Pang Manhui, Martinez Ariel F, Fernandez Isabel, Balkan Wayne, Troen Bruce R
Geriatrics Research, Education and Clinical Center & Research Service, Miami Veterans Affairs Medical Center, Miami FL 33125, United States.
Gene. 2007 Nov 15;403(1-2):151-8. doi: 10.1016/j.gene.2007.08.007. Epub 2007 Aug 25.
Cathepsin K (CTSK) is a secreted protease that plays an essential role in osteoclastic bone resorption, and CTSK levels increase with osteoclast differentiation and activation, a process that is controlled by a complex physiological network of hormones and cytokines. A critical regulator of this process is receptor activator of NF-kappaB ligand (RANKL), a member of the tumor necrosis factor (TNF) superfamily of cytokines that can act via the TNF receptor activating factor (TRAF6)/AP-1 signaling pathway. However, the mechanism whereby RANKL modulates CTSK expression is not fully understood. Therefore, we investigated the regulation of CTSK expression and promoter activity in RAW 264.7 osteoclast precursor cells, which can be readily differentiated to osteoclasts upon RANKL stimulation. Western blot analysis, real-time RT-PCR and luciferase reporter gene assays revealed that RANKL stimulated CTSK expression and promoter activity in a dose- and time-dependent manner and that this activation was inhibited by either dominant negative (DN) TRAF6 or DN-c-fos. TRAF6 stimulated the basal activity of a truncated CTSK promoter, and DN-c-fos blocked this stimulation. JunB alone also stimulated basal CTSK promoter activity, whereas c-jun, JunD or c-fos alone did not. However, co-transfection of any of these jun-family members with c-fos (AP-1) significantly increased CTSK promoter expression. siRNA targeted against c-jun or junB suppressed RANKL-mediated CTSK expression. Therefore, both TRAF6 and AP-1 help regulate the basal and RANKL-mediated stimulation of CTSK gene expression in RAW 264.7 cells.
组织蛋白酶K（CTSK）是一种分泌型蛋白酶，在破骨细胞介导的骨吸收过程中发挥着重要作用，且CTSK水平会随着破骨细胞的分化和激活而升高，这一过程受激素和细胞因子构成的复杂生理网络调控。该过程的一个关键调节因子是核因子κB受体活化因子配体（RANKL），它是肿瘤坏死因子（TNF）细胞因子超家族的成员，可通过TNF受体激活因子（TRAF6）/AP-1信号通路发挥作用。然而，RANKL调节CTSK表达的机制尚未完全明确。因此，我们研究了RANKL刺激后可轻易分化为破骨细胞的RAW 264.7破骨细胞前体细胞中CTSK表达及启动子活性的调控情况。蛋白质免疫印迹分析、实时逆转录聚合酶链反应和荧光素酶报告基因检测显示，RANKL以剂量和时间依赖性方式刺激CTSK表达及启动子活性，且这种激活作用可被显性负性（DN）TRAF6或DN-c-fos抑制。TRAF6刺激截短型CTSK启动子的基础活性，而DN-c-fos可阻断这种刺激。单独的JunB也可刺激CTSK启动子基础活性，而单独的c-jun、JunD或c-fos则无此作用。然而，这些jun家族成员中的任何一个与c-fos（AP-1）共转染均可显著增加CTSK启动子表达。针对c-jun或junB的小干扰RNA（siRNA）可抑制RANKL介导的CTSK表达。因此，TRAF6和AP-1均有助于调节RAW 264.7细胞中CTSK基因表达的基础水平及RANKL介导的刺激作用。
