N-terminal domains of CCN family 2/connective tissue growth factor bind to aggrecan
N-terminal domains of CCN family 2/connective tissue growth factor bind to aggrecan
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DOI：
10.1042/bj20081991
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发表时间：
2009-06-15
影响因子：
4.1
通讯作者：
Takigawa, Masaharu
中科院分区：
文献类型：
--
作者：
Aoyama, Eriko;Hattori, Takako;Takigawa, Masaharu
CCN 2/CTGF（CCN家族2/结缔组织生长因子）是具有广泛活性的多细胞蛋白。它调节许多细胞功能，包括增殖、迁移、粘附和细胞外基质产生，因此参与许多生物学和病理学过程。特别是，CCN 2/CTGF对正常骨骼发育至关重要。为了鉴定能够调节其在软骨中的作用的CCN 2/CTGF相互作用蛋白，我们使用CCN 2/CTGF肽作为诱饵和来自软骨细胞系HCS-2/8的cDNA文库进行酵母双杂交筛选。在本论文中，我们报告的鉴定聚集蛋白聚糖，这是一个主要的蛋白多糖的细胞外基质中的软骨，它的CCN 2/CTGF结合蛋白。在CCN 2/CTGF的四个结构域中，IGFBP [IGF（胰岛素样生长因子）结合蛋白样]和/或VWC（血管性血友病因子C型）结构域在酵母双杂交测定中与聚集蛋白聚糖直接相互作用。使用聚集蛋白聚糖包被的板的固相结合测定的结果也显示以剂量依赖性方式与重组CCN 2/CTGF结合。与rTSP 1（重组血小板反应蛋白I型重复序列）和rCT（重组C末端胱氨酸结）模块肽相比，rIGFBP（重组IGFBP）和rVWC（重组VWC）模块肽与聚集蛋白聚糖的结合更强。表面等离子体共振（SPR）分析显示CCN 2/CTGF与聚集蛋白聚糖之间存在直接相互作用。并且异位过表达的CCN 2/CTGF和AgG 3（聚集蛋白聚糖的G3结构域）证实了它们在体内的结合。间接免疫荧光分析表明，CCN 2/CTGF与聚集蛋白聚糖在HCS-2/8细胞上存在细胞外共定位。与rTSP-rCT肽相比，rIGFBP-rVWC肽有效地增强了软骨细胞中聚集蛋白聚糖的产生和释放。这些结果表明CCN 2/CTGF通过其N-末端IGFBP和VWC模块结合聚集蛋白聚糖。这种结合可能与CCN 2/CTGF增强软骨细胞聚集蛋白聚糖的产生和分泌有关。
CCN2/CTGF (CCN family 2/connective tissue growth factor) is a multi-cellular protein with a broad range of activities. It modulates many cellular functions, including proliferation, migration, adhesion and extracellular matrix production, and it is thus involved in many biological and pathological processes. In particular, CCN2/CTGF is essential for normal skeletal development. To identify CCN2/CTGF-interactive proteins capable of modulating its action in cartilage, we carried Out a yeast two-hybrid screening using CCN2/CTGF peptide as a bait and a cDNA library from a chondrocytic cell line, HCS-2/8. In the present paper, we report the identification of aggrecan, which is a major proteoglycan of the extracellular matrix in cartilage, its a CCN2/CTGF-binding protein. Among the four domains of CCN2/CTGF, the IGFBP [IGF (insulin-like growth factor)-binding protein-like] and/or VWC (von Willebrand factor type C) domains had a direct interaction with aggrecan in a yeast two-hybrid assay. The results of a solid-phase-binding assay using aggrecan-coated plates also showed binding to recombinant CCN2/CTGF in a dose-dependent manner. rIGFBP (recombinant IGFBP) and rVWC (recombinant VWC) module peptides had stronger binding to aggrecan compared with rTSP1 (recombinant thrombospondin type I repeat) and rCT (recombinant C-terminal cystine knot) module peptides. SPR (surface plasmon resonance) analysis showed the direct interaction between the CCN2/CTGF and aggrecan. and ectopically overexpressed CCN2/CTGF and AgG3 (G3 domain of aggrecan) confirmed their binding in vivo. Indirect immunofluorescence analysis indicated that CCN2/CTGF was extracellularly co-localized with aggrecan on HCS-2/8 cells. The rIGFBP-rVWC peptide effectively enhanced tire production and release of aggrecan compared with the rTSP-rCT peptide in chondrocytes. These results indicate that CCN2/CTGF binds to aggrecan through its N-terminal IGFBP and VWC Modules. and this binding may be related to the CCN2/CTGF-enhanced production and secretion of aggrecan by chondrocytes.