Hinoi Eiichi, Fujimori Sayumi, Wang Liyang, Hojo Hironori, Uno Kyosuke, Yoneda Yukio
Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School of Natural Science and Technology, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan.
J Biol Chem. 2006 Jun 30;281(26):18015-24. doi: 10.1074/jbc.M600603200. Epub 2006 Apr 12.
Nrf2 (nuclear factor E2 p45-related factor 2) is believed to be a transcription factor essential for the regulation of many detoxifying and antioxidative genes in different tissues. In the present study, we investigated the role of Nrf2 in the regulation of osteoblastic differentiation. nrf2 mRNA expression was significantly up-regulated in femur isolated from ovariectomized mice, whereas in situ hybridization analysis revealed that up-regulation of nrf2 mRNA was mainly found in osteoblasts attached on cancellous bone in femur of ovariectomized mice. Expression of Nrf2 protein was also seen in osteoblasts in neonatal mouse tibia and calvaria. In osteoblastic MC3T3-E1 cells stably transfected with nrf2 expression vector, significant inhibition was seen in the maturation-dependent increase in alkaline phosphatase activity as well as the mineralized matrix formation. Stable overexpression of nrf2 significantly impaired Runx2 (runt-related transcription factor 2)-dependent stimulation of osteocalcin promoter activity and recruitment of Runx2 on osteocalcin promoter without affecting the expression of runx2 mRNA. Coimmunoprecipitation and mammalian two-hybrid assay revealed a physical interaction between Runx2 and Nrf2, whereas cellular distribution of endogenous Runx2 was not apparently changed by nrf2 overexpression in MC3T3-E1 cells. Alternatively, Nrf2 bound to antioxidant-responsive element-like-2 sequence of osteocalcin promoter. The inhibition by nrf2 on runx2-dependent osteocalcin promoter activity was partially prevented by the introduction of reporter of deletion mutant for ARE-like-2 sequence of osteocalcin promoter. These data suggest that Nrf2 may negatively regulate cellular differentiation through inhibition of the Runx2-dependent transcriptional activity in osteoblasts.
Nrf2（核因子E2 p45相关因子2）被认为是一种转录因子，对不同组织中许多解毒和抗氧化基因的调控至关重要。在本研究中，我们研究了Nrf2在成骨细胞分化调控中的作用。去卵巢小鼠股骨中nrf2 mRNA表达显著上调，而原位杂交分析显示，nrf2 mRNA的上调主要见于去卵巢小鼠股骨松质骨上附着的成骨细胞。在新生小鼠胫骨和颅骨的成骨细胞中也可见Nrf2蛋白表达。在稳定转染nrf2表达载体的成骨MC3T3-E1细胞中，碱性磷酸酶活性的成熟依赖性增加以及矿化基质形成均受到显著抑制。nrf2的稳定过表达显著损害了Runx2（ runt相关转录因子2）依赖性的骨钙素启动子活性刺激以及Runx2在骨钙素启动子上的募集，而不影响runx2 mRNA的表达。免疫共沉淀和哺乳动物双杂交试验揭示了Runx2与Nrf2之间存在物理相互作用，而在MC3T3-E1细胞中，nrf2过表达并未明显改变内源性Runx2的细胞分布。另外，Nrf2与骨钙素启动子的抗氧化反应元件样-2序列结合。通过引入骨钙素启动子ARE样-2序列缺失突变体报告基因，部分阻止了nrf2对Runx2依赖性骨钙素启动子活性的抑制。这些数据表明，Nrf2可能通过抑制成骨细胞中Runx2依赖性转录活性来负向调节细胞分化。
