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J. Biol. Chem., Vol. 275, Issue 28, 21255-21261, July 14, 2000

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Tandem Repeats Are Involved in G1 Domain Inhibition of Versican Expression and Secretion and the G3 Domain Enhances Glycosaminoglycan Modification and Product Secretion via the Complement-binding Protein-like Motif^*

Bing L. Yang, Liu Cao, Chris Kiani, Vivian Lee, Yaou Zhang, Mark E. Adams^§, and Burton B. Yang^¶

From the Sunnybrook & Women's College Health Sciences Centre and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M4N 3M5, Canada

Received for publication, February 22, 2000, and in revised form, May 3, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The large aggregating chondroitin sulfate proteoglycans, including aggrecan, versican (PG-M), neurocan, and brevican, are characterized by N-terminal and C-terminal globular (or selectin-like) domains known as the G1 and G3 domains, respectively. For this study, we generated a series of expression constructs containing various combinations of chicken versican/PG-M domains and a leading peptide of link protein in order to examine the roles of the G1 and G3 domains in versican function. In transfection studies, we observed that the presence of the G1 domain was sufficient to inhibit product secretion, while the G3 domain enhanced this process. We also demonstrated that the G1 domain inhibited the attachment of glycosaminoglycan chains to the core proteins, while the G3 domain enhanced this process. Further studies revealed that inhibition of secretion by G1 was mediated by its two tandem repeats, while G3's promotion of glycosaminoglycan chain attachment was apparently dependent on G3's complement-binding protein (CBP)-like motif. The modulatory effects of these two molecular domains may contribute to versican's biological activities.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Versican, a large aggregating chondroitin sulfate proteoglycan, was initially isolated from human fibroblasts (1, 2). The homologous molecule in chicken, PG-M, was first cloned from developing mesenchymes (3) and exists in at least four isoforms as a result of alternative splicing (4-6). Versican is expressed in a wide variety of tissues, including embryonic tissues (1, 7-9), central and peripheral nervous system, the luminal surface of glandular epithelia (10), blood vessels in normal (11) and tumor (12) tissues, dermis, and the proliferative zone of the epidermis (13). Its expression can be tightly regulated; e.g. during hair growth, versican expression is regulated cyclically, correlated to the hair growth cycle itself (14).

Characteristic structural features of this molecular family include a globular domain (G1) at the amino terminus directly following signal peptide, and a globular domain known as the G3 (or the selectin-like) domain at the carboxyl terminus (15). A large sequence named for its role in chondroitin sulfate (CS) chain attachment is situated between G1 and G3. The G1 and G3 domains are highly conserved (between species and within this proteoglycan family). The G1 domain is composed of one immunoglobulin (IgG)-like motif and two tandem repeats. This domain has the same structure as link protein, which binds hyaluronan (16, 17). The G3 domain is composed of two alternatively spliced epidermal growth factor (EGF)¹-like motif(s), one lectin (also known as carbohydrate recognition domain or CRD)-like motif, one complement-binding protein (CBP)-like motif, and a short carboxyl-terminal tail.

Given their ubiquitousness and their high degree of conservation, it is likely that G1 and G3 play vital roles in proteoglycan functions, but the details of these roles are only beginning to be elucidated. In the fatal chicken disorder known as nanomelia, for instance, the core protein of the proteoglycan aggrecan contains a premature stop codon N-terminal to the G3 domain. This truncated aggrecan does not contain any glycosaminoglycan (GAG) chains and is poorly secreted, suggesting that the presence of a G3 domain is crucial for proteoglycan processing and function. This study was designed to investigate the roles of G1 and G3 domains in chicken versican/PG-M synthesis and activities. We have generated a number of recombinant constructs to examine these effects. We observed that the presence of the G1 domain inhibits the attachment of glycosaminoglycan chains and secretion of the products, while the G3 domain enhances both processes. Furthermore, on the subdomain level, we demonstrated that the tandem repeats of G1 are in a large part responsible for its inhibition of versican secretion, and it is G3's CBP motif that seems to enhance GAG attachment to versican.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- The reverse transcription-PCR kit was from CLONTECH. Taq DNA polymerase, T4 DNA ligase, and restriction endonucleases were from Roche Molecular Biochemicals. Bacterial growth medium was from Difco. The Prep-A-Gene DNA purification kit, prestained protein markers, and protein assay kit were from Bio-Rad. The DNA Mini-prep kit was from Bio/Can Scientific, and the DNA Midi-prep kit was from Qiagen Inc. Lipofectin, Geneticin (G418), Dulbecco's modified Eagle's medium (DMEM) growth medium, fetal bovine serum (FBS), Hanks' balanced salt solution, and trypsin/EDTA were from Life Technologies, Inc. The ECL Western blot detection kit was from Amersham Pharmacia Biotech. Horseradish peroxidase-conjugated goat anti-mouse IgG was from Sigma. 24- and 96-well tissue culture plates were from Nunc Inc. All chemicals were from Sigma.

Generation of Recombinant Chicken Versican Constructs-- The following constructs were described previously (18, 19): miniversican, mutant miniversican (versicanEGF), G1, G3, mutant G3 (G3EGF), miniversican lacking the G3 domain (G1CS), miniversican lacking the G1 domain (CSG3), and aggrecan G3 (aG3). Each construct contains a leading peptide (60 amino acids) originally obtained from link protein (17), which contains a signal peptide and an epitope recognized by the monoclonal antibody 4B6 (20). In the original cloning procedure, the G1 domain was synthesized using primers G1N and G1Ca, the CS fragment was synthesized by CSNa and CSC, and G3 was synthesized by G3Na and G3C in reverse transcription-PCR reactions. The sequences of these primers are given in Table I. The identity of each fragment was confirmed by sequencing. For this study, eight new constructs containing different combinations of motifs from versican were also produced, for a total of 16 recombinant constructs. These new constructs included G1CSCRD, G1CSCBP, G1G3, IgG, TR, CBP, CS, and CRD. Using the protocol described by us previously (18, 19, 21, 22), we generated these constructs by subcloning and PCR methods employing the primers described in Table I.

Briefly, to generate the G1CSCRD construct, CRDN and CRDC were used as primers in a PCR using the G3 domain as a template. The PCR products were purified by agarose gel electrophoresis and digested with XhoI and XbaI. The digested products were purified again and used to replace the G3 fragment of the miniversican construct that had been digested with XhoI and XbaI (a cloning site in the vector) to remove the G3 fragment. In the same way, CBP fragment was synthesized with CBPN and G3C as primers and G3 domain as a template in a PCR. The PCR products were digested with XhoI and SphI and purified. The purified products were used to replace the G3 domain of the miniversican construct, which had been digested with XhoI and SphI (a cloning site in the vector).

To produce the G1G3 construct, LPN and G1Cc were used as primers and the miniversican as a template in a PCR. The PCR products were digested with EcoRI and XhoI and purified. The purified products were inserted into an EcoRI- (a cloning site in the vector) and XhoI-digested miniversican construct, from which the leading peptide, the G1 domain, and CS sequence had been removed by EcoRI and XhoI digestion. In a similar way, LPN and IgGC were used as primers in a PCR using the miniversican as template. The PCR products were digested with EcoRI and XbaI, and the purified products were inserted into EcoRI- and XbaI-digested pcDNA3 plasmid to obtain the IgG construct.

To generate the TR and CBP constructs, TR was synthesized using TRN and G1Cb as primers, while CBP was synthesized using CBPN and G3C as primers. Both products were digested with XhoI and SphI. The leading peptide was synthesized with LPN and LP60b to generate an EcoRI restriction site at the amino terminus and an XhoI site at the carboxyl terminus of the leading peptide. Thus, the leading peptide, one TR fragment, and one CBP fragment were inserted into EcoRI- and SphI-digested pcDNA1 plasmid.

To generate the CS construct, CS sequence was synthesized using CSNb and CSC as primers in a PCR. The PCR products were digested with BamHI and XhoI. The leading peptide was synthesized with LPN and LP60a and digested with EcoRI and BamHI. Both fragments were inserted into EcoRI- and XhoI-digested pcDNA3 plasmid.

To produce the CRD construct, CRD was synthesized using CRDN and CRDC as primers in a PCR. The PCR products were digested with XhoI and XbaI and purified. The purified products were inserted into an XhoI- and XbaI-digested CBP construct, in which the XhoI site was situated between the leading peptide and the CBP fragment, while the XbaI site was located 3' of the SphI site.

Expression of Recombinant Constructs-- COS-7 cells, astrocytoma cell line U87, or NIH3T3 fibroblasts, cultured in six-well plates at a density of 1.5 × 10⁵ cells/well, were allowed to attach and grow overnight in DMEM supplemented with 5% FBS. The following day, Lipofectin (10 µl) was incubated with plasmid DNA (2 µg) for 15 min in 200 µl of DMEM followed by the addition of 800 µl of DMEM. The Lipofectin-DNA mixture was added to washed cultures, and the cultures were incubated in an incubator at 37 °C. After 5 h, the DNA/Lipofectin mixture was replaced with 2 ml of DMEM supplemented with 5% FBS. The culture medium and cell lysate were harvested separately after 3 days, and samples were frozen at 20 °C until analysis.

Analysis of Recombinant Gene Products on Western Blot-- Cell lysate and growth medium that contained recombinant gene products were harvested from transfected cultures and subjected to SDS-PAGE. Immunoblotting was carried out as described previously (18, 19). All products were detected using the 4B6 antibody.

Densitometry Analysis-- Densitometer readings were used to estimate relative protein concentration from the intensity of the chemiluminescent signal, according to the manufacturer's instructions (Molecular Dynamics Inc., Sunnyvale, CA). Results were reported below each Western blot analyzed.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The G1 Domain of Versican Inhibits Glycosaminoglycan Chain Attachment and Secretion of Recombinant Gene Products via the Tandem Repeats-- This study is designed to investigate the roles of the G1 domain and the G3 domain in versican biosynthesis and secretion. We have previously observed that the versican signal peptide is not sufficient for secretion of the G1 domain (data not shown). Replacing the native versican signal peptide with the leading peptide of link protein resulted in secretion of G1. For reasons of consistency, we have substituted the link protein leading peptide for the endogenous signal peptide in every construct used in this study (Fig. 1).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Diagram of recombinant genes used in this study. The strategy for construction of the miniversican gene and other constructs is shown. The versican G1 domain includes nucleotides 145-1182 of mature versican; the truncated CS region includes nucleotides 1183-2424; and the G3 domain includes nucleotides 9904-10830. The leading peptide added to all constructs was obtained from link protein (nucleotides 1-180). The miniversican contains two EGF-like motifs. Numbers above the schematic correspond to nucleotides in the sequence of full-length versican.

To investigate the role of the G1 domain of versican in product secretion, the CS and G1 constructs (Fig. 1) were expressed in COS-7 cells as described under "Experimental Procedures." Growth medium was collected and analyzed on Western blot probed with the monoclonal antibody 4B6, which recognizes an epitope within the leading peptide. G1 and CS products were produced and secreted to the media as expected (Fig. 2A), except that CS expression was significantly higher than that of G1.

View larger version (68K): [in this window] [in a new window]   Fig. 2.   The G1 domain inhibits products secretion and glycosaminoglycan chain attachment. A, COS-7 cells were transiently transfected with the recombinant constructs CS and G1. The cultures were maintained in DMEM, 5% FBS. Culture medium was harvested as described under "Experimental Procedures," subjected to SDS-PAGE (2, 5, 10, and 30 µl per well as indicated) in a 7% gel. The separated proteins were then electroblotted onto a nitrocellulose membrane and probed with 4B6 antibody, which recognizes an epitope in the link protein leading peptide. In this and all other figures, numbers below the Western blot correspond to the densitometric analysis of each band, performed using software from Molecular Dynamics. The intensity of bands representing the expressed products was much higher for the CS products than for the G1 products. Due to saturation of the film, the readings do not correlate proportionally to increases in volume loaded in each well. B, COS-7 cells were transiently transfected with the G1CS and CS constructs as described in A. Culture medium was harvested, and varying amounts (as shown) were subjected to 5% SDS-PAGE and immunoblotting using the 4B6 antibody. Note that G1CS exhibits reduced GAG chain attachment to the core proteins than that of the CS construct, as indicated by the weak smears present in the G1CS samples (and confirmed by densitometric analysis).

Using these constructs, we then compared glycosaminoglycan chain attachment to the CS-bearing sequence of the core protein. COS-7 cells were transfected with G1CS and CS constructs, and the growth medium was analyzed on Western blot. Both constructs produced a characteristic proteoglycan smear after immunoblotting (Fig. 2B), indicating that the core proteins of the recombinant constructs were modified by GAG chain attachment. However, the CS construct exhibited higher levels of GAG modification than did the G1CS construct.

In view of the reduced secretion of the G1 product, we sought to identify specific motif(s) in the G1 domain responsible for inhibition of secretion. We thus generated an IgG construct and a TR construct containing two tandem repeats (Fig. 1) and expressed them in COS-7 cells. Cell lysate and culture medium were harvested and analyzed on Western blot. Both constructs were well expressed, and significant amounts of products were found in the cell lysate (Fig. 3). However, while the product of the IgG construct was secreted into the medium at high levels, the product of the TR construct was not observed in the media at all. Prolonged exposure of the Western blot revealed two weak bands in the lane containing the greatest amount of loaded media, suggesting glycosylation of the products. We further investigated the time course of biosynthesis and secretion of TR products. COS-7 cells were transfected with the TR construct and cultured for 2, 3, or 5 days after transfection. Western blot analysis showed that TR products were expressed after only 2 days of transfection, but secretion was weak even 5 days after transfection (Fig. 4).

View larger version (60K): [in this window] [in a new window]   Fig. 3.   The tandem repeats in G1 inhibit product secretion. COS-7 cells were transiently transfected with the recombinant constructs IgG and TR. After 3 days, cell lysate and culture media were harvested and subjected to SDS-PAGE on a 10% gel in the amounts indicated. After electroblotting, samples were probed with 4B6. The products of IgG were well secreted to the culture medium, but the products of TR was hardly detected in the medium. Two weak bands only appeared when large amounts of TR were loaded, and the membrane was exposed to film for a prolonged time.

View larger version (53K): [in this window] [in a new window]   Fig. 4.   A time course of TR product secretion. COS-7 cells were transiently transfected with the TR construct and cultures were maintained in DMEM, 5% FBS for different periods as indicated in the figure (2, 3, or 5 days). Cell lysate and culture media were harvested, and 10 or 20 µl was analyzed on Western blot probed with 4B6. Secretion of TR products to the culture media was readily detected after 3 days.

The G3 Domain of Versican Enhances GAG Chain Attachment and Promotes Secretion of Recombinant Gene Products via the CBP Motif-- The work done in this laboratory and others has suggested that the G3 domain has important physiological functions (18, 19, 23-26). This led us to investigate the role of the versican G3 domain in biosynthesis and product secretion. We transfected COS-7 cells with the G1 and G3 constructs. Gene products were analyzed as above. Transfected cells secreted significantly greater amounts of G3 than G1 (Fig. 5A). Cell lysate obtained from G3-transfected cells also contained a higher level of the G3 product. A similar level of secretion was observed in cells transfected with the CSG3 construct, implying that the G3 domain of the recombinant gene plays an important role in biosynthesis and secretion of proteoglycans. Further confirmation came from comparison of the G1CS and CSG3 constructs. COS-7 cells transfected with CSG3 construct produced significantly higher levels of products, and these products were secreted at greater levels than were G1CS gene products (Fig. 5B). This experiment also showed that the levels of GAG chain modification on the G1CS and CSG3 core proteins were very different. The sizes of the core proteins of G1CS and CSG3 constructs are similar, so the fact that the smear bands in CSG3-transfected cells (Fig. 5B, medium) were much more intense than in G1CS-transfected cells suggested that there were more GAG chains attached to the CS sequence of CSG3 construct than to G1CS construct and that more products of CSG3 were secreted to the medium than G1CS products. These results suggested again that the G3 domain promoted GAG attachment.

View larger version (58K): [in this window] [in a new window]   Fig. 5.   The G3 domain enhances glycosaminoglycan chain attachment and product secretion. A, COS-7 cells were transiently transfected with the recombinant construct G1CS, CSG3, G1, or G3 and maintained as above. To examine product secretion, 10 or 20 µl of culture media and cell lysate were subjected to SDS-PAGE on a 7% gel. Probing the electroblotted samples with 4B6 revealed that G3 and CSG3 exhibited higher levels of secretion, as compared with G1. B, to test the effect of G1 and G3 on GAG chain modification, culture medium and cell lysate were harvested from cells transfected with G1CS and CSG3 constructs. The level of GAG chain attachment to CSG3 was significantly higher than that to G1CS.

In view of the significant but distinct effects of these two domains, we generated a G1G3 construct to examine interdomain interaction. Its expression and secretion were analyzed as above. More G1G3 product was observed in the cell lysate than in the culture medium (Fig. 6). This suggested that secretion of the G3 product is inhibited by G1 when the two domains are physically linked.

View larger version (50K): [in this window] [in a new window]   Fig. 6.   G1 inhibits G3 secretion. Culture medium and cell lysate from COS-7 cells transfected with G1G3 were harvested and analyzed on Western blot in various amounts (indicated in µl) probed with 4B6. Significantly more G1G3 products were observed in the cell lysate than in the culture medium.

The G3 domain is composed of two EGF-like motifs, a CRD-like motif and a CBP-like motif. When the two EGF-like motifs were deleted from the G3 domain, the resulting G3EGF construct was synthesized by transfected cells and secreted to the medium at the same levels as the G3 product itself (Fig. 7A). Aggrecan G3 domain, which does not contain any EGF-like motifs, showed similar behavior. Consistent results were also observed when the two EGF-like motifs were removed from the miniversican construct. Miniversican and versicanEGF showed comparable levels of biosynthesis, GAG chain attachment, and product secretion (Fig. 7B).

View larger version (47K): [in this window] [in a new window]   Fig. 7.   The EGF-like motifs have no effect on biosynthesis and product secretion. A, versican G3 and G3 lacking the EGF-like motifs (G3EGF) were transiently expressed in COS-7 cells along with aggrecan G3 (aG3). Growth medium and cell lysate were analyzed on Western blot probed with 4B6. The products of all constructs were well secreted. B, 15 or 30 µl of growth medium and cell lysate from cells transfected with the versican minigene or versicanEGF was analyzed on Western blot. Both constructs migrated as proteoglycan smears, as expected, and no significant differences in GAG attachment and product secretion in both constructs were observed. Due to its smaller size, versicanEGF migrated slightly faster than miniversican.

When CRD and CBP constructs were transfected into COS-7 cells, their products were secreted to the culture media (Fig. 8A). However, CBP was synthesized and secreted in greater amounts than was CRD. Concomitant with this, CBP products showed higher levels of modification by glycosaminoglycan chains and product secretion, resulting in more intense smears on the blot. To test the effects of the CRD and CBP motifs on GAG chain attachment to the CS sequence, COS-7 cells were transfected with constructs lacking EGF-like motif plus one of these two motifs (G1CSCRD and G1CSCBP constructs). Three days after transfection, culture media and cell lysate were prepared and analyzed on Western blot. The results revealed that GAG chains attached to the core proteins of both constructs; however, the levels of GAG chain modification were significantly higher on the G1CSCBP products than on G1CSCRD products (Fig. 8B).

View larger version (54K): [in this window] [in a new window]   Fig. 8.   CBP enhances the addition of glycosaminoglycan chains. A, to examine product secretion of CRD and CBP products, COS-7 cells were transiently transfected with these constructs, and 5 and 10 µl of growth medium and cell lysate were analyzed on Western blot probed with 4B6. Although both were well expressed and could be easily detected, the levels of CBP products were higher in both cell lysate and culture medium. As well, CBP was modified by glycosaminoglycan chain attachment, resulting in diffuse bands in the lysate and media while CRD was less modified. B, to test the effect of CBP motif on GAG attachment to the CS sequence, COS-7 cells were transiently transfected with G1CSCRD and G1CSCBP constructs and the products were analyzed as above. Note that in both cases, CBP-containing bands were more diffuse, indicating higher levels of GAG chain modification.

The synthesis and secretion of the G1 and G3 constructs were also examined in cell types other than COS-7 cells. Both constructs were transiently expressed in astrocytoma cell line U87 and NIH3T3 fibroblasts. Analysis of culture medium and cell lysate from transfected cultures indicated that the products of G1 were poorly secreted as compared with G3 products, although both constructs were well synthesized (Fig. 9).

View larger version (43K): [in this window] [in a new window]   Fig. 9.   Synthesis and secretion of G1 and G3 constructs in other cell types. Culture medium and cell lysate from NIH3T3 fibroblasts (A) and astrocytoma cell line U87 (B) transfected with G1, G3, and a control vector were harvested and analyzed on Western blot probed with 4B6. More G3 products were detected in culture medium than G1 products, although the levels of G1 and G3 products were similar in cell lysate. Due to the poor secretion, 20 µl of culture medium from G1-transfected cells was loaded, while the rest of the samples were loaded at 10 µ l/well.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Versican, also known as PG-M, is a member of the large aggregating chondroitin sulfate proteoglycan family (1, 3). This family also includes aggrecan (23), neurocan (27), and brevican (28). We have previously demonstrated that versican reduced cell adhesion (29, 30) and stimulated cell migration (31) via the G1 domain and promoted cell proliferation (18) and inhibited cell differentiation (19) via the G3 domain. This study was motivated by reports indicating that the G1 and G3 domains of aggrecan are also important in the synthesis and secretion of this proteoglycan (25, 32). The roles of versican G1 and G3 domains are not yet defined, but since versican expression is highly regulated during tissue development and maturation, we investigated the possibility that the G1 and G3 domains of versican play a part in this regulation.

To perform this study, we found it necessary to substitute the link protein leading peptide for the versican signal peptide, to permit maximal secretion of secretable products, since preliminary studies showed that constructs containing the versican signal peptide were retained within the cells. The presence of this peptide also allowed detection of all constructs using a single antibody (4B6, which recognizes an epitope in the link protein leading peptide). We observed that products of the G1 and G3 constructs containing the link protein leading peptide were able to secrete to the culture media. However, comparison of the levels of G1 and G3 products in the culture medium and in the cell lysate indicated that product secretion of G1 was inhibited. This result was confirmed in studies comparing a miniversican construct with a miniversican construct lacking the G1 domain (CSG3) and comparing a G1CS construct with a CS construct. In all cases, expression and secretion of the G1-containing constructs were reduced compared with controls. The inhibition of G1 secretion and promotion of G3 secretion were also observed in NIH3T3 fibroblasts and astrocytoma cell line U87.

Given that the CS sequence by itself was well synthesized, modified by GAG chains, and secreted, the addition of G1 to this sequence had dramatic inhibitory effects on its synthesis, GAG chain attachment, and product secretion. On the other hand, G3's stimulatory effects on synthesis and secretion were not evident in the CSG3 construct, since CS was already synthesized and secreted at high levels. The effects of G3 were seen by contrasting the G1CS construct and G1CSG3 (miniversican) construct, since the inclusion of G3 led to increased GAG chain attachment and secretion.

Although versican is expressed in a variety of tissues, its expression is strictly regulated. For example, versican expression is highly elevated in the early stages of development (7, 8). As a result, developing mesenchymes contain high levels of versican. In these stages, versican may enhance cell proliferation and the growth of limb buds as we have demonstrated that versican stimulated growth of NIH3T3 fibroblast (18) and chicken chondrocytes (30). However, during maturation, versican expression is significantly down-regulated (13), and the mature cartilage contains small amounts of versican that are only detected with reverse transcription-PCR techniques (33). Versican expression may be down-regulated to allow cell differentiation. We earlier demonstrated that versican inhibits mesenchymal chondrogenesis (19), and versican expression has been shown to be up- and down-regulated during hair growth cycles (14); versican expression is up-regulated during hair growth and down-regulated just before hair enters the next cycle (14). We speculate that versican is subjected to dual modes of regulation, mediated via G1 and G3 domains. Cells may have developed pathways that allow versican up- or down-regulation (via G3 or G1, respectively) during various stages, to promote or inhibit cell growth and differentiation. In tissues where moderate levels of versican are required, both G1- and G3-regulating pathways would function to permit moderate expression and maintain a balance in versican expression.

The significance of the effects of G1 and G3 on GAG chain attachment are not immediately clear. Two lines of evidence suggest that regulation of GAG chain attachment may be important during tissue development. It has been reported that attachment of GAG chains changes during tissue growth, and the types and numbers of GAG chains vary during different growth stages (34). Furthermore, the GAG chains of versican have been reported to be involved in reducing cell adhesion (35). Since a decrease in cell adhesion is required for cell proliferation, cell migration, and tissue outgrowth, it seems that G3's promotion of versican synthesis, GAG chain attachment, and product secretion are relevant to tissue growth. G1's ability to inhibit these processes may be required for cell differentiation and tissue maturation. Although the concentration of versican may change to meet the requirements for tissue growth and maturation, it is not known if G1 and G3 are directly involved in the synthesis and attachment of GAG chains to versican core protein during tissue growth and differentiation. In vitro observations could well differ from in vivo effects. The significance of G1-mediated inhibition and G3-mediated enhancement of GAG chain attachment in cell culture awaits confirmation in tissue studies.

	ACKNOWLEDGEMENT

We thank Dr. Paul F. Goetinck for the 4B6 monoclonal antibody.

	FOOTNOTES

^* This work was supported by Medical Research Council of Canada Grant MOP-13730 (to B. B. Y.).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.

The first two authors contributed equally to this study.

^§ On sabbatical from the University of Calgary.

^¶ Scholar of the Arthritis Society of Canada. To whom correspondence should be addressed: Research Bldg., Sunnybrook & Women's College Health Sciences Centre, 2075 Bayview Ave., Toronto, Ontario M4N 3M5 Canada. Tel.: 416-480-5874; Fax: 416-480-5737; E-mail: Burton.Yang@swchsc.on.ca.

Published, JBC Papers in Press, May 5, 2000, DOI 10.1074/jbc.M001443200

	ABBREVIATIONS

The abbreviations used are: EGF, epidermal growth factor; CRD, carbohydrate recognition domain; CBP, complement-binding protein; GAG, glycosaminoglycan; CS, chondroitin sulfate; TR, tandem repeat; versicanEGF, a miniversican construct lacking two EGF-like motifs; G3EGF, versican G3 domain lacking two EGF-like motifs; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				Y. Wu, L. Chen, P.-S. Zheng, and B. B. Yang beta 1-Integrin-mediated Glioma Cell Adhesion and Free Radical-induced Apoptosis Are Regulated by Binding to a C-terminal Domain of PG-M/Versican J. Biol. Chem., March 29, 2002; 277(14): 12294 - 12301. [Abstract] [Full Text] [PDF]

				M. Oleszewski, P. Gutwein, W. von der Lieth, U. Rauch, and P. Altevogt Characterization of the L1-Neurocan-binding Site. IMPLICATIONS FOR L1-L1 HOMOPHILIC BINDING J. Biol. Chem., October 27, 2000; 275(44): 34478 - 34485. [Abstract] [Full Text] [PDF]

				J. D. Sandy, J. Westling, R. D. Kenagy, M. L. Iruela-Arispe, C. Verscharen, J. C. Rodriguez-Mazaneque, D. R. Zimmermann, J. M. Lemire, J. W. Fischer, T. N. Wight, et al. Versican V1 Proteolysis in Human Aorta in Vivo Occurs at the Glu441-Ala442 Bond, a Site That Is Cleaved by Recombinant ADAMTS-1 and ADAMTS-4 J. Biol. Chem., April 13, 2001; 276(16): 13372 - 13378. [Abstract] [Full Text] [PDF]

				Y. Wu, Y. Zhang, L. Cao, L. Chen, V. Lee, P.-S. Zheng, C. Kiani, M. E. Adams, L. C. Ang, F. Paiwand, et al. Identification of the Motif in Versican G3 Domain That Plays a Dominant-negative Effect on Astrocytoma Cell Proliferation through Inhibiting Versican Secretion and Binding J. Biol. Chem., April 20, 2001; 276(17): 14178 - 14186. [Abstract] [Full Text] [PDF]

				L. Chen, Y. Wu, V. Lee, C. Kiani, M. E. Adams, Y. Yao, and B. B. Yang The Folded Modules of Aggrecan G3 Domain Exert Two Separable Functions in Glycosaminoglycan Modification and Product Secretion J. Biol. Chem., January 18, 2002; 277(4): 2657 - 2665. [Abstract] [Full Text] [PDF]

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