Versican/PG-M Regulates Chondrogenesis as an Extracellular Matrix Molecule Crucial for Mesenchymal Condensation

Nobuhiro Kamiya,Hideto Watanabe,Hiroko Habuchi,Hidekazu Takagi,Tamayuki Shinomura,Katsuji Shimizu,Koji Kimata

doi:10.1074/jbc.m509341200

Nobuhiro Kamiya, Hideto Watanabe + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m509341200

Copy DOI

Abstract

Mesenchymal cell condensation is an essential step for cartilage development. Versican/PG-M, a large chondroitin sulfate proteoglycan, is one of the major molecules expressed in the extracellular matrix during condensation. However, its role, especially as an environment for cells being condensed, has not been elucidated. Here we showed several lines of evidence for essential roles of versican/PG-M in chondrogenic condensation using a new chondrocytic cell line, N1511. Chondrogenic stimuli (treatment with parathyroid hormone, dexamethasone, 10% serum) induced a marked increase in the transcription and protein synthesis of versican/PG-M. Stable antisense clones for versican/PG-M, depending on suppression of the expression of versican/PG-M, showed different capacities for chondrogenesis, as indicated by the expression and deposition of aggrecan, a major chondrocytic cell product. The cells in the early stages of the culture only expressed V0 and V1 forms, having more chondroitin sulfate chains among the four variants of versican/PG-M, and treatment of those cells with chondroitinase ABC suppressed subsequent chondrogenesis. Furthermore, treatment with beta-xyloside, an artificial chain initiator of chondroitin sulfate synthesis to consequently inhibit the synthesis on the core proteins, suppressed chondrogenesis. In addition, forced expression of the variant V3, which has no chondroitin sulfate chain, disrupted the deposition and organization of native versican/PG-M (V0/V1) and other extracellular matrix molecules known to be expressed during the mesenchymal condensation and resulted in the inhibition of subsequent chondrogenesis. These results suggest that versican/PG-M is involved in positively regulating the formation of the mesenchymal matrix and the onset of chondrocyte differentiation through the attached chondroitin sulfate chains.

Highlights

Versican/PG-M was originally isolated as PG-M from the core of chick embryonic limb bud at stage 23 [10]
Because cell density is known to be a key factor for chondrogenesis, which may reflect the effect of mesenchymal condensation, the possible mechanism could be explained at least in part by the high level expression of versican/PG-M in the cells at high density
The results suggest that the chondrogenic effects of the treatment and cell density on N1511 cells involve the strong expression and deposition of versican/PG-M in the early phase

Summary

EXPERIMENTAL PROCEDURES

Cell Line and Culture Conditions—N1511 cells, a cell line established from chondrocytic cells in the rib cage of a 4-week-old male p53-null mouse, reproduce each step of chondrogenesis [38]. The cultured medium was combined from day 1 to day 5 and precipitated with 3 volumes of cold ethanol containing 1.3% potassium acetate These precipitates and cell layers were suspended in 2 or 1 ml of 0.2 M NaOH, respectively, incubated for 16 h at room temperature, neutralized with 4 M acetic acid added to a final concentration of 10 mM MgCl2, and treated with DNase (100 ␮g) and RNase (100 ␮g) at 37 °C for 2 h. Constructions of Vectors for Antisense Inhibition and for Expression of V3—The versican/PG-M antisense vector was constructed using the Ϫ99 to ϩ359 portion of the cDNA region as described previously [32] The fragment containing this region was amplified by PCR using primers (sense, 5Ј-GCTTCCTATGTGATCTTCCG-3Ј, and antisense, 5Ј-AGTTTGACCATGGTGAGGGA-3Ј), purified by agarose gel electrophoresis, and ligated into a SmaI site of the expression vector pKCR with the SV40 promoter.

RESULTS

Group III

DISCUSSION