Duan Li, de Vos Paul, Fan Mingwen, Ren Yijin
Department of Orthodontics, University Medical Centre Groningen, University of Groningen, The Netherlands.
Front Biosci (Elite Ed). 2010 Jun 1;2(3):1065-72. doi: 10.2741/e164.
During osteoclast differentiation and resorption, both NF-kappaB and Notch signalling are activated. This study defines the mechanism about the influence of NFkappaB on Notch. To this end, IkappaBalphaM and Wild-type-IkappaBalpha were transfected into RAW 264.7 cells. The number of cells that differentiated into osteoclasts was quantified. The resorption area was measured. NF-kappaB transcriptional activity was determined by EMSA. Hes1, DC-STAMP and MMP-9 mRNA expression levels were determined by RT-PCR. Hes1 protein expression was determined by western blots. ChIP was used to study binding of IkappaBalpha to the Hes1 promoter. NF-kappaB inactivation inhibited the differentiation and resorption ability of osteoclasts. Compared with Wild-type cells, NF-kappaB inactivation resulted in an up-regulation of Hes1 expression, and a down-regulation of DC-STAMP and MMP-9 expression. Moreover, in response to RANKL, NF-kappaB inactivation resulted in a down-regulation of DC-STAMP and MMP-9 expression compared with Wild-type cells. The Hes1 promoter was detected by ChIP using IkappaBalpha antibody. In conclusion, our data suggest IkappaBalpha regulates Hes1-mediated activity in osteoclast differentiation and resorption, which supports a cross-talk between NF-kappaB and Notch in osteoclast activity.
在破骨细胞分化和吸收过程中，NF-κB和Notch信号通路均被激活。本研究确定了NF-κB对Notch影响的机制。为此，将IkappaBalphaM和野生型IkappaBalpha转染至RAW 264.7细胞中。对分化为破骨细胞的细胞数量进行定量。测量吸收面积。通过电泳迁移率变动分析（EMSA）测定NF-κB转录活性。通过逆转录聚合酶链反应（RT-PCR）测定Hes1、DC-STAMP和MMP-9 mRNA表达水平。通过蛋白质免疫印迹法测定Hes1蛋白表达。采用染色质免疫沉淀法（ChIP）研究IkappaBalpha与Hes1启动子的结合情况。NF-κB失活抑制破骨细胞的分化和吸收能力。与野生型细胞相比。NF-κB失活导致Hes1表达上调，DC-STAMP和MMP-9表达下调。此外，在RANKL刺激下，与野生型细胞相比，NF-κB失活导致DC-STAMP和MMP-9表达下调。使用IkappaBalpha抗体通过ChIP检测Hes1启动子。总之，我们的数据表明IkappaBalpha在破骨细胞分化和吸收过程中调节Hes1介导的活性，这支持了破骨细胞活性中NF-κB和Notch之间的相互作用。
