CASEIN KINASE AND CD44 FUNCTION IN HUMAN CARTILAGE

酪蛋白激酶和 CD44 在人类软骨中的功能

• 批准号: 6478059
• 负责人: KATHLEEN T ROUSCHE
• 金额: $ 4.02万
• 依托单位: RUSH UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6478059
• 关键词: CD44 molecule; cartilage development; cartilage disorder; cartilage metabolism; casein kinase; chondrocytes; enzyme mechanism; human tissue; immunoprecipitation; interleukin 1; osteoarthritis; phosphorylation; polymerase chain reaction; tissue /cell culture; western blottings

Osteoarthritis (0A) is characterized by the lack of retention of extracellular matrix components in articular cartilage. OA is likely manifested in part by the inability of the chondrocyte to retain matrix at the cell surface. CD44 is a transmembrane glycoprotein receptor that links the matrix to the chondrocyte, thereby controlling matrix assembly and retention It is hypothesized that phosphorylation of CD44 directly regulates CD44-mediated cell/matrix interactions, a process not yet fully understood. investigators have recently shown that casein kinase II alpha (CKIIalpha') phosphorylates the cytoplasmic domain of CD44 in human breast cancer cells. It is highly probable that CKII plays a critical role in cartilage metabolism via the same mechanism. We hypothesize that CKII functions in cartilage to indirectly regulate matrix assembly and retention by mediating CD44 phosphorylation. Aims set forth in this proposal include (1) determining the presence and distribution of CKII in chondrocytes in all zones of human articular cartilage. Immunohistochemistry and in situ hybridization will be conducted on cartilage explants or cultured chondrocytes derived from normal, damaged and OA tissue. Levels of CKII and CD44 expression will be compared using competitive RT-PCR and Western blot techniques. Experiments will then (2) determine the ability of CKII to mediate CD44 phosphorylation in cultured explants and chondrocytes. RT-PCR, immunoprecipitation/Western blot and particle exclusion techniques will assess the ability of CKII to regulate CD44 phosphorylation and subsequent pericellular matrix formation following CKIIalpha antisense oligonucleotide or matrix degrading enzyme treatment Finally, it will be (3) determined whether any of these parameters are altered following treatment with interleukin-1, a cartilage matrix degrading cytokine. Findings will establish the role of CKII in both normal and pathogenic cartilage.

骨关节炎（OA）的特征在于关节软骨中缺乏细胞外基质成分的保留。OA可能部分表现为软骨细胞不能将基质保留在细胞表面。CD 44是一种跨膜糖蛋白受体，它将基质连接到软骨细胞，从而控制基质组装和保留。假设CD 44的磷酸化直接调节CD 44介导的细胞/基质相互作用，这一过程尚未完全了解。研究者最近已经表明酪蛋白激酶II α（CKII α ′）磷酸化人乳腺癌细胞中的CD 44的胞质结构域。CKII很可能通过相同的机制在软骨代谢中起关键作用。我们假设CKII在软骨中的功能是通过介导CD 44磷酸化来间接调节基质组装和保留。本发明的目的包括（1）确定人关节软骨所有区域的软骨细胞中CKII的存在和分布。将对软骨外植体或来自正常、受损和OA组织的培养软骨细胞进行免疫组织化学和原位杂交。将使用竞争性RT-PCR和Western印迹技术比较CKII和CD 44表达水平。然后实验将（2）确定CKII介导培养的外植体和软骨细胞中的CD 44磷酸化的能力。RT-PCR、免疫沉淀/蛋白质印迹和颗粒排除技术将评估CKII α反义寡核苷酸或基质降解酶处理后CKII调节CD 44磷酸化和随后的细胞周围基质形成的能力。最后，将（3）确定用白细胞介素-1（一种软骨基质降解细胞因子）处理后这些参数中的任何一个是否改变。研究结果将确定CKII在正常和致病软骨中的作用。