Cloning of Mouse Chondromodulin-I cDNA and Localization of the Gene Transcripts

小鼠软骨调节素-I cDNA 的克隆和基因转录本的定位

• 批准号: 09671892
• 负责人: HIRAKI Yuji
• 金额: $ 1.86万
• 依托单位: KYOTO UNIVERSITY (1998)Osaka University (1997)
• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671892/
• 关键词: Chondrocytes; Cell Differentiation; Angiogenesis; Chondromodulin-I; Vascular Endothelial Cell; in situ Hybridization; Morphogenesis; Bone Formation

Studies of chondrocytes have been carried out by using primary cultures of chondrocytes that were isolated from mammalian cartilaginous tissue. However, it is theoretically not feasible to study the mechanism of chondrogenic differentiation using primary cultures of differentiated chondrocytes. Moreover, passages of primary chondrocytes result in a rapid loss of differentiated phenotype of cells. Therefore, we attempted to construct an in vitro chondrogenic culture system using a clonal EC cell line ATDC5. We demonstrated that the culture of ATDC5 cells kept track of the early-phase and late-phase differentiation that includes formation of type II collagen expressing proliferating chondrocytes and the subsequent cellular hypertrophy and mineralization.Then, we isolated a full length cDNA for mouse chondromodulin-I (ChM-I) from cDNA prepared from the differentiated ATDC5 cell culture by a PCR cloning method. The nucleotide sequence of mouse ChM-I cDNA revealed that mouse ChM-I precursor … More protein contained 334 amino acid residues. The Ch-SP domain of the mouse ChM-I precursor exhibited about 90% sequence identity, compared to the human counterpart. The mature ChM-I domain contained 120 amino acid residues as the human mature ChM-I did. The sequence identity in this mature domain was 86%. Most substitutions of amino acids were found in the N-terminal domain (40 residues). However, the N-linked oligosaccharide attachment site (Asn29) was concerved. In contrast, the C-terminal domain (about 80 residues from Phe42 to Val120) was completely conserved, except for the substitution from Ilel 16 to VaIl 16.Expression of ChM-I mRNA was induced along with the early-phase chondrogenic differentiation of ATDC5 cells and withdrawn as the late-phase differentiation proceeded. In situ hybridization in the ATDC5 cultures revealed the localized expression of ChM-I mRNA in cartilage nodules. In mouse embryo, cartilage formation was first observed on day 11. ChM-I transcripts were specifically detected at the sites of cartilage formation. On day 16, ChM-l expression was specifically abolished in the late-hypertrophic and mineralizing cartilage. This pattern of expression was well compatible with the functional role of ChM-I protein as "angioinhibin." Less

软骨细胞的研究已经通过使用分离自哺乳动物软骨组织的软骨细胞的原代培养物进行。然而，用原代培养的分化软骨细胞研究软骨分化的机制在理论上是不可行的。此外，原代软骨细胞的传代导致细胞分化表型的快速丧失。因此，我们试图构建一个在体外软骨培养系统，使用克隆EC细胞系ATDC 5。我们证明ATDC 5细胞的培养跟踪早期和晚期分化，包括形成表达II型胶原的增殖软骨细胞和随后的细胞肥大和矿化，然后，我们通过PCR克隆方法从分化的ATDC 5细胞培养物制备的cDNA中分离小鼠软骨调节素-I（ChM-I）的全长cDNA。小鼠ChM-I cDNA的核苷酸序列显示，小鼠ChM-I前体 ...更多信息 蛋白质含有334个氨基酸残基。小鼠ChM-I前体的Ch-SP结构域与人类对应物相比表现出约90%的序列同一性。成熟ChM-I结构域与人成熟ChM-I结构域相同，由120个氨基酸残基组成。该成熟结构域的序列同一性为86%。大部分的氨基酸替换被发现在N-末端结构域（40个残基）。而N-连接寡糖的连接位点（Asn 29）则受到关注。而C端结构域（从Phe 42到Val 120约80个残基）则完全保守，只有Ile 16被替换为Val 16。ChM-Ⅰ mRNA的表达沿着ATDC 5细胞向软骨细胞分化的早期被诱导，随着分化的晚期被撤回。ATDC 5培养物中的原位杂交显示ChM-Ⅰ mRNA在软骨结节中的局部表达。在小鼠胚胎中，在第11天首次观察到软骨形成。ChM-I转录本在软骨形成的位点特异性检测。在第16天，ChM-1表达在晚期肥大和矿化软骨中特异性消除。这种表达模式与ChM-I蛋白作为“血管生成素”的功能作用非常一致。“减

项目成果

Guiming Cai: "Mutational analysis of the DTDST gene in a Japanese patient with achondrogenesis type IB." Am.J.Med.Gene.78. 58-60 (1998)

蔡桂明：“日本 IB 型软骨发育不全患者 DTDST 基因突变分析”。

Yuji Hiraki: "Inhibition of DNA synthesis and tube morphogenesis of cultured vascular endothelial cells by chondromodulin-I." FEBS Lett.415. 321-324 (1997)

Yuji Hiraki：“软骨调节蛋白-I 对培养的血管内皮细胞的 DNA 合成和管形态发生的抑制。”

Chisa Shukunami: "Spatiotemporal pattern of the mouse chondromodulin-I gene expression and its regulatory role in vascular invasion into cartilage during endochondral bone formation" Int.J.Dev.Biol.43. 39-49 (1999)

Chisa Shukunami：“小鼠软骨调节蛋白-I 基因表达的时空模式及其在软骨内骨形成过程中血管侵入软骨中的调节作用”Int.J.Dev.Biol.43。

Yutaka Otsuka: "Requirement of FGF Signaling for Regeneration of Epiphyseal Morphology in Rabbit Full-Thickness Defects of Articular Cartilage." Dev.Growth Diff.39. 143-156 (1997)

Yutaka Otsuka：“FGF 信号传导对兔关节软骨全层缺损骨骺形态再生的要求。”

Chisa, Shukunami: "Sequential progression of the differentiation program by bone morphogenetic protein-2 in chondrogenic cell line ATDC5." Exp.Cell Res.241. 1-11 (1998)

Chisa, Shukunami：“软骨形成细胞系 ATDC5 中骨形态发生蛋白 2 分化程序的顺序进展。”