Hepatocyte Growth Factor-induced Ectodomain Shedding of Cell Adhesion Molecule L1
2004; Elsevier BV; Volume: 279; Issue: 30 Linguagem: Inglês
10.1074/jbc.m403587200
ISSN1083-351X
AutoresMonika Heiz, Jürgen Grünberg, P. August Schubiger, Ilse Novak‐Hofer,
Tópico(s)Axon Guidance and Neuronal Signaling
ResumoThe L1 cell adhesion molecule and its soluble form are tumor-associated proteins and potential markers for tumor staging as well as targets for therapeutic intervention. Soluble L1 is produced by metalloprotease-mediated ectodomain shedding of L1. We investigated effects of hepatocyte growth factor (HGF), a growth factor shown to increase invasiveness of renal carcinoma cells, on ectodomain shedding of L1 from these cells. All of the tested L1-positive renal carcinoma cell lines released a 180-kDa form of L1 into the medium. In the presence of serum, addition of HGF led to a dose-dependent increase in L1 shedding with a maximum reached at 5 ng/ml. In contrast, L1 shedding was inhibited by glial cell line-derived neurotrophic factor (GDNF). The tyrosine kinase inhibitor Genistein reduced basal and HGF-stimulated L1 shedding, indicating that protein phosphorylation is involved. To investigate the role of the L1 intracellular domain, two mutants of the L1 cytoplasmic part were constructed. L1trun lacking the complete intracellular domain showed enhanced basal shedding. In a L1YH mutant, containing the mutation tyrosine 1229 to histidine that deletes the ankyrin binding motif of L1, basal shedding was reduced. Disruption of actin assembly by cytochalasin D also reduced shedding of L1. These results indicate that the cytoplasmic domain regulates basal shedding of L1, and association with the cytoskeleton through the L1 ankyrin binding site is involved. HGF stimulated L1 shedding in both mutants, indicating that receptor-mediated phosphorylation in the L1 cytoplasmic domain is not required for HGF-stimulated shedding. The L1 cell adhesion molecule and its soluble form are tumor-associated proteins and potential markers for tumor staging as well as targets for therapeutic intervention. Soluble L1 is produced by metalloprotease-mediated ectodomain shedding of L1. We investigated effects of hepatocyte growth factor (HGF), a growth factor shown to increase invasiveness of renal carcinoma cells, on ectodomain shedding of L1 from these cells. All of the tested L1-positive renal carcinoma cell lines released a 180-kDa form of L1 into the medium. In the presence of serum, addition of HGF led to a dose-dependent increase in L1 shedding with a maximum reached at 5 ng/ml. In contrast, L1 shedding was inhibited by glial cell line-derived neurotrophic factor (GDNF). The tyrosine kinase inhibitor Genistein reduced basal and HGF-stimulated L1 shedding, indicating that protein phosphorylation is involved. To investigate the role of the L1 intracellular domain, two mutants of the L1 cytoplasmic part were constructed. L1trun lacking the complete intracellular domain showed enhanced basal shedding. In a L1YH mutant, containing the mutation tyrosine 1229 to histidine that deletes the ankyrin binding motif of L1, basal shedding was reduced. Disruption of actin assembly by cytochalasin D also reduced shedding of L1. These results indicate that the cytoplasmic domain regulates basal shedding of L1, and association with the cytoskeleton through the L1 ankyrin binding site is involved. HGF stimulated L1 shedding in both mutants, indicating that receptor-mediated phosphorylation in the L1 cytoplasmic domain is not required for HGF-stimulated shedding. The L1 cell adhesion molecule (L1) 1The abbreviations used are: L1, L1 cell adhesion molecule; HGF, hepatocyte growth factor; GDNF, glial cell line-derived neurotrophic factor; HEK, human embryonic kidney; PBS, phosphate-buffered saline; FCS, fetal calf serum; BSA, bovine serum albumin; mAb, monoclonal antibody. was originally described as a protein of the nervous system, and mutated forms of L1 are linked to severe neurological pathologies referred to as MASA syndrome (1Kenwrick S. Watkins A. De Angelis E. Hum. Mol. Genet. 2000; 9: 879-886Google Scholar). A non-neuronal isoform of L1 lacking exons 2 and 27 is expressed at low levels in the normal adult human kidney (2Meli M.L. Carrel F. Waibel R. Amstutz H. Crompton N. Jaussi R. Moch H. Schubiger P.A. Novak-Hofer I. Int. J. Cancer. 1999; 83: 401-408Google Scholar) as well as during renal development (3Debiec H. Christensen E.I. Ronco P.M. J. Cell Biol. 1998; 143: 2067-2079Google Scholar). In contrast, high expression of L1 was found in 8 of 12 renal tumor cell lines and in some renal tumors (2Meli M.L. Carrel F. Waibel R. Amstutz H. Crompton N. Jaussi R. Moch H. Schubiger P.A. Novak-Hofer I. Int. J. Cancer. 1999; 83: 401-408Google Scholar). 2I. Novak-Hofer, M. Heiz, J. Grünberg, and G. Thalman, unpublished data. It was shown that the cell-bound form of L1 can serve as a target for radioimmunodiagnosis of metastatic neuroblastoma (4Hoefnagel C.A. Rutgers M. Buitenhuis C.K. Smets L.A. de Kraker J. Meli M. Carrel F. Amstutz H. Schubiger P.A. Novak-Hofer I. Eur. J. Nucl. Med. 2001; 28: 359-368Google Scholar), and L1 may also be a marker and therapeutic target for certain renal cancers. In cultured cell lines originating from a number of different tumors the L1 ectodomain was found to be cleaved proximal to the cell membrane by metalloproteases, followed by release of soluble L1 into the medium (5Beer S. Oleszewski M. Gutwein P. Geiger C. Altevogt P. J. Cell Sci. 1999; 112: 2667-2675Google Scholar, 6Gutwein P. Oleszewski M. Mechtersheimer S. Agmon-Levin N. Krauss K. Altevogt P. J. Biol. Chem. 2000; 275: 15490-15497Google Scholar, 7Kalus I. Schnegelsberg B. Seidah N.G. Kleene R. Schachner M. J. Biol. Chem. 2003; 278: 10381-10388Google Scholar). Ectodomain shedding of L1 could be stimulated by phorbol esters and vanadate, indicating that phosphorylation events regulate L1 release (6Gutwein P. Oleszewski M. Mechtersheimer S. Agmon-Levin N. Krauss K. Altevogt P. J. Biol. Chem. 2000; 275: 15490-15497Google Scholar). In cell culture soluble L1 substrate was found to stimulate cellular motility and migration (8Mechtersheimer S. Gutwein P. Agmon-Levin N. Stoeck A. Oleszewski M. Riedle S. Postina R. Fahrenholz F. Fogel M. Lemmon V. Altevogt P. J. Cell Biol. 2001; 155: 661-673Google Scholar). Release of soluble L1 is not restricted to cells in culture as soluble L1 was recently detected in serum samples of patients with ovarian and uterine tumors and is believed to lead to increased cell migration and metastatic spread in these malignancies (9Fogel M. Gutwein P. Mechtersheimer S. Riedle S. Stoeck A. Smirnov A. Edler L. Ben-Arie A. Huszar M. Altevogt P. Lancet. 2003; 362: 869-875Google Scholar). Here we investigated whether hepatocyte growth factor (HGF) and glial cell line-derived neurotrophic factor (GDNF) influence shedding of L1 from renal carcinoma cells. Changes in the normal expression patterns of HGF and GDNF are associated with abnormal proliferation in the human renal system. While in the normal human kidney, HGF and c-Met are expressed at very low levels, increased HGF and c-Met expression is found in about 90% of renal cell carcinomas (10Natali P.G. Prat M. Nicotra M.R. Bigotti A. Olivero M. Comoglio P.M. Di Renzo M.F. Int. J. Cancer. 1996; 69: 212-217Google Scholar, 11Horie S. Aruga S. Kawamata H. Okui N. Kakizoe T. Kitamura T. J. Urol. 1999; 161: 990-997Google Scholar, 12Oh R.R. Park J.Y. Lee J.H. Shin M.S. Kim H.S. Lee S.K. Kim Y.S. Lee S.H. Lee S.N. Yang Y.M. Yoo N.J. Lee J.Y. Park W.S. Apmis. 2002; 110: 229-238Google Scholar), and overexpression of HGF/c-Met enhances cellular motility of renal carcinoma cells in vitro and in vivo (13Nakamura T. Kanda S. Yamamoto K. Kohno T. Maeda K. Matsuyama T. Kanetake H. Oncogene. 2001; 20: 7610-7623Google Scholar, 14Miyata Y. Ashida S. Nakamura T. Mochizuki Y. Koga S. Kanetake H. Shuin T. Kanda S. Biochem. Biophys. Res. Commun. 2003; 302: 892-897Google Scholar). GDNF is overexpressed in renal dysplasia (15El-Ghoneimi A. Berrebi D. Levacher B. Nepote V. Infante M. Paris R. Simonneau M. Aigrain Y. Peuchmaur M. J. Urol. 2002; 168: 2624-2628Google Scholar) and in collecting duct cysts (16Lee D.C. Chan K.W. Chan S.Y. Oncogene. 2002; 21: 5582-5592Google Scholar). During kidney organogenesis, HGF and GDNF, as well as L1 are important for ureteric bud outgrowth and branching morphogenesis (17Davies J. J. Anat. 2001; 198: 257-264Google Scholar). In vivo, L1 knock-out mice show improper growth and branching patterns of ureteral branches (18Debiec H. Kutsche M. Schachner M. Ronco P. Nephrol. Dial. Transplant. 2002; 17: 42-44Google Scholar), and blocking of HGF function inhibits proliferation and branching in kidney organ cultures (17Davies J. J. Anat. 2001; 198: 257-264Google Scholar). Effects of growth factors on L1 could be mediated by direct interaction of receptors with the L1 extracellular domain. This is suggested by the findings that L1-mediated cell adhesion activates the fibroblast growth factor (FGF) receptor (19Saffell J.L. Williams E.J. Mason I.J. Walsh F.S. Doherty P. Neuron. 1997; 18: 231-242Google Scholar) and the epidermal growth factor (EGF) receptor (20Islam R. Kristiansen L.V. Romani S. Garcia-Alonso L. Hortsch M. Mol. Biol. Cell. 2004; 15: 2003-2012Google Scholar) tyrosine phosphorylation, respectively, the latter an effect which appears to involve L1/EGF receptor trans interactions (20Islam R. Kristiansen L.V. Romani S. Garcia-Alonso L. Hortsch M. Mol. Biol. Cell. 2004; 15: 2003-2012Google Scholar). On the other hand, the cytoplasmic domain of L1 contains several phosphorylation sites and it was shown before that phosphorylation events induced by growth factors regulate the interaction of the L1 family member neurofascin with the ankyrin/actin network and L1 lateral movement (21Kamiguchi H. Lemmon V. Curr. Opin. Cell Biol. 2000; 12: 598-605Google Scholar, 22Garver T.D. Ren Q. Tuvia S. Bennett V. J. Cell Biol. 1997; 137: 703-714Google Scholar, 23Gil O.D. Sakurai T. Bradley A.E. Fink M.Y. Cassella M.R. Kuo J.A. Felsenfeld D.P. J. Cell Biol. 2003; 162: 719-730Google Scholar). We show here that shedding of L1 is a regulated process, triggered by the physiological factor HGF, which is overexpressed in renal carcinomas. When we investigated the effect of mutations in the intracellular domain of L1 on basal and HGF-stimulated release of soluble L1, we found that the cytoplasmic domain of L1 and its interaction with the actin cytoskeleton regulate basal, but not HGF-stimulated L1 shedding. Cell Culture—The human embryonic kidney cell line HEK293 and the human renal carcinoma cell lines A-498 and Caki-2 were obtained from the German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany. The human renal carcinoma cell line Foehn was obtained from the Institute of Pathology, University of Zurich, Switzerland. KTCTL-2 and KTCTL-53 were from the German Center for Tumor Research, Heidelberg, Germany. The cell lines RCC-W1, RCC-W2, RCC-GW, RCC-FG2, and RCC-MF were obtained from the Cell Lines Service and Cellbank in Eppelheim, Germany. The human embryonic kidney cell line 293T, originally referred to as 293tsA1609neo (24Pear W.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Google Scholar, 25DuBridge R.B. Tang P. Hsia H.C. Leong P.M. Miller J.H. Calos M.P. Mol. Cell. Biol. 1987; 7: 379-387Google Scholar), was kindly provided by Prof. K. Ballmer, PSI, Villigen, Switzerland. This cell line is a derivative of HEK293 cells into which the gene for the temperature-sensitive SV40 T-antigen mutant tsA1609 has been inserted, which leads to a large production of replication-competent T antigen at 37 °C (25DuBridge R.B. Tang P. Hsia H.C. Leong P.M. Miller J.H. Calos M.P. Mol. Cell. Biol. 1987; 7: 379-387Google Scholar). HEK293 and 293T cells were maintained in Dulbecco's modified Eagle's medium, Foehn, KTCTL-2, and KTCTL-53 cells in RPMI medium, A498 cells in MEM containing 1 mm sodium pyruvate, and all other cell lines in McCoy medium at 37 °C and 7.5% CO2. All media were supplemented with 10% fetal calf serum, 2 mm glutamine, and antibiotics (100 units/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml fungizone). All media and additives were obtained from BioConcept (Allschwil, Switzerland). Reagents and Antibodies—Human recombinant HGF and GDNF and cytochalasin D were obtained from Sigma. Genistein was purchased from Calbiochem (Juro, Lucerne, Switzerland). The anti-L1 antibody chCE7 directed against the ectodomain of L1 was constructed, expressed, and purified as previously described (26Amstutz H. Rytz C. Novak-Hofer I. Spycher M. Schubiger P.A. Blaser K. Morgenthaler J.J. Int. J. Cancer. 1993; 53: 147-152Google Scholar, 27Grunberg J. Knogler K. Waibel R. Novak-Hofer I. BioTechniques. 2003; 34: 968-972Google Scholar). The anti-L1 antibody UJ127.11 directed against the ectodomain of L1 was purchased from Neo Markers (P. H. Stehelin, Basel, Switzerland). The anti-L1 antibody 74–5H7 directed against the cytoplasmic domain of L1 was from CRP Inc. (Eurogentech, Seraing, Belgium). The anti-c-Met antibody C-12 and the anti-c-Ret antibody C-19 were obtained from Santa Cruz Biotechnology (LabForce, Nunningen, Switzerland). The anti-mouse IgG-horseradish peroxidase antibody conjugate W402B was from Promega (Catalys, Wallisellen, Switzerland). The anti-rabbit IgG-horseradish peroxidase and anti-goat IgG-horseradish peroxidase antibody conjugates were from Santa Cruz Biotechnology (LabForce). Protein G-Sepharose 4 FF was obtained from Amersham Biosciences. All other chemicals were purchased from Fluka (Buchs, Switzerland). Cell Lysis and Immunoprecipitation—Cells were washed twice with PBS and detached with 1 mm EDTA in PBS. After centrifugation for 5 min at 500 × g, cells were lysed in 50 mm Tris-HCl, pH 7.5, 50 mm NaCl, 1% Nonidet P-40 (lysis buffer) containing Complete™ Protease Inhibitor Mixture (Roche Diagnostics) and incubated on ice for 10 min. Cell lysates were centrifuged for 10 min at 16,000 × g at 4 °C, and the protein concentration of the supernatant was measured using the DC Protein Assay from Bio-Rad. For immunoprecipitation of L1 and c-Met, 500 μg of protein in 300 μl of lysis buffer were precleared with 20 μl of protein-G-Sepharose suspension (10% in lysis buffer, PGS) for 30 min at 4 °C. After preclearing, lysates were incubated with 20 μl of PGS and the corresponding antibody (2 μg of chCE7 for L1, 1 μg of C-12 for c-Met) for 2 h at 4 °C. Protein G-Sepharose beads were washed twice with lysis buffer and once with Tris-HCl, pH 7.5 and analyzed by Western blotting. For analysis of soluble L1 in the medium, cell supernatant was precleared with 50 μl of PGS for 30 min at 4 °C. Immunoprecipitation of soluble L1 was performed overnight with 6 μg of chCE7 and 50 μl of PGS at 4 °C. Samples were centrifuged for 5 min at 1700 × g and 4 °C, Protein-G-Sepharose beads were washed as described and analyzed by immunoblotting using mAb UJ127.11. Separation of Triton X-100-soluble and -insoluble Membrane Fraction—Isolation of Triton X-100-insoluble membrane fractions was performed as described in Ref. 28Dihne M. Bernreuther C. Sibbe M. Paulus W. Schachner M. J. Neurosci. 2003; 23: 6638-6650Google Scholar with modifications. Foehn cells on 15-cm plates were detached with 1 mm EDTA in PBS, and cells were centrifuged at 500 × g for 5 min at 4 °C. Cells were homogenized on ice in TNE buffer (25 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm EDTA) including Complete™ Protease Inhibitor Mixture using a Dounce homogenizer. Homogenates were centrifuged at 200 × g and 4 °C for 5 min. The supernatants were centrifuged at 100,000 × g for 1 h at 4 °C. The pellets were resuspended in 200 μl of TNE buffer, pH 11, containing 1% Triton X-100 and incubated on ice for 30 min followed by centrifugation at 100,000 × g for 1 h at 4 °C. The Triton X-100-insoluble material was dissolved in 50 μl of TNE buffer containing 1% SDS, and 450 μl of TNE buffer was added. For analysis of L1 and c-Met distribution, both supernatant and dissolved pellet were subjected to immunoprecipitation and Western blotting. For analysis of L1 distribution after HGF stimulation, Foehn cell cultures with a confluency of 80% were serum-starved overnight. Cells were stimulated for 30 min with starve-medium containing 10 ng/ml HGF or 10% FCS as control, and analysis of Triton X-100-soluble and -insoluble membrane fractions was performed as described above. Western Blot Analysis—Protein samples or immunoprecipitates were mixed with sample buffer and boiled for 5 min. Samples were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions on 7.5% gels, except for detection with mAb chCE7 where non-reducing conditions are required. The proteins were transferred to polyvinylidene difluoride membranes (Millipore) by semi-dry electroblotting, and the membranes were blocked for 1 h at room temperature with 1% bovine serum albumin (BSA) in Tris-buffered saline (TBST, 20 mm Tris-HCl, 500 mm NaCl, 0.05% Tween-20, pH 7.5). The membranes were incubated with the respective primary antibody in 1% BSA in TBST for 1 h at room temperature or overnight at 4 °C, washed three times with TBST for 15 min, and probed with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:20,000 in 1% BSA in TBST) for 30 min at room temperature. After three washes with TBST for 15 min, immunodetection was performed using the enhanced chemiluminescence kit (Pierce). Primary antibodies were used in a 1:1000 (chCE7), 1:100 (UJ127.11), 1:200 (74–5H7, c-Met, c-Ret) dilution. L1 Cell Surface Expression—Cell surface expression of L1 in transfected 293T cells was determined by measuring binding sites for mAb chCE7. mAb chCE7 was labeled with 125I as described previously (2Meli M.L. Carrel F. Waibel R. Amstutz H. Crompton N. Jaussi R. Moch H. Schubiger P.A. Novak-Hofer I. Int. J. Cancer. 1999; 83: 401-408Google Scholar). 24 h after transfection of 293T cells, medium was changed, and cells were incubated for 3 h. 0.5 × 106 cells/ml were incubated in a total volume of 0.5 ml of 0.5% BSA in PBS, 100,000 cpm of 125I-chCE7 and increasing concentrations of unlabeled chCE7 (75–1200 ng) overnight at 4 °C on a shaking platform. Cells were washed twice with 0.5% BSA in PBS, and cell-associated radioactivity was counted in a γ-counter. Saturation curves were analyzed by the Scatchard method. Proliferation Assay—RCC-GW cells were seeded in triplicate at 50,000 cells per well on 6-well plates in 2 ml of serum-free medium. Either HGF (20 ng/ml) or GDNF (10 ng/ml) were added, and cells were incubated for the indicated time points. Cells were removed with 0.05% trypsin, 0.5 mm EDTA in PBS and counted in a hemocytometer. To evaluate statistical significance of the obtained data, two-tailed Student's t test was performed. Scattering Assay—100,000 RCC-GW cells per well were plated on 6-well plates either in full medium alone or supplemented with 20 ng/ml HGF and 10 ng/ml GDNF, respectively. Cells were incubated for 48–96 h, and scattering was documented using a microscope with digital equipment. Analysis of L1 Shedding—For analysis of L1 release from carcinoma cell lines, the indicated cells were seeded on 10-cm plates and incubated for 5–17 h. Medium was changed and eventually supplemented with either HGF at the indicated concentrations, 50 ng/ml GDNF or 100 μm Genistein, respectively. Medium was collected at the indicated time points and passed to analysis. Cloning of L1 Mutants—The cDNA for human full-length L1 (a gift from Dr. V. Lemmon, University of Miami, FL) was cloned into the EcoRI/XhoI restriction site of pcDNA3.1+ (Invitrogen, Basel, Switzerland). All mutations were introduced in the cDNA of L1 by high fidelity PCR and standard molecular biology techniques (29Sambrook J. Russel D. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001Google Scholar). All PCR products started shortly upstream of the SacI restriction site of the L1 cDNA and ended with a XhoI restriction site after the stop codon of the C terminus or after the stop codon introduced behind Ser1147 for the L1trun mutant, respectively. The point mutation Tyr1229 to His was created by overlap extension. The SacI/XhoI DNA fragment of the L1wt was replaced by mutated SacI/XhoI fragments. All mutations were confirmed by DNA sequencing (Microsynth, Balgach, Switzerland). The sequences of oligonucleotides used for mutagenesis in this study are available on request. Transient DNA Transfection and Analysis of L1 Shedding—Transient DNA transfection was performed as described before (27Grunberg J. Knogler K. Waibel R. Novak-Hofer I. BioTechniques. 2003; 34: 968-972Google Scholar). 293T cells were seeded on 10-cm plates and incubated for 7 h. Transfection was performed using the calcium phosphate method. In brief, 5 μg of cDNA was dissolved in 400 μl of H2O and 61 μl of 2 m CaCl2 were added. The DNA mixture was dropped into 500 μlof2× HBS (HEPES-buffered saline: 50 mm HEPES pH 7.1, 280 mm NaCl, 1.5 mm Na2HPO4), and the calcium phosphate DNA precipitate was allowed to stand at room temperature for 20 min. 10 ml of fresh complete medium was added to the cells before the transfection mixture was added, and cells were incubated overnight. Cells were washed twice with PBS, split into four 6-cm plates and incubated for 24 h. 2 ml of fresh medium was added and either supplemented with 10 ng/ml HGF or 2 μm cytochalasin D, respectively. Cells were incubated for 3 h and medium was collected and filtered through a sterile filter. Immunoprecipitation and analysis of soluble L1 was performed as described before. Expression of L1, c-Met, and c-Ret in Human Renal Carcinoma Cells—To find suitable renal carcinoma cell lines to study the effects of HGF on production of soluble L1, a number of human renal carcinoma cell lines and human embryonic kidney cells were screened for expression of L1, using Western blot analysis with mAb chCE7. Of the 11 tested cell lines 7 were found to express L1, some of them to a high extent (Fig. 1A). In some cell lines, the 140-kDa cleavage product of L1 was found to be associated with full-length L1, as described before by Kalus et al. (7Kalus I. Schnegelsberg B. Seidah N.G. Kleene R. Schachner M. J. Biol. Chem. 2003; 278: 10381-10388Google Scholar). Renal carcinoma cell lines and embryonic kidney cells (HEK293 and 293T) were then screened by Western blotting for the presence of c-Met and c-Ret to find L1-positive cells which also respond to HGF and GDNF. The extent of c-Met- and c-Ret expression varied widely in different renal carcinoma cell lines (Fig. 1, B and C). In some cells (Fig. 1B, lanes 3, 4, 7, and 8) the 170 kDa proform of c-Met (c-Met pro) could be detected in addition to the 145-kDa β-subunit of mature c-Met (c-Met β), indicating strong overexpression of c-Met in these cell lines. As L1 is known to associate with cytoskeletal proteins through its intracellular domain, we investigated the distribution of L1 between the Triton X-100 soluble and insoluble membrane fractions in Foehn renal carcinoma cells. Immunoprecipitation showed that most of L1 partitions in the Triton X-100-soluble membrane fraction (S) and a smaller amount is present in the insoluble fraction (P), which includes cytoskeletal proteins and lipid rafts (Fig. 1D, lanes 1 and 2). No significant changes in this distribution pattern of L1 could be observed after stimulation of serum-starved Foehn cells with 10 ng/ml HGF for 30 min (Fig. 1D, lanes 3 and 4). Similar to L1, expression of the β-subunit of c-Met in Foehn cells was found to be stronger in the Triton X-100 soluble membrane fraction (S) than in the insoluble fraction (P) whereas the proform of c-Met was shown to be expressed to a similar extent in the soluble and insoluble membrane fraction (Fig. 1E). Effects of HGF and GDNF on Proliferation and Scattering of RCC-GW Cells—In order to test cellular responses to HGF and GDNF, the effect of these factors on proliferation and scattering of the L1- and c-Met/c-Ret receptor-positive cell line RCC-GW was investigated. It is known that serum can mask the proliferative effect of HGF (30Cantley L.G. Barros E.J. Gandhi M. Rauchman M. Nigam S.K. Am. J. Physiol. 1994; 267: F271-F280Google Scholar), therefore cell growth was measured in three independent experiments over a time period of 96 h in serum free medium alone or in the presence of 20 ng/ml HGF and 10 ng/ml GDNF, respectively. Fig. 2A shows in a typical experiment that both factors led to a significant increase in cell number, however the effect of GDNF appeared at later times than the effect of HGF. HGF is also known as "scatter factor," and addition of 20 ng/ml HGF to complete medium led to a strong scattering effect and elongation of RCC-GW cells within 24 h (Fig. 2, B, panel 2), whereas 10 ng/ml GDNF showed no scattering over a time period of 96 h (Fig. 2, B, panel 3). These results show that RCC-GW cells do not only express the HGF and GDNF receptors, but also respond to these growth factors. Influence of Growth Factors on L1 Shedding from Renal Carcinoma Cells—L1 is known to be released from the cell surface of various tumor cell lines due to membrane proximal cleavage by metalloproteases (5Beer S. Oleszewski M. Gutwein P. Geiger C. Altevogt P. J. Cell Sci. 1999; 112: 2667-2675Google Scholar, 7Kalus I. Schnegelsberg B. Seidah N.G. Kleene R. Schachner M. J. Biol. Chem. 2003; 278: 10381-10388Google Scholar, 31Montgomery A.M. 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: 475-485Google Scholar). To analyze shedding of L1 from the cell surface of renal tumor cells, immunoprecipitation of soluble L1 from cell culture medium with mAb chCE7 followed by immunoblotting with mAb UJ127.11 was performed. RCC-GW cells expressing full-length L1 (200 kDa) were used as a control (Fig. 3, A and B, lane 1). All of the tested cell lines released a 180-kDa form of L1 into the medium (sL1) and the amount of soluble L1 correlated with the extent of L1 expression (Fig. 3A). Shedding of L1 was also observed in the L1-expressing neuroblastoma cell line SK-N-AS. A time course of L1 release from RCC-GW cells showed that when the cells were supplied with fresh culture medium, soluble L1 was faintly detectable in the medium after 1h and shedding increased over 48 h (Fig. 3B). No concomitant decrease in L1 expression on the cell surface was seen over the whole time period (data not shown). To evaluate effects of HGF and GDNF on L1 shedding, RCC-GW cells were stimulated by 100 ng/ml HGF or 50 ng/ml GDNF for 24 h. Addition of HGF to complete medium including 10% FCS strongly stimulated the release of soluble L1 into the medium (Fig. 3C), accompanied by a decrease of cell surface expression of L1 (data not shown). Densitometric analysis of the intensities of the 180-kDa Western blot bands using Aida© software (Raytest GmbH, Straubenhardt, Germany) revealed a 2-fold increase of L1 shedding after HGF stimulation compared with basal levels (n = 3, av = 1.96, S.D. = 0.23, p = 0.02). When GDNF (50 ng/ml) was added to complete medium inhibition of L1 shedding was observed (Fig. 3C). Increasing concentrations of HGF in a range from 1 to 50 ng/ml led to a dose-dependent increase of L1 shedding, with a maximum reached at 5 ng/ml (Fig. 3D), as confirmed by three independent experiments. Basal shedding of L1 was found to be similar in serum free and complete medium (Fig. 3E, lanes 1 and 2), while no stimulation of L1 shedding by HGF in serum-free medium was observed (Fig. 3E, lanes 3 and 4), indicating that components present in the serum are involved in HGF-stimulated shedding of L1 but not in basal L1 release. These results demonstrate, that L1 is shed from the cell surface of renal carcinoma cells and that release of L1 is stimulated by physiological concentrations of HGF but not by GDNF. In order to investigate if shedding of L1 is mediated by tyrosine phosphorylation, we analyzed basal and HGF-stimulated L1 shedding from Foehn cells in the presence of the broad range tyrosine kinase inhibitor Genistein. We found that both basal and HGF-stimulated shedding of L1 was suppressed after addition of 100 μm Genistein for 3 h (Fig. 3F). The inhibition of L1 shedding was not due to a toxic effect on the cells, because cell viability measured by the trypan blue assay after 3 h of incubation with Genistein was found to be similar to that in control cells (85%). These results indicate that protein phosphorylation regulates the release of soluble L1 from the cell surface. Influence of Mutations in the L1 Cytoplasmic Domain on L1 Shedding—We constructed L1 mutants and transiently expressed them in 293T cells (Fig. 4) to address the role of the intracellular domain in L1 shedding. In contrast to HEK293 cells, these cells express replication-competent T antigen, which leads to high expression of transfected L1 mutants and high amounts of soluble L1 in the medium. A first mutant was cloned missing all but 3 amino acids (KRS) of the cytoplasmic domain (L1trun). A second mutant with an exchange of tyrosine 1229 to histidine (L1YH) was generated. This mutation known from the MASA syndrome abolishes the tyrosine phosphorylation-mediated intracellular binding of L1 to ankyrin (32Needham L.K. Thelen K. Maness P.F. J. Neurosci. 2001; 21: 1490-1500Google Scholar) and enhances lateral motility of L1 (23Gil O.D. Sakurai T. Bradley A.E. Fink M.Y. Cassella M.R. Kuo J.A. Felsenfeld D.P. J. Cell Biol. 2003; 162: 719-730Google Scholar). All L1 mutant proteins were expressed in equal amounts in 293T cells as verified by immunoblotting of cell lysates with mAb UJ127.11 24 h after transfection (Fig. 5A, L1ecto). Analysis of 293T cell lysates with mAb 74–5H7 directed against the cytoplasmic part of L1 revealed strong reactivity with full-length L1 (L1wt) and L1YH (Fig 5A, L1cyto). As expected, no reactivity with L1 lacking the whole intracellular domain (L1trun) was detectable. No endogenous L1 expression was detectable in 293T cells transfected with the vector pcDNA3.1+ (Fig. 5A, lane 1). To
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