Shi J, Schmitt-Talbot E, DiMattia D A, Dullea R G
Department of Antibacterial, Immunology, and Inflammation, Pfizer Global Research and Development, Groton, CT 06340, USA.
Inflamm Res. 2004 Aug;53(8):377-89. doi: 10.1007/s00011-004-1271-3. Epub 2004 Aug 10.
Interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and matrix metalloproteinases (MMPs) play important roles in the pathogenesis of osteoarthritis (OA). In the present study, using Affymetrix oligonucleotide array technology and real-time quantitative RT-PCR we have investigated the molecular mechanisms underlying the differential effect of IL-1 and TNF-alpha on gene expression in the human chondrosarcoma cell line, SW1353.
SW1353 cells were stimulated singularly with IL-1alpha, TNF-alpha, Phorbol 12-myristate 13-acetate (PMA), or treated with the combination of cytokine and PMA. Total RNA was collected at multiple time points over a 24-h period followed by biotinylated cRNA target preparation and hybridization onto the Affymetrix HG-U95Av2 array. The differential expression patterns of several cytokine and MMP genes were further confirmed by real time quantitative RT-PCR, Western blot, and ELISA.
Our microarray experiments have broadly confirmed previously published data on chondrocyte gene expression regulated by IL-1 and TNF-alpha. The expression pattern of proIL-1beta, MMP-1, and MMP-13 in chondrocytes is differentially regulated when stimulated with proinflammatory cytokines. IL-1, but not TNF-alpha, can induce IL-6, bone morphogenic protein 2 (BMP-2), and cyclooxygenase (COX-2) expression in SW1353 cells. Additionally, our Western blot results provide the first evidence that IL-1beta is produced in the proform in IL-1alpha-activated chondrosarcoma cells and that additional signals are required for its posttranslational processing/activation.
IL-1 and TNF-alpha each activate a distinct set of genes in chondrosarcoma cells, and gene expression in these cells is regulated by groups of genes related in part by their function. Chondrocyte IL-1alpha appears to serve an important role in the pathogenesis OA contributing to joint inflammation and cartilage destruction.
白细胞介素-1（IL-1）、肿瘤坏死因子-α（TNF-α）和基质金属蛋白酶（MMPs）在骨关节炎（OA）的发病机制中起重要作用。在本研究中，我们使用Affymetrix寡核苷酸阵列技术和实时定量逆转录-聚合酶链反应（RT-PCR），研究了IL-1和TNF-α对人软骨肉瘤细胞系SW1353基因表达产生差异影响的分子机制。
用IL-1α、TNF-α、佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）单独刺激SW1353细胞，或用细胞因子与PMA的组合处理细胞。在24小时内的多个时间点收集总RNA，随后制备生物素化的cRNA靶标并与Affymetrix HG-U95Av2阵列杂交。通过实时定量RT-PCR、蛋白质印迹法和酶联免疫吸附测定（ELISA）进一步确认几种细胞因子和MMP基因的差异表达模式。
我们的微阵列实验广泛证实了先前发表的关于IL-1和TNF-α调节软骨细胞基因表达的数据。当用促炎细胞因子刺激时，软骨细胞中proIL-1β、MMP-1和MMP-13的表达模式受到差异调节。IL-1而非TNF-α可诱导SW1353细胞中IL-6、骨形态发生蛋白2（BMP-2）和环氧化酶（COX-2）的表达。此外，我们的蛋白质印迹结果首次证明，IL-1β在IL-1α激活的软骨肉瘤细胞中以前体形式产生，其翻译后加工/激活需要额外的信号。
IL-1和TNF-α各自在软骨肉瘤细胞中激活一组不同的基因，这些细胞中的基因表达由部分因其功能相关的基因群调节。软骨细胞IL-1α似乎在OA的发病机制中起重要作用，导致关节炎症和软骨破坏。
