and α1 and were then analyzed by FACS analysis. A comparison of FACS histograms confirmed that stable expression of L1 in 3T3 cells is associated with a significant increase in the surface expression of αvβ3 (Fig. 3B). No change in the levels of the αv-subunit, α1β1, α2β1, or α6β1 was observed (Fig. 3, C–F), and only a modest increase in the expression of α5β1 and the β1-subunit was found (Fig. 3, G and H). Induction of αvβ3 expression was also confirmed in L1-transfected K1735 cells (Fig. 3I), and reverse transcription-PCR analysis, using primers specific for the murine β3-subunit, established that the increased surface expression of αvβ3 in the 3T3 cells is associated with a significant increase in message for the β3-subunit (Fig. 3J). Increased expression of the αvβ3-heterodimer (Fig. 3B), without a change in the overall levels of the αv-subunit (Fig. 3C), has been reported (28Woods D. Cherwinski H. Venetsanakos E. Bhat A. Gysin S. Humbert M. Bray P.F. Saylor V.L. McMahon M. Mol. Cell. Biol. 2001; 21: 3192-3205Crossref PubMed Scopus (111) Google Scholar) and is believed to occur at the expense of other αv-heterodimers such as αvβ5 (29Koistinen P. Heino J. J. Biol. Chem. 2002; 277: 24835-24841Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). To confirm that induction of β3 expression in our models is due to activation of the ERK pathway, L1-transfected 3T3 cells were treated with the inhibitor U0126 prior to FACS analysis. Consistent with a role for ERK, this inhibitor significantly decreased levels of β3 expression after 72 h (Fig. 3K). Long-term treatment with U0126 was not observed to impact cell viability (data not shown). The association between ERK activation and induction of β3 expression was further tested in 3T3 cells infected to express a ΔB-RAF:ER fusion protein (30McCarthy S.A. Samuels M.L. Pritchrad C.A. Abraham J.A. McMahon M. Genes and Dev. 1995; 9: 1953-1964Crossref PubMed Scopus (170) Google Scholar). The kinase activity of this fusion protein is selectively and conditionally activated by the addition of 4-hydroxytamoxifen (4HT) resulting in the selective and sustained activation of the ERK pathway within 10–15 min (30McCarthy S.A. Samuels M.L. Pritchrad C.A. Abraham J.A. McMahon M. Genes and Dev. 1995; 9: 1953-1964Crossref PubMed Scopus (170) Google Scholar, 31Samuels M.A. Weber M.J. Bishop M. McMahon M. Mol. Cell. Biol. 1993; 13: 6241-6252Crossref PubMed Scopus (323) Google Scholar). Treatment with 4HT and concomitant ERK activation in these 3T3 cells resulted in a marked increase in β3 expression (Fig. 3L), and this induction could be blocked using the ERK pathway inhibitor U0126 (Fig. 3M). The increased αvβ3 expression in this model was only evident after 12–18 h of treatment with 4HT (Fig. 3N). Based on these results, we propose that L1 induces αvβ3 expression by virtue of inducing a sustained ERK response. L1 Induces the Expression of Multiple ERK-regulated Gene Products—A cDNA microarray that allows the simultaneous detection of 96 genes intimately associated with motility and invasion (Mouse metastasis GE array Q series, SuperArray Inc.) was utilized to determine whether ectopic L1 expression induces more global changes in the expression of pro-invasive or pro-migratory gene products. A full listing of the genes on this array are available on the company web site. 2Available on the World Wide Web at www.superarray.com/cancer.php. L1-dependent gene expression was assessed by comparing mRNA samples derived from mock- or L1-transfected 3T3 or K1735-C11 cells cultured under identical conditions. Based upon the observation that L1 can induce sustained ERK activation, it was further determined if L1-induced genes are also expressed as a result of conditional ERK activation. ERK-regulated genes were identified by comparing mRNA samples derived from 3T3ΔRAF:ER cells treated with 4HT or vehicle alone. Fig. 4 lists multiple gene products that were significantly induced in 3T3 and K1735 cells both as a result of L1 expression and conditional ERK activation. Only those genes that were consistently induced in repeat experiments are listed. Notable examples include the cysteine proteases cathepsin-L and -B, the small GTPases RhoC and Rac-1, the matrix component osteopontin, and the adhesion receptor CD44 (Fig. 4). Despite the distinct histological origin of 3T3 and K1735 cell lines, six of the nine genes identified in the 3T3 model were also confirmed in the K1735 model (Fig. 4). Although
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