Department of Biochemistry and Genetics, University of Navarra, Pamplona, Spain.
Department of Biochemistry and Genetics, University of Navarra, Pamplona, Spain; IdiSNA, Navarra Institute for Health Research, Spain.
Free Radic Biol Med. 2018 Oct;126:15-26. doi: 10.1016/j.freeradbiomed.2018.07.013. Epub 2018 Jul 21.
NADPH oxidase (Nox) variants Nox1, Nox2 and Nox4 are implicated in the progression of liver fibrosis. However, the role of Nox5 is not yet known, mainly due to the lack of this enzyme in rat and mouse genomes. Here we describe the expression and functional relevance of Nox5 in the human cell line of hepatic stellate cells (HSC) LX-2. Under basal conditions, three long (Nox5-L: Nox5α, -β, and -δ) and a short (Nox5-S or Nox5ε) splice variants were detected, which were silenced with specific siRNAs for Nox5. The most abundant isoform was Nox5-S, accounting for more than 90% of Nox5 protein. Overexpression of Nox5β generated reactive oxygen species (ROS) in the presence of calcium, as judged by the production of hydrogen peroxide, L-012 luminescence and cytochrome c reduction. Nox5ε did not generated ROS under these conditions, and a reduced ROS production was observed when co-expressed with Nox5β. In contrast, dihydroethidium oxidation was increased by Nox5β or Nox5ε, suggesting that Nox5ε induced intracellular oxidative stress by an unknown mechanism. Functional studies showed that both Nox5β and Nox5ε stimulated the proliferation of LX-2 cells and the collagen type I levels, while Nox5 siRNAs inhibited these effects. Interestingly, TGF-β and angiotensin II upregulated Nox5 expression, which was reduced in cells pre-incubated with catalase. Further studies silencing Nox5 in TGF-β-treated cells resulted in a reduction of collagen levels via p38 MAPK. Collectively, these results show for the first time that Nox5 can play a relevant role in the proliferation and fibrosis on human HSC.
NADPH 氧化酶 (Nox) 变体 Nox1、Nox2 和 Nox4 被认为与肝纤维化的进展有关。然而，Nox5 的作用尚不清楚，主要是因为这种酶在大鼠和小鼠基因组中缺失。在这里，我们描述了 Nox5 在人肝星状细胞 (HSC) LX-2 细胞系中的表达和功能相关性。在基础条件下，检测到三种长 (Nox5-L：Nox5α、-β 和 -δ) 和一种短 (Nox5-S 或 Nox5ε) 剪接变体，这些变体被特异性 Nox5 siRNA 沉默。最丰富的亚型是 Nox5-S，占 Nox5 蛋白的 90%以上。在存在钙的情况下，过表达 Nox5β 会产生活性氧物质 (ROS)，这可以通过产生过氧化氢、L-012 发光和细胞色素 c 还原来判断。在这些条件下，Nox5ε 不会产生 ROS，并且当与 Nox5β 共表达时，观察到 ROS 产生减少。相比之下，二氢乙啶氧化增加了 Nox5β 或 Nox5ε，这表明 Nox5ε 通过未知机制引起细胞内氧化应激。功能研究表明，Nox5β 和 Nox5ε 均刺激 LX-2 细胞的增殖和胶原 I 水平，而 Nox5 siRNA 抑制了这些作用。有趣的是，TGF-β 和血管紧张素 II 上调了 Nox5 的表达，而过氧化氢酶预处理的细胞中这种表达减少。进一步研究表明，在 TGF-β 处理的细胞中沉默 Nox5 会通过 p38 MAPK 减少胶原水平。总之，这些结果首次表明 Nox5 可以在人 HSC 的增殖和纤维化中发挥重要作用。
