Abstract
Versican is a highly expressed proteoglycan in zones of developing tissues. To investigate whether versican plays a role in cell differentiation, we studied its role in mesenchymal condensation and chondrogenesis. Here we report that a mini-versican gene product inhibits mesenchymal chondrogenesis but not condensation. The mini-versican-treated mesenchymal cultures form fewer, smaller cartilaginous nodules and produced lower levels of link protein and type II collagen. The versican G3 domain alone, but not G1, was sufficient to inhibit mesenchymal chondrogenesis. Deletion of two epidermal growth factor (EGF)-like motifs in the G3 domain abolished the effect of versican. The G3 domain of aggrecan, which does not contain an EGF-like motif, did not inhibit mesenchymal chondrogenesis. We also generated a chimera construct containing the two EGF-like motifs of versican and the G3 domain of aggrecan, and we observed that this chimera construct inhibited chondrogenesis to a lesser extent than did the full-length versican G3 construct. Direct transfection of mesenchymal cells with different constructs produced similar results. Furthermore, treatment with versican antisense oligonucleotides and transfection with a versican antisense construct promoted chondrogenesis. Taken together, our results strongly suggest that versican inhibits mesenchymal chondrogenesis via its EGF-like motifs.
Highlights
Versican is expressed in a variety of tissues
Whereas selectins play a role in mediating cell adhesion, no such function has been identified for the G3 domain of versican
Versican Inhibits Mesenchymal Chondrogenesis—It has been reported that versican is highly expressed in chicken mesenchymal cells in the early developmental stages [2, 3] and is restricted to the zone of keratinocyte proliferation in the epidermis [8]
Summary
Materials—Chicken eggs (products of Hamburger and Hamilton strains) were purchased from Brampton Chick Hatchery Co. The PCR product was digested with BamHI (restriction site present at nucleotides 244 –249) [9] and SphI This fragment and the leading peptide of link protein were ligated into pcDNA1. To delete the EGF-like motifs from the mini-versican, a site for the restriction enzyme XhoI was engineered at the 5Ј end of carbohydrate recognition domain This was performed by using 5Ј-AAA CTC GAG C(10129)AA GAC ACA GAG ACT-3Ј (located in nucleotides 10129 – 10143 of versican sequence) and 5Ј-AAA GCA TGC G(10830)CG CCT TGA GTC CTG CCA CGT-3Ј (complementary to nucleotides 10810 – 10830) as primers in a PCR reaction using the mini-versican as a template.
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