Department of Pathology, University of Texas Health Science Center at San Antonio, TX 78229, USA.
Biochem J. 2011 Jan 15;433(2):393-402. doi: 10.1042/BJ20100357.
BMP-2 (bone morphogenetic protein-2) promotes differentiation of osteoblast precursor cells to mature osteoblasts that form healthy bone. In the present study, we demonstrate a novel mechanism of BMP-2-induced osteoblast differentiation. The antioxidant NAC (N-acetyl-L-cysteine) and the flavoprotein enzyme NAD(P)H oxidase inhibitor DPI (diphenyleneiodonium) prevented BMP-2-stimulated alkaline phosphatase expression and mineralized bone nodule formation in mouse 2T3 pre-osteoblasts. BMP-2 elicited a rapid generation of ROS (reactive oxygen species) concomitant with increased activation of NAD(P)H oxidase. NAC and DPI inhibited BMP-2-induced ROS production and NAD(P)H oxidase activity respectively. NAD(P)H oxidases display structurally similar catalytic subunits (Nox1-5) with differential expression in various cells. We demonstrate that 2T3 pre-osteoblasts predominantly express the Nox4 isotype of NAD(P)H oxidase. To extend this finding, we tested the functional effects of Nox4. Adenovirus-mediated expression of dominant-negative Nox4 inhibited BMP-2-induced alkaline phosphatase expression. BMP-2 promotes expression of BMP-2 for maintenance of the osteoblast phenotype. NAC and DPI significantly blocked BMP-2-stimulated expression of BMP2 mRNA and protein due to a decrease in BMP2 gene transcription. Dominant-negative Nox4 also mimicked this effect of NAC and DPI. Our results provide the first evidence for a new signalling pathway linking BMP-2-stimulated Nox4-derived physiological ROS to BMP-2 expression and osteoblast differentiation.
骨形态发生蛋白 2（BMP-2）可促进成骨细胞前体细胞向成熟成骨细胞分化，从而形成健康的骨骼。在本研究中，我们揭示了 BMP-2 诱导成骨细胞分化的新机制。抗氧化剂 NAC（N-乙酰-L-半胱氨酸）和黄素蛋白酶 NAD（P）H 氧化酶抑制剂 DPI（二苯基碘）可阻止 BMP-2 刺激的碱性磷酸酶表达和矿化骨结节形成。BMP-2 迅速产生 ROS（活性氧），同时 NAD（P）H 氧化酶的激活增加。NAC 和 DPI 分别抑制 BMP-2 诱导的 ROS 产生和 NAD（P）H 氧化酶活性。NAD（P）H 氧化酶具有结构相似的催化亚基（Nox1-5），在不同细胞中表达不同。我们证明 2T3 前成骨细胞主要表达 NAD（P）H 氧化酶的 Nox4 同工型。为了扩展这一发现，我们测试了 Nox4 的功能影响。腺病毒介导的显性负性 Nox4 抑制 BMP-2 诱导的碱性磷酸酶表达。BMP-2 促进 BMP-2 的表达以维持成骨细胞表型。NAC 和 DPI 由于 BMP2 基因转录减少而显著阻断 BMP-2 刺激的 BMP2 mRNA 和蛋白表达。显性负性 Nox4 也模拟了 NAC 和 DPI 的这种作用。我们的研究结果为新的信号通路提供了首个证据，该信号通路将 BMP-2 刺激的 Nox4 衍生的生理性 ROS 与 BMP-2 表达和成骨细胞分化联系起来。
