Alteration of chondroitin sulfate composition on proteoglycan produced by knock-in mouse embryonic fibroblasts whose versican lacks the A subdomain

Versican/proteoglycan-mesenchymal (PG-M) is a large chondroitin sulfate (CS) proteoglycan of the extracellular matrix (ECM) that is constitutively expressed in adult tissues such as dermis and blood vessels. It serves as a structural macromolecule of the ECM, while in embryonic tissue it is transiently expressed at high levels and regulates cell adhesion, migration, proliferation, and differentiation. Knock-in mouse embryonic (Cspg2Δ3/Δ3) fibroblasts whose versican lack the A subdomain of the G1 domain exhibit low proliferation rates and acquire senescence. It was suspected that chondroitin sulfate on versican core protein would be altered when the A subdomain was disrupted, so fibroblasts were made from homozygous Cspg2Δ3/Δ3 mouse embryos to investigate the hypothesis. Analysis of the resulting versican deposition demonstrated that the total versican deposited in the Cspg2Δ3/Δ3 fibroblasts culture was approximately 50% of that of the wild type (WT), while the versican deposited in the ECM of Cspg2Δ3/Δ3 fibroblasts culture was 35% of that of the WT, demonstrating the lower capacity of mutant (Cspg2Δ3/Δ3) versican deposited in the ECM. The analysis of CS expression in the Cspg2Δ3/Δ3 fibroblasts culture compared with wild-type fibroblasts showed that the composition of the non-sulfate chondroitin sulfate isomer on the versican core protein increased in the cell layer but decreased in the culture medium. Interestingly, chondroitin sulfate E isomer was found in the culture medium. The amount of CS in the Cspg2Δ3/Δ3 cell layer of fibroblasts with mutant versican was dramatically decreased, contrasted to the amount in the culture medium, which increased. It was concluded that the disruption of the A subdomain of the versican molecule leads to lowering of the amount of versican deposited in the ECM and the alteration of the composition and content of CS on the versican molecule.


Introduction
Versican/PG-M is a large chondroitin sulfate (CS) proteoglycan of the extracellular matrix (ECM), mainly synthesized by fibroblasts and vascular smooth muscle cells. Its core protein consists of two globular domains G1 and G3 at the N-and C-termini, respectively, and two CS attachment domains, CSa and CSb, between the two globular domains. As many as 23 CS chains can be attached to these domains, causing the molecular mass of versican to reach up to 1,000 kDa. The N-terminal G1 domain consists of A, B, B? looped subdomains.
Versican exhibits two distinct expression patterns. In adult tissues it is constitutively expressed in epithelial and connective tissues and serves as a structural macromolecule of the ECM, while in embryonic tissue it is transiently expressed at high levels and regulates cell behavior such as adhesion (9Á11), migration (12Á14), proliferation (15Á17), and differentiation (18,19). The G3 domain of versican has been shown to enhance the proliferation of NIH3T3 fibroblasts through an epidermal growth factor (EGF)-like motif (20). The G3 domain without the EGF-like motif enhances the interaction of EGF receptor (EGFR) and b1-integrin, impairing the growth of U87 astrocytoma cells (21). The V1 variant enhances proliferation and inhibits the apoptosis of NIH3T3 fibroblasts, while the V2 variant does the opposite (17). Chondroitin sulfate chains on the versican molecule bind to several biological molecules such as chemokines, CD44, and selectins. Serreno et al. reported that the short V3 isoform of versican that lacks CS chains could reverse the malignant phenotype of melanoma cells (22).
Recently, we generated knock-in mice (Cspg2 D3/D3 ) whose versican lacks the A subdomain of the G1 domain. These mice, expressing the mutant versican at a level of Â50% (compared with normal versican in wild-type mice), exhibit abnormalities in cardiovascular systems and skin, contrasting to versican-null hdf mice which die at E9.5 from severe cardiac defects (23). As hdf heterozygotes expressing Â50% normal versican are viable with no obvious phenotype, the abnormalities of embryos are likely due to decreased levels of versican deposition in the ECM by the absence of the A subdomain.
Embryonic fibroblasts obtained from Cspg2 D3/D3 mice exhibited unusual characteristics. Within 20 passages, the rate of proliferation of the Cspg2 D3/D3 fibroblasts slowed, and the cells exhibited senescence. Whereas the extracellular matrix of the wild-type fibroblasts exhibited a network structure of hyaluronan and versican, that of the Cspg2 D3/D3 fibroblasts exhibited Â30% and Â85% deposition of versican and hyaluronan (HA) without such a structure. In this experiment we examined the chondroitin sulfate composition in proteoglycan molecules from Cspg2 D3/D3 fibroblasts culture comparing them with wild-type (WT) fibroblasts culture.

Materials and methods
WT and the Cspg2 D3/D3 mouse embryonic fibroblast culture preparation Female Cspg2 WT/D3 and male Cspg2 WT/D3 mice were mated and allowed to gestate. These mice produced WT heterozygous (Cspg2 WT/D3 ) and homozygous (Cspg2 D3/D3 ) offspring whose versican lacked the A subdomain. After 12 days, the pregnant mice were killed using carbon dioxide gas. The placentas were removed, and each embryo was separated and placed in a Petri dish containing 1% penicillin/ streptomycin phosphate buffer saline (PBS) solution. Each embryo's tail was cut and collected in a microcentrifuge tube for further genotyping. The rest of the embryo was roughly cut into small pieces using scissors. Trypsin/EDTA was added to those pieces, and then incubated for 15Á30 minutes. Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin was added to stop the reaction. The medium was removed by suction, and the remaining tissue was passed through a cell strainer to get single cells. Medium was then added, and the cells were cultured until they reached confluence. At the first and second passages, fibroblasts in the culture were collected and stored in liquid nitrogen for future use.

Mouse embryo genotyping
Each mouse embryo tail was soaked in proteinase K overnight. Genomic DNA was then extracted using a DNeasy kit (Qiagen). The extracted DNA was analyzed by the genomic polymerase chain reaction (PCR) method using PCR mixtures for genomic PCR composed of 10X Extaq buffer, 2.5 mM dNTP, deionized water, Extaq polymerase, and primers for WT/mutant allele. To detect the mutant allele, a sense primer, 5?-CTGAAGAAGGAGAT-CAGCAGCCTC-3?, and an antisense primer, 5?-GTGTGGGTTCCAGGAATCGTACTGAG-3?, were used. To detect the WT allele, a sense primer, 5?-ATTAATTCTAGACTGGAACTTAGAGC-3?, and an antisense primer, 5?-TCTTACAGATCT-GAATGCCTTACCATCC-3?, were used. Exactly 9 mL of PCR mixture was aliquoted to a PCR tube, then 1 mL of embryo DNA was added. The PCR reaction test for WT and mutant alleles was performed. The PCR products were applied to 1% agarose gel, and electrophoresis was performed. Visual bands, produced using ethidium bromide, were used to identify embryo alleles.

[ 35 S] incorporation assay
Semi-confluent mouse embryonic fibroblasts were labeled with [ 35 S]-sulfate (100 mCi/mL) in 100 mm dishes for 24 hours. The medium and cell layer were collected separately and extracted using 4 M Guanidinium Hydrochloride (GuHCl) as previously described (24). Proteoglycans were precipitated with 3 volumes of 95% (v/v) ethanol containing 1.3% (w/v) potassium acetate. The precipitate was dissolved in buffer A (7 M urea, 50 mM Tris-HCl pH 7.5, 0.01% Triton X) and then rotated at 48C overnight. The sample was applied to a DEAE-Sephacel column. Eluate fractions by 2 M NaCl were collected and combined. The eluted sample was applied to a gel filtration (Superose6) column. The proteoglycan fraction was collected, and its radioactivity was measured using a Beckman Coulter TM LS6500 multi-purpose scintillation counter.

Chondroitin sulfate composition analysis
Fibroblasts were cultured in 60 mm culture dishes up to confluence, after which the medium and cells were collected. Cell lysate, prepared using Cytobuster reagent (Novagens), was centrifuged at 13,000 g at 48C for 10 minutes, after which the supernatant was collected. A sample of supernatant and culture medium were each applied to a separate 0.3 mL DEAE-Sephacel column and equilibrated with the equilibration buffer (50 mM Tris-HCl pH 7.2, 0.1 M NaCl). After washing with 3 mL of the equilibration buffer, the proteoglycan fraction was eluted with elution buffer (50 mM Tris-HCl pH 7.2, 2 M NaCl). Three volumes of 95% ethanol containing 1.3% potassium acetate were added to the eluate. The solution was chilled at (208C overnight and then centrifuged at 13,000 g for 30 minutes at 48C. The resulting pellet of proteoglycan was washed twice with 98% ethanol and then dried. After the pellet was reconstituted in deionized water, the protein concentration in the precipitated pellet of proteoglycan was determined by bicinchoninic acid (BCA) assay. Equal amounts of extracted proteoglycan from WT and Cspg2 D3/D3 fibroblasts were dissolved in chondroitinase ABC buffer (20 mM Tris-HCl pH 8.0, 20 mM sodium acetate, 0.02% bovine serum albumin (BSA)) and digested with chondroitinase ABC (5 mU/mL) at 378C for 3 hours. The reaction was stopped by boiling at 1008C for 5 minutes.
The resulting solution was centrifuged at 14,000 rpm for 15 minutes. (Prior to centrifuging the solution, the centrifuge was prepared by adding 100 mL of distilled water (high performance liquid chromatography grade) to the centrifugal filter and centrifuging it twice for 4 minutes each at 14,000 rpm, removing the flow-through each time.) The flow-through was collected for further analysis by high performance liquid chromatography (HPLC). The sample was added to a HPLC vial tube, and a postcolumn reaction HPLC was performed. The unsaturated CS disaccharides product was analyzed by fluorometric postcolumn HPLC, and its content was calculated from the standard curve of chondroitin sulfate-derived disaccharides.

Protein assay
The bicinchoninic acid (BCA) assay procedure (Pierce) was used to determine the concentration of protein in the sample. First, standard albumin was prepared to establish a calibration curve (2 mg/mL bovine serum albumin was diluted to get concentration ranges from 0.5 to 200 mg/mL). Then reagent A (sodium carbonate, sodium bicarbonate, and sodium tartrate in 0.2 M sodium hydroxide), reagent B (4% bicinchoninic acid), and reagent C (4% cupric sulfate pentahydrate) were thoroughly mixed at the ratio of 50:48:2, respectively, to make the working reagent. Each 25 mL of the standard or unknown sample was placed on a microplate and combined with 200 mL of the BCA working reagent. The combination was mixed thoroughly on the plate shaker for 30 seconds. The plate was then covered and incubated at 378C for 30 minutes, then cooled and measured at absorbance 562 nm on the plate reader. The sample concentration was calculated from the calibration curve of standard bovine serum albumin.

Genotyping of mouse embryos
To characterize the genotypes of the mouse embryos, 22 embryos were isolated from heterozygous pregnant Cspg2 WT/D3 mice, embryo tails were collected, and genomic DNA was extracted. The genotype of each embryo was characterized by using the genomic PCR technique. Two primer sets were used for genotype characterization including WT, heterozygous mutant, and homozygous mutant alleles. The PCR products from the WTallele revealed a band for the WT primers set ( Figure 1). The PCR products from the homozygous mutant allele revealed a band for the mutant primers set. The PCR products from the heterozygous mutant allele revealed bands for both the WT and mutant primers sets. The results indicated that embryos number 1, 4, 8, 15, and 21 were WTallele, embryos number 6, 12, 19, and 22 were homozygous allele, and the remaining embryos were heterozygous allele. These results were used for choosing fibroblast culture to perform further experiments.
We observed that the heterozygous Cspg2 WT/D3 mice were viable, fertile, and without any obvious abnormalities like WT mice, but the Cspg2 D3/D3 homozygotes died at the embryonic stage. Therefore, to study the abnormalities of Cspg2 D3/D3 homozygotes, homozygote alleles (Cspg2 D3/D3 and Cspg2 WT/WT ) were chosen to make fibroblast cultures. The morphology of WT fibroblasts differed from that of the Cspg2 D3/D3 fibroblasts. The Cspg2 D3/D3 fibroblasts appeared larger, flatter, and more spread out than WT fibroblasts ( Figure 2). In previous experiments, we found that the proliferation rate of the Cspg2 D3/D3 fibroblasts was slower than that of the WT fibroblasts, and that the versican expression level was reduced in the Cspg2 D3/D3 fibroblast cultures. In vitro studies done by Miquel-Serra et al. (25) on the expression of versican isoform lacking the chondroitin sulfate chain showed negative effects on primary cell growth, similar to our results. Therefore, we suspected that the characteristics of the chondroitin sulfate on the versican molecules of Cspg2 D3/D3 fibroblasts might explain some of the differences shown. For our next experiment, we determined the chondroitin sulfate composition in versican extracted from Cspg2 D3/D3 fibroblasts and compared it with WT versican.  (26), we expected that the extracted chondroitin sulfate proteoglycan from mouse embryonic fibroblast cultures in this experiment would represent as versican. However, fibroblasts are also able to produce decorin (27), a CS chain bearing chondroitin sulfate proteoglycan. To verify whether the mouse embryonic fibroblasts cultures in this study produce only versican molecules, Western blot analysis of versican and decorin molecules was performed by using antibodies against versican and decorin molecules, respectively. The results revealed that only versican was detected in the fibroblast cultures (data not shown). We can conclude, therefore, that mouse embryonic fibroblasts in the study produce only versican as chondroitin sulfate proteoglycan.

Decrease of chondroitin sulfate proteoglycan deposition in
[ 35 S]-sulfate incorporation studies revealed that the total versican (culture medium and cell layer) secreted by Cspg2 D3/D3 fibroblasts was approximately 50% of the total secreted by WT (Figure 3). Of the total secreted versican of the WT, 70% was deposited in the cell layer, while only 35% of the total secreted versican of the Cspg2 D3/D3 was deposited in the cell layer. This result clearly demonstrated the lower capacity of mutant (Cspg2 D3/D3 ) fibroblasts to deposit versican in the cell layer.   major isomer (59%), whereas non-sulfate chondroitin sulfate (33%) and chondroitin 6-sulfate (8%) were the minor isomers. The culture medium of Cspg2 D3/D3 fibroblasts contained chondroitin 4sulfate (59%), non-sulfate chondroitin sulfate (28%), and chondroitin 6-sulfate (12%). Remarkably, a trace amount (1%) of chondroitin sulfate E was also found. Thus, our results showed that the percentage of the chondroitin 4-sulfate isomer in the culture medium of Cspg2 D3/D3 fibroblasts was the same as that in the culture medium of WT fibroblasts, but the percentage of the non-sulfate chondroitin sulfate tended to be lower than that of the culture medium of WT fibroblasts. We also found that the negative charge of the chondroitin sulfate isomers in the culture medium of Cspg2 D3/D3 fibroblasts appeared to be higher than that of the culture medium of WT fibroblasts.
We found that the cell layer of the WT fibroblasts culture contained 14.1 ng/mL of chondroitin sulfate from the versican molecule, while the cell layer of the Cspg2 D3/D3 fibroblast cultures contained 5.0 ng/mL of chondroitin sulfate, nearly three times less (Figure 4). The WT fibroblast culture medium contained 12.1 ng/mL of chondroitin sulfate on the versican molecule, while the Cspg2 D3/D3 fibroblast culture medium contained 19.0 ng/mL, one-and-a-half times more ( Figure 4). These data showed that the amount of chondroitin sulfate on the mutant versican molecule in the cell layer was dramatically lower than that in the WT cell layer, while the amount in the culture medium was higher than in the WT medium.
We also found that the cell layer of the WT fibroblasts culture contained 12.3 ng/mL of chondroitin 4-sulfate on the versican molecule, while the cell layer of the Cspg2 D3/D3 fibroblast cultures contained 4.0 ng/mL, three times less. This demonstrates that versican molecules in Cspg2 D3/D3 fibroblast cultures contain less negatively charged chondroitin sulfate in the extracellular matrix. Interestingly, the negatively charged chondroitin sulfate increased in the Cspg2 D3/D3 fibroblast culture medium. We concluded that the disruption of the A subdomain of the versican molecule leads to the alteration of the composition and content of chondroitin sulfate on the versican molecule.

Discussion
In this study we compared certain characteristics of fibroblasts from Cspg2 D3/D3 mouse embryos with those of WT mouse embryos, which were studied  Our analysis of chondroitin sulfate was as we suspected. When the A subdomain was disrupted, chondroitin sulfate composition on the versican core protein was changed. Non-sulfate chondroitin sulfate isomer tended to increase in the cell layer but to decrease in the culture medium. This demonstrated that the negative charge on versican molecules decreased in the ECM but increased in the culture medium. Interestingly we found chondroitin sulfate E (di-sulfated chondroitin sulfate) on the versican molecule in the culture medium. The chondroitin sulfate chain has a negative charge which makes it possible for proteoglycans to bind various positively charged molecules such as growth factors, cytokines, and chemokines (29). Moreover it allows proteoglycans in the ECM or on the cell surface to generate immobilized gradients of certain growth factors and present them to their receptors; chondroitin sulfate chains also protect proteoglycans from proteolytic degradation (30). We found that both the charge and the content of chondroitin sulfate on the versican molecule were decreased in the cell layer of the Cspg2 D3/D3 fibroblast cultures. Other studies have shown that the charge of chondroitin sulfate regulates cell activity. For example, the number of neurites decreased with high disulfation chondroitin sulfate (31), and chondroitin sulfate tetrasaccharide stimulated the growth and differentiation of neurons (32).
Fluctuation in chondroitin sulfate content has been found to affect biological activity. An increase in chondroitin sulfate chains in cephalic neural crest migratory routes not only disrupts their migration, but also impedes detachment of the rhombencephalic neuroepithelium neural crest cells (33). Evidence suggests, then, that the decrease of the total charge and deposition of versican molecules in the Cspg2 D3/D3 fibroblasts cell layer will affect the proliferation and morphology of fibroblasts. We can conclude, then, that the Cspg2 D3/D3 mutant versican molecule, which contains a lower negative charge and less chondroitin sulfate on its core protein than normal versican, does not maintain growth factors and provides other essential molecules to serve cells, and thus leads to the alteration of cell behavior, such as a slow growth rate and the acquisition of senescence in the Cspg2 D3/D3 fibroblasts.
In addition, Matsumoto et al. (34) showed that the chondroitin sulfate composition on the versican molecule of WT mouse embryo fibroblasts differed from the composition in mouse cartilage. They found more non-sulfated chondroitin sulfate than chondroitin 4-sulfate in mouse cartilage, but more chondroitin 4-sulfate than non-sulfated chondroitin in mouse fibroblasts. This is more evidence that, even though versican is distributed throughout the body, the composition of chondroitin sulfate on the versican molecule is different for different tissues.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.