Department of Orthopedic Surgery, Kobe University Graduate School of Medicine, Kobe 650‑0017, Japan.
Int J Mol Med. 2018 Sep;42(3):1548-1558. doi: 10.3892/ijmm.2018.3729. Epub 2018 Jun 13.
Aquaporins (AQPs) are small integral membrane proteins that are essential for water transport across membranes. AQP9, one of the 13 mammalian AQPs (including AQP0 to 12), has been reported to be highly expressed in hydrarthrosis and synovitis patients. Given that several studies have identified signal transduction as an additional function of AQPs, it is hypothesized that AQP9 may modulate inflammatory signal transduction in chondrocytes. Therefore, the present study used a model of interleukin (IL)‑1β‑induced inflammation to determine the mechanisms associated with AQP9 functions in chondrocytes. Osteoarthritis (OA) and normal cartilage samples were subjected to immunohistological analysis. In addition, matrix metalloproteinase (MMP)3, MMP13 and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS‑5) mRNA and protein analysis was conducted in normal human articular chondrocytes from the knee (NHAC‑Kn) stimulated with IL‑1β by reverse transcription‑polymerase chain reaction (RT‑qPCR) and western blotting, respectively. AQP9 knockdown was also performed by transfection of AQP9‑specific small interfering RNA using Lipofectamine. AQP1, 3, 7, 9 and 11 mRNA expression levels were detected in OA human chondrocytes and in IL‑1β‑treated normal human chondrocytes. The levels of AQP9, MMP‑3, MMP‑13 and ADAMTS‑5 mRNA were increased by treatment with 10 ng/ml IL‑1β in a time‑dependent manner, while knockdown of AQP9 expression significantly decreased the mRNA levels of the MMP3, MMP13 and ADAMTS‑5 genes, as well as the phosphorylation of IκB kinase (IKK). Treatment with a specific IKK inhibitor also significantly decreased the expression levels of MMP‑3, MMP‑13 and ADAMTS‑5 in response to IL‑1β stimulation. Furthermore, immunohistochemical analysis demonstrated that AQP9 and inflammatory markers were highly expressed in OA cartilage. In addition, the downregulation of AQP9 in cultured chondrocytes decreased the catabolic gene expression in response to IL‑1β stimulation through nuclear factor‑κB signaling. Therefore, AQP9 may be a promising target for the treatment of OA.
水通道蛋白（AQP）是一种小的完整膜蛋白，对于跨膜的水转运至关重要。AQP9 是哺乳动物 13 种 AQP 之一（包括 AQP0 至 12），已报道在滑液和滑膜炎患者中高度表达。鉴于几项研究已将信号转导鉴定为 AQP 的另一种功能，因此假设 AQP9 可能调节软骨细胞中的炎症信号转导。因此，本研究使用白细胞介素（IL）-1β诱导的炎症模型来确定与软骨细胞中 AQP9 功能相关的机制。对骨关节炎（OA）和正常软骨样本进行免疫组织化学分析。此外，通过逆转录-聚合酶链反应（RT-qPCR）和 Western blot 分别分析正常人类膝关节关节软骨细胞（NHAC-Kn）在 IL-1β刺激下的基质金属蛋白酶（MMP）3、MMP13 和 a 型血小板反应蛋白 5（ADAMTS-5）mRNA 和蛋白。还通过使用 Lipofectamine 转染 AQP9 特异性小干扰 RNA 来进行 AQP9 敲低。在 OA 人类软骨细胞和 IL-1β处理的正常人类软骨细胞中检测到 AQP1、3、7、9 和 11 mRNA 表达水平。AQP9 表达水平在 10 ng/ml IL-1β处理下呈时间依赖性增加，而 AQP9 表达的敲低显著降低了 MMP3、MMP13 和 ADAMTS-5 基因的 mRNA 水平，以及 IκB 激酶（IKK）的磷酸化。用特异性 IKK 抑制剂处理也显著降低了 IL-1β刺激下 MMP-3、MMP-13 和 ADAMTS-5 的表达水平。此外，免疫组织化学分析表明 AQP9 和炎症标志物在 OA 软骨中高表达。此外，在培养的软骨细胞中下调 AQP9 通过核因子-κB 信号降低了对 IL-1β刺激的分解代谢基因表达。因此，AQP9 可能是治疗 OA 的有前途的靶点。
