Nox4 is a novel inducible source of reactive oxygen species in monocytes and macrophages and mediates oxidized low density lipoprotein-induced macrophage death

Abstract

Rationale: The enhanced formation of intracellular reactive oxygen species (ROS) induced by oxidized low-density lipoprotein (OxLDL) promotes macrophage death, a process likely to contribute to the formation of necrotic cores and the progression of atherosclerotic lesions. Yet macrophage deficiency of phagocytic NADPH oxidase (Nox2), the primary source of ROS in macrophages, does not reduce atherosclerotic lesion development in mice. This suggests an as yet unidentified NADPH oxidase may be present in macrophages and responsible for the intracellular ROS formation induced by OxLDL.

Objective: The aim of this study was to identify the source of intracellular ROS involved in macrophage death.

Methods and results: Nox4 was expressed in human monocytes and mature macrophages, and was localized to the endoplasmic reticulum and to defined foci within the nucleus. Nox4 colocalized with p22(phox), and both proteins were upregulated in response to OxLDL stimulation, whereas Nox2/gp91(phox) levels remained unchanged. Induction of Nox4 expression, intracellular ROS formation and macrophage cytotoxicity induced by OxLDL were blocked by MEK1/2 inhibition, but not by inhibitors of p38-MAPK (mitogen-activated protein kinase), JNK (Jun N-terminal kinase), or JAK2 (Janus kinase 2). Small interfering RNA knockdown of Nox4 inhibited both intracellular ROS production and macrophage cytotoxicity induced by OxLDL, whereas Nox4 overexpression enhanced both OxLDL-stimulated ROS formation and macrophage death.

Conclusions: Nox4 is a novel source of intracellular ROS in human monocytes and macrophages. Induction of Nox4 by OxLDL is mediated by the MEK1/ERK pathway and required for OxLDL cytotoxicity in human macrophages, implicating monocytic Nox4 in atherogenesis.

Figure 1. Human blood monocytes and mature human monocyte-derived macrophages (HMDM) express Nox4 mRNA

Human mononuclear cells were isolated by Ficoll gradient centrifugation. Human monocytes were allowed to adhere for 1 h and non-adhering cells were removed. Mature HMDM were generated from the same mononuclear cell fraction and allowed to differentiate as described under “Methods”. Expression profiling was performed by semi-quantitative RT-PCR using the gene specific primers listed in Table 1. Representative images of three independent experiments are shown. Mono: human monocytes; HMDM: human monocyte-derived macrophages.

Figure 2. Nox4 protein colocalizes with p22^phox and is localized in the endoplasmic reticulum and the nucleus of HMDM

Immunohistochemistry and confocal microscopy was performed on HMDM stained with our new rabbit monoclonal antibody directed against Nox4 (red) and p22^phox (Panel A; green) or the ER-specific marker PDI (Panel B; green), or DAPI (Panel C; blue; arrows identify discrete Nox4 loci). The overlays show the extent of co-localization (yellow) of Nox4 with each of these markers. Bar = 10 μm. Panel D: HMDM lysates were prepared and Nox4 protein was immunoprecipitated as described under “Methods”. Immunoprecipitates were subjected to Western blot analysis and probed for Nox4 and of p22^phox.

Figure 3. OxLDL induces Nox4 and p22^phox protein expression

HMDM were stimulated with OxLDL for 18 h at the concentrations indicated. Expression levels of Nox1 through 5 mRNA were determined were determined by RT-PCR (Panel A and B). Nox4 protein levels were determined by Western blot analysis (Panel C). Nox2 protein expression was not increased by OxLDL under any of the conditions tested (see Supplemental Figure IIA). Panel D: Time course of Nox4 and p22^phox protein expression in response to OxLDL (75μg/ml). Quantitative results from Western blot analyses are shown as mean ± standard deviation from 3 independent experiments. *: P<0.01 versus vehicle or 0 h (n=3)

Figure 4. Inhibition ofMEK1/2 blocks OxLDL-induced Nox4 expression, intracellular ROS formation and cytotoxicity

HMDM were pre-incubated for 2 h with either vehicle (DMSO), U0126 (U, 5μM), SB203580 (SB, 5μM), or SP600125 (SP, 10μM) and then exposed to OxLDL (75 μg/ml) for 6 h (protein expression, ROS formation) or 18 h (cytotoxicity). For each experiment, we determined Nox4 protein expression by Western blot analysis (Panel A and B), intracellular ROS production (Panel C), and cytotoxicity (Panel D). For each parameter measured, data were normalized to the OxLDL-induced signal determined in vehicle-treated HMDM versus vehicle-treated cells incubated in the absence of OxLDL, and that signal was set at 1.0 to allow for comparisons of all three signals measured. Nox4 protein levels were normalized against β-actin. *: P <0.01 versus DMSO (n=3).

Figure 5. Knockdown of Nox4 attenuates intracellular ROS formation and macrophage death induced by OxLDL whereas overexpression of Nox4 sensitizes macrophages to OxLDL-induced ROS formation and cell injury

For the knockdown experiments, HMDM were infected overnight with adenoviruses carrying either siRNA directed against either Nox4 (Nox4i) or a random sequence (Scr) (Panel A, B, C). To overexpress Nox4, HMDM were infected with adenoviruses carrying a doxycycline-inducible construct with the sequence for human Nox4 (Panel D, E, F). Nox4 expression was induced by adding 1 μg/ml of doxycycline (Dox) to the culture medium. HMDM were stimulated with OxLDL (75μg/ml) for 6 h to measure Nox4 protein expression and ROS production, and for 18 h to determine cytotoxicity. Nox4 protein levels were normalized against β-actin. Representative Western blots are shown in panels A and D. To allow for a direct comparison of the effect of Nox4 knockdown or overexpression on Nox4 expression levels (Panels A and D), ROS production (Panels B and E) and cytotoxicity (Panels C and F), data were normalized as described in Figure 4. *: P <0.05 versus Scr (n=3).

Figure 6. Hypothetical model for the role of Nox4 in OxLDL-mediated macrophage cytotoxicity

OxLDL induces Nox4 expression via the MEK/ERK pathway and is blocked by MEK inhibitors UO126 and PD98059. Knock-down of Nox4 (Nox4 RNAi) protected macrophages from increased ROS production and cell injury. The putative site of action for the peroxyl radical scavenger Trolox is indicated. Overexpression of Nox4 (pAdNox4) does not affect basal ROS production or macrophage viability, but sensitizes macrophages to OxLDL-induced ROS formation and cell death.