Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M404075200 on May 5, 2004

J. Biol. Chem., Vol. 279, Issue 28, 28880-28888, July 9, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/28/28880    most recent
M404075200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Silletti, S.
Right arrow Articles by Montgomery, A. M. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Silletti, S.
Right arrow Articles by Montgomery, A. M. P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Extracellular Signal-regulated Kinase (ERK)-dependent Gene Expression Contributes to L1 Cell Adhesion Molecule-dependent Motility and Invasion*

Steve Silletti{ddagger}, Mayra Yebra§, Brandon Perez§, Vincenzo Cirulli§, Martin McMahon¶, and Anthony M. P. Montgomery§||

From the §Department of Pediatrics, The Whittier Institute, and {ddagger}Moores Comprehensive Cancer Center, University of California at San Diego, La Jolla, California 92037 and Mount Zion Comprehensive Cancer Center, University of California, San Francisco, California 94143

Received for publication, April 13, 2004 , and in revised form, May 5, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {beta}3-integrin subunit, small GTPases, and the cysteine proteases cathepsin-L and -B. Induction of integrin {alpha}v{beta}3 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (1, 2). L1 was first described as a neural cell adhesion molecule based upon a restricted distribution that included post-mitotic neurons (3). 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 (4–10).

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 (3, 11–13). Function-blocking antibodies to L1 have been shown to abrogate the migration of granule precursors in cerebellar explant cultures (3, 14), and the migration and positioning of dopaminergic neuronal precursors is disrupted in L1-deficient mice (13). Additional studies have linked L1 to motile processes involved in tumor cell extravasation and glioma dissemination in the brain (15, 16). Several recent reports have also now linked L1 expression to melanoma and prostate metastasis (17–20). For example, Thies et al. (18) 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 (11). However, L1 is also now known to have a significant impact upon cell-matrix interactions required for haptotaxis and matrix remodeling (9, 21, 22). Two recent reports (21, 22) have shown that ectopic L1 expression strongly potentiates {alpha}5{beta}1-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 (22) or involve L1-shedding and autocrinal binding via {alpha}v{beta}5 (21). 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 (21, 22).

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)1 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Anti-mouse integrin antibodies to {beta}1 (Ha2/5), {alpha}1 (Ha31/8), {alpha}2 (HMa2), {alpha}5 (5H10–27), {alpha}6 (GoH3), {beta}3 (2C9.G2), and {alpha}v (H9.2b8) were purchased from BD Biosciences. A goat polyclonal antibody (pAb) to {alpha}5{beta}1 (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 (23). 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) (24). Control cells received empty vector, and all cells were cotransfected with a {beta}-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-{beta}-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 x 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 20x 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 x 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-{beta}1 (CD29,Ha2/5), anti-{alpha}1 (CD49a, Ha31/8), anti-{alpha}2 (CD49b, HMa2), anti-{alpha}6 (CD49f, GoH3), anti-{beta}3 (CD61, 2C9.G2), and anti-{alpha}v (CD51, H9.2b8). Anti-{alpha}2, {alpha}6, and {beta}3 antibodies were all conjugated directly to phycoerythrin. Phycoerythrin-conjugated non-binding isotype-matched antibodies were used as controls. Unlabelled anti-integrin antibodies to {beta}1, {alpha}1, and {alpha}v 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 x 105 or 1 x 106 cells/well. After 24 h, the cells seeded at 1 x 106/well were cultured in 0.5% serum for 48 h followed by 1 h without serum. Cells at 5 x 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 x 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 RedTM 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 {beta}3 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-{beta}3 and {beta}-actin gene products. For analysis of mouse integrin {beta}3 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 (6, 25–27). 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).



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 1.
L1 induces sustained ERK activation. A, FACS analysis showing L1 expression by mock- or L1-transfected 3T3 or K1735-C11 cells. Cells were stained using an anti-L1 pAb (L1-ECD) or control rabbit immunoglobulin. B, levels of activated ERK in stable mock- or L1-transfected 3T3 or K1735-C11 cells. Cells were grown in the presence of serum and were harvested at 80% confluence. C, levels of activated ERK in wild-type 3T3 cells transiently transfected with full-length L1 cDNA or empty vector (Mock). The cells were harvested after 72 h, and L1 expression was confirmed by using antibody UJ127. D, mock- or L1-transfected 3T3 cells were plated in six-well plates at 0.6 x 105 or 2.5 x 105 cells/well and were grown for 48 h prior to the addition of fresh growth media for 1 h and harvesting to assess levels of ERK activation. E, densitometric quantification of relative amounts of ERK-1/2 phosphorylation at the different seeding densities. Mock- or L1-transfected 3T3 cells were plated in six-well plates at densities from 0.6 to 5 x 105 cells/well and were grown for 48 h prior to the addition of fresh growth media for 1 h and harvesting. F, mock- or L1-transfected 3T3 cells were plated in six-well plates at 2.5 x 105 cells/well and were grown for 24 h prior to harvesting (0 hrs.); additional cells were then harvested 12, 18, 24, and 36 h later. To prevent media depletion, the cultures received fresh growth media every 12 h. B–F, cell lysates were subjected to Western blot analysis with an anti-phospho-ERK mAb (p-ERK-1/2, E10) and an anti-ERK2 pAb (ERK-2, C14) as a loading control.

 
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 x 105/well). Differences in ERK activation were only evident at higher seeding densities (e.g. 2.5 x 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).



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 2.
Sustained L1-mediated ERK activation requires serum or growth factor co-operation. A, mock- or L1-transfected 3T3 cells were plated in six-well plates and were maintained in 10% serum or were serum-starved prior to harvesting to assess ERK activation. B, mock- or L1-transfected 3T3 cells were plated into six-well plates coated with vitronectin at 1.2 x 106 cells/well. After 5 h without serum, some wells received PDGF (50 ng/ml) for 30 or 120 min prior to harvesting to assess ERK activation. C, densitometric quantification of relative amounts of ERK-1/2 phosphorylation after the addition of PDGF. Results are expressed as mean values ± 1 S.D. (n = 2). A and B, cell lysates were subjected to Western blot analysis with an anti-phospho-ERK mAb (p-ERK-1/2, E10) and an anti-ERK2 pAb (ERK-2, C14) as a loading control.

 
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 {alpha}v{beta}3—A prior report (28) has shown that sustained activation of the ERK pathway induces the expression of select integrins, including {alpha}5{beta}1 and {alpha}v{beta}3. 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 {alpha}v, {beta}3, {beta}1, {alpha}6, {alpha}5, {alpha}2, and {alpha}1 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 {alpha}v{beta}3 (Fig. 3B). No change in the levels of the {alpha}v-subunit, {alpha}1{beta}1, {alpha}2{beta}1, or {alpha}6{beta}1 was observed (Fig. 3, C–F), and only a modest increase in the expression of {alpha}5{beta}1 and the {beta}1-subunit was found (Fig. 3, G and H). Induction of {alpha}v{beta}3 expression was also confirmed in L1-transfected K1735 cells (Fig. 3I), and reverse transcription-PCR analysis, using primers specific for the murine {beta}3-subunit, established that the increased surface expression of {alpha}v{beta}3 in the 3T3 cells is associated with a significant increase in message for the {beta}3-subunit (Fig. 3J). Increased expression of the {alpha}v{beta}3-heterodimer (Fig. 3B), without a change in the overall levels of the {alpha}v-subunit (Fig. 3C), has been reported (28) and is believed to occur at the expense of other {alpha}v-heterodimers such as {alpha}v{beta}5 (29).



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 3.
L1 induces the expression of integrin {alpha}v{beta}3. A–H, the integrin expression profiles of mock- or L1-transfected 3T3 cells were compared by FACS analysis using antibodies to {alpha}1 (Ha31/8), {alpha}2 (HMa2), {alpha}5 (5H10–27), {alpha}6 (GoH3), {beta}1 (pAb FNR), {beta}3 (2C9.G2), and {alpha}v (H9.2b8). I, comparison of {beta}3 expression by mock- or L1-transfected K1735-C11 cells. J, reverse transcription-PCR analysis showing {beta}3 mRNA levels in mock- and L1-transfected 3T3 cells. Actin mRNA levels are shown to confirm equal loading. K, impact of blocking the ERK pathway on {beta}3 expression by L1-transfected 3T3 cells. Cells were treated with U0126 (25 µM) or Me2SO vehicle alone for 72 h prior to FACS analysis. Fresh inhibitor was added every 24 h. L, impact of activating the ERK pathway on {beta}3 expression by 3T3 cells. 3T3{Delta}RAF:ER cells were treated with 4HT (50 ng/ml) or vehicle alone (EtOH) for 24 h prior to FACS analysis. M, 3T3{Delta}RAF:ER cells were pretreated with the ERK inhibitor U0126 (25 µM) for 1 h prior to the addition of 4HT (15 ng/ml) for a further 24 h. N, 3T3{Delta}RAF:ER cells were treated with 4HT (50 ng/ml) or vehicle alone (EtOH) for 6, 12, 18, and 24 h prior to assessing {beta}3 expression.

 
To confirm that induction of {beta}3 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 {beta}3 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 {beta}3 expression was further tested in 3T3 cells infected to express a {Delta}B-RAF:ER fusion protein (30). 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 (30, 31). Treatment with 4HT and concomitant ERK activation in these 3T3 cells resulted in a marked increase in {beta}3 expression (Fig. 3L), and this induction could be blocked using the ERK pathway inhibitor U0126 (Fig. 3M). The increased {alpha}v{beta}3 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 {alpha}v{beta}3 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.2

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 3T3{Delta}RAF: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).



View larger version (37K):
[in this window]
[in a new window]
  		FIG. 4.
Induction of ERK-regulated genes following ectopic L1 expression in NIH-3T3 and K1735-C11 cells. cDNA microarray analysis demonstrating L1 and ERK-induced genes. *, levels of membrane hybridization for genes induced 2-fold or more are shown for mock- or L1-transfected 3T3 or K1735-C11 cells. Housekeeping genes glyceraldehyde-3-phosphate dehydrogenase and {beta}-actin are shown to confirm equal loading. {dagger}, average fold induction of genes following ectopic L1 expression. {ddagger}, average fold induction following the treatment of 3T3{Delta}RAF:ER cells with 4HT (100 ng/ml for 24 h). Bckd., background levels; AIP, apoptosis inhibitory protein.

 
Although ERK activation can account for the majority of the L1-induced gene products, this may not be the only mechanism by which L1 can induce changes in gene expression. Thus, several additional genes were induced as a result of L1 expression which were not significantly induced after conditional activation of the ERK pathway. In the 3T3 model, these genes include colony-stimulating factor-1, cystatin-C, thrombospondin-2, TIMP-2, and S100A4 (data not shown). Induction of colony-stimulating factor-1 and cystatin-C was also confirmed in the K1735 cells (data not shown). Although the microarray used can detect up to 96 different genes, it may only be useful for assessing genes expressed at high levels and, therefore, could result in an underestimation of the number of genes induced by L1. The {beta}3-integrin subunit, for example, is represented on the array, but no significant hybridization signal could be detected for this gene. Together, these data show that L1 can induce global changes in gene expression, and this can be attributed in large part to activation of the ERK pathway.

ERK-regulated Gene Expression Contributes Directly to L1-mediated Motility—Several of the L1- and ERK-regulated gene products identified in this study, including integrin {alpha}v{beta}3 (32) and the GTPase rac-1 (33), have been strongly linked to motility. Studies were performed to determine whether induction of these gene products by L1 contributes directly to the expression of a motile phenotype.

To determine whether L1 induces a motile phenotype, we compared the migration of mock- or L1-transfected 3T3 cells to a variety of extracellular matrix components. Confirming that L1 induces cell motility, ectopic L1 expression in 3T3 cells was found to markedly enhance migration to a variety of substrates, including vitronectin, denatured type I collagen, laminin-1, and fibronectin (Fig. 5A). Consistent with a prior report (22), L1 was not observed to increase migration to native collagen.



View larger version (40K):
[in this window]
[in a new window]
  		FIG. 5.
Integrin {alpha}v{beta}3 and the GTPase Rac-1 contribute to L1-dependent motility. A, migration of mock- or L1-transfected 3T3 cells toward vitronectin (6 h), native and denatured type I collagen (6 h), laminin (18 h), and fibronectin (4 h). B and C, migration of mock- or L1-transfected 3T3 cells to vitronectin or denatured collagen (type I) in the presence or absence of an antibody to {alpha}v{beta}3 (2C9.G2, 40 µg/ml). D, migration of transfected K1735-C11 cells toward denatured collagen (type IV, 18 h) in the presence or absence of an antibody to {alpha}v{beta}3 (2C9.G2, 40 µg/ml). E, migration of L1-transfected 3T3 cells to vitronectin was assessed after transient transfection with the dominant-negative Rac-1 construct Rac-N17. Control cells (Mock) received empty vector. A {beta}-galactosidase reporter vector was used to identify transfected cells, and the migration of transfected 3T3 cells to vitronectin was assessed in a 6-h assay. F, Western blot analysis of mock- or L1-transfected 3T3 lysates using anti-Rac-1 mAb Cl.102. Actin levels were determined to confirm equal loading. G, 3T3{Delta}RAF:ER cells were pretreated with 4HT (15 ng/ml) or vehicle control in serum-free media for 18 h prior to harvesting and assessing migration to denatured collagen over 6 h. Additional cells were treated with U0126 (25 µM) for 1 h prior to the addition of 4HT. H, 3T3{Delta}RAF:ER cells were pretreated with actinomycin-D (Act., 0.5 µg/ml) or vehicle control (Me2SO) for 2 h prior to washing and the addition of 4HT (50 ng/ml) for a further 18 h. Migration of pretreated cells to denatured collagen was then assessed in a 6-h assay.

 
A function-blocking antibody to the {beta}3-integrin subunit was used to determine whether increased expression of integrin {alpha}v{beta}3 contributes to the L1-dependent haptotaxis observed. Using this antibody, it was confirmed that the enhanced 3T3 cell migration to both vitronectin and denatured collagen can be attributed to increased or de novo utilization of integrin {alpha}v{beta}3 (Fig. 5, B and C). Induction of {alpha}v{beta}3-mediated haptotaxis was also confirmed after ectopic L1-expression in the K1735-C11 melanoma line (Fig. 5D). The {alpha}v{beta}3-mediated migration observed was not significantly inhibited by the addition of a variety of anti-L1 monoclonal or polyclonal antibodies to the migration assays (data not shown). This finding suggests that direct L1 participation may not be required and that increased expression of {alpha}v{beta}3 suffices to induce the migration observed. The enhanced L1-dependent migration to laminin and fibronectin (Fig. 5A) was inhibited using antibodies to {alpha}6{beta}1 and {alpha}5{beta}1 respectively (data not shown). The contribution of these integrins to L1-mediated haptotaxis is consistent with prior reports (21, 22).

Rac-1 has been shown to be an important signaling intermediate in cell motility (33), and this GTPase was induced 3-fold after ectopic L1 expression in 3T3 cells (Fig. 4). To determine whether rac-1 contributes to L1-dependent motility, L1-transfected 3T3 cells were transiently transfected with a vector encoding dominant-negative rac (Rac-N17) (24). Control cells received empty vector.

Expression of Rac-N17 did not have a significant impact on 3T3 adhesion or viability (data not shown) but did significantly reduce L1-dependent migration to vitronectin (Fig. 5E). In confirmation of the gene array data, increased levels of rac-1 protein were detected in lysates of L1-transfected 3T3 cells (Fig. 5F).

Our findings suggest that the induction of ERK-regulated gene products (e.g. {alpha}v{beta}3 and rac-1) by L1 contributes to the induction of 3T3 cell motility. To confirm that ERK-dependent gene transcription suffices to promote 3T3 migration, we assessed the impact of conditional ERK activation and gene transcription on the migration of 3T3{Delta}RAF:ER cells (30, 31). After inducing ERK-activation with 4HT (18 h pretreatment) we observed a 1.8–2-fold induction of migration to denatured collagen (Fig. 5G). Induction of this migration was blocked both by the ERK pathway inhibitor U0126 and by the transcriptional inhibitor actinomycin-D (Fig. 5, G and H). At the concentration used (0.5 µg/ml for 2 h), actinomycin-D was not observed to affect 3T3 viability (data not shown). In timecourse studies, it was observed that ERK activation had to be sustained for at least 12 h to induce 3T3 motility (data not shown). Based on these observations, we conclude that sustained ERK activation and concomitant gene transcription is both necessary and sufficient to induce 3T3 migration.

Induction of Cathepsin Activity by L1 Contributes to Matrix Invasion—Cathepsins-L and -B have both been implicated in matrix invasion (34), and these cysteine proteases were strongly induced as a result of L1 expression in both 3T3 and K1735 cells (Fig. 4). To determine whether L1 induces an invasive phenotype, we utilized Transwell inserts coated with a barrier of reconstituted basement membrane (RBM) (matrigel, BD Biosciences). PDGF was added to lower chambers to serve as a chemoattractant and to activate L1-mediated motility. A comparison of RBM invasion by mock- or L1-transfected 3T3 cells confirmed the induction of an invasive phenotype with ectopic L1 expression (Fig. 6, A and B).



View larger version (36K):
[in this window]
[in a new window]
  		FIG. 6.
L1 induces cathepsin activity and an invasive phenotype. A and B, mock- or L1-transfected 3T3 cells were added to RBM invasion chambers, and the invasion of cells to lower chambers was assessed after 42 h. PDGF was added to lower chambers at 50 ng/ml. A, photomicrographs demonstrating relative invasion between mock- and L1-transfected cells. Bar, 100 µM. B, quantification of invasion by mock- or L1-transfected 3T3. Error bars represent ± 1 S.D. C, impact of peptide inhibitors CA-064 Me and E-64-d on RBM invasion by L1-transfected 3T3 cells. Peptides were added to both upper and lower chambers, and invasion was assessed after 42 h. D and E, intracellular cathepsin-L and -B activity in mock- or L1-transfected 3T3 cells were assessed using specific Magic Red fluorogenic substrates (Immunochemistry Technologies) and FACS analysis. Histograms (solid lines) show fluorescence generated as a result of intracellular enzymatic cleavage. Left, dashed lines, histograms show levels of background fluorescence for mock- or L1-transfected cells in the absence of fluorogenic substrate. Insets, additional cells were treated with the ERK-pathway inhibitor U0126 or Me2SO vehicle alone for 72 h prior to measuring intracellular cathepsin-L and -B activity.

 
Cell permeable peptide inhibitors of cathepsins-L and B were used to determine whether these cysteine proteases contribute to the L1-mediated invasion observed. Peptide CA-064 Me (selective inhibitor of procathepsin-B) and peptide E-64-d (inhibitor of cathepsins-L and -B) both significantly inhibited invasion by L1-transfected 3T3 cells (Fig. 6C). At the concentrations used, neither peptide was observed to significantly affect 3T3 viability, and neither peptide significantly inhibited migration to RBM (data not shown). Induction of cathepsin expression and activity as a result of L1 expression was further confirmed using Magic Red fluorogenic substrates specific for cathepsins-L or B (Immunochemistry Technologies). Intracellular fluorescence, generated as a result of substrate cleavage, was assessed by flow cytometry, and this approach confirmed significantly higher levels of cathepsin-L and -B activity in the L1-transfectants (Fig. 6, D and E). Underscoring the importance of L1-mediated ERK activation, the expression and activity of both enzymes was significantly reduced after pretreating L1-transfected 3T3 cells with the ERK pathway inhibitor U0126 (Fig. 6, D and E, insets).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Prior reports (21, 22) have documented the potentiation of integrin-mediated haptotaxis after ectopic L1 expression and have provided evidence for non-translational mechanisms involving direct L1-integrin ligation and the induction of transient integrin signaling and endocytosis. In this study, we provide evidence that L1 can induce both motility and invasion via an additional novel mechanism that involves sustained ERK activation and the concomitant induction of motility-associated gene products. L1 expression, in two histologically distinct cell lines, is shown to induce higher constitutive levels of ERK activation and the concomitant expression of gene products that contribute directly to increased L1-dependent motility or invasion. Induction of the integrin {alpha}v{beta}3 and the small GTPase rac-1 is shown to facilitate L1-dependent haptotaxis, whereas induction of cathepsin-L and -B promote matrix invasion. The ability of L1 to promote sustained ERK activation is shown to be critically dependent upon cell density and upon co-operation with serum or serum growth factors.

Although prior reports (35, 36) have shown that L1 cross-linking induces transient ERK activation, this is the first report that L1 can induce sustained ERK activation under normal culture conditions. This is an important distinction because sustained, rather than transient, activation of this pathway is often required for optimal gene transcription (28, 37). In this regard, we found that induction of {alpha}v{beta}3 expression in 3T3 cells was only evident after ERK activation had been maintained for at least 12 h. Consistent with our observations, many of the L1- and ERK-induced genes identified in this study have previously been shown to be regulated by the ERK pathway, including cathepsins-L and -B, osteopontin, RhoC, CD44, and {alpha}v{beta}3 (28, 38–40). Induction of ERK-dependent gene transcription by L1 has not been documented previously. However, other members of the immunoglobulin superfamily have been shown to induce ERK-dependent gene transcription. Neural cell adhesion molecule, for example, has been shown to promote neuritogenesis and gene transcription by inducing the sequential activation of ERK-1/2 and the transcription factor cAMP-response element-binding protein (41). Interestingly, induction of cAMP-response element-binding protein-dependent gene expression is known to require sustained ERK activation (42), and neural cell adhesion molecule has been shown to support sustained ERK activation in both hippocampal neurons and PC12 cells (43).

Using 3T3{Delta}RAF:ER cells (30), we observed that sustained ERK activation and gene transcription is sufficient to induce 3T3 cell motility to denatured collagen. Accordingly, L1 may induce migration via a translational mechanism without direct L1-integrin interaction and signaling (21, 22). However, L1 was also found to promote {alpha}5{beta}1-dependent migration to fibronectin as described (21, 22), and we found no evidence that gene transcription is sufficient for this response. Thus, {alpha}5{beta}1 levels were only minimally affected by L1 expression, and conditional ERK activation in 3T3{Delta}RAF:ER failed to induce significant migration to fibronectin (data not shown). These findings suggest that both translational and non-translational mechanisms potentiate L1-mediated migration, depending upon which integrin or substrate is engaged.

The novel observation that L1 promotes the expression and utilization of integrin {alpha}v{beta}3 has broad mechanistic ramifications for the participation of L1 in a variety of normal and pathological processes. Together with L1, this integrin has been shown to promote neurite extension (44–46), extravasation (16, 47, 48), myelination (49, 50), and metastasis (17, 32). In addition, L1 has recently been shown to promote fibroblast-matrix interactions in vivo, a process that could also involve {alpha}v{beta}3 (9). Accordingly, a wide variety of L1-driven processes could involve the increased expression and utilization of {alpha}v{beta}3. It is also noteworthy that L1 can also serve as a direct ligand for {alpha}v{beta}3 (6), and that direct L1-{alpha}v{beta}3 pairing has been shown to influence both neurite extension and extravasation (16, 44).

The contribution of L1 to normal central nervous system development could also be influenced by the transcription and expression of cathepsins-B and -L and rac-1. Cathepsins-L and -B have been shown to regulate neurite formation and the integrity of the postnatal central nervous system (51, 52), and rac-1 is a critical regulator of actin dynamics governing growth cone motility and neurite extension (53). Rac1 has also been shown to be an important intermediate in L1-mediated signaling (35).

Our novel finding that L1 induces matrix (RBM) invasion could account for reports documenting a strong association between L1 expression and metastasis (17–20). Induction of {alpha}v{beta}3 expression in the K1735-C11 melanoma cell line is particularly salient, because this integrin has been implicated in many aspects of melanoma progression including tumorigenicity, survival, invasion, and transendothelial migration (54–56). It is also salient that L1 induces the expression of cathepsins-L and -B, because these cysteine proteases have been implicated in invasion and metastasis by multiple cell types, including NIH-3T3 cells (34, 38). Finally, the ability of L1 to induce sustained activation of the ERK pathway is significant because constitutive activation of this pathway has repeatedly been linked to expression of a malignant phenotype (38, 57, 58).

Sustained L1-dependent ERK activation in our 3T3 model required growth factor co-operation, and it is unclear whether this is due to a convergence of L1 and growth factor signaling pathways or whether L1 can directly or indirectly impact the activity of growth factor receptors. In support of the latter, it has recently been shown that L1-type molecules are able to interact directly with the EGF receptor to induce tyrosine kinase activity (59). It is also conceivable that L1 can influence growth factor receptor signaling by serving as a cellular ligand for integrins (21, 22). Thus, integrin ligation and clustering is known to result in the recruitment and activation of select growth factor receptors (60). Induction of {alpha}v{beta}3 expression may also be significant because this integrin is known to collaborate directly with PDGF and its receptor to promote ERK activation (61).

This report provides the first evidence that L1 can induce global changes in gene expression resulting in a more motile or invasive phenotype. Such alterations in gene expression may be particularly important for stabilizing or maintaining changes in cellular phenotype that occur during normal development and tumorigenesis.


   FOOTNOTES
 
* This work was supported by NCI/National Institutes of Health RO1 Grant CA69112-01, NHLBI/National Institutes of Health RO1 Grant HL62477-01 (to A. M. P. M.), and NIDDK/National Institutes of Health R01 Grants DK55183 and DK55183S1 (to V. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Dept. of Pediatrics, The Whittier Institute, University of California at San Diego, 9894 Genesee Ave., La Jolla, CA 92037. Tel.: 858-550-2909; Fax: 858-558-3495; E-mail: ammontgo{at}ucsd.edu.

1 The 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. Back

2 Available on the World Wide Web at www.superarray.com/cancer.php. Back


   ACKNOWLEDGMENTS
 
We thank Thomas Kaido for comments on this manuscript and Lillian Meyer for assistance with the FACS analysis.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Brummendorf, T., and Rathjen, F. G. (1993) J. Neurochem. 61, 1207–1219[Medline] [Order article via Infotrieve]
  2. Hortsch, M. (1996) Neuron 17, 587–593[CrossRef][Medline] [Order article via Infotrieve]
  3. Lindner, J., Rathjen, F. G., and Schachner, M. (1983) Nature 305, 427–430[CrossRef][Medline] [Order article via Infotrieve]
  4. Thor, G., Probstmeier, R., and Schachner, M. (1987) EMBO J. 6, 2581–2586[Medline] [Order article via Infotrieve]
  5. Ebeling, O., Duczmal, A., Aigner, S., Geiger, C., Schollhammer, S., Kemshead, J. T., Moller, P., Schwartz-Albiez, R., and Altevogt, P. (1996) Eur. J. Immunol. 26, 2508–2516[Medline] [Order article via Infotrieve]
  6. Montgomery, A. M. P., Becker, J. C., Siu, C.-H., Lemmon, V. P., Cheresh, D. A., Pancook, J. D., Zhao, X., and Reisfeld, R. A. (1996) J. Cell Biol. 132, 475–485[Abstract/Free Full Text]
  7. Pancook, J. D., Reisfeld, R. A., Varki, N., Vitiello, A., Fox, R. I., and Montgomery, A. M. P. (1997) J. Immunol. 158, 4413–4421[Abstract]
  8. Debiec, H., Christensen, E. I., and Ronco, P. M. (1998) J. Cell Biol. 143, 2067–2079[Abstract/Free Full Text]
  9. Di Sciullo, G., Donahue, T., Schachner, M., and Bogen, S. A. (1998) J. Exp. Med. 187, 1953–1963[Abstract/Free Full Text]
  10. Nolte, C., Moos, M., and Schachner, M. (1999) Cell Tissue Res. 298, 261–273[CrossRef][Medline] [Order article via Infotrieve]
  11. Miura, M., Asou, H., Kobayashi, M., and Uyemura, K. (1992) J. Biol. Chem. 267, 10752–10758[Abstract/Free Full Text]
  12. Dahme, M., Bartsch, U., Martini, R., Anliker, B., Schachner, M., and Mantei, N. (1997) Nat. Genet. 17, 346–349[CrossRef][Medline] [Order article via Infotrieve]
  13. Demyanenko, G. P., Tsai, A. Y., and Maness, P. F. (1999) J. Neurosci. 19, 4907–4920[Abstract/Free Full Text]
  14. Crossin, K. L., Prieto, A. L., Hoffman, S., Jones, F. S., and Friedlander, D. R. (1990) Exp. Neurol. 109, 6–18[CrossRef][Medline] [Order article via Infotrieve]
  15. Izumoto, S., Ohnishi, T., Arita, N., Hirage, S., Taki, T., and Hayakawa, T. (1996) Cancer Res. 56, 1440–1444[Abstract/Free Full Text]
  16. Voura, E. B., Ramjeesingh, R. A., Montgomery, A. M. P., and Siu, C.-H. (2001) Mol. Biol. Cell. 12, 2699–2710[Abstract/Free Full Text]
  17. Linnemann, D., Raz, A., and Bock, E. (1989) Int. J. Cancer 43, 709–712[Medline] [Order article via Infotrieve]
  18. Thies, A., Schachner, M., Moll, I., Berger, J., Schulze, H.-J., Brunner, G., and Schumacher, U. (2002) Eur. J. Cancer 38, 1708–1716[CrossRef][Medline] [Order article via Infotrieve]
  19. Calvo, A., Xiao, N., Kang, J., Best, C. J. M., Leiva, I., Emmert-Buck, M. R., Jorcyk, C., and Green, J. E. (2002) Cancer Res. 62, 5325–5335[Abstract/Free Full Text]
  20. Fogel, M., Mechtersheimer, S., Huszar, M., Smirnov, A., Abu-Dahi, A., Tilgen, W., Reichrath, J., Georg, T., Altevogt, P., and Gutwein, P. (2003) Cancer Lett. 189, 237–247[CrossRef][Medline] [Order article via Infotrieve]
  21. Mechtersheimer, S., Gutwein, P., Agmon-Levin, N., Stoeck, A., Oleszewski, M., Riedle, S., Fogel, M., Lemmon, V., and Altevogt, P. (2001) J. Cell Biol. 155, 661–673[Abstract/Free Full Text]
  22. Thelen, K., Keder, V., Panicker, A. K., Schmid, R.-S., Midkiff, B. R., and Maness, P. F. (2002) J. Neurosci. 22, 4918–4931[Abstract/Free Full Text]
  23. Kamiguchi, H., Long, K. E., Pendergast, M., Schaefer, A. W., Rapoport, I., Kirchhausen, T., and Lemmon, V. (1998) J. Neurosci. 18, 5311–5321[Abstract/Free Full Text]
  24. Minden, A., Lin, A., Claret, F., Abo, A., and Karin, M. (1995) Cell 81, 1147–1157[CrossRef][Medline] [Order article via Infotrieve]
  25. Nayeem, N., Silletti, S., Yang, X.-M., Lemmon, V. P., Reisfeld, R. A., Stallcup, W. B., and Montgomery, A. M. P. (1999) J. Cell Sci. 112, 4739–4749[Abstract]
  26. Balaian, L., Moelher, T., and Montgomery, A. M. P. (2000) Eur. J. Immunol. 30, 938–943[CrossRef][Medline] [Order article via Infotrieve]
  27. Primiano, T., Baig, M., Maliyekkkel, A., Cahng, B. D., Fellars, S., Sadhu, J., Axenovich, S. A., Holzayer, T. A., and Roninson, I. B. (2003) Cancer Cells 4, 415–423
  28. Woods, D., Cherwinski, H., Venetsanakos, E., Bhat, A., Gysin, S., Humbert, M., Bray, P. F., Saylor, V. L., and McMahon, M. (2001) Mol. Cell. Biol. 21, 3192–3205[Abstract/Free Full Text]
  29. Koistinen, P., and Heino, J. (2002) J. Biol. Chem. 277, 24835–24841[Abstract/Free Full Text]
  30. McCarthy, S. A., Samuels, M. L., Pritchrad, C. A., Abraham, J. A., and McMahon, M. (1995) Genes and Dev. 9, 1953–1964[Abstract/Free Full Text]
  31. Samuels, M. A., Weber, M. J., Bishop, M., and McMahon, M. (1993) Mol. Cell. Biol. 13, 6241–6252[Abstract/Free Full Text]
  32. Varner, J. A., and Cheresh, D. A. (1996) Curr. Opin. Cell Biol. 8, 724–730[CrossRef][Medline] [Order article via Infotrieve]
  33. Ridley, A. J., Allen, W. E., Peppelenbosch, M., and Jones, G. E. (1999) Biochem. Soc. Symp. 65, 111–123[Medline] [Order article via Infotrieve]
  34. Janulis, M., Silberman, S., Ambegaokar, A., Gutkind, J. S., and Schultz R. M. (1999) J. Biol. Chem. 274, 801–813[Abstract/Free Full Text]
  35. Schmid, R.-S., Pruitt, W. M., and Maness, P. F. (2000) J. Neurosci. 20, 4177–4188[Abstract/Free Full Text]
  36. Schaefer, A. W., Kamiguchi, H., Wong, E. V., Beach, C. M., Landreth, G., and Lemmon, V. (1999) J. Biol. Chem. 274, 37965–37973[Abstract/Free Full Text]
  37. Hazzalin, C. A., and Mahadevan, L. C. (2002) Nat. Rev. Mol. Cell Biol. 3, 30–40[CrossRef][Medline] [Order article via Infotrieve]
  38. Welch, D. R., Sakamaki, T., Pioquinto, R., Leonard, T. O., Goldberg, S. F., Hon, Q., Erikson, R. L., Rieber, M., Reiber, M. S., Hicks, D. J., Bonventre, J. V., and Alessandrini, A. (2000) Cancer Res. 60, 1552–1556[Abstract/Free Full Text]
  39. Moses, S., Franzen, A., Lovdahl, C., and Hultgardh-Nilsson, A. (2001) Arch. Biochem. Biophys. 396, 133–137[CrossRef][Medline] [Order article via Infotrieve]
  40. Zuber, J., Tchernitsa, O. I., Hinzmann, B., Schmitz, A. C., Grips, M., Hellriegel, M., Sers, C., Rosenthal, A., and Schafer, R. (2000) Nat. Genet. 2, 144–152
  41. Jessen, U., Novitskaya, V., Pedersen, N., Serup, P., Berezin, V., and Bock, E. (2001) J. Neurochem. 79, 1149–1160[CrossRef][Medline] [Order article via Infotrieve]
  42. Bito, H., Deisseroth, K., and Tsien, R. W. (1996) Cell 87, 1203–1214[CrossRef][Medline] [Order article via Infotrieve]
  43. Kolkova, K., Novitskaya, V., Pedersen, N., Berezin, V., and Bock, E. (2000) J. Neurosci. 20, 2238–2246[Abstract/Free Full Text]
  44. Yip, P. M., Zhao, X., Montgomery, A. M., and Siu, C.-H. (1998) Mol. Biol. Cell 9, 277–290[Abstract/Free Full Text]
  45. Ivins, J. K., Yurchenco, P. D., and Lander, A. D. (2000) J. Neurosci. 17, 6551–6560
  46. Altetsee, C., Mullen, L., Kim, D., Pak, K., Brors, D., Dazert, S., and Ryan, A. F. (2001) Audiol. Neurootol. 2, 57–65[CrossRef]
  47. Hangan, D., Morris, V. L., Boeters, L., von Ballestem, C., Uniyal, S., and Chan, B. M. (1997) Cancer Res. 57, 3812–3817[Abstract/Free Full Text]
  48. Thompson, R. D., Wakelin, M. W., Larbi, K. Y., Dewar, A., Asimakopoulos, G., Horton, M. A., Nakada, M. T., and Nourshargh, S. (2000) J. Immunol. 165, 426–434[Abstract/Free Full Text]
  49. Relvas, J. B., Setzu, A., Baron, W., Buttery, P. C., LaFlamme, S. E., Franklin, R. J., and French-Constant, C. (2001) Curr. Biol. 6, 1445–1455
  50. Bartsch, U. (2003) Front. Biosci. 8, D477–D490[Medline] [Order article via Infotrieve]
  51. Bednarski, E., Ribak, C. E., and Lynch, G. (1997) J. Neurosci. 17, 4006–4021[Abstract/Free Full Text]
  52. Felbor, U., Kessler, B., Mothes, W., Goebel, H. H., Ploegh, H. L., Bronson, R. T., and Bjorn, R. O. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7883–7888[Abstract/Free Full Text]
  53. Meyer, G., and Feldman, E. L. (2002) J. Neurochem. 83, 490–503[CrossRef][Medline] [Order article via Infotrieve]
  54. Albeda, S. M., Mette, S. A., Elder, D. E., Stewart, R., Damjanovich, L., Herlyn, M., and Buck, C. A. (1991) Cancer Res. 50, 6757–6764
  55. Hsu, M. Y., Shih, D. T., Meier, F. E., Van Belle, P., Hsu, J. Y., Elder, D. E., Buck, C. A., and Herlyn, M. (1998) Am. J. Pathol. 153, 1435–1442[Abstract/Free Full Text]
  56. Petitclerc, E., Stromblad, S., Von Schalscha, T. L., Mitjans, F., Piulats, J., Montgomery, A. M. P., Cheresh, D. A., and Brooks, P. C. (1999) Cancer Res. 59, 2724–2730[Abstract/Free Full Text]
  57. Webb, C. P., Van Aelst, A., Wigler, M. H., and Woude, G. F. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8773–8778[Abstract/Free Full Text]
  58. Govindarajan, B., Bai, X., Cohen, C., Zhong, H., Kilroy, S., Louis, G., Moses, M., and Arbiser, J. L. (2003) J. Biol. Chem. 278, 9790–9795[Abstract/Free Full Text]
  59. Islam, R., Kristiansen, L. V., Romani, S., Garcia-Alonso, L., and Hortsch, M. (2004) Mol. Biol. Cell 15, 2003–2012[Abstract/Free Full Text]
  60. Yamada, K. M., and Even-Ram, S. (2002) Nat. Cell Biol. 4, E75–76[CrossRef][Medline] [Order article via Infotrieve]
  61. Schneller, M., Vuori, K., and Ruoslahti, E. (1997) EMBO J. 16, 5600–5607[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Cancer Res.Home page
C. Geismann, M. Morscheck, D. Koch, F. Bergmann, H. Ungefroren, A. Arlt, M.-S. Tsao, M. G. Bachem, P. Altevogt, B. Sipos, et al.
Up-regulation of L1CAM in Pancreatic Duct Cells Is Transforming Growth Factor {beta}1- and Slug-Dependent: Role in Malignant Transformation of Pancreatic Cancer
Cancer Res., May 15, 2009; 69(10): 4517 - 4526.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
A. V. Ivanova, C. M.V. Goparaju, S. V. Ivanov, D. Nonaka, C. Cruz, A. Beck, F. Lonardo, A. Wali, and H. I. Pass
Protumorigenic Role of HAPLN1 and Its IgV Domain in Malignant Pleural Mesothelioma
Clin. Cancer Res., April 15, 2009; 15(8): 2602 - 2611.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
M.-R. Du, W.-H. Zhou, L. Dong, X.-Y. Zhu, Y.-Y. He, J.-Y. Yang, and D.-J. Li
Cyclosporin A Promotes Growth and Invasiveness In Vitro of Human First-Trimester Trophoblast Cells Via MAPK3/MAPK1-Mediated AP1 and Ca2+/Calcineurin/NFAT Signaling Pathways
Biol Reprod, June 1, 2008; 78(6): 1102 - 1110.
[Abstract] [Full Text] [PDF]

Home page
 Ann. Surg. Oncol.Home page
Y.-J. Boo, J.-M. Park, J. Kim, Y.-S. Chae, B.-W. Min, J.-W. Um, and H.-Y. Moon
L1 Expression as a Marker for Poor Prognosis, Tumor Progression, and Short Survival in Patients with Colorectal Cancer
Ann. Surg. Oncol., May 1, 2007; 14(5): 1703 - 1711.
[Abstract] [Full Text] [PDF]

Home page
 BrainHome page
J. Chen, J. Wu, I. Apostolova, M. Skup, A. Irintchev, S. Kugler, and M. Schachner
Adeno-associated virus-mediated L1 expression promotes functional recovery after spinal cord injury
Brain, April 1, 2007; 130(4): 954 - 969.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
K. Knogler, J. Grunberg, K. Zimmermann, S. Cohrs, M. Honer, S. Ametamey, P. Altevogt, M. Fogel, P. A. Schubiger, and I. Novak-Hofer
Copper-67 Radioimmunotherapy and Growth Inhibition by Anti-L1-Cell Adhesion Molecule Monoclonal Antibodies in a Therapy Model of Ovarian Cancer Metastasis
Clin. Cancer Res., January 15, 2007; 13(2): 603 - 611.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
K. Szczur, H. Xu, S. Atkinson, Y. Zheng, and M.-D. Filippi
Rho GTPase CDC42 regulates directionality and random movement via distinct MAPK pathways in neutrophils
Blood, December 15, 2006; 108(13): 4205 - 4213.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
W.-S. Wu, R. K. Tsai, C. H. Chang, S. Wang, J.-R. Wu, and Y.-X. Chang
Reactive Oxygen Species Mediated Sustained Activation of Protein Kinase C {alpha} and Extracellular Signal-Regulated Kinase for Migration of Human Hepatoma Cell Hepg2
Mol. Cancer Res., October 1, 2006; 4(10): 747 - 758.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
S. Guo, J. Lu, A. Subramanian, and G. E. Sonenshein
Microarray-Assisted Pathway Analysis Identifies Mitogen-Activated Protein Kinase Signaling as a Mediator of Resistance to the Green Tea Polyphenol Epigallocatechin 3-Gallate in Her-2/neu-Overexpressing Breast Cancer Cells.
Cancer Res., May 15, 2006; 66(10): 5322 - 5329.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. J.E. Arlt, I. Novak-Hofer, D. Gast, V. Gschwend, G. Moldenhauer, J. Grunberg, M. Honer, P. A. Schubiger, P. Altevogt, and A. Kruger
Efficient Inhibition of Intra-Peritoneal Tumor Growth and Dissemination of Human Ovarian Carcinoma Cells in Nude Mice by Anti-L1-Cell Adhesion Molecule Monoclonal Antibody Treatment
Cancer Res., January 15, 2006; 66(2): 936 - 943.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. Conacci-Sorrell, A. Kaplan, S. Raveh, N. Gavert, T. Sakurai, and A. Ben-Ze'ev
The Shed Ectodomain of Nr-CAM Stimulates Cell Proliferation and Motility, and Confers Cell Transformation
Cancer Res., December 15, 2005; 65(24): 11605 - 11612.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
T. Maretzky, M. Schulte, A. Ludwig, S. Rose-John, C. Blobel, D. Hartmann, P. Altevogt, P. Saftig, and K. Reiss
L1 Is Sequentially Processed by Two Differently Activated Metalloproteases and Presenilin/{gamma}-Secretase and Regulates Neural Cell Adhesion, Cell Migration, and Neurite Outgrowth
Mol. Cell. Biol., October 15, 2005; 25(20): 9040 - 9053.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
P. Gutwein, A. Stoeck, S. Riedle, D. Gast, S. Runz, T. P. Condon, A. Marme, M.-C. Phong, O. Linderkamp, A. Skorokhod, et al.
Cleavage of L1 in Exosomes and Apoptotic Membrane Vesicles Released from Ovarian Carcinoma Cells
Clin. Cancer Res., April 1, 2005; 11(7): 2492 - 2501.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
N. Gavert, M. Conacci-Sorrell, D. Gast, A. Schneider, P. Altevogt, T. Brabletz, and A. Ben-Ze'ev
L1, a novel target of {beta}-catenin signaling, transforms cells and is expressed at the invasive front of colon cancers
J. Cell Biol., February 14, 2005; 168(4): 633 - 642.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Pathol.Home page
T Suzuki, S Izumoto, Y Fujimoto, M Maruno, Y Ito, and T Yoshimine
Clinicopathological study of cellular proliferation and invasion in gliomatosis cerebri: important role of neural cell adhesion molecule L1 in tumour invasion
J. Clin. Pathol., February 1, 2005; 58(2): 166 - 171.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/28/28880    most recent
M404075200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Silletti, S.
Right arrow Articles by Montgomery, A. M. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Silletti, S.
Right arrow Articles by Montgomery, A. M. P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement