Abstract
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.
Highlights
Versican [1, 2], or PG-M, is a member of the large aggregating chondroitin sulfate proteoglycan family, which includes aggrecan, neurocan, and brevican [3]
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
Fibroblasts were seeded into 96-well tissue culture plates, and the cultures were incubated for 3 days with antisense and sense oligonucleotides of mouse EGF receptor (EGFR), in the presence or absence of purified miniversican products. (Cells not treated with purified mini-versican were incubated in a column elute from vector-transfected cells only.) The antisense oligonucleotides are complementary to sequences located at the 5Ј and 3Ј of the gene [29]
Summary
Materials—The reverse transcription-PCR kit was from CLONTECH. Taq DNA polymerase, T4 DNA ligase, and restriction endonucleases were from Boehringer Mannheim. 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]. To delete the EGF-like motifs from the G3 domain of versican, a site for the restriction enzyme XhoI was engineered at the 5Ј end of the CRD This was performed by using CRDNXhoI (AAA CTC GAG C10129AA GAC ACA GAG ACT) and G3CSphI (5Ј-AAA GCA TGC G10830CG CCT TGA GTC CTG CCA CGT) as primers in a PCR reaction. Cell lysate and growth medium that contained recombinant gene products were subjected to SDS-PAGE electrophoresis. 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).
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