Cathepsin B expression and down-regulation by gene silencing and antisense DNA in human chondrocytes.
Cathepsin B expression and down-regulation by gene silencing and antisense DNA in human chondrocytes.
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人软骨细胞中组织蛋白酶 B 的表达以及基因沉默和反义 DNA 的下调。
DOI：
10.1042/bj20020210
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
Baici,Antonio
中科院分区：
文献类型：
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作者：
Zwicky,Roman;Muntener,Kathrin;Goldring,MaryB;Baici,Antonio
组织蛋白酶B是去分化软骨细胞表型的标志物，在骨关节炎中促进软骨破坏，在类风湿性关节炎和癌症中促进病理性蛋白水解。在寻找可能的手段中和这种酶的作用，我们比较了它的表达，生物合成和分布在关节软骨细胞和两个线的永生化的人软骨细胞。原代培养的天然关节软骨细胞和多克隆T/C-28 a2软骨细胞系在内体和溶酶体的数量、三种选择性剪接的组织蛋白酶B mRNA形式的分布和组织蛋白酶B活性方面相似。相比之下，克隆C-28/I2细胞系含有4倍更高水平的细胞内组织蛋白酶B活性，略高数量的内体和溶酶体，以及所有三种组织蛋白酶B转录物的均匀分布，因此类似于在去分化的早期阶段的传代培养的软骨细胞。用双链组织蛋白酶B mRNA转染T/C-28 a2软骨细胞，由于RNA干扰，导致组织蛋白酶B生物合成的抑制高达70%，并且各种大小的单链反义DNA使组织蛋白酶B生物合成的抑制高达78%。设计成与组织蛋白酶B的外显子1的末端和外显子3的开始杂交的反义寡核苷酸成功地特异性抑制缺少外显子2的mRNA剪接变体。这些结果表明，组织蛋白酶B的表达和活性可能是靶向的基因沉默的RNA干扰和反义DNA在软骨细胞中。此外，组织蛋白酶B的差异表达和分布和存在的基因沉默的永生化的人软骨细胞系中的必要的分子装置表明，它们可以作为一个有用的模型，用于研究相关酶在软骨病理学的功能。
Cathepsin B, a marker of the dedifferentiated chondrocyte phenotype, contributes to cartilage destruction in osteoarthritis and pathological proteolysis in rheumatoid arthritis and cancer. In search of possible means for neutralizing the action of this enzyme, we compared its expression, biosynthesis and distribution in articular chondrocytes and two lines of immortalized human chondrocytes. Native articular chondrocytes in primary culture and the polyclonal T/C-28a2 chondrocyte cell line were similar with respect to the number of endosomes and lysosomes, the distribution of three alternatively spliced cathepsin B mRNA forms, and the cathepsin B activity. In contrast, the clonal C-28/I2 cell line contained four times higher levels of intracellular cathepsin B activity, slightly higher numbers of endosomes and lysosomes, and uniform distribution of all three cathepsin B transcripts and thus resembled subcultured chondrocytes at an early stage of dedifferentiation. Transfection of T/C-28a2 chondrocytes with double-stranded cathepsin B mRNA resulted in inhibition of cathepsin B biosynthesis by up to 70% due to RNA interference, and single-stranded antisense DNAs of various sizes decreased cathepsin B biosynthesis by up to 78%. An antisense oligonucleotide designed to hybridize to the end of cathepsin B's exons 1 and the beginning of exon 3 was successful in specifically inhibiting the mRNA splice variant lacking exon 2. These results indicate that cathepsin B expression and activity may be targeted for gene silencing by RNA interference and antisense DNA in chondrocytes. Furthermore, the differential expression and distribution of cathepsin B and presence of the necessary molecular apparatus for gene silencing in the immortalized human chondrocyte cell lines indicate that they may serve as a useful model for studying the function of relevant enzymes in cartilage pathologies.