Kamiya Nobuhiro, Watanabe Hideto, Habuchi Hiroko, Takagi Hidekazu, Shinomura Tamayuki, Shimizu Katsuji, Kimata Koji
Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-1195, Japan.
J Biol Chem. 2006 Jan 27;281(4):2390-400. doi: 10.1074/jbc.M509341200. Epub 2005 Oct 28.
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.
间充质细胞凝聚是软骨发育的关键步骤。多功能蛋白聚糖/PG-M是一种大型硫酸软骨素蛋白聚糖，是凝聚过程中细胞外基质中表达的主要分子之一。然而，其作用，尤其是作为细胞凝聚环境的作用尚未阐明。在这里，我们使用一种新的软骨细胞系N1511展示了几条证据，证明多功能蛋白聚糖/PG-M在软骨生成凝聚中起重要作用。软骨生成刺激（用甲状旁腺激素、地塞米松、10%血清处理）导致多功能蛋白聚糖/PG-M的转录和蛋白质合成显著增加。多功能蛋白聚糖/PG-M的稳定反义克隆，取决于对多功能蛋白聚糖/PG-M表达的抑制，表现出不同的软骨生成能力，这通过主要软骨细胞产物聚集蛋白聚糖的表达和沉积来表明。培养早期的细胞仅表达V0和V1形式，在多功能蛋白聚糖/PG-M的四种变体中具有更多的硫酸软骨素链，用硫酸软骨素酶ABC处理这些细胞会抑制随后的软骨生成。此外，用β-木糖苷处理，一种硫酸软骨素合成的人工链引发剂，从而抑制核心蛋白上的合成，抑制了软骨生成。此外，无硫酸软骨素链的变体V3的强制表达破坏了天然多功能蛋白聚糖/PG-M（V0/V1）和已知在间充质凝聚过程中表达的其他细胞外基质分子的沉积和组织，并导致随后软骨生成的抑制。这些结果表明，多功能蛋白聚糖/PG-M通过附着的硫酸软骨素链积极参与间充质基质的形成和软骨细胞分化的起始调节。
