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Expression and function of RGD-CAP conatining RGD-motif in cartilage

含有RGD基序的RGD-CAP在软骨中的表达和功能

基本信息

批准号：
09671894
负责人：
NOSHIRO Mitsuhide
金额：
$ 2.24万
依托单位：
Grant-in-Aid for Scientific Research (C)(2)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

The following results were obtained to clarify the role of RGD-CAP as the mediator in the interaction of cells and matrix concerning the growth and development of chondrocytes.(1) A cDNA clone for RGD-CAP (collagen-associated protein containing the RGD sequence) was isolated from chick embryo chondrocyte cDNA library. The deduced amino acid sequence (680 amino acid residues) contained four internal repeat domains and two highly conserved sequences (Hi and H2) in each repeat. The protein contains an integrin recognition site, the RGD motif in the fourth domain of four repetitive structures.(2) We examined the effect of recombinant chick RGD-CAP on the adhesion of chondrocytes and fibroblasts using RGD-CAP-coated dishes. When rabbit chondrocytes or human fibroblasts were seeded on plastic dishes coated with RGD- CAP, cell adhesion was enhanced compared with control dishes.(3) RGD-CAP produced a synergistic stimulation of cell adhesion in thepresence of type II or III collagen, and an additive stimulation in the presence of type I or V collagen.(4) The effect of RGD-CAP on cell adhesion required divalent cations (Mg^<2+> or Mn^<2+>) , and was reduced by EDTA and by the GRGDS peptide.(5) Monoclonal antibodies to the human integrin alpha1, alpha5, or beta1 subunit but not alpha2, alpha3, or beta2 decreased the RGD-CAP-induced cell adhesion. These findings suggest that RGD-CAP is a novel ligand for integrin alpha1beta1/alpha5beta1, and that a site other than the RGD sequence on RGD-CAP is also involved in cell adhesion.(6) An RGD-CAP fragment containing two repeating domains was also capable of enhancing efficiently the adhesion of chondrocytes indicating the importance of the repetition of two domains.

为了阐明RGD-CAP在软骨细胞生长发育过程中作为细胞与基质相互作用的中介的作用，我们获得了以下结果。(1)从鸡胚软骨细胞cDNA文库中分离到RGD- cap（含RGD序列的胶原相关蛋白）cDNA克隆。推导出的氨基酸序列（680个氨基酸残基）包含4个内部重复结构域和2个高度保守序列（Hi和H2）。该蛋白包含一个整合素识别位点，RGD基序位于四个重复结构的第四个结构域。(2)利用RGD-CAP包被培养皿检测重组鸡RGD-CAP对软骨细胞和成纤维细胞粘附的影响。将兔软骨细胞或人成纤维细胞接种于包被RGD- CAP的塑料培养皿中，细胞粘附性较对照培养皿增强。(3) RGD-CAP在II型或III型胶原存在时对细胞粘附产生协同刺激，在I型或V型胶原存在时产生加性刺激。(4) RGD-CAP对细胞粘附的影响需要二价阳离子（Mg^<2+>或Mn^<2+>）， EDTA和GRGDS肽可以降低RGD-CAP对细胞粘附的影响。(5)人整合素α 1、α 5或β 1亚基单克隆抗体，而α 2、α 3或β 2单克隆抗体则不降低rgd - cap诱导的细胞粘附。这些发现表明，RGD- cap是整合素alpha1beta1/alpha5beta1的新型配体，RGD- cap上的RGD序列之外的一个位点也参与了细胞粘附。(6)含有两个重复结构域的RGD-CAP片段也能够有效地增强软骨细胞的粘附，这表明两个重复结构域的重要性。