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• W2077895679 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 RAC1Q61L 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. 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 RAC1Q61L 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. The Rho GTPase RAC1 is a pleiotropic regulator of many cellular processes. It is a well established that RAC1 is a mediator in execution of the inflammatory program of the innate immune system (1Arbibe L. Mira J.P. Teusch N. Kline L. Guha M. Mackman N. Godowski P.J. Ulevitch R.J. Knaus U.G. Toll-like receptor 2-mediated NF-κB activation requires a Rac1-dependent pathway.Nat. Immunol. 2000; 1: 533-540Crossref PubMed Scopus (567) Google Scholar, 2Bokoch G.M. Regulation of innate immunity by Rho GTPases.Trends Cell Biol. 2005; 15: 163-171Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 3Ohsawa K. Imai Y. Kanazawa H. Sasaki Y. Kohsaka S. Involvement of Iba1 in membrane ruffling and phagocytosis of macrophages/microglia.J. Cell Sci. 2000; 113: 3073-3084Crossref PubMed Google Scholar). Among several roles assigned to RAC1 in inflammation, its participation in cytoskeleton remodeling and chemotaxis as well as in NADPH oxidase-dependent production of superoxide have been well established (4Sanlioglu S. Williams C.M. Samavati L. Butler N.S. Wang G. McCray Jr., P.B. Ritchie T.C. Hunninghake G.W. Zandi E. Engelhardt J.F. Lipopolysaccharide induces Rac1-dependent reactive oxygen species formation and coordinates tumor necrosis factor-α secretion through IKK regulation of NF-κB.J. Biol. Chem. 2001; 276: 30188-30198Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, 5Bosco E.E. Mulloy J.C. Zheng Y. Rac1 GTPase: a “Rac” of all trades.Cell. Mol. Life Sci. 2009; 66: 370-374Crossref PubMed Scopus (230) Google Scholar). Moreover, RAC1 leads to activation of the transcription factor NF-κB and its target genes, including several proinflammatory cytokines such as TNF (4Sanlioglu S. Williams C.M. Samavati L. Butler N.S. Wang G. McCray Jr., P.B. Ritchie T.C. Hunninghake G.W. Zandi E. Engelhardt J.F. Lipopolysaccharide induces Rac1-dependent reactive oxygen species formation and coordinates tumor necrosis factor-α secretion through IKK regulation of NF-κB.J. Biol. Chem. 2001; 276: 30188-30198Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, 6Utsugi M. Dobashi K. Ishizuka T. Kawata T. Hisada T. Shimizu Y. Ono A. Mori M. Rac1 negatively regulates lipopolysaccharide-induced IL-23 p19 expression in human macrophages and dendritic cells and NF-κB p65 transactivation plays a novel role.J. Immunol. 2006; 177: 4550-4557Crossref PubMed Scopus (33) Google Scholar). However, it is not known how cells activated by RAC1 ultimately change their gene expression back to a “resting” state or toward a resolution profile. One of the pathways implicated in the control of inflammation is the transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2), 2The abbreviations used are:NRF2nuclear factor erythroid 2-related factor 2AREantioxidant response elementHO-1heme oxygenase-1SFNsulforaphaneSODsuperoxide dismutaseDNdominant-negativeROSreactive oxygen speciesMEFmouse embryo fibroblastXIAPX-chromosome-linked inhibitor of apoptosisqRTquantitative real-timeKeap1Kelch-like ECH-associated protein 1. which is a master regulator of redox homeostasis (7Bryan H.K. Olayanju A. Goldring C.E. Park B.K. The Nrf2 cell defence pathway: Keap1-dependent and -independent mechanisms of regulation.Biochem. Pharmacol. 2013; 85: 705-717Crossref PubMed Scopus (780) Google Scholar). NRF2 regulates the expression of a battery of cytoprotective genes that share in common a cis-acting enhancer sequence termed antioxidant response element (ARE) (8Innamorato N.G. Lastres-Becker I. Cuadrado A. Role of microglial redox balance in modulation of neuroinflammation.Curr. Opin. Neurol. 2009; 22: 308-314Crossref PubMed Scopus (88) Google Scholar, 9Jazwa A. Cuadrado A. Targeting heme oxygenase-1 for neuroprotection and neuroinflammation in neurodegenerative diseases.Curr. Drug Targets. 2010; 11: 1517-1531Crossref PubMed Scopus (178) Google Scholar). These genes include those coding the antioxidant enzymes heme oxygenase-1 (HO-1) and NADP(H) quinone oxidoreductase (NQO1), enzymes of glutathione metabolism and protein degradation, through the proteasome and autophagy routes (10Joshi G. Johnson J.A. The Nrf2-ARE pathway: a valuable therapeutic target for the treatment of neurodegenerative diseases.Recent Pat. CNS Drug Discov. 2012; 7: 218-229Crossref PubMed Scopus (179) Google Scholar, 11Hayes J.D. Dinkova-Kostova A.T. The Nrf2 regulatory network provides an interface between redox and intermediary metabolism.Trends Biochem. Sci. 2014; 39: 199-218Abstract Full Text Full Text PDF PubMed Scopus (1263) Google Scholar). Previous data from our group indicated that NRF2 knock-out mice are hypersensitive to the inflammation induced by LPS (12Innamorato N.G. Rojo A.I. García-Yagüe A.J. Yamamoto M. de Ceballos M.L. Cuadrado A. The transcription factor Nrf2 is a therapeutic target against brain inflammation.J. Immunol. 2008; 181: 680-689Crossref PubMed Scopus (385) Google Scholar) and exhibit exacerbated activation of the brain resident macrophage, microglia, in comparison to their wild type littermates. Interestingly, the treatment with sulforaphane, a compound that induces NRF2-dependent gene expression, attenuated the LPS-induced microglial infiltration to the hippocampus, reinforcing the idea of NRF2 anti-inflammatory properties. This evidence has been corroborated in other recent reports by analyzing other cells of the monocyte-macrophage linage (8Innamorato N.G. Lastres-Becker I. Cuadrado A. Role of microglial redox balance in modulation of neuroinflammation.Curr. Opin. Neurol. 2009; 22: 308-314Crossref PubMed Scopus (88) Google Scholar, 13Rojo A.I. Innamorato N.G. Martín-Moreno A.M. De Ceballos M.L. Yamamoto M. Cuadrado A. Nrf2 regulates microglial dynamics and neuroinflammation in experimental Parkinson's disease.Glia. 2010; 58: 588-598Crossref PubMed Scopus (256) Google Scholar, 14Lastres-Becker I. Ulusoy A. Innamorato N.G. Sahin G. Rábano A. Kirik D. Cuadrado A. α-Synuclein expression and Nrf2 deficiency cooperate to aggravate protein aggregation, neuronal death, and inflammation in early-stage Parkinson's disease.Hum. Mol. Genet. 2012; 21: 3173-3192Crossref PubMed Scopus (178) Google Scholar, 15Brandenburg L.O. Kipp M. Lucius R. Pufe T. Wruck C.J. Sulforaphane suppresses LPS-induced inflammation in primary rat microglia.Inflamm. Res. 2010; 59: 443-450Crossref PubMed Scopus (102) Google Scholar). On the other hand, the transcription factor nuclear factor κB (NF-κB) plays a crucial role in immune responses through the regulation of genes encoding proinflammatory cytokines, adhesion molecules, chemokines, growth factors, and inducible enzymes (16Li Q. Verma I.M. NF-κB regulation in the immune system.Nat. Rev. Immunol. 2002; 2: 725-734Crossref PubMed Scopus (3307) Google Scholar). It is remarkable that NRF2 and NF-κB influence each other and coordinate the final fate of innate immune cells (17Wakabayashi N. Slocum S.L. Skoko J.J. Shin S. Kensler T.W. When NRF2 talks, who's listening?.Antioxid. Redox Signal. 2010; 13: 1649-1663Crossref PubMed Scopus (486) Google Scholar), but it is not known how this interconnection takes place. nuclear factor erythroid 2-related factor 2 antioxidant response element heme oxygenase-1 sulforaphane superoxide dismutase dominant-negative reactive oxygen species mouse embryo fibroblast X-chromosome-linked inhibitor of apoptosis quantitative real-time Kelch-like ECH-associated protein 1. In this study we report that RAC1 induces the anti-inflammatory NRF2/HO-1 pathway. We also provide evidence that NF-κB activity is induced by active RAC1 and that NRF2 can modulate this effect. These results uncover a new mechanism of regulation of inflammatory events trough a RAC1/NRF2/HO-1 axis. Human embryonic kidney (HEK) 293T cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 80 μg/ml gentamycin. Transient transfections were performed with calcium phosphate using reagents from Sigma. BV-2 microglial cells were cultured in RPMI 1640 medium supplemented with 10% FCS and 80 μg/ml gentamicin. Cells were changed to serum-free RPMI without antibiotics 16 h before the addition of LPS (Sigma). HEK-TLR4-MD2/CD14 cells were kindly provided by Dr. Manuel Fresno (Centro de Biología Molecular Severo Ochoa (CBMSO), Madrid, Spain). These cells were grown in DMEM containing 4.5 g/liter glucose, 4 mm l-glutamine, 10% fetal bovine serum, 50 units/ml penicillin, 50 μg/ml streptomycin, 10 μg/ml blasticidin and 50 μg/ml Hygrogold. The p65−/− mouse embryo fibroblasts (MEFs) and their corresponding wild type p65+/+ MEFs were kindly provided by Dr. Jianping Ye (Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, LA) (18Gao Z. Yin J. Zhang J. He Q. McGuinness O.P. Ye J. Inactivation of NF-κB p50 leads to insulin sensitization in liver through post-translational inhibition of p70S6K.J. Biol. Chem. 2009; 284: 18368-18376Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The NRF2−/− and NRF2+/+ MEFs were isolated around embryonal day 17. Skin pieces were minced in 3-cm Petri dishes and underwent enzymatic digestion at 37 °C for 30 min in Falcon tubes with 1 ml of HyQTase (Hyclone). The digested tissue was passed through a 160-μm Nitex filter into 3-cm Petri dishes, and the enzymes were neutralized with DMEM plus 15% FCS (Sigma). The resulting suspension was centrifuged at 450 × g for 5 min, and the cells were plated at a density of 1 × 106 cells/cm2 in culture flasks. The fibroblasts were placed in an incubator at 37 °C supplemented with 5% CO2, and medium was changed daily for the first 3 days. After near confluence, cells were detached with HyQTase and replated at a density of 1 × 106 cells/cm2 in 6-cm plates every third day. MEFs were grown in DMEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 2 mm l-glutamine. Sulforaphane (SFN) (LKT Laboratories, St. Paul, MN) was used at 14 μm for 6 h. The RAC1 binding domain from GST fusion of the PAK1 p21 binding domain (19Teramoto H. Coso O.A. Miyata H. Igishi T. Miki T. Gutkind J.S. Signaling from the small GTP-binding proteins Rac1 and Cdc42 to the c-Jun N-terminal kinase/stress-activated protein kinase pathway. A role for mixed lineage kinase 3/protein-tyrosine kinase 1, a novel member of the mixed lineage kinase family.J. Biol. Chem. 1996; 271: 27225-27228Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar) was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. BV-2 were treated for different times with LPS (500 ng/ml) and then lysed in a buffer composed of 50 mm Tris-HCl, pH 7.5, 1% Triton X-100, 100 mm NaCl, 10 mm MgCl2, 5% glycerol, 1 mm Na3VO4, and protease inhibitors (20 μg/ml aprotinin; pepstatin and leupeptin (1 μg/ml each). Lysates were centrifuged at 15,000 × g for 10 min, and the Triton X-100-soluble fraction was recovered. A bacterial lysate containing the GST-PAK1 fusion protein was added to BV-2 lysates together with glutathione-Sepharose beads. After 1 h the beads were collected by centrifugation and washed 3 times in 25 mm Tris-HCl, pH 7.5, 1% Triton X-100, 1 mm dithiothreitol, 100 mm NaCl, and 30 mm MgCl2. The beads were then resuspended in Laemmli sample buffer and boiled under reducing conditions for 5 min. The precipitated proteins were subjected to 12% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked in TBS supplemented with 0.2% Tween 20 and 5% milk and then incubated for 1 h with a primary antibody (anti-RAC1) and thereafter washed 3 times for 5 min in TBS supplemented with 0.2% Tween 20. The membranes were subsequently incubated for 1 h with peroxidase-conjugated anti-mouse IgGs (1:10,000) in TBS supplemented with 0.2% Tween 20. The blots were extensively washed, and antibody binding was visualized by enhanced chemiluminescence. In another set of experiments we confirmed RAC1 activation with anti-active GTP-RAC1 antibody (#26903, NewEast Biosciences). Expression vectors pcDNA3.1/V5HisB-mNRF2 and pcDNA3.1/V5HisB-mNRF2ΔETGE are described in McMahon et al. (20McMahon M. Thomas N. Itoh K. Yamamoto M. Hayes J.D. Redox-regulated turnover of Nrf2 is determined by at least two separate protein domains, the redox-sensitive Neh2 degron and the redox-insensitive Neh6 degron.J. Biol. Chem. 2004; 279: 31556-31567Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). For luciferase assays, transient transfections of HEK293T cells were performed with the expression vectors pHO1–15-LUC, ARE-LUC, pEF-ΔNRF2(DN), and ARE-mut-LUC (Dr. J. Alam, Dept. of Molecular Genetics, Ochsner Clinic Foundation, New Orleans, LA). pcDNA3-IκBS32A/S36A was described in Traenckner et al. (21Traenckner E.B. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. Phosphorylation of human IκB-α on serines 32 and 36 controls I κ B-α proteolysis and NF-κB activation in response to diverse stimuli.EMBO J. 1995; 14: 2876-2883Crossref PubMed Scopus (931) Google Scholar), and pCEFL-AU5-RAC1Q61L was kindly provided by Dr. Silvio Gutkind, NIDCR, National Institutes of Health, Bethesda, MD. pCCMV-p65, pCCMV-p50, and NF-κB-dependent (−453/+80)-HIV-LUC are described in Rojo et al. (22Rojo A.I. Salinas M. Martín D. Perona R. Cuadrado A. Regulation of Cu/Zn-superoxide dismutase expression via the phosphatidylinositol 3 kinase/Akt pathway and nuclear factor-κB.J. Neurosci. 2004; 24: 7324-7334Crossref PubMed Scopus (170) Google Scholar). pcDNA3-Gal4-NRF2 was generated in our laboratory and described in Espada et al. (23Espada S. Rojo A.I. Salinas M. Cuadrado A. The muscarinic M1 receptor activates Nrf2 through a signaling cascade that involves protein kinase C and inhibition of GSK-3β: connecting neurotransmission with neuroprotection.J. Neurochem. 2009; 110: 1107-1119Crossref PubMed Scopus (52) Google Scholar). Transient transfections of HEK293T cells were performed with the expression vectors for TK-Renilla (Promega, Madison, CA) and the corresponding firefly luciferase reporters. Cells were seeded on 24-well plates (100,000 cells per well), cultured for 16 h, and transfected using calcium phosphate. After 24 h of recovery from transfection, the cells were lysed and assayed with a dual-luciferase assay system (Promega) according to the manufacturer's instructions. Relative light units were measured in a GloMax 96 microplate luminometer with dual injectors (Promega). Cells were washed once with cold PBS and lysed on ice with radioimmune precipitation assay lysis buffer (150 mm NaCl, 25 mm Tris-HCl, pH 7.5, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 1 mm phenylmethylsulfonyl fluoride, 1 mm NaF, 1 mm sodium pyrophosphate, 1 mm sodium orthovanadate, 1 μg/ml leupeptin, 1 mm EGTA). Precleared cell lysates were resolved in SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Billerica, MA). The primary antibodies used were anti-V5 (Invitrogen), anti-GAPDH (Merck), anti-NF-κB (p65, RelA) (Calbiochem, Merck), anti-β-actin (sc-1616) and anti-lamin B (sc-6217) (Santa Cruz Biotechnology), anti-HO-1 (AB1284, Millipore), anti-AU5 (Covance, Berkeley, CA), anti-IBA1 (ab5076, Abcam, Cambridge, UK), anti-NRF2 (1:1000, Abyntek, Spain), anti-MnSOD (Stressgen), and anti-XIAP (Sigma). These membranes were analyzed using the primary antibodies indicated above and the appropriate peroxidase-conjugated secondary antibodies. Proteins were detected by enhanced chemiluminescence (GE Healthcare). HEK293T and HEK-TLR4-MD2/CD14 cells were seeded in p100 plates (2 × 106 cells/plate) and transfected with calcium phosphate or treated with 1 mg/ml LPS, respectively. Cytosolic and nuclear fractions were prepared as described previously (22Rojo A.I. Salinas M. Martín D. Perona R. Cuadrado A. Regulation of Cu/Zn-superoxide dismutase expression via the phosphatidylinositol 3 kinase/Akt pathway and nuclear factor-κB.J. Neurosci. 2004; 24: 7324-7334Crossref PubMed Scopus (170) Google Scholar). Briefly, cells were washed with cold PBS and harvested by centrifugation at 1100 rpm for 10 min. The cell pellet was resuspended in 3 pellet volumes of cold buffer A (20 mm HEPES, pH 7.0, 0.15 mm EDTA, 0.015 mm EGTA, 10 mm KCl, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 20 mm NaF, 1 mm sodium pyrophosphate, 1 mm sodium orthovanadate, 1 μg/ml leupeptin) and incubated in ice for 30 min. Then the homogenate was centrifuged at 500 × g for 5 min. The supernatants were taken as the cytosolic fraction. The nuclear pellet was resuspended in 5 volumes of cold buffer B (10 mm HEPES, pH 8.0, 0.1 mm EDTA, 0.1 mm NaCl, 25% glycerol, 1 mm phenylmethylsulfonyl fluoride, 20 mm NaF, 1 mm sodium pyrophosphate, 1 mm sodium orthovanadate, 1 μg/ml leupeptin). After centrifugation in the same conditions indicated above, the nuclei were resuspended in loading buffer containing 0.5% SDS. The cytosolic and nuclear fractions were resolved in SDS-PAGE and immunoblotted with the indicated antibodies. Total RNA from microglial primary cultures and from BV-2 cells was extracted using TRIzol reagent according to the manufacturer's instructions (Invitrogen). One microgram of RNA from each experimental condition was treated with DNase (Invitrogen) and reverse-transcribed using 4 μl of High capacity RNA-to-cDNA Master Mix (Applied Biosystems, Foster City, CA). For real-time PCR analysis, we performed the method previously described by Rojo et al. (13Rojo A.I. Innamorato N.G. Martín-Moreno A.M. De Ceballos M.L. Yamamoto M. Cuadrado A. Nrf2 regulates microglial dynamics and neuroinflammation in experimental Parkinson's disease.Glia. 2010; 58: 588-598Crossref PubMed Scopus (256) Google Scholar). Primer sequences are shown in Table 1. To ensure that equal amounts of cDNA were added to the PCR, the β-actin housekeeping gene was amplified. Data analysis is based on the ΔΔCt method with normalization of the raw data to housekeeping genes as described in the manufacturer's manual (Applied Biosystems, Invitrogen). All PCRs were performed in triplicate.TABLE 1Genes and primers for quantitative PCR amplificationGene productForward primerReverse primerGpx5′-GGACTACACCGAGATGAACG-3′5′-GATGTACTTGGGGTCGGTCA-3′Gstm35′-GAGAAGCAGAAGCCAGAGT-3′5′-ATACGATACTGGTCAAGAAT-3′Ho-15′-CACAGATGGCGTCACTTCGTC-3′5′-GTGAGGACCCACTGGAGGAG-3′IL-1β5′-CTGGTGTGTGACGTTCCCATTA-3′5′-CCGACAGCACGAGGCTTT-3′Mn-SOD5′-AACAATCTCAACGCCACCGA-3′5′-GATTAGAGCAGGCAGCAATC-3′NRF25′-CCCGAAGCACGCTGAAGGCA-3′5′-CCAGGCGGTGGGTCTCCGTA-3′NQO15′-GGTAGCGGCTCCATGTACTC-3′5′-CATCCTTCCAGGATCTGCAT-3′p655′-GGCCTCATCCACATGAACTT-3′5′-CACTGTCACCTGGAAGCAGA-3′TNF5′-CATCTTCTCAAAATTCGAGTGACAA-3′5′-TGGGAGTAGACAAGGTACAACCC-3′β-Actin5′-TCCTTCCTGGGCATGGAG-3′5′-AGGAGGAGCAATGATCTTGATCTT-3′ Open table in a new tab The fluorescent probe 2′-7′-dihydrodichlorofluorescein diacetate was used to assess ROS production (24Hernández-Fonseca K. Cárdenas-Rodríguez N. Pedraza-Chaverri J. Massieu L. Calcium-dependent production of reactive oxygen species is involved in neuronal damage induced during glycolysis inhibition in cultured hippocampal neurons.J. Neurosci Res. 2008; 86: 1768-1780Crossref PubMed Scopus (63) Google Scholar). Cells were transfected, and after 24 h of recovery from transfection, the cells were incubated with 2′-7′-dihydrodichlorofluorescein diacetate 100 mm for 1 h. Fluorescence intensity was measured in a Synergy fluorescence microplate reader (Bio-Tek Instruments, Inc., Winooski, VT). The double-stranded oligonucleotide used for the NF-κB probe was 5′-AGTTGAGGGACTTTCCCAGGC-3′ (25Pan Q. Bao L.W. Merajver S.D. Tetrathiomolybdate inhibits angiogenesis and metastasis through suppression of the NFκB signaling cascade.Mol. Cancer Res. 2003; 1: 701-706PubMed Google Scholar) and for Nrf2 probe was 5′-TTTTATGCTGTGTCATGGTT-3′ (26Stewart D. Killeen E. Naquin R. Alam S. Alam J. Degradation of transcription factor Nrf2 via the ubiquitin-proteasome pathway and stabilization by cadmium.J. Biol. Chem. 2003; 278: 2396-2402Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Then 250 ng/μl concentrations of each oligonucleotide was annealed by incubation in STE buffer (100 mm NaCl, 10 mm Tris-HCl, pH 8.0, and 1 mm EDTA) at 80 °C for 2 min. The mixture was cooled down slowly to 4 °C, with a thermal profile of 1 °C/min. Annealed oligonucleotides were diluted to 25 ng/μl in STE buffer. 5′-End labeling was performed with T4 polynucleotide kinase (Promega) using 25 ng of double-stranded oligonucleotide and 25 μCi of [γ-32P]ATP (3000 Ci/mmol; Amersham Biosciences). The labeled probes were purified in a G-25 spin column (Amersham Biosciences). The binding reaction mixture contained 5 μg of nuclear protein extract diluted in a buffer containing, at final concentration, 40 mm HEPES, pH 8.0, 50 mm KCl, 0.05% Nonidet P-40, 1% dithiothreitol, and 10 μg/ml poly(dI-dC) in a total volume of 20 μl. Unlabeled competitor probe (25 ng) was added, and the reaction was incubated for 1 h at 25 °C. Labeled DNA (0.25 ng) was added to the mixture and was submitted to an additional 20 min incubation at 25 °C. Samples were resolved at 4 °C in a 6% non-denaturing polyacrylamide gel in 0.5× Tris borate/EDTA buffer. After electrophoresis the gel was dried and autoradiographed. Data are presented as the mean ± S.E. One and two-way analysis of variance with post hoc Newman-Keuls test and Bonferroni's test were used as appropriate. Student's t test was used to assess differences between groups. The Prism Version 5.03 software for Windows (GraphPad, San Diego, CA) was used as described in each figure legend. RAC1 activity was analyzed in cell lysates of BV-2 cells treated with LPS in a GST pulldown assay. This assay was based on the use of a chimeric protein made of glutathione S-transferase fused to the RAC1 interactive binding domain of p21-activated kinase 1 (Fig. 1A). We found that LPS (500 ng/ml) increased the levels of active RAC1 within 5 min and lasted for at least 40 min (Fig. 1B), indicating that RAC1 is implicated in LPS signaling. Moreover, LPS induced the expression of the antioxidant enzyme HO-1 in a dose-dependent fashion after 6 h (Fig. 1, C and D) and more markedly after 24 h (Fig. 1, E and D). To control that LPS was inducing an inflammatory response in BV-2 microglial cells, we determined the protein levels of IBA1, which is expressed in microglia and is up-regulated during the activation of these cells. LPS induced a dose-dependent and time-dependent increase of IBA1 protein levels (Fig. 1, C, E, and F), which correlated with the induction of HO-1. We also measured messenger RNA (mRNA) levels by qRT-PCR of two NRF2-regulated genes coding HO-1 and NAD(P)H dehydrogenase, quinone 1(NQO1) and two proinflammatory cytokines, IL-1β and TNF (Fig. 1, G–J, respectively) and found that treatment with LPS for 24 h increased the mRNA expression of NRF2-regulated genes and proinflammatory cytokines. These results indicate that RAC1 is activated very shortly after LPS stimulation leading to long term transcriptional modifications that include increased expression of HO-1. LPS stimulation of HEK-TLR4-MD2/CD14 cells (human HEK 293T cells are completely unresponsive to LPS but acquire high LPS sensitivity after transient transfection with CD14, TLR4, and MD-2) induced the expression of protein and mRNA levels of HO-1 (Fig. 2, A–C) after 6 and 24 h. Increased TNF mRNA levels confirmed the induction of inflammation due to LPS treatment (Fig. 2D). Besides, LPS induced nuclear translocation of p65 (Fig. 2, E and F) and NRF2 (Fig. 2, E and G) in a time-dependent manner and reached a maximum after 60 min. As observed in BV2 cells, LPS increased the levels of endogenous active RAC1 (Fig. 2, H and I), indicating that RAC1 activation is an essential effector of LPS inflammatory process. To analyze in depth the relevance of RAC1 activation on the NRF2 and NF-κB pathways, we transfected HEK293T cells with a constitutively active mutant of RAC1 (AU5-tagged RAC1Q61L) and analyzed the endogenous protein levels of HO-1 as a reporter of NRF2 transcriptional activity. Our results showed that RAC1Q61L increased HO-1 levels in a dose-response manner (Fig. 2, J and K), suggesting a direct connection between RAC1 activation and expression of the gene coding HO-1 (HMOX1). To further confirm these data, we analyzed the induction of two firefly luciferase reporters containing either 15 kb of the mouse Hmox1 promoter or three tandem sequences of the NRF2-dependent antioxidant response element (ARE-LUC) derived from the Hmox1 promoter. We found a dose-dependent activation of both reporters (Fig. 2, L and M). These results indicate that NRF2 mediates the RAC1-dependent up-regulation of HO-1 expression. To determine if RAC1Q61L leads to an increase in NRF2 protein levels, we co-transfected HEK293T cells with different concentrations of RAC1Q61L and a constant concentration of a V5-tagged wild type form of NRF2 (NRF2-V5) or a stable mutant of NRF2, NRF2ΔETGE-V5, that lacks four residues (ETGE) essential for recognition by the E3 ligase complex Cul3/Keap1. The results indicated that RAC1Q61L produced a dose-dependent accumulation of both wild type NRF2-V5 and mutant NRF2ΔETGE-V5 (Fig. 3, A and B). Moreover, we observed by subcellular fractionation that the overexpression of RAC1Q61L induced a very strong increase in NRF2 protein levels in both cytosol and nucleus (Fig. 3C). Moreover, the implication of NRF2 in RAC1-induced HO-1 expression was analyzed with two different strategies. First, HEK293T cells were co-transfected with the expression vector for RAC1Q61L, a dominant-negative mutant of NRF2 (DN-NRF2) lacking the transcriptional activation domain and the luciferase reporter HO-1-LUC. We observed that DN-NRF2 abolished the RAC1Q61L-mediated induction of HO-1-LUC reporter (Fig. 3D). Second, we used a one-hybrid assay to study the transactivating activity of NRF2 in the presence of RAC1Q61L. The assay consisted of the use of an expression vector for a fusion protein containing the DNA binding domain of the yeast transcription factor Gal4 and NRF2 (Gal4-NRF2). HEK293T cells were co-transfected with RAC1Q61L, Gal4-NRF2, and the Gal4-LUC reporter plasmid as indicated in Fig. 3E. After 16 h, cells co-transfected with RAC1Q61L and Gal4-NRF2 exhibited a very strong increase in luciferase activity compared with that of cells co-transfected with Gal4-LUC and empty vector. Interestingly, RAC1Q61L highly increased the transactivating activity of NRF2 compared with NRF2 alone (Fig. 3E), indicating that RAC1Q61L activates NRF2 by targeting its transactivation domain. Taken together, all these data showed that active RAC1 led to accumulation of NRF2, resulting in increased ARE gene expression." @default.
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• W2077895679 title "Transcription Factors NRF2 and NF-κB Are Coordinated Effectors of the Rho Family, GTP-binding Protein RAC1 during Inflammation" @default.
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