Transcription factors NRF2 and NF-κB are coordinated effectors of the Rho family, GTP-binding protein RAC1 during inflammation

Abstract

The small GTPase protein RAC1 participates in innate immunity by activating a complex program that includes cytoskeleton remodeling, chemotaxis, activation of NADPH oxidase, and modulation of gene expression. However, its role in regulating the transcriptional signatures that in term control the cellular inflammatory profiles are not well defined. Here we investigated the functional and mechanistic connection between RAC1 and the transcription factor NRF2 (nuclear factor erythroid 2-related factor 2), master regulator of the anti-oxidant response. Lipopolysaccharide and constitutively active RAC1(Q61L) mutant induced the anti-oxidant enzyme heme-oxygenase-1 (HO-1) through activation of NRF2. The use of KEAP1-insensitive NRF2 mutants indicated that RAC1 regulation of NRF2 is KEAP1-independent. Interestingly, NRF2 overexpression inhibited, whereas a dominant-negative mutant of NRF2 exacerbated RAC1-dependent activation of nuclear factor-κB (NF-κB), suggesting that NRF2 has an antagonistic effect on the NF-κB pathway. Moreover, we found that RAC1 acts through NF-κB to induce NRF2 because either expression of a dominant negative mutant of IκBα that leads to NF-κB degradation or the use of p65-NF-κB-deficient cells demonstrated lower NRF2 protein levels and basally impaired NRF2 signature compared with control cells. In contrast, NRF2-deficient cells showed increased p65-NF-κB protein levels, although the mRNA levels remain unchanged, indicating post-translational alterations. Our results demonstrate a new mechanism of modulation of RAC1 inflammatory pathway through a cross-talk between NF-κB and NRF2.

Keywords: Inflammation; Lipopolysaccharide (LPS); Microglia; NF-κ B (NF-KB); Nuclear Factor 2 (Erythroid-derived 2-like Factor) (NFE2L2) (Nrf2); Oxidative Stress; Ras-related C3 Botulinum Toxin Substrate 1 (Rac1); Rho GTPases.

FIGURE 1.

LPS activates RAC1 and increases HO-1 protein levels in microglia. A, BV-2 cells were treated with LPS (500 ng/ml) for 5, 10, 20, and 40 min, and cell lysates were used to perform a GST-PAK1-PBD pulldown assay as described under “Experimental Procedures.” As a negative control we used the same cell lysate from 40 min of treatment but with a GST empty vector. Immunoblots with anti-RAC1 antibody: upper panel, active RAC1; lower panel, total RAC1. B, quantification of immunoblots from two independent experiments for active RAC1 and total RAC1. One-way ANOVA followed by Newman-Keuls test was used to assess differences among groups. Asterisks denote significant differences. ***, p < 0.001, comparing the basal group to the indicated groups. C, BV-2 cells were treated with different doses of LPS for 6 h. Upper panel, immunoblots with anti-HO-1 antibody; middle panel, immunoblots with anti-IBA1 antibody as a control for microglial activation after LPS exposure; lower panel, anti-β-actin as protein load control. D, quantification of immunoblots for HO-1 at 6 and 24 h after LPS treatment. One-way ANOVA followed by Newman-Keuls test was used to assess differences among groups. Asterisks and plus symbols denote significant differences. *, p < 0.05; **, p < 0.01; ***, p < 0.001 comparing the indicated groups. E, BV-2 cells were treated with different doses of LPS for 24 h. Upper panel, immunoblots with anti-HO-1 antibody; middle panel, immunoblots with anti-IBA1 antibody as a control for microglial activation after LPS exposure; lower panel, anti-β-actin as protein load control. F, quantification of immunoblots for IBA1 at 6 and 24 h after LPS treatment. The statistical analysis was performed as in D. G–J, qRT-PCR determination of mRNA levels of Ho-1 (G), Nqo1 (H), Il-1β (I), and Tnf (J) normalized by β-actin levels. Student's t test was used to assess differences between groups. Asterisks denote statistically significant differences with: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 2.

RAC1 activates the ARE/HO-1 pathway. A, HEK-TLR4-MD2/CD14 cells were treated with 1 μg/ml LPS for 6 and 24 h. Upper panel, immunoblots with anti-HO-1 antibody; lower panel, anti-β-actin as protein load control. B, quantification of immunoblots for HO-1 at 6 and 24 h after LPS treatment. Student's t test was used to assess differences between groups. Asterisks denote statistically significant differences with: *, p < 0.05; **, p < 0.01. C and D, qRT-PCR determination of mRNA levels of Ho-1 (C) and Tnf (D) normalized by β-actin levels. One-way ANOVA followed by Newman-Keuls test was used to assess differences among groups. Asterisks denote significant differences (**, p < 0.01; ***, p < 0.001) comparing the basal group to the indicated groups. E, LPS induces p65 and NRF2 nuclear translocation. HEK-TLR4-MD2/CD14 cells were treated with 1 μg/ml for 15, 30, 60, 120, and 180 min, and subcellular fractionation was performed. Upper panels, immunoblots with anti-p65 or NRF2 antibodies; middle panel, immunoblots with GAPDH antibody as a control for cytosolic protein load; lower panel, anti-lamin B (B) as a control for nuclear protein load. F and G, quantification of immunoblots for p65 (F) or NRF2 (G) after LPS treatment. One-way ANOVA followed by Newman-Keuls test was used to assess differences among groups. Asterisks and plus denote significant differences: *, p < 0.05; **, p < 0.01; ***, p < 0.001 comparing the indicated groups. H, HEK-TLR4-MD2/CD14 cells were treated with LPS (1 μg/ml) for 5, 10, 20, and 40 min, and cell lysates were used to analyze active RAC1 by an active-RAC1-specific antibody (see “Experimental Procedures”). Immunoblots: upper panel, active RAC1; middle panel, total RAC1; lower panel, GAPDH as protein load control. I, quantification of immunoblots from two independent experiments for active RAC1 and total RAC1. One-way ANOVA followed by Newman-Keuls test was used to assess differences among groups. Asterisks denote significant differences (**p < 0.01; ***, p < 0.001) comparing the basal group to the indicated groups. J, HEK293T cells were transfected with different concentrations of the expression vector for AU5-tagged RAC1^Q61L. Upper panel, HO-1; middle panel, AU5; lower panel, anti-GAPDH as protein loading control. K, quantification of immunoblots for HO-1. The statistical analysis was performed as in I. L and M, HEK293T cells were transfected with the HO1–15-LUC or ARE-LUC and TK-Renilla control vectors, with different amounts of RAC1^Q61L vector. One-way ANOVA followed by Newman-Keuls test was used to assess differences among groups. Asterisks denote significant differences (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

FIGURE 3.

RAC1 induces NRF2 signaling pathway. A, RAC1 induces NRF2 expression in a dose-dependent manner. Cells were transfected with different doses of RAC1^Q61L and a constant amount of V5-tagged NRF2 or NRF2^ΔETGE-mutant plasmids. Upper panels, immunoblots with anti-AU5 antibody; middle panel, immunoblots with anti-V5; lower panel, anti-GAPDH as protein load control. B, quantification of immunoblots for NRF2-WT-V5 and NRF2-ΔETGE-V5. One-way ANOVA followed by Newman-Keuls test was used to assess differences among groups as previously. C, RAC1 induces NRF2 nuclear translocation. Cells were co-transfected with or without RAC1^Q61L and a constant dose of NRF2^ΔETGE-V5 plasmid, and subcellular fractionation was performed. Upper panels, immunoblots with anti-AU5 or V5 antibodies; middle panel, immunoblots with GAPDH antibody as a control for cytosolic protein load; lower panel, anti-lamin B as a control for nuclear protein load. D, RAC1 requires NRF2 to activate the HO-1 promoter. HEK293T cells were co-transfected with RAC1^Q61L expression vector, pHO1–15-LUC, Renilla control vectors, and either empty vector or dominant negative NRF2, (DN)-NRF2, expression vector. E, RAC1 uses the transactivating activity of NRF2 to induce AREs. Cells were co-transfected with Gal4-LUC (or pGL3-basic as a control) and either empty vector of expression vectors for Gal4NRF2 and RAC1 as indicated. Luciferase experiments were performed at least three times using three-four samples per group. The values in graphs correspond to the mean ± S.E. Student's t test was used to assess differences between groups or one-way ANOVA followed by a Newman-Keuls post-test (C). Asterisks denote statistically significant differences with: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 4.

RAC1 activates NF-κB pathways and NRF2 acts as modulator. A, RAC1 overexpression induces generation of intracellular ROS. HEK293T cells were transfected with different doses of RAC1^Q61L or empty (Ctrl) vectors, and after 16 h cells were incubated with 10 μm 2′-7′-dihydrodichlorofluorescein diacetate (H₂DCFDA) for 1 h before fluorescence measurement. 2 mm H₂O₂ was used as positive control. B, RAC1 induces NF-κB pathway. Cells were co-transfected with NF-κB-dependent (−453/+80) HIV-LUC reporter and different concentrations of RAC1^Q61L vector in absence or presence of p65/p50 plasmids. C, NRF2 inhibits NF-κB pathway. Cells were co-transfected with HIV-LUC reporter, p50, and increasing amounts of p65 in the absence or presence of NRF2^ΔETGE-V5. D, p65/p50 increased the ARE activity mediated by NRF2. Cells were co-transfected with 3XARE-LUC, p50, and different concentrations of p65 in absence or presence of NRF2^ΔETGE-V5. E, cells were co-transfected with HIV-LUC reporter, RAC1^Q61L, NRF2^ΔETGE-V5, or both in the absence (white bars) or presence (black bars) of p65/p50. F, cells were co-transfected with HIV-LUC reporter, RAC1^Q61L, (DN)-NRF2, or both in the absence (white bars) or presence (black bars) of p65/p50. One-way ANOVA followed by a Newman-Keuls post-test was used to assess significant differences among groups. Asterisks denote statistically significant differences with: ***, p < 0.001; ##, p < 0.01; ###, p < 0.001 (respect to their control).

FIGURE 5.

IκBα mediates the activation of NRF2 and NF-κB induced by RAC1. A, a dominant-negative mutant of IκBα (IκBα^S32A/S36A) inhibits RAC1-dependent induction of the NF-κB pathway. HEK293T cells were co-transfected with HIV-LUC reporter, RAC1^Q61L, IκBα^S32A/S36A, or both in the absence (white bars) or presence (black bars) of p65/p50. B, IκBα is implicated in the induction of NRF2 by RAC1. Cells were co-transfected with ARE-LUC promoter, RAC1^Q61L, IκBα^S32A/S36A, or both in the absence (white bars) or presence (black bars) of p65/p50. One-way ANOVA followed by a Newman-Keuls post-test was used to assess significant differences among groups. Asterisks denote statistically significant differences with: *, p < 0.05; **, p < 0.005; ***, p < 0.001; ###, p < 0.001 (with respect to their control). C, Western blot analysis of cells co-transfected with RAC1^Q61L, IκBα^S32A/S36A, and NRF2^ΔETGE-V5 and their respective controls. Upper panel, immunoblot with anti-V5 antibody; middle panel, immunoblot with anti-AU5; lower panel, anti-GAPDH as protein load control. D, EMSA, using a double-stranded oligonucleotide containing the core ARE sequence and nuclear extracts from HEK-TLR4-MD2/CD14 cells treated with 1 μg/ml LPS for 1 h. E, EMSA, using a double-stranded oligonucleotide containing the core ARE sequence and nuclear extracts from HEK293T cells expressing RAC1^Q61L ± IκBα^S32A/S36A. F, EMSA, using a double-stranded oligonucleotide containing the core ARE sequence and nuclear extracts from HEK293T cells expressing RAC1^Q61L ± DN-NRF2. In all cases the concentration of unlabeled oligonucleotide was 100× in the competition binding between labeled and unlabeled ARE probe. G, EMSA, using a double-stranded oligonucleotide containing the NF-κB binding element and nuclear extracts from HEK-TLR4-MD2/CD14 cells treated with 1 μg/ml LPS for 1 h. H, EMSA, using a double-stranded oligonucleotide containing the NF-κB promoter sequence and nuclear extracts from HEK293T cells expressing RAC1^Q61L ± IκB^S32A/S36A. I, EMSA, using a double-stranded oligonucleotide containing the NF-κB promoter sequence and nuclear extracts from HEK293T cells expressing RAC1^Q61L ± DN-NRF2. In all cases the concentration of unlabeled oligonucleotide was 100× in the competition binding between labeled and unlabeled NF-κB probe. IkB* stands for IkB S32A/S36A mutations (a mutated plasmid). + indicates that the complete sample is Rac1Q61L + DN-NRF2, for example.

FIGURE 6.

NRF2/ARE pathway is modulated by p65-NF-κB. A and B, NRF2 expression is decreased in p65^−/− MEFs. A, qRT-PCR determination of mRNA levels of Nrf2 normalized by β-actin levels. B, immunoblot analysis of NRF2 protein levels, GAPDH was used as load control. C, quantification of immunoblots for NRF2 protein levels. Decreased levels of phase two enzymes in p65^−/− MEFs. qRT-PCR determination of mRNA levels of Gstm3 (D), Gpx (E), and Ho-1 (F) normalized by β-actin levels. Dpx, glutathione peroxidase. G, immunoblot analysis of HO-1 protein levels, GAPDH was used as load control. H, quantification of immunoblots for HO-1 protein levels. Student's t test was used to assess differences between groups. Asterisks denote statistically significant differences with: *, p < 0.05; **, p < 0.01; ***, p < 0.001. p65^+/+ and p65^−/− MEF were incubated in a serum-free medium with 14 μm of SFN for 6 h. I, qRT-PCR determination of mRNA levels of Ho-1 normalized by β-actin levels. J, immunoblot analysis of HO-1 protein levels; GAPDH was used as load control. K, quantification of immunoblots for HO-1 protein levels. Two-way ANOVA followed by a Bonferroni post-test was used to assess significant differences among groups. Asterisks denote statistically significant differences with: *, p < 0.05; ***, p < 0.001.

FIGURE 7.

NRF2 deficiency increased p65-NF-κB protein levels. A and B, qRT-PCR determination of mRNA levels of p65-NF-κB (A) or MnSOD (B) normalized by β-actin levels in NRF2^+/+ and NRF2^−/− MEFs at basal conditions and after 6 h of TNF-α (20 ng/ml) treatment. C, immunoblots analysis of p65, MnSOD, and XIAP protein levels GAPDH was used as load control in NRF2^+/+ and NRF2^−/− MEFs at basal conditions and after 6 h TNF-α (20 ng/ml) treatment. D--F, quantification of immunoblots for p65-NF-κB (D), MnSOD (E), and XIAP (F) protein levels. Two-way ANOVA followed by a Bonferroni post-test was used to assess significant differences among groups. Asterisks denote statistically significant differences with: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 8.

Implication of RAC1 in NF-κB and NRF2 transcriptional activities mediated by IκBα. A, RAC1 activation induces NF-κB and NRF2 transcription factors through IκBα implicated in different processes like oxidative stress, inflammation, or phagocytosis. But there are other pathways that could be implicated in these activation like PI3K and MAPK or the generation of ROS generated by NAPDH oxidase. B, in the absence of p65 there is a suboptimal expression of the Nrf2 and ARE-regulated genes, which are unable to regulate properly inflammation. The Nrf2 gene has a NF-κB binding site at the +270 bp from the TSS to which p65/p50 heterodimer is recruited (11). C, in the absence of NRF2 there is no proper modulation of redox homeostasis, which could lead to IκBα degradation, and therefore, we observed increased p65 protein levels, which could induce inflammation.