HIV-Tat Induces the Nrf2/ARE Pathway through NMDA Receptor-Elicited Spermine Oxidase Activation in Human Neuroblastoma Cells

Abstract

Previously, we reported that HIV-Tat elicits spermine oxidase (SMO) activity upregulation through NMDA receptor (NMDAR) stimulation in human SH-SY5Y neuroblastoma cells, thus increasing ROS generation, which in turn leads to GSH depletion, oxidative stress, and reduced cell viability. In several cell types, ROS can trigger an antioxidant cell response through the transcriptional induction of oxidative stress-responsive genes regulated by the nuclear factor erythroid 2-related factor 2 (Nrf2). Here, we demonstrate that Tat induces both antioxidant gene expression and Nrf2 activation in SH-SY5Y cells, mediated by SMO activity. Furthermore, NMDAR is involved in Tat-induced Nrf2 activation. These findings suggest that the NMDAR/SMO/Nrf2 pathway is an important target for protection against HIV-associated neurocognitive disorders.

Fig 1. Effects of Tat on ARE-driven gene expression in SH-SY5Y cells.

Cells grown on 35-mm-diameter tissue culture dishes were treated with Tat (200 ng/ml) for 4, 8, and 24 h. After incubation at 37°C, the cells were homogenized and total RNA has been purified to assess mRNA levels of several genes (NQO1, CAT, SOD1, SOD2, HO-1) by RT-qPCR. Data are calculated relative to the internal housekeeping gene (GAPDH) and are expressed as the mean fold change compared with control. Each value represents the mean ± SEM of three independent experiments. One-way ANOVA, followed by Bonferroni's test, was used to determine significant differences. NQO1: * p≤0.01 vs CTRL, ** p≤0.01 vs CTRL, *** Not significant vs CTRL; CAT: * p≤0.01 vs CTRL, ** p≤0.05 vs CTRL, *** Not significant vs CTRL; SOD1: * p≤0.01 vs CTRL, ** p≤0.05 vs CTRL, *** Not significant vs CTRL; SOD2: * p≤0.01 vs CTRL, ** p≤0.05 vs CTRL, *** Not significant vs CTRL; HO-1: * p≤0.05 vs CTRL, ** p≤0.01 vs CTRL, *** Not significant vs CTRL.

Fig 2. Effects of Tat on spermine oxidase activity.

SH-SY5Y cells (1×10⁶) were treated with Tat (200 ng/ml) for 15 and 60 min. At the end of incubation cell extracts were prepared as described in Materials and methods and analyzed for SMO activity. The data shown are the means of three independent experiments. One-way ANOVA, followed by Bonferroni's test, was used to determine significant differences. ** p≤0.05 vs CTRL, * p≤0.01 vs CTRL.

Fig 3. Effects of SMO inhibition on Tat-induced gene expression in SH-SY5Y cells.

Cells grown on 35-mm-diameter tissue culture dishes were overnight pretreated with CHL (10 nM) or medium alone before the addition of Tat (200 ng/ml) for 4 h. After incubation at 37°C, the cells were homogenized and total RNA has been purified to assess mRNA levels of NQO1, CAT, SOD1, SOD2, HO-1 genes by RT-qPCR. Data are calculated relative to GAPDH and are expressed as the mean fold change compared with control. Each value represents the mean ± SEM of three independent experiments. One-way ANOVA, followed by Bonferroni's test, was used to determine significant differences. NQO1: * p≤0.01 vs CTRL, ^ p≤0.01 vs Tat; CAT: * p≤0.01 vs CTRL, ^ p≤0.01 vs Tat; SOD1: * p≤0.01 vs CTRL, ^ p≤0.01 vs Tat; SOD2: * p≤0.01 vs CTRL, ^ p≤0.01 vs Tat; HO-1: * p≤0.01 vs CTRL, ^ p≤0.01 vs Tat.

Fig 4. Effects of Tat on Nrf2 nuclear translocation in SH-SY5Y cells.

Cells grown on 100-mm-diameter tissue culture dishes were treated with Tat (200 ng/ml) for the indicated time points. After incubation at 37°C, cells were mechanically harvested, and the nuclear extracts were prepared as specified in the Materials and Methods section to assess Nrf2 levels by western blot analysis. The histogram shows the densitometric analysis of the western blots for each sample. Values are calculated relative to the nuclear Lamin B content and are the means ± SEM from three separate experiments, each performed in duplicate. One-way ANOVA, followed by Bonferroni's test, was used to determine significant differences. ** p≤0.05 vs CTRL, * p≤0.01 vs CTRL.

Fig 5. Effects of SMO inhibition on Tat-induced Nrf2 activation in SH-SY5Y cells.

Cells grown on 100-mm-diameter tissue culture dishes were overnight pretreated with CHL (10 nM) or medium alone before the addition of Tat (200 ng/ml) for 15 min. After incubation at 37°C, cells were mechanically harvested, and the nuclear extracts were prepared as specified in Methods to assess Nrf2 levels by western blot analysis. The histogram shows the densitometric analysis of the western blots for each sample. Values are calculated relative to the nuclear Lamin B content and are the means ± SEM from three separate experiments, each performed in duplicate. One-way ANOVA, followed by Bonferroni's test, was used to determine significant differences. * p≤0.01 vs CTRL, ^ p≤0.01 vs Tat, § Not significant vs Tat + CHL.

Fig 6. Role of NMDA receptor in Tat-induced Nrf2 activation.

SH-SY5Y cells grown on 35-mm-diameter tissue culture dishes were pretreated for 2 h with MK-801 (10 μM), NAC (2 mM), or medium alone before the addition of Tat (200 ng/ml) for 15 min. After incubation at 37°C, the cells were homogenized, and Nrf2 activation was quantified by TransAM assay as detailed in the Materials and Methods section. Data points are the means ± S.E.M. from 3 separate experiments, each performed in duplicate. One-way ANOVA, followed by Bonferroni's test, was used to determine significant differences. * p≤0.01 vs CTRL, ^ p≤0.01 vs Tat, § p≤0.01 vs CTRL.

Fig 7. Proposed model of the role of NMDAR-elicited SMO activation in the Tat-induced Nrf2 pathway.

On the one hand, HIV-1 Tat induces neuronal cell death through the production of H₂O₂ and 3-AP by a mechanism involving NMDAR-induced SMO activation. On the other hand, the same pathways are able to trigger an antioxidant response through the transcriptional induction of Nrf2-dependent ARE genes. For more details see text. Abbreviations: 3-AP, aldehyde 3-aminopropanal; ARE, antioxidant-response element; CAT, catalase; CHL, Chlorhexidine digluconate; DMF, dimethyl fumarate; H₂O₂, hydrogen peroxide; HIV, human immunodeficiency virus; NQO1, NAD(P)H:quinone oxidoreductase type 1; HO, heme-oxygenase; NAC, N-acetylcysteine; NMDA, N-methyl-D-aspartate; Nrf2, nuclear factor erythroid 2-related factor 2; SMO, spermine oxidase; SOD, superoxide dismutase.