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: 6534397
• 负责人: KATHLEEN T ROUSCHE
• 金额: $ 4.57万
• 依托单位: RUSH UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6534397
• 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)的特征是关节软骨细胞外基质成分缺乏滞留。骨关节炎可能部分表现为软骨细胞不能将基质保留在细胞表面。CD44是一种跨膜糖蛋白受体，将基质与软骨细胞连接起来，从而控制基质的组装和滞留。假设CD44的磷酸化直接调节CD44介导的细胞/基质相互作用，这一过程尚未完全了解。研究人员最近发现，酪蛋白激酶IIα(CKIIpha‘)可磷酸化人乳腺癌细胞中CD44的胞浆结构域。CKII很可能通过同样的机制在软骨代谢中发挥重要作用。我们假设CKII在软骨中的作用是通过介导CD44的磷酸化来间接调节基质的组装和滞留。本提案提出的目标包括(1)确定CKII在人类关节软骨所有区域的软骨细胞中的存在和分布。将对软骨外植体或来自正常、受损和骨关节炎组织的培养软骨细胞进行免疫组织化学和原位杂交。CKII和CD44的表达水平将使用竞争性RT-PCR和Western印迹技术进行比较。然后，实验将(2)确定CKII在培养的外植体和软骨细胞中介导CD44磷酸化的能力。RT-PCR、免疫沉淀/Western印迹和颗粒排斥技术将评估CKII在CKIIα反义寡核苷酸或基质降解酶处理后调节CD44磷酸化和随后的细胞周围基质形成的能力。最后，将确定这些参数中的任何一个在IL-1处理后是否发生改变。研究结果将确定CKII在正常和致病软骨中的作用。