Oxidative stress has been identified as a key mechanism of hepatitis C virus (HCV)-induced pathogenesis. Studies suggest that HCV increases the generation of hydroxyl radical and peroxynitrite close to the cell nucleus, inflicting DNA damage, but the source of reactive oxygen species (ROS) remains incompletely characterized. We hypothesized that HCV increased the generation of superoxide and H2O2 close to the hepatocyte nucleus and that this source of ROS was Nox4. Huh7 human hepatoma cells and telomerase-reconstituted primary human hepatocytes, transfected or infected with virus-producing HCV strains of genotype 2a and 1b, were examined for mRNA, protein, and subcellular localization of Nox proteins, along with human liver. We found that genotype 2a HCV induced persistent elevation of Nox1 and Nox4 mRNA and proteins in Huh7 cells. Genotype 1b HCV likewise elevated the levels of Nox1 and Nox4 in telomerase-reconstituted primary human hepatocytes. Furthermore, Nox1 and Nox4 proteins were increased in HCV-infected human liver compared to uninfected liver. Unlike Nox1, Nox4 was prominent in the nuclear compartment of these cells as well as human liver, particularly in the presence of HCV. HCV-induced ROS and nuclear nitrotyrosine could be decreased with siRNAs to Nox1 and Nox4. Finally, HCV increased the level of transforming growth factor beta-1 (TGFβ1). TGFβ1 could elevate Nox4 expression in the presence of infectious HCV, and HCV increased Nox4 at least in part through TGFβ1. Conclusion HCV induced a persistent elevation of Nox1 and Nox4 and increased nuclear localization of Nox4 in hepatocytes in vitro and in human liver. Hepatocyte Nox proteins are likely to act as a persistent, endogenous source of ROS during HCV-induced pathogenesis.

Transforming growth factor ␤ (TGF-␤) stimulates reactive oxygen species (ROS) production in various cell types, which mediates many of the effects of TGF-␤. The molecular mechanisms whereby TGF-␤ increases ROS production and ROS modulate the signaling processes of TGF-␤, however, remain poorly defined. In this study, we show that TGF-␤1 stimulates NADPH oxidase 4 (Nox4) expression and ROS generation in the nucleus of murine embryo fibroblasts (NIH3T3 cells). This is associated with an increase in protein thiol modification and inactivation of MAPK phosphatase 1 (MKP-1), a nuclear phosphatase. Furthermore, knockdown of MKP-1 using small interfering RNA enhances TGF-␤1-induced phosphorylation of JNK and p38 as well as the expression of plasminogen activator inhibitor 1 (PAI-1), a TGF-␤-responsive gene involved in the pathogenesis of many diseases. Knockdown of Nox4 with Nox4 small interfering RNA, on the other hand, reduces TGF-␤1-stimulated ROS production, p38 phosphorylation, and PAI-1 expression. TGF-␤ also increased the nuclear level of Nox4 protein as well as PAI-1 expression in human lung fibroblasts (CCL-210 cells), suggesting that TGF-␤ may induce PAI-1 expression by a similar mechanism in human lung fibroblasts. In summary, in this study we have identified nuclear MAPK phosphatase MKP-1 as a novel molecular target of ROS in TGF-␤ signaling pathways. Our data suggest that increased generation of ROS by Nox4 mediates TGF-␤1-induced PAI-1 gene expression at least in part through oxidative modification and inhibition of MKP-1 leading to a sustained activation of JNK and p38 MAPKs.

Objective We investigated atheroprotective properties of apoE beyond its ability to lower plasma cholesterol. We hypothesized that apoE reduces atherosclerosis by decreasing lipid accumulation in circulating monocytes and the inflammatory state of monocytes and the vascular endothelium. Methods and Results We developed mice with spontaneous hyperlipidemia with and without plasma apoE: Hypomorphic apoE mice deficient in low-density lipoprotein receptor (Apoeh/hLdlr–/–) were compared to Apoe–/–Ldlr–/– mice. Despite 4-fold more plasma apoE than WT mice, Apoeh/hLdlr–/– mice displayed similar plasma cholesterol as Apoe–/–Ldlr–/– mice but developed 4-fold less atherosclerotic lesions by 5 months of age. The aortic arch of Apoeh/hLdlr–/– mice showed decreased endothelial expression of ICAM-1, PECAM-1, and JAM-A. In addition, Apoeh/hLdlr–/– mice had less circulating leukocytes and pro-inflammatory Ly6Chigh monocytes. These monocytes had decreased neutral lipid content and reduced surface expression of ICAM-1, VLA-4, and L-Selectin. Apoeh/hLdlr–/– mice displayed increased levels of apoA1-rich HDL that were potent in promoting cellular cholesterol efflux. Conclusions Our findings suggest that apoE reduces atherosclerosis in the setting of hyperlipidemia by increasing plasma apoA1-HDL that likely contribute to reduce intracellular lipid accumulation and thereby the activation of circulating leukocytes and the vascular endothelium.

A critical event in mammalian embryo development is construction of an inner cell mass surrounded by a trophoectoderm (a shell of cells that later form extraembryonic structures). We utilize multi-scale, stochastic modeling to investigate the design principles responsible for robust establishment of these structures. This investigation makes three predictions, each supported by our quantitative imaging. First, stochasticity in the expression of critical genes promotes cell plasticity and has a critical role in accurately organizing the developing mouse blastocyst. Second, asymmetry in the levels of noise variation (expression fluctuation) of Cdx2 and Oct4 provides a means to gain the benefits of noise-mediated plasticity while ameliorating the potentially detrimental effects of stochasticity. Finally, by controlling the timing and pace of cell fate specification, the embryo temporally modulates plasticity and creates a time window during which each cell can continually read its environment and adjusts its fate. These results suggest noise has a crucial role in maintaining cellular plasticity and organizing the blastocyst.