From the Trauma Research Program and Department of Laboratory Medicine and Pathobiology, Sunnybrook Health Science Centre, University of Toronto, Toronto, Ontario M4N 3M5, Canada
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ABSTRACT |
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Versican is a member of the large aggregating chondroitin sulfate proteoglycan family. We have expressed in NIH3T3 fibroblasts a recombinant versican mini-gene comprising the G1 and G3 domains and 15% of the CS domain. We observed that expression of the mini-versican gene stimulated cell proliferation as determined by cell counting and cell cycle analysis. Addition of exogenous mini-versican protein to cultured cells produced the same result. The effects of the mini-versican were greatly reduced when the G3 domain was deleted. Expression of the G3 domain alone promotes cell proliferation, and addition of purified G3 gene products to NIH3T3 fibroblasts and cultured chicken fibroblasts enhances cell growth. Further, deletion of the epidermal growth factor (EGF)-like motifs in the versican G3 domain reduced the effects of the mini-versican on cell proliferation. In the presence of the purified mini-versican protein, antisense oligonucleotides to the EGF receptor inhibited proliferation of NIH3T3 fibroblasts, compared with control sense oligonucleotides. Taken together, these results imply that versican enhances cell proliferation, and this effect is mediated, at least in part, by the action of versican EGF-like motifs on endogenous EGF receptor.
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INTRODUCTION |
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Versican (1, 2), or PG-M, is a member of the large aggregating
chondroitin sulfate proteoglycan family, which also includes aggrecan,
neurocan, and brevican (3). The core proteins of these large
proteoglycans have sizes ranging from 200 to 400 kDa. Common features
in this family include the presence of N-terminal G1 and C-terminal G3
domains, and a large chondroitin sulfate side chain-bearing sequence
localized in the middle region. In versican, 12-15 chondroitin sulfate
side chains are covalently attached to this central sequence (2, 4),
which also contains two alternatively spliced domains named GAG- and
GAG-
(5, 6). The G1 domain of versican binds hyaluronan (7). The G3
domain consists of a set of lectin- (also called carbohydrate recognition domain or CRD), epidermal growth factor
(EGF)1-, and
complement-binding protein-like subdomains. In this respect, G3 is
similar to the selectin family (8) except that the G3 domain contains
only one complement-binding protein-like subdomain while selectins
contain at least two. Functionally, selectins play a role in mediating
cell adhesion, but no such role has been identified for the G3
domain.
The versican gene (named as CSPG2) (9, 10) is expressed in a variety of tissues. As a result of alternative splicing, the protein has many isoforms, which may express different functions. Versican is suspected to play a role in cellular attachment, migration, and proliferation by interacting with cell surfaces and extracellular matrix molecules (2). It has been reported that versican interferes with the attachment of cells to various extracellular matrix components such as collagen I, fibronectin, and laminin (11) and inhibits cell adhesion (12, 13). It may also be involved in the formation of tissues that act as barriers to migratory neural crest cells and outgrowing axons during embryonic development (14). Furthermore, versican appears to regulate cell differentiation in hair follicles (15).
The tissues that contain high concentrations of versican include embryonic tissues such as human embryonic lung fibroblasts (2), the mesenchymal cell condensation area of limb buds (16, 17), the perinotochordal mesenchyme between the notochord and neural tube, and basement membranes facing the neuroepithelial cells of chicks (18). It is also distributed in embryonic aorta, lung, cornea, and skeletal muscle (18). In adult tissues, versican is detected in the loose connective tissue of various organs including the central and peripheral nervous system, the luminal surface of glandular epithelia (19), blood vessels (20), vessels of brain tumors (21), dermis, and in the proliferative zone of the epidermis (22). Since versican is highly expressed in fast growing tissues and cells, it has been suggested that versican plays a role in cell proliferation. Using molecular biological techniques, we show in this report that the expression of a truncated versican gene, or exogenous addition of a truncated versican gene product, promotes cell proliferation. Expression of the versican G3 domain alone achieves the same effect, but deletion of the EGF-like motifs greatly reduces the ability of versican to stimulate growth. Antisense oligonucleotides to EGF receptor (EGFR) inhibited cell proliferation, and the effect was greater in the recombinant versican-transfected cell line than in the vector-transfected cell line. These results suggest that the EGF-like motifs in versican G3 domain may promote cell proliferation through a direct or indirect interaction with EGFR.
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EXPERIMENTAL PROCEDURES |
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Materials--
The reverse transcription-PCR kit was from
CLONTECH. Taq DNA polymerase, T4 DNA
ligase, and restriction endonucleases were from Boehringer Mannheim.
Bacterial growth medium was from Difco. Prep-A-Gene DNA purification
kit, prestained protein markers, and the protein assay kit were from
Bio-Rad. DNA mini-prep kit was from Bio/Can Scientific. Lipofectin,
Geneticin (G418), DMEM growth medium, fetal bovine serum, Hank's
balanced salt solution, trypsin/EDTA, and
isopropyl-1-thio--D-galactopyranoside were from Life
Technologies, Inc. ECL Western blot detection kit was from Amersham
Phamacia Biotech. Goat anti-mouse IgG horseradish peroxidase and goat
anti-rabbit horseradish peroxidase were from Sigma. DNA Midi-prep kit,
Ni-NTA-agarose and MRGS·His antibody were from
Qiagen Inc. (Chatsworth, CA). [3H]Thymidine was from
Amersham. 24-well and 96-well tissue culture plates were from Nunc Inc.
All other chemicals were from Sigma. Four-day old chicken embryos
(products of Hamburger and Hamilton strains and purchased from Brampton
Chick Hatchery Co. Ltd., Brampton, Ontario, Canada) were used to
isolate mesenchymal cells from limb buds.
Construction and Expression of Recombinant Genes in Mammalian Cells-- To construct a recombinant versican gene, the G1 domain, 15% of the CS domain, and the G3 domain were individually cloned into pUC19. Briefly, a HindIII site and an MluI site were added at the 5' and 3' ends of the G1 domain using G1NHindIII (AAA AAG CTT GCC GCC ACC ATG TTG TTA AAC ATA AAA AGC) and G1CMluI (AAA ACG CGT TTC GTA GCA GTA GGC ATC AAA) as primers in a reverse transcription-PCR reaction. These primers were used to amplify nucleotides 145-1182 in the versican cDNA sequence published by Shinomura et al. (1). Total RNA was extracted from the sterna of 14-day-old-chicken embryos and used as template for reverse transcription-PCR. The PCR product was purified from agarose gel following electrophoresis using the Prep-A-Gene kit. This generated a cDNA of 1038 base pairs corresponding to the G1 domain of the published sequence (nucleotides 145-1182) (1). The G3 fragment was prepared in a similar manner: the restriction sites for XhoI and SphI were created using primers G3NXhoI (5'-AAA CTC GAG GGA CAG GAT CCA TGC AAA AGT) and G3CSphI (5'-AAA GCA TGC GCG CCT TGA GTC CTG CCA CGT). These primers are located in the nucleotide sequences 9904-9924 and 10810-10830 and amplify a 927-base pair cDNA corresponding to the G3 domain of versican (1). To clone a section of the CS sequence, chicken genomic DNA was used as template since the CS sequence is in a large exon. CSNMluI (5'-AAA ACG CGT C1183 GT AAA AAA ATT GTA TCA GAG) and CNCXhoI (5'-AAA CTC GAG A2424CT CAT TTC TGG CTC CTT TGT) were used as primers to create MluI and XhoI sites at the 5' and 3' ends of the CS sequence. These primers generate a cDNA of 1242 base pairs, containing nucleotides 1183-2424 of versican. This represents the portion of the CS sequence immediately 3' of the G1 domain, and contains a high proportion of potential sites for glycosaminoglycan chain attachment. We chose not to clone the whole 8-kilobase pair CS sequence because we are primarily interested in studying the functions of the G1 and G3 domains. All constructs obtained by PCR in the study were sequenced to confirm their identity. Finally, the three DNA fragments prepared above were ligated together, as shown in Fig. 1A, into pcDNA1 (Invitrogen). The recombinant versican gene (G1CSG3) was 3.2 kilobase pairs, and was expected to yield a core protein of 150 kDa. With the attachment of glycosaminoglycan chains, we expected that the recombinant proteoglycan would migrate on SDS-PAGE gel as a smear, at around 200 kDa or higher.
In generating the G3 construct, the leading peptide of link protein (nucleotides 1-180) (23) was joined with the versican G3 domain in order to allow secretion of the G3 gene product. The leading peptide of link protein was amplified with the primers 5'-AAA GAA TTC GCC GCC ACC ATG GCA AGT CTA CTC TTT CTG and 5'-AAA CTC GAG AGG CAG TGT GAC GTT GCC in a PCR reaction. The PCR product was purified and digested with EcoRI and XhoI. The G3 domain was derived from the G1CSG3 construct using the restriction enzymes XhoI and SphI. The G3 fragment and link protein leading peptide were ligated into the pcDNA1 vector digested with EcoRI and SphI. As well, a MRGS·His (Qiagen) epitope (MRGSHHHHHH) was generated at the C terminus of the G3 construct for staining and purification with the monoclonal antibody MRGS·His. This was obtained using a primer (AAA TCT AGA GTG ATG GTG ATG GTG ATG AGA TCC TCT CAT TGA GTC CTG CCA CGT CCT) (1), which is complementary to the 3' end of the G3 domain and contains a sequence encoding the MRGS·His epitope, and a second primer located in the 5' terminus of the G3 domain, in a PCR reaction. The reaction generated the G3 construct containing the MRGS·His epitope. The G1CS construct shown in Fig. 1A was generated by insertion of the link protein leading peptide and G1CS sequence between the EcoRI and XhoI sites of pcDNA1. The leading peptide contains an EcoRI site at its 5' end and a BamHI site at its 3' end, while the G1CS sequence contains a BamHI site at its 5' end (nucleotides 244-249) (1) and a XhoI site at its 3' end created as described above. To delete the EGF-like motifs from the G3 domain of versican, a site for the restriction enzyme XhoI was engineered at the 5' end of the CRD. This was performed by using CRDNXhoI (AAA CTC GAG C10129AA GAC ACA GAG ACT) and G3CSphI (5'-AAA GCA TGC G10830CG CCT TGA GTC CTG CCA CGT) as primers in a PCR reaction. The product, a G3 fragment lacking the EGF-like motifs (G3Western Blot Assays for Proteoglycans-- Electrophoresis of recombinant proteoglycans was performed in SDS-PAGE-Western blot assay. Cell lysate and growth medium that contained recombinant gene products were subjected to SDS-PAGE electrophoresis. The stacking gel contained 4% polyacrylamide. A 5% separating gel was used for the mini-versican product and a 12% separating gel for the G3 domain. The buffer system is 1x TG (Tris-glycine buffer, Amresco product) containing 1% SDS. Proteins separated on SDS-PAGE were transblotted onto a nitrocellulose membrane (Bio-Rad) in 1x TG buffer containing 20% methanol. The membrane was blocked in TBST (10 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) containing 10% non-fat dry milk powder (TBSTM) for 1 h at room temperature, and then incubated at 4 °C overnight with anti-G3 polyclonal antibody (1:1000 dilution) in TBSTM. The membranes were washed with TBST (three times for 30 min each) and then incubated for 1 h with goat anti-rabbit antibody conjugated to horseradish peroxidase in TBSTM. After washing as above, the bound antibody was visualized with an ECL kit according to the manufacturer's instructions (Amersham).
Electrophoresis of endogenous versican was performed in agarose gel (agarose-Western blot assay). The agarose gel (4-cm height containing 1.5% agarose in a barbital buffer containing 0.124 M Tris-Cl, 27 mM barbituric acid, 1 mM EDTA, pH 8.7) was made on top of a 1-cm conventional 10% polyacrylamide gel. The polyacrylamide gel was used only to seal the bottom of the casting stand and acted as a base for the agarose separating gel. The barbital buffer was also used as a running buffer and the electrophoresis took place at 40 V for 5 h at room temperature. Molecules, up to 2 million Da in size (blue dextran 2000), are able to enter the agarose gel. Growth media from chicken mesenchymal culture and NIH3T3 fibroblast culture of equal protein concentrations were analyzed in the gel. To allow transfer of such large molecules onto the nitrocellulose membrane, the blotting took place in TG buffer at 20 V overnight at 4 °C. The rest of the procedure was similar to the Western blot protocol described above.Digestion of Glycosaminoglycan Chains with Chondroitinase ABC-- The conditioned medium collected from the mini-versican-transfected cells was dialyzed overnight against 10 volumes of reaction buffer (100 mM Tris-HCl, pH 8.0, 30 mM sodium acetate). After a buffer change, the mixture was further dialyzed for 1 h. Meanwhile, chondroitinase ABC (Sigma, catalog no. C2905) was dissolved in the reaction buffer at a concentration of 1.33 units/ml. This enzyme was added to the dialyzed versican solution (1:1), and the mixture was incubated at 37 °C overnight to digest the glycosaminoglycan chains. In some samples, chondroitinase ABC was replaced by hyaluronidase, which was used as a control. The enzyme-digested products were subjected to SDS-PAGE and analyzed on Western blot.
Antigen Preparation and Antibody Generation-- Strategy for insertion of the G3 domain into pQE30 (Qiagen Inc., catalog no. 32149) is shown in Fig. 1A. pQE30, a bacterial expression vector, contains an epitope (MRGSHHHHHH) at its amino terminus recognized by the monoclonal antibody anti-MRGS·His. The six histidines in this epitope bind nickel and allow purification of fusion proteins on a Ni-NTA affinity column. The epitope is followed by multiple cloning sites. The G3 domain of the recombinant versican was ligated into the first cloning site, BamHI, producing an epitope-tagged G3 gene product. The G3 domain was generated using two primers G3NBamHI (AAA GGA TCC GGA CAG GAT CCA TGC AAA) and G3CSphI (as above) in a PCR reaction, as above, and ligated into the bacterial expression vector pQE30. The resulting construct contained an N-terminal MRGS·His tag and was expressed in Escherichia coli strain M15. Gene products produced by bacteria were purified on a Ni-NTA affinity column (Qiagen, catalog no. 30230) according to the manufacturer's instructions. To eliminate trace amounts of contamination that may occur during column purification, the column-purified product was subjected to preparative SDS-PAGE. To produce polyclonal anti-G3 antibody, adult New Zealand White rabbits were immunized with the SDS-PAGE purified G3 domain peptide, according to methods previously described (26). The antiserum obtained was used to monitor the expression of the recombinant genes on Western blot.
Proliferation Assays-- 1 × 104 fibroblasts were seeded into each well of 24-well plates and cultured with standard medium (DMEM supplemented with 5% fetal bovine serum). Cell numbers were determined at day 1, 2, and 3 using a cytometer.
In the chicken fibroblast proliferation assay, the fibroblasts were isolated from 14-day-old chicken embryos. Chicken skin was removed and rinsed with phosphate-buffered saline. The skin was then cut into pieces and incubated at 37 °C for 10 min in dissociation medium containing 0.1% trypsin and 0.3% collagenase (Sigma, catalog no. C-6885) dissolved in Hanks' balanced salt solution. The dissociation medium was changed, and the tissue was incubated for a further 2 h. An equal volume of growth medium was added to stop the enzyme reaction. Cells were collected by passing the mixture through a filter (70-µm nylon) and centrifuging the filtrate at 1100 rpm. The cells were then resuspended in growth medium and plated in tissue culture dishes. The cells could be used for assay after 1 week of culture. Proliferation of chicken fibroblasts was assayed by counting as above.Cell Cycle Analysis-- Flow cytometry of labeled DNA was used to analyze cell cycles. The mini-versican-transfected and vector-transfected stable cell lines were cultured in DMEM supplemented with 5% fetal bovine serum for 1-3 days. Cells were harvested with 10 mM EDTA dissolved in DMEM and collected by centrifugation at 200 × g. The cell pellets were resuspended gently in 1 ml of hypotonic propidium iodide (Sigma) solution (50 µg/ml) prepared in 0.1% sodium citrate plus 0.1% Triton X-100 for DNA staining. The samples were analyzed with a FACScan (Becton Dickinson) as described previously (27).
Purification of G3 and Mini-versican Gene Products-- To study the effect of G3 gene products on cell proliferation, G3 was purified from the G3-transfected COS-7 cells and G3-expressing bacteria. Briefly, conditioned medium from COS-7 cells that had been transfected with G3 construct, and cell lysate from G3-transformed bacteria, were dialyzed against equilibration buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 10 mM imidazole). This buffer was also used to equilibrate a Ni-NTA column. The dialyzed samples were incubated with the Ni-NTA column and washed extensively with washing buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 20 mM imidazole, 1 mM phenylmethylsulfonyl fluoride). G3 peptide was eluted with elution buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 250 mM imidazole). The volume of elution buffer used in each sample was the same as the volume of the original cultures. The purified products were dialyzed against phosphate-buffered saline, and then analyzed on SDS-PAGE and visualized with Coomassie Blue dye or silver staining.
Purification of the mini-versican products obtained from mini-versican-transfected COS-7 cells was performed as above. Lysate from vector-transfected COS-7 cells was subjected to the same purification procedure to serve as a control in order to estimate the effect of any contaminants that were bound to the column and eluted by the elution buffer. The elution buffer containing purified mini-versican and the control elution buffer were dialyzed against phosphate-buffered saline overnight with two changes of phosphate-buffered saline.Normalization of Gene Products in Conditioned Media-- To evaluate the concentration of gene product in the growth media, both Western blot and ELISA were used. Purified link protein was used as a standard in Western blots. Link protein contains an epitope recognized by a monoclonal antibody (4B6) (28) in its leading peptide, and we added this leading peptide to the G3 construct. This served a dual purpose; it allowed secretion of the G3 gene product and allowed detection by the 4B6 antibody. We transiently transfected COS-7 cells with the G3 construct and the control vector alone in order to produce the G3 gene product. Expression of the G3 construct was demonstrated with Western blot assay, using cell growth medium. Briefly, 10 µl of G3-containing growth medium were run on SDS-PAGE alongside a series of link protein dilutions (1-20 ng), electrotransferred, and probed with the 4B6 antibody. Since the antibody recognizes both link protein and the G3 construct product with the same affinity, and since both link protein and G3 proteins are approximately the same size, similar protein concentrations would produce similar band intensities after ECL development. This technique provides a reasonable estimate of gene product concentration.
For evaluation of the concentration of versican in growth medium, Western blot was not appropriate because versican migrates as a smear in SDS-PAGE, making determination of band density difficult. The ELISA assay provided a useful means of evaluating versican's concentration. In ELISA, growth medium (100 µl) from vector- and recombinant versican-transfected chicken fibroblasts was coated onto each well of a 96-well plate and incubated at 4 °C overnight. The medium was then removed and the plate was blocked with TBSTM (200 µl/well) at room temperature for 1 h. Washes and incubation with primary and secondary antibody were similar to Western blot assay. The primary antibody was rabbit anti-G3 antibody, and the secondary antibody was peroxidase-conjugated goat anti-rabbit antibody. TMB (Amersham, code RPN 2718) was used for color development. Optical density was determined using an ELISA reader set to 570 nm according to the manufacturer's instructions. The optical density value in the medium from the vector-transfected cells was assigned a value of 100%.Cell Proliferation Affected by Antisense Oligonucleotides-- To test the possible role of the EGFR in versican's effects on cell proliferation, 200 µl of NIH3T3 fibroblast suspension at a concentration of 3 × 104 cells/ml were inoculated into each well of 96-well tissue culture plates followed by addition of 50 µl of purified mini-versican solution at an estimated concentration of approximately 0.4 ng/µl. The control solution obtained from the purification was used as a negative control. Human EGF (Sigma, catalog no. E-9644) at a final concentration of 15 ng/well was used as a positive control. As well, 15 µl of sense (ATG CGA CCC TCA GGG ACC GCG, located in the 5' end of the coding sequence of EGFR) (29) or antisense (antisense 1, CGC GGT CCC TGA GGG TCG CAT, 5' end of EGFR; and antisense 2, TGC TCC AAT AAA CTC ACT GCT, 3' end of EGFR) oligonucleotides were added to the cultures at a final concentration of 3.2 µM. Cells without oligonucleotide treatment served as controls. Cultures were incubated at 37 °C in a tissue culture incubator. After 3 days, the fibroblasts were harvested by incubating the cells with 10 mM EDTA for 10 min. Cell number was determined by cell counting as described above. Each treatment was done in quadruplicate and analyzed statistically.
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RESULTS |
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Expression of a Mini-versican Gene Stimulated Cell Proliferation-- The mini-versican gene, containing the G1 domain, a portion of the central sequence, and the G3 domain, was constructed (Fig. 1A) in pcDNA1 for transient expression and in pcDNA3 for stable expression. To monitor the expression of the recombinant constructs, we raised antibody against the G3 domain. The G3 construct was expressed in E. coli M15 using the vector pQE30 (Fig. 1A) and purified on a Ni-NTA affinity column. The antibody specifically recognizes versican and the G3 construct (see below).
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Versican G3 Domain Promotes Fibroblast Proliferation-- Versican consists of a G1 domain, a G3 domain, and a central region for glycosaminoglycan chain attachment. We suspected that the G3 domain is important in stimulating cell proliferation since it has been shown that the EGF motifs in laminin and thrombomodulin are responsible for cell proliferation (30, 31), and versican's G3 domain contains two EGF-like motifs.
To determine whether recombinant G3 peptide alone was sufficient to stimulate cell proliferation, COS-7 cells were transiently transfected with the G3 construct. Expression of the G3 construct was evaluated using link protein as a standard because both the G3 and link protein contain the same epitope (4B6). The assay showed that a band produced by 10 µl of the growth medium has a density similar to 10 ng of link protein (Fig. 4A), indicating that the growth medium from G3-transfected cells contained approximately 1 ng of G3 protein per microliter of medium. Growth medium from G3-transfected COS-7 cells was mixed with fresh growth medium in different proportions, and the mixture was introduced into fibroblast cultures to test its effect on cell proliferation. The assay showed that medium containing the G3 gene product promoted cell proliferation in a dose-dependent manner, but medium from vector-transfected cells did not (Fig. 4B).
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Deletion of the G3 Domain Suppresses the Effects of Versican on Cell Proliferation-- We removed the G3 domain from our mini-versican gene making a new construct, G1CS, in pcDNA3. COS-7 cells were transiently transfected with the mini-versican, G3 and G1CS constructs, and the vector, under the same conditions. Growth media from different transfections were examined for their effects on cell proliferation. Mini-versican and G3 alone stimulated cell proliferation to a similar degree. However, the G1CS product showed a significant reduction of cell proliferation when compared with the effects of versican (Fig. 7). These results further demonstrated that the effects of the mini-versican on cell proliferation are mediated, at least in part, by the activity of the G3 domain.
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Deletion of the EGF-like Motifs Greatly Reduced the Ability of
Versican to Stimulate Cell Proliferation--
Given the importance of
the versican G3 domain in the observed proliferation effects, we sought
to characterize the effects of this domain in detail. G3 contains two
EGF-like motifs, and to investigate their role in this phenomenon, we
deleted them from the mini-versican gene to obtain the versicanEGF
construct as described under "Experimental Procedures" and shown in
Fig. 1A. Fibroblasts were transfected with the
versican
EGF construct and cell lines stably expressing the
versican
EGF construct were isolated. Growth medium collected from
these cell lines was analyzed for versican
EGF expression and
secretion, while growth medium from the mini-versican-transfected cells
was used as control. We verified that deletion of the EGF-like motifs
resulted in smaller bands which migrated slightly faster than
mini-versican on Western blot (Fig. 8
A).
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DISCUSSION |
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The large chondroitin sulfate proteoglycan versican is highly expressed in various tissues where the cells are actively metabolizing and proliferating such as in mesenchymal cells. In epidermis, versican is restricted to the proliferating zone. In cell culture, versican is highly expressed in both dermal fibroblasts and keratinocytes while the cells are actively proliferating. Once confluent, the growth rate of these cells slows and the expression of versican is turned off (22). Therefore, it is thought that versican plays a role in cell proliferation.
Chicken mesenchymal cells provide an ideal model for the study of cell proliferation and differentiation. We have previously established an in vitro model of chicken chondrogenesis2 in order to study the role of versican in chicken mesenchymal condensation and chondrogenesis. However, stable transfection of genes into chicken mesenchymal cells is not possible since chicken mesenchymal cells cannot be maintained in culture. We therefore used NIH3T3 fibroblasts as a model to investigate the role of versican. It has been well documented that integration of transfected genes into a genome is a random event (32, 33). We reported that cell lines transfected with a recombinant mini-versican grew faster, incorporated more [3H]thymidine into DNA, and exhibited faster cell-cycle turnover. Our experiments performed on at least three stably transfected fibroblast cell lines demonstrated that the enhanced cell growth was a result of expression of the mini-versican gene rather than a disruption of genes controlling cell proliferation in the genome. Furthermore, we found that the exogenous addition of recombinant gene product had the same effects on cell proliferation. As versican is a component of extracellular matrix, addition of versican into cell culture may mimic the effect of this molecule when it is secreted by the cells. This indicates that we can study the role of versican in chondrogenesis by introducing gene products generated in NIH3T3 fibroblasts into chicken mesenchymal cells. In other studies, we demonstrated that addition of matrix molecules to the cultures plays a role in mediating cell activities (34). Such an approach is useful in the investigation of the role of matrix molecules.
Versican is a large molecule and its domains exhibit characteristic properties. For example, the G1 domain, which resides at the N terminus of versican, demonstrates hyaluronan binding activity (7). The hyaluronan-binding property of the gene may be important in cell-cell and cell-matrix interactions such as those that involve CD44 (35). The G1 domain may also be involved in cell migration, since receptor for hyaluronic acid-mediated motility, another hyaluronan-binding receptor, is known to regulate cell migration (36). The central sequence is a large region situated between the G1 and G3 domains for the attachment of chondroitin sulfate side chains. It has been reported that the chondroitin sulfate chains are involved in cell adhesion (11). The lectin-like motif in the G3 domain binds tenascin-R (37, 38).
In this study, we demonstrated that the ability of a mini-versican to stimulate cell proliferation was due to the G3 domain. The G3 domain is composed of a lectin-like subdomain, a complement binding protein-like motif (2), and one or two EGF-like repeats (depending on splicing). Specifically, we demonstrated that deletion of the G3 domain significantly abolished the effect of the mini-versican on cell proliferation and that the G3 domain alone was as effective as the intact mini-versican.
The G3 domain of versican we studied contains two EGF-like repeats. The EGF-like unit is a sequence of approximately 40 amino acid residues which has a significant homology to epidermal growth factor. It contains six conserved cysteine residues which form three disulfide bonds, folding the sequence into its active form. Other amino acids in this motif are also highly conserved. Many proteins contain EGF-like regions in single or multiple copies, including urokinase; laminin B1; low density lipoprotein receptor; tissue plasminogen activator; coagulation factors IIV, IX, X, and XII; protein C, S, and Z; fibronectin; thrombomodulin; transforming growth factor; and other proteins (39). All of these proteins are known or thought to take part in protein-protein or protein-cell interactions. For example, transforming growth factor, fibronectin, laminin, and thrombomodulin are able to stimulate cell proliferation (30, 31, 40). A fragment containing the inner rodlike segments from the short arm of laminin, which are composed of EGF-like repeats, stimulated thymidine incorporation in cultured cells possessing EGF receptors but had no effect on a cell line lacking this receptor. The recombinant thrombomodulin peptide, which is composed of six EGF-like structures, induced proliferation of Swiss 3T3 cells and accelerated [3H]thymidine uptake into their DNA (30).
Based on these observations, we hypothesized that the effects of G3 on cell proliferation were mediated by the EGF-like motifs. To investigate this, we deleted the EGF-like motifs from the mini-versican and observed that deletion of the EGF-like motifs greatly, but not completely, reduced the effect of versican on cell proliferation.
It has been reported that NIH3T3 fibroblasts express low levels of functional EGF receptors and respond to EGF stimulation by exhibiting denser growth in monolayer cultures and increased DNA synthesis (41). Overexpression of EGFR conferred on these and other EGFR-negative cells heightened the response to EGF stimulation. In our study, deletion of the EGF-like motifs greatly reduced versican's ability to stimulate NIH3T3 cell proliferation, suggesting that the EGF-like motifs play a role in versican's enhancement of cell growth, perhaps by interacting with the EGFR on the cell surface. Evidence for this was provided by antisense studies in which the growth of NIH3T3 fibroblasts was inhibited by treatment with antisense oligonucleotides to mouse EGFR. However, the levels of growth inhibition of cells treated with purified mini-versican were greater as compared with the control. This suggests that EGFR is involved in the mini-versican-induced promotion of cell proliferation. The growth of control cells was also reduced after the treatment with antisense oligonucleotides, indicating that antisense treatment also interfered with basal proliferation levels generated by endogenous versican and EGFR-binding growth factors. Since deletion of EGF-like motifs did not completely abolish versican's ability to stimulate cell proliferation, it seems that this effect is not mediated entirely by these motifs. Further studies will likely lead to the identification of other elements in the versican molecule which play a role in enhancing cell proliferation.
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ACKNOWLEDGEMENTS |
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We thank Dr. M. Johnston and Dr. J. Filmus for helpful suggestions, and Jodi Braunton for preparation of this manuscript.
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FOOTNOTES |
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* This work was supported by Grant MT-13730 from Medical Research Council of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Postdoctoral Fellow of Sunnybrook Trust.
§ Scholar of the Arthritis Society of Canada. To whom correspondence should be addressed: Sunnybrook Health Science Centre, University of Toronto, 2075 Bayview Ave., Toronto, Ontario M4N 3M5 Canada. Tel.: 416-480-5874; Fax: 416-480-5737; E-mail: byang{at}srcl.sunnybrook.utoronto.ca.
The abbreviations used are: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; CRD, carbohydrate recognition domain; NTA, nitrilotriacetic acid; ELISA, enzyme-linked immunosorbent assay.
2 Y. Zhang, L. Cao, B. L. Yang, and B. B. Yang, manuscript submitted for publication.
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REFERENCES |
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