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5 Chain Globular Domain That Binds to CD44 and Inhibits Malignancy
1 Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, NIH, Bethesda, Maryland; 2 Fourth Department of Internal Medicine, Nippon Medical School, Tokyo, Japan; 3 Respiratory Division of Internal Medicine, Tokyo Metropolitan Komagome Hospital, Tokyo, Japan; and 4 Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5 chain is a component of laminin-10 (5ß1
1) and -11 (5ß21). In this study, we have screened 113 overlapping synthetic peptides from the laminin 5 globular domain (G-domain) for cell attachment activity with B16-F10 cells using peptide-coated dishes. Eleven attachment-active peptides were identified. In vivo experimental B16-F10 pulmonary metastasis and primary tumor growth assays found that 4 of the 11 peptides inhibited tumor metastasis and growth and increased apoptosis. These four peptides also blocked tumor cell migration, invasion, and angiogenesis. Two of the peptides were highly homologous and showed significant similarity to sequences in collagens. We sought to identify the B16-F10 cell surface receptors for each of the four active peptides using peptide affinity chromatography. Only one peptide recognized a cell surface protein. Peptide A5G27 (RLVSYNGIIFFLK, residues 2892–2904) bound a diffuse Mr 
120,000–180,000 band that eluted with 2 M NaCl. Glycosidase digestion of the 2 M eluate yielded protein bands of Mr 90,000 and 60,000 that reacted in Western blot analysis with antibodies to CD44. Immunoprecipitation of the A5G27-bound membrane proteins with various cell surface proteoglycan antibodies confirmed CD44 as the surface receptor for A5G27. Finally, attachment assays to A5G27 in the presence of soluble glycosaminoglycans (GAGs) identified the GAGs of CD44 as the binding sites for A5G27. Our results suggest that A5G27 binds to the CD44 receptor of B16-F10 melanoma cells via the GAGs on CD44 and, thus, inhibits tumor cell migration, invasion, and angiogenesis in a dominant-negative manner. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, one ß, and one chain, such that laminin-1 is composed of 1ß11, laminin-2 is composed of 2ß11, and so forth. There are five , three ß, and three chains identified to date. These trimeric molecules form a family that has at least 15 laminin isoforms. Laminins have multiple biological activities, including cell adhesion, migration, angiogenesis, differentiation, tumor growth, and metastasis (1)
. The reason for the large number of family members is unclear, but each molecule likely has a tissue-, time-, and cell-specific function. Previously, cell adhesive sequences on laminin-1 were screened using 700 overlapping 12-mer synthetic peptides covering the entire protein. More than 20 functional sequences from laminin-1 have been identified (2, 3, 4, 5, 6)
. Some of these peptides regulate the malignant phenotype (7, 8, 9)
. Many laboratories are now beginning to identify additional active sites on the homologous laminin chains (10
, 11)
. Most of the active peptides are localized in the globular domains (G-domains) and play a critical role in binding to cell surface receptors in a peptide- and cell type-specific manner (12, 13, 14, 15, 16)
. Several receptors for these active sequences have been identified. Two peptides in the G-domains interact with syndecans (5
, 10)
. One is a laminin 1 chain peptide, AG73 (RKRLQVQLSIRT, residues 2719–2730), that promotes cell adhesion, and salivary gland cell differentiation by binding to the heparin-like glycosaminoglycans (GAGs) on syndecan-1. A laminin 3 chain peptide, A3G75aR (NSFMALYLSKGR, residues 1412–1423), promotes cell adhesion via binding to syndecan-2 and -4.
The laminin chains are generally large (Mr, 400,000) and contain a COOH-terminal G-domain consisting of five modules LG1–LG5. The globular modules on the chains are of particular interest because of their biological activity. The 5 chain is a component of laminin-10 (5ß11) and -11 (5ß21), which are important in malignancy. Previously, 113 overlapping synthetic peptides from the 5 chain G-domain were screened for cell attachment activity with HT1080 cells, and 21 cell-binding sites were identified (11)
. Heparin was able to block attachment to 16 of these peptides, suggesting that this G-domain interacts with proteoglycans.
Here, the active sites for malignancy have been identified and characterized in the laminin 5 chain G-domain. First, 113 overlapping synthetic peptides of the laminin 5 chain were screened for cell attachment activity with B16-F10 mouse melanoma cells, and 11 active peptides were identified. Next, the effect of the 11 active peptides on B16-F10 mouse melanoma lung metastasis was examined, and four peptides were found to reduce lung colonization, with the remainder being inactive. These four peptides were further studied to determine the mechanisms involved in metastasis inhibition and their cellular receptors. All four active peptides inhibited primary tumor growth in vivo, angiogenesis, migration, and invasion, but they did not affect proliferation in vitro. One of the four peptides, A5G27 (RLVSYNGIIFFLK, residues 2892–2904), interacted with CD44, a receptor important in metastasis. Two of the other peptides, A5G81 (AGQWHRVSVRWG, residues 3337–3348) and A5G101 (DGRWHRVAVIMG, residues 3516–3527), were highly homologous to each other, showed strong identity of sequence in the other laminin chains, and showed significant homology with certain collagen sequences. We conclude that the active 5 G-domain peptides serve important biological functions in tumor metastasis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation and Culture of Endothelial Cells.
Human umbilical vein endothelial cells (HUVECs) were obtained from freshly delivered umbilical cords by treatment with 0.1% collagenase. Cells were grown in RPMI 1640 supplemented with 20% defined bovine calf serum (BCS; HyClone Laboratories, Inc., Logan, UT), 5 units/ml heparin (Fisher Scientific, Pittsburgh, PA), 200 µg/ml endothelial cell growth supplement (ECGS; Collaborative Research, Bedford, MA), 100 units/ml penicillin, 100 µg/ml streptomycin, 50 µg/ml gentamicin, and 2.5 µg/ml amphotericin B (Life Technologies, Inc. Gaithersburg, MD; Ref. 18
) at 37°C and 5% CO2. Only those cells from passages 3–5 were used.
Culture of B16-F10 Melanoma Cells.
B16-F10 melanoma cells (19)
were cultured in DMEM (Life Technologies, Inc., Rockville, MD), containing 10% fetal bovine serum (HyClone, Logan, UT), 100 units/ml penicillin, 100 µg/ml streptomycin, and Nonessential Amino Acids solution (Life Technologies, Inc.) at 37°C, 5% CO2.
Attachment Assay Using Peptide-Coated Plates.
Tumor cell attachment was assayed in U-bottomed 96-well plates coated overnight with synthetic peptides (0–5 µg). Wells were washed with PBS, blocked with 1% BSA in PBS, and washed again with PBS. Cells, detached by 0.02% EDTA in PBS and resuspended in DMEM containing 0.1% BSA, were added (5 x 104 cells/100 µl) to each well and incubated for 1 h at 37°C, 5% CO2. The attached cells were stained with 50 µl of 0.2% crystal violet aqueous solution in 20% methanol for 10 min. Dishes were extensively washed, and bound dye was solubilized in 2% SDS and quantitated at 600 nm. Assays were performed in triplicate at least three times.
Inhibition of Cell Attachment.
Plates were coated with 0.5 µg of peptide in 50 µl of H2O and were dried overnight. The wells were blocked and washed as described above. The wells were then preincubated with 5 µg of the GAGs in 25 µl of DMEM containing 0.1% BSA for 30 min at 37°C. Heparin, heparan sulfate (purity 100%), chondroitin sulfate A (CS-A; purity 70%), CS-B (purity >99%), and CS-C (purity 90%), and hyaluronic acid (Sigma, St. Louis, MO) were tested. B16-F10 cells (5 x 104) were added in 25 µl of DMEM containing 0.1% BSA resulting in a final volume of 50 µl. Attachment was measured as described above.
In Vivo Experimental Pulmonary Metastasis Assay.
For the in vivo experimental pulmonary metastasis assay, 0.2 ml of Versene-detached B16-F10 cells (1 x 106 cells/ml in DMEM) were injected into the tail veins of C57BL/6J mice (5–6 weeks old), and 1.0 mg peptide solution (250 µl) was immediately injected i.p. Control mice received saline i.p. The mice were sacrificed 10 days after injection. The lungs were removed, and visible colonies on the surface of the lungs were counted.
This experiment was repeated three times using six mice per data point.
Subcutaneous Tumor Growth.
B16-F10 cells (1 x 105 cells/0.1 ml), mixed with 0.4 ml of Matrigel and 0.5 mg of peptide in a final volume of 0.5 ml, were injected s.c. into C57BL/6J mice. Tumor growth was monitored with a caliper, and the volume was determined using the formula width2 x length x 0.52. Tumors were removed, weighed, and fixed with formalin 12 days postinjection. The number of vessels was determined by staining with a rat polyclonal anti-CD31/PECAM-1 antibody that recognizes platelet-endothelial cell adhesion molecule-1 (PECAM-1) on endothelial cells (1:100 dilution; PharMingen, San Diego, CA). Antibody binding was detected with the use of an EnVision+ peroxidase system (DAKO, Carpinteria, California), and the number of blood vessels was counted in six fields per three randomly chosen sections. The number of apoptotic cells was determined by terminal deoxynucleotidyltransferase (TdT) labeling of fragmented DNA [TdT-mediated nick end labeling (TUNEL); Paragon Bioservices, Inc., Baltimore, MD]. Six x100 fields were counted from at least three different tumors for each experimental condition. This experiment was repeated twice using six mice per data point.
Cell Proliferation.
Proliferation of B16-F10 cells was quantified using a Cell Titer 96 Aqueous Cell Proliferation assay kit (Promega, Madison, WI). Cells were plated on four 96-well dishes at 5 x 103 cells/well and cultured in AIM-V serum-free medium (Life Technologies, Inc., Gaithersburg, MD). After 1 h, peptides were added at a final concentration of 100 µg/ml. Separate dishes were used to quantitate proliferation at 2, 24, 48, and 72 h by reading the absorbance at 490 nm on a plate reader. Each experiment was done in triplicate at least two times.
Chick Chorioallantoic Membrane Assay.
The chorioallantoic membrane (CAM) assay was performed using embryonated eggs (CBT, Chestertown, MD) as described previously (20)
. On embryonal day 3, 4 ml of ovalbumin was removed from each egg. After opening windows on embryonal day 10, A5G peptides in 5 µl of distilled water were applied to the CAM after drying on 13-mm-diameter quartered Thermanox plastic coverslips (NUNC International, Naperville, IL). Three days later, the eggs were scored for a response and photographed. The positive control was 50 ng basic fibroblast growth factor (bFGF) and the negative control was the vehicle water. This experiment was repeated twice using a minimum of 10 eggs for each data point.
Migration and Invasion Assays.
Migration was quantified using a QCM Chemotaxis 96-Well Cell Migration assay kit (Chemicon, Temecula, CA). B16-F10 cells (5 x 104) were suspended in 100 µl of DMEM and then were placed in the upper chambers. One hundred fifty µl (100 µg/ml) of each peptide in DMEM were added to the lower chambers. The plate was incubated for 12 h at 37°C in 5% CO2 and was read with a computer-based fluorescence reader. The invasion assay was performed in a similar way, except that the upper layer of the Cell Migration Chamber Plate was coated with 20 µl of 0.1 mg/ml Matrigel (BD Biosciences, Bedford, MA) before the cells were added to the upper chambers.
Peptide-Affinity Chromatography.
Affi-gel 10 (Bio-Rad, Hercules, CA) laminin peptide affinity columns (1 ml) were prepared according to the manufacturer’s instructions and were run as described previously (5
, 6)
. The A5G27 affinity column was run in parallel with the A5G27S-negative control (scrambled peptide) column. The columns were equilibrated in running buffer containing 6.0 M urea, 1% Triton X-100, and Complete Protease Inhibitor mixture (Boehringer Mannheim, Indianapolis, IN) in Tris-buffered saline (pH 7.4). For each experiment, three 150-mm plates of subconfluent tumor cells were surface-biotinylated using sulfo-NHS-biotin (Pierce, Rockford, IL). A crude cell membrane fraction was prepared by hypo-osmotic lysis in 20 mM Tris (pH 7.4) containing 10 mM KCl, 0.1% ß-mercaptoethanol, and 1 mM EDTA. After Dounce homogenization, the nuclei were removed by centrifugation at 1,500 x g for 5 min. The NaCl concentration of the remaining supernatant was increased to 150 mM, and the cell membranes were pelleted at 50,000 x g for 30 min at 4°C. The cell membrane pellet was solubilized in Tris-buffered saline containing 2 ml of 8.0 M urea, 1% Triton X-100, 0.5 M KCl, and Complete Protease Inhibitor mixture. The insoluble material was removed by centrifugation at 14,000 x g for 20 min, and the volume was increased to 10 ml with running buffer. A 500-µl aliquot of the crude cell membrane fraction (700 µg of total protein) was incubated with either the peptide or a control peptide affinity column for 2 h at 4°C. The columns were washed with running buffer (60 ml) and then sequentially eluted with 2-ml aliquots of running buffer containing either 20 mM EDTA, 250 mM (1.0 M), or 2.0 M NaCl. Material in the eluted fractions was precipitated with acetone, washed in 80% ethanol, and air dried. Proteins were separated by SDS-PAGE (4–12% gels) and transferred to nitrocellulose filters (Novex, San Diego, CA). The filters were blocked in Tris-buffered saline with Tween 20 (TBS-T) containing 5% nonfat milk, and then were washed, incubated with streptavidin-horseradish peroxidase in TBS-T for 1 h, and then were washed three times for 10 min each in TBS-T. The biotinylated material was visualized by enhanced chemiluminescence (Amersham, Arlington Heights, IL). This experiment was repeated three times.
GAG Digestion and Western Blot.
Material eluted from the peptide affinity columns with 2.0 M NaCl was precipitated with acetone and was digested for 2 h with either chondroitinase ABC (1.0 units/ml), heparitinase (0.1 units/ml), or both, together in the presence of complete protease inhibitor mixture, as described previously (6)
. The digested material was separated by SDS- PAGE, transferred to nitrocellulose filters, and processed as above with streptavidin-horseradish peroxidase. For Western blot analysis, the same filters were blocked with 5% nonfat milk in TBS-T (0.1% Tween 20), washed, incubated with CD44 (1:500 dilution; Cymbus Biotechnology Ltd, Chandlers Ford, United Kingdom) and syndecan-1, -2, -3, and -4 (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies in 1% nonfat milk in TBS-T for 1 h. The filters were washed three times for 10 min each in TBS-T and then were incubated with a species-appropriate horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature. The filters were visualized by enhanced chemiluminescence. These experiments were performed at least three times.
Immunoprecipitation.
Immunoprecipitation of proteins isolated from the peptide affinity chromatography columns was performed using CD44 and syndecan-1, -2, -3, and -4 antibodies as specified by the manufacturers. A B16-F10 cell pellet was collected and incubated for 1 h on ice with 1 ml of 0.5% NP40 in PBS (NP-PBS) with complete protease inhibitor mixture. The supernatant was collected after centrifugation for 15 min at 11,000 x g at 4°C. Protein G beads were incubated twice, 2 h each time, with PBS containing 0.5% NP40 (NP-PBS) containing the unlabeled B16-F10 cell protein extract to prevent nonspecific binding. Beads were then incubated with control rat IgG and CD44 and syndecan 1–4 antibodies for 2 h in NP-PBS containing 0.1% BSA. Then, the precipitated 2.0 M NaCl eluant from the affinity column, digested with chondroitinase ABC and with heparitinase, was incubated with the beads in the presence of 0.1% BSA and 0.5% NP40 for 12 h. The beads were washed five times with RIPA buffer (0.15 M NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) and once in Tris buffer. Immunoprecipitated proteins were removed from the beads by boiling in sample buffer (Invitrogen, Carlsbad, CA) and then were separated on SDS-PAGE, transferred to nitrocellulose filters, and processed as above.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5 Chain G-Domain Peptides.5 G-domain peptides for cell attachment activity with B16-F10 cells to identify sites important in malignancy. As a positive control, active laminin 1 (AG73) and 1 (C16) chain peptides were used. Eleven of the synthetic peptides from the laminin 5 G-domain chain showed cell attachment activity (Table 1)
. As previously determined, positive control peptide AG73 showed strong cell attachment activity, and positive control peptide C16 showed moderate cell attachment activity. Three peptides (A5G6, -64, and -71) showed strong cell attachment activity similar to that of AG73. Three peptides (A5G3, -27, and -73) showed weaker activity comparable with that observed with the less active C16 peptide. Five peptides (A5G28, -33, -77, -81, and -101) showed much weaker cell attachment activity (Table 1)
. The other 102 synthetic peptides showed little or no attachment activity (data not shown). A scrambled version of one of the peptides, A5G27, was also inactive for cell adhesion.
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5 Chain G-Domain Peptides on In Vivo Lung Colonization.
; Fig. 1
). The other seven synthetic peptides showed no activity in vivo.
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. The tumors were examined by histology, and all four peptides inhibited tumor angiogenesis by 25–30% (Fig. 2C)
. All four of the peptides also increased apoptosis in the tumor by 4–8-fold (Fig. 2D)
, with A5G27, A5G81, and A5G101 being more active than A5G73. A proliferation assay was performed in the presence of 100 µg/ml of soluble peptides to determine whether these peptides directly affected tumor cell proliferation in vitro (Fig. 3)
. None of the four peptides affected B16-F10 cell growth in vitro. These data suggest that peptide inhibition of tumor growth is due to an effect on angiogenesis rather than a direct effect on tumor cell proliferation.
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, panel b). When mixtures containing 50 ng of bFGF and 1.0 µg of each of the four active peptides were tested in the CAM assay, all four peptides inhibited FGF-induced angiogenesis by 37 to 50% (Fig. 4)
. Peptide A5G27 (1.0 µg) alone did not affect angiogenesis (Fig. 4A)
. These data demonstrate that all four of the peptides inhibit bFGF-induced angiogenesis in vivo.
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15% with only A5G27 and A5G81 having statistical significance in the migration assay (Fig. 5)
. We conclude that the four active peptides reduce tumor cell migration and invasion.
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, Lanes 2–6) and on control scrambled peptide A5G27S-coupled Affi-gel 10 columns (Fig. 6A
, Lanes 7–11). Fractions were collected containing the buffer wash (Fig. 6A
, Lanes 2 and 7), and the 20 mM EDTA (Fig. 6A
, Lanes 3 and 8), 250 mM NaCl (Fig. 6A
, Lanes 4 and 9), 1 M NaCl (Fig. 6A
, Lanes 5 and 10), and 2 M NaCl (Fig. 6A
, Lanes 6 and 11) eluates. No proteins bound to the scrambled peptide A5G27S column. Multiple protein bands appeared in the EDTA-eluted fraction from the A5G27 column, but these bands and those in the 1 M NaCl eluate appeared to be nonspecific. The 2-M NaCl eluate demonstrated a broad, high molecular weight band (Mr 120,000–180,000), suggestive of proteoglycans, which typically run as a smear because of variations in glycosylation. Therefore, we next performed digestion of this material with glycosidases. Proteins in the 2 M NaCl affinity-purified fraction (Fig. 6B
, Lane 1) were digested with heparitinase (Fig. 6B
, Lane 2), chondroitinase ABC (Fig. 6B
, Lane 3), or a combination of both (Fig. 6B
, Lane 4). The band treated with heparitinase migrated slightly faster than the untreated material, suggesting some degradation. Treatment with chondroitinase ABC and a combination of both glycosidases showed almost complete digestion of the high molecular weight material, resulting in Mr 90,000 and 60,000 core protein bands. Using this same membrane with the various glycosidase digests, we probed by Western blot for cell surface proteoglycans CD44 and syndecans. CD44 antibody stained the core proteins (Mr 90,000 and 60,000; Fig. 6C
, Lanes 3 and 4), but did not stain the high molecular weight band (Fig. 6C
, Lane 1 and 2). Syndecan-1, -2, -3, and -4 antibodies did not stain any bands (data not shown). To further confirm the identity of the cell surface ligand for A5G27, immunoprecipitation of A5G27-bound membrane proteins in the 2-M NaCl eluate was performed with syndecan-1 (Fig. 6D
, Lane 2), -2, -3, and -4 (data not shown), and CD44 antibodies (Fig. 6D
, Lane 3). Beads alone were used as a negative control (Fig. 6D
, Lane 1). Only the CD44 antibody immunoprecipitated the Mr 90,000 and 60,000 bands. We conclude that the B16-F10 surface receptor for A5G27 is CD44. Because heparin blocks cell binding to this peptide (11)
, it is likely that the GAG side chains of CD44 bind directly to the peptide.
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60% inhibition. CS-A reduced cell attachment by 40% and hyaluronic acid (HA) was inactive (Fig. 7)
. These data demonstrate that B16-F10 cells bind to the laminin A5G27 peptide via the chondroitin and heparin-like GAG side chains of CD44.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5 chain, a component of laminin-10 (5ß11) and -11 (5ß21), is expressed at higher levels in adult tissues, such as lung, heart, bone marrow, pancreas, and kidney, with low levels in the brain and skeletal muscle and even lower levels in the liver, gut, and skin (21)
. Laminin-10/11 is recognized by several different receptors, including integrins 3ß1, 6ß1, 6ß4, Lutheran blood group glycoprotein, and dystroglycan (22, 23, 24)
. Furthermore, human colon carcinoma cell migration on laminin-10 is mediated by integrins 3ß1, 6ß4 (25)
.
Here, we have focused on identifying active sites for malignancy on the COOH-terminal G-domain of the 5 chain, because this region has been found to be important for the biological activity of laminins. We screened 113 synthetic peptides encompassing the entire G-domain and found 11 peptides active for B16-F10 melanoma cell adhesion in vitro. Four of the 11 peptides blocked lung colonization in vivo. The four peptides that inhibited lung colonization also blocked s.c. tumor growth and angiogenesis and increased apoptosis in vivo. These peptides also reduced B16-F10 cell invasion and migration in vitro. No effect on cell growth in vitro was observed.
Two of the peptides identified in this screening of the A5G domain, A5G81 and 101, are of interest. Both were among the most active peptides previously observed with HT1080 cells and these peptides are highly homologous to each other (Tables 2
and 3)
, with 7 of their 12 amino acids identical in the mouse and 5 homologous in the human. In addition, there is considerable homology of this sequence among the other laminin chains. Unexpectedly, the A5G101 sequence is also present in several collagen chains, in which there is 75% homology in 4(V) and 3(V) and 80% homology in 1 (IX) collagen. The conserved sequences across laminin and collagen chains suggest important functions.
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5 chain G-domain active peptides, but we were able to identify only one cellular receptor. Peptide affinity chromatography followed by immunoprecipitation and Western blot analysis identified CD44 as the cell surface receptor for peptide A5G27. The finding of a cell surface proteoglycan as the receptor for A5G27 was not unexpected, because laminin is known to bind proteoglycans, and adhesion to this peptide had previously been shown to be blocked by heparin. Furthermore, the G-domain has several heparin-binding sites (11)
. We further determined that A5G27 binds to the GAG side chains of CD44. In addition, one other peptide from the G-domain, peptide AG73, from the 1 chain has been shown to bind to syndecan-1 on a human submandibular gland cell line via its heparan sulfate side chains (5)
. The finding that peptide A5G27 binds CD44 is the first demonstration of a laminin peptide binding to CD44. Because laminin has not been previously shown to bind CD44, our data would suggest that the A5G27 site may be cryptic in the intact molecule, but this has not yet been demonstrated.
Previously, this region of the 5 G-domain has been studied using this peptide approach with HT-1080 and PC12 cells, and 21 active sites for cell adhesion were identified, with peptides A5G27, -65, -71, -73, -81, and -101 being the most active (11)
. We find that the 5 G-domain had fewer active peptides for B16-F10 melanoma cell adhesion (11 versus 21), and four peptides that were active in vivo were identified. These active peptides include A5G27, -73, -81, and -101, which were considered the most active with the HT-1080 cells. Adhesion to these four peptides was inhibited by heparin and other GAGs, but not by hyaluronic acid. Interestingly, all except A5G27 promoted neurite outgrowth (11)
. These data suggest that the peptides identified with HT-1080 and PC12 cells, and now with B16-F10 cells, are highly active. We found that the four 5 chain peptides inhibited lung colonization. In all of the screens of laminin for antimetastatic activity, only one other peptide, YIGSR from the ß1 chain (residues 929–933), strongly inhibited lung colonization (26)
. The receptor for the YIGSR peptide is a Mr 67,000 protein whose levels correlate with malignancy (27)
. Likewise, the receptor for A5G27 is CD44, a cell surface proteoglycan known to be important in malignancy. CD44 levels also correlate with malignancy. It is likely that both YIGSR and A5G27 peptides, when injected into mice, inhibit lung colonization by binding to their respective receptors and blocking tumor arrest. CD44 has been found to bind to many molecules, including osteonectin and matrix metalloproteinases, but the physiological role for these interactions in vivo has not been demonstrated (28)
. CD44 has also been implicated in many normal processes from axon guidance to limb bud development and atherosclerosis. Its role in malignancy is the most well studied. CD44 functions as a specialized surface ligand for matrix metalloproteinases and growth factors, which may be part of the mechanism by which it promotes tumor invasion and growth and angiogenesis (29, 30, 31)
. Several proteases known to bind CD44 can promote invasion and metastasis by multiple mechanisms. For example, such proteases can activate transforming growth factor ß (TGF-ß) and cleave collagen IV, potentially generating angiogenic or antiangiogenic fragments (32
, 33)
. Interestingly, it has also been shown that the binding of hyaluronan to cell surface CD44 reduces tumor cell apoptosis (34)
. CD44 is present on endothelial cells (35
, 36)
, and our finding of A5G27-reducing bFGF-induced angiogenesis suggests that this peptide also affects angiogenesis via its binding to CD44. Thus, CD44 can promote malignancy by multiple pathways. CD44 is a single gene, but alternate splicing generates several different proteins. In addition, there is heterogeneity in the GAG side chains. Proteolytic cleavage of CD44 also results in additional forms of the proteoglycan. Many of the different activities of CD44 are likely dependent on the structures and posttranslational modifications. Our finding of a broad band of material in the 2.0-M NaCl eluate of the peptide affinity column indicates that the material that binds to the peptide is heterogeneous in its GAG content. Furthermore, we identified two major protein cores, Mr, 90,000 and 60,000, suggesting that possibly two different splice variants bind to the peptide. Because binding is via the covalently attached GAG chains, it is not unexpected that if different splice variants are present, they could both bind to B16-F10 melanoma cells.
Clearly, the sequences in peptides A5G27, -73, -81, and -101 are important active sites in laminin. Their ability to reduce tumor cell metastasis and angiogenesis and to increase apoptosis may be important in the development of therapeutics for malignancy and possibly for other biological processes regulated by CD44.
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FOOTNOTES |
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Requests for reprints: Hynda K. Kleinman, Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, NIH, Building 30, Room 433, 30 Convent Drive, MSC 4370, Bethesda, MD 20892. Phone: 301-496-4069; Fax: 301-402-0897; E-mail: hkleinman{at}dir.nidcr.nih.gov
Received 1/14/04. Revised 4/ 1/04. Accepted 5/12/04.
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1 chain. J Biol Chem, 273: 32491-9, 1998.-1 chain by systematic peptide screening. J Biol Chem, 272: 32198-205, 1997. chain. J Biol Chem, 276: 22077-85, 2001.3ß1, 6ß1 and 6ß4 integrins. J Cell Sci, 113: 869-76, 2000.[Abstract]
5 through expression of chimeric laminin chains in vivo.. J. Biol Chem, 277: 44864-9, 2002.3ß1, 6ß4 integrins. Exp Cell Res, 266: 1-10, 2000.
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, Control; 
, A5G27; 
, A5G73; 
, A5G81; 
, A5G101. B, A5G peptides inhibited B16-F10 melanoma tumor weight. Tumors were removed and weighed at 12 days postinjection. *, P < 0.05; **, P < 0.025; ***, P < 0.01. C, A5G peptides inhibited B16-F10 melanoma angiogenesis in s.c. tumors. Vessels were counted from six x10 fields from three different tumors. *, P < 0.025; **, P < 0.01. D, A5G peptides increased the apoptotic index of tumor cells. The apoptotic index [terminal deoxynucleotidyltransferase-mediated nick end labeling(TUNEL)] was determined by the percentage of stained cells scored from six x100 fields from three different tumors for each peptide. *, P < 0.025; **, P < 0.005; ***, P < 0.001.
