Department of Clinical Biochemistry, Ministry of Health Numune Training and Research Hospital, Ankara, Turkey.
Cell Biochem Funct. 2013 Oct;31(7):539-44. doi: 10.1002/cbf.2932. Epub 2012 Nov 23.
The pathways involved in the regulation of a disintegrin and metalloproteinase with thrombospondin motifs 9 (ADAMTS9) expression have not yet been elucidated. Therefore, the aim of this study was to investigate the involvement of nuclear factor-κB (NF-κB), mitogen activated protein kinases (MAPK) and Phosphatidylinositol 3-kinase (PI3 kinase) in ADAMTS9 gene regulation, with special focus on the involvement of NF-κB in IL-1β-induced ADAMTS9 expression. The OUMS-27 chondrosarcoma cells were exposed to IL-1β. They were pretreated with 20 μM PD98059 (specific inhibitor of p44/42 kinase), 10 μM SB203580 (specific inhibitor of p38 kinase), 20 μM SB600125 (MAPK inhibitor), and 1 μM Wortmannin and 10 μM LY294002 (specific inhibitors of PI3 kinase) for 30 min and subsequently incubated with IL-1β. For the effects of NF-κB and IκB inhibitors, cells were pretreated with curcumin or BAY117085 for 30 min and subsequently incubated with IL-1β. BAY117085 and different concentrations of curcumin were applied to the cells just after the first experiment to determine their concentration effect on ADAMTS9 gene expression. After total RNA was extracted, they were reversely transcribed with random primers and then real-time polymerase chain reaction (PCR) was performed on cDNA samples. There was a significant difference between control and stimulated cells in terms of ADAMTS9/β-actin ratio. Wortmannin and LY294002 did not have any repressive effect on the OUMS-27 whereas SB203580 and SP600125 were found to decrease the expression of ADAMTS9 gene. BAY 117085 and curcumin, which are two NF-κB inhibitors, led to a decrease in the ratio of ADAMTS9/β-actin. As a conclusion, the pathways MAPK and NF-κB were thought to be responsible pathways for the induction of ADAMTS9 gene.
调控解整合素金属蛋白酶 9（ADAMTS9）表达的途径尚未阐明。因此，本研究旨在探讨核因子-κB（NF-κB）、丝裂原活化蛋白激酶（MAPK）和磷脂酰肌醇 3-激酶（PI3 激酶）在 ADAMTS9 基因调控中的作用，特别关注 NF-κB 在 IL-1β诱导的 ADAMTS9 表达中的作用。将 OUMS-27 软骨肉瘤细胞暴露于 IL-1β中。用 20 μM PD98059（p44/42 激酶特异性抑制剂）、10 μM SB203580（p38 激酶特异性抑制剂）、20 μM SB600125（MAPK 抑制剂）和 1 μM Wortmannin 和 10 μM LY294002（PI3 激酶特异性抑制剂）预处理 30 分钟，然后用 IL-1β孵育。对于 NF-κB 和 IκB 抑制剂的作用，细胞用姜黄素或 BAY117085 预处理 30 分钟，然后用 IL-1β孵育。BAY117085 和不同浓度的姜黄素在第一次实验后立即应用于细胞，以确定它们对 ADAMTS9 基因表达的浓度效应。提取总 RNA 后，用随机引物反转录，然后对 cDNA 样品进行实时聚合酶链反应（PCR）。ADAMTS9/β-肌动蛋白比值在对照组和刺激组之间有显著差异。Wortmannin 和 LY294002 对 OUMS-27 没有任何抑制作用，而 SB203580 和 SP600125 则发现降低了 ADAMTS9 基因的表达。BAY 117085 和姜黄素，两种 NF-κB 抑制剂，导致 ADAMTS9/β-肌动蛋白的比值降低。总之，MAPK 和 NF-κB 途径被认为是诱导 ADAMTS9 基因表达的途径。
